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Glucosamine and Females

1: GLUT2 is a high affinity glucosamine transporter.

FEBS Lett 2002 Jul 31;524(1-3):199-203

Uldry M, Ibberson M, Hosokawa M, Thorens B.

When expressed in Xenopus oocytes, GLUT1, 2 and 4 transport glucosamine with V(max) values that are three- to four-fold lower than for glucose. The K(m)s for glucosamine and glucose of GLUT1 and GLUT4 were similar. In contrast, GLUT2 had a much higher apparent affinity for glucosamine than for glucose (K(m)=0.8+/-0.1 mM vs. approximately 17-20 mM). Glucosamine transport by GLUT2 was confirmed in mammalian cells and, using hepatocytes from control or GLUT2-null mice, HgCl(2)-inhibitable glucosamine uptake by liver was shown to be exclusively through GLUT2. These data have implications for glucosamine effects on impaired glucose metabolism and for structure-function studies of transporter sugar binding sites.

2: Use of galactosyltransferase to assess the biological function of O-linked N-acetyl-d-glucosamine: a potential role for O-GlcNAc during cell division.

Exp Cell Res 2001 Feb 15;263(2):243-53

Fang B, Miller MW.

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl-d-glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes beta1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation. Copyright 2001 Academic Press.

3: Secretion of paracrine factors enabling expansion of cumulus cells is developmentally regulated in pig oocytes.

Biol Reprod 2000 Oct;63(4):1149-56

Nagyova E, Vanderhyden BC, Prochazka R.

To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metaphase I (MI), and metaphase II (MII) oocytes were prepared by culture of oocyte-cumulus complexes (OCC) for 0, 22, 27, and 42 h, respectively. To block GV breakdown, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I (50 microM). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of >90% of mouse OOX, whereas oocytes in MI and MII caused disintegration of mouse OOX without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse OOX were cultured in the conditioned media in the presence of 2.5 &mgr;Ci of D-[6-(3)H]glucosamine hydrochloride. After 18 h, incorporation of the [(3)H]glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse OOX was not different from that of intact OCC. However, oocytes in GV, LD, and after butyrolactone I treatment enabled mouse OOX to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that secretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation.

4: Evolution, structure, and expression of GNPI/Oscillin orthologous genes.

Genomics 2000 Sep 1;68(2):179-86

Nakamura Y, Miura K, Fujino Y, Iwao H, Ogita S, Yamanaka S.

Oscillin was identified from hamster sperm as a factor responsible for oocyte calcium oscillations. However, its high level of homology with the bacterial glucosamine-6-phosphate isomerase suggests that it may play more fundamental roles. In the current study, we identified Oscillin orthologs from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Their amino acid identities with hamster oscillin were 67.0, 72.3, 97.6, and 95.5%, respectively. No Oscillin orthologs were found in Saccharomyces cerevisiae. The human Oscillin gene (HGMW-approved symbol GNPI) spans 12.4 kb and consists of eight exons. The position of the fourth intron was conserved in other species. The human Oscillin promoter has features characteristic of housekeeping genes, including a GC-rich content, multiple SP1 binding sites, and the absence of a TATA motif. Human and mouse Oscillin genes were ubiquitously expressed in all tissues examined. These data showed that Oscillin is a housekeeping gene conserved throughout evolution and do not support the notion that Oscillin is the sperm-specific factor responsible for calcium oscillations. Copyright 2000 Academic Press.

5: Interspecific variation of zona pellucida glycoconjugates in several species of marsupial.

J Reprod Fertil 2000 May;119(1):111-20

Chapman JA, Wiebkin OW, Breed WG.

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.

6: Cloning, sequencing, and expression analysis of mouse glucosamine-6-phosphate deaminase (GNPDA/oscillin).

Mol Reprod Dev 2000 Jul;56(3):424-35

Amireault P, Dube F.

It was reported that a hamster protein, called "oscillin," with a sequence related to that of an Escherichia coli GNPDA triggered Ca(2+) oscillations in mammalian oocytes when introduced into their cytoplasm upon fertilization. Recently, it was shown that GNPDA/oscillin is ubiquitously expressed in rat tissues and that a recombinant hamster GNPDA/oscillin protein does not exhibit oscillin activity when injected into oocytes. In the mouse, the nature and role of such a GNPDA/oscillin is not known, but another candidate protein, tr-kit, has been proposed as a sperm factor causing oocyte activation. In order to clarify this issue, we have characterized the mouse homolog of hamster and human GNPDA/oscillin, and examined its expression along with that of tr-kit, in parallel. We report here the molecular cloning and sequencing of mouse GNPDA/oscillin, which shows over 96% identity with the hamster and human homologs. Using specific primers, we performed an RT-PCR analysis to determine the tissue distribution of mouse GNPDA/oscillin mRNA. Unlike tr-kit mRNA which is expressed solely in mouse testis, GNPDA/oscillin mRNA is detected in unfertilized oocytes and in all tissues examined including testis, heart, thymus, liver, ovary, uterus, kidney, spleen, and lung. The protein itself is also detected in all tissues examined by Western blots. Indirect immunofluorescence studies, using an antibody raised against hamster GNPDA, demonstrate that GNPDA is lost with the acrosome reaction of mouse spermatozoa, is localized in the equatorial and neck regions of the human spermatozoa and the post-acrosomal region of the hamster spermatozoa. Our results thus indicate that mouse GNPDA/oscillin, the homolog of hamster oscillin, unlike tr-kit, does not exhibit some of the required characteristics expected from a putative sperm-derived oocyte-activating factor. Copyright 2000 Wiley-Liss, Inc.

7: Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.

Biol Reprod 1999 Sep;61(3):569-74

Nagyova E, Prochazka R, Vanderhyden BC.

Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium.

8: Partial characterization of the calcium-releasing activity of porcine sperm cytosolic extracts.

Dev Biol 1998 Nov 15;203(2):369-81

Wu H, He CL, Jehn B, Black SJ, Fissore RA.

Injection of sperm cytosolic extracts into mammalian eggs has been shown to elicit intracellular calcium ([Ca2+]i) oscillations that are similar in amplitude, duration, and frequency to those observed following fertilization. Thus, to characterize the Ca2+-release component(s) in porcine sperm cytosolic extracts, a combination of fractionation techniques was used. The fraction with Ca2+ releasing activity was precipitated by 50% saturating solutions of ammonium sulfate and Western blot analysis showed that the pellets contained glucosamine-6-phosphate deaminase (gpd)/oscillin, a protein which has been suggested to be the sperm's active component. Single and double isoelectrofocusing (IEF) of porcine sperm extracts generated fractions with different Ca2+-releasing activities. Fractions with maximal Ca2+-releasing activity did not contain material that was immunoreactive with antibodies against gpd/oscillin; adjacent fractions containing gpd/oscillin had no Ca2+-releasing activity. These findings were confirmed by IEF coupled with size exclusion chromatography on Superose 12 and with hydroxyapatite chromatography. These procedures predict an isoelectric point of our active component of 6.5-7.0 and a relative molecular weight ranging from 29 to 68 kDa. In summary, the data show that the Ca2+ release-inducing component(s) of porcine sperm extracts can be fractionated and that gpd/oscillin is not the pig sperm Ca2+ oscillogen. Copyright 1998 Academic Press.

9: Effect of oocytectomy on glycosaminoglycan composition during cumulus expansion of porcine cumulus-oocyte complexes cultured in vitro.

Biol Reprod 1996 Dec;55(6):1299-304

Nakayama T, Inoue M, Sato E.

To investigate the role of oocytes in the accumulation of glycosaminoglycans during cumulus expansion, we analyzed the amount and composition of glycosaminoglycans in porcine intact and oocytectomized cumulus-oocyte complexes. Follicular fluid induced cumulus expansion in intact and oocytectomized cumulus-oocyte complexes cultured for over 24 h, but the degree of expansion in oocytectomized complexes reached only 76% of that in intact complexes after 24 h in culture. There were no differences in the amounts of [3H]glucosamine incorporated into each type of complex. Metabolic labeling studies on glycosaminoglycans with [3H]glucosamine and [35S]sulfate showed that the amounts of 3H- and 35S-labeled glycosaminoglycans increased, and rapidly so, from 16 h in culture. The 3H-labeled glycosaminoglycans at these high levels were digested by Streptomyces hyaluronidase or chondroitinase ABC, and the rate of increase in 3H-labeled glycosaminoglycans was reduced by oocytectomy. In contrast, the increased levels of 35S-labeled glycosaminoglycans were affected only by chondroitinase ABC, and oocytectomy was ineffective. In conclusion, follicular fluid promotes cumulus expansion, and the oocyte plays an essential role in the acute synthesis of hyaluronic acid, but not chondroitin sulfate, during cumulus expansion stimulated in vitro.

10: Characterization of the complex carbohydrates in the zona pellucida of mammalian oocytes using lectin histochemistry.

Vet Res Commun 1996;20(3):225-36

Parillo F, Stradaioli G, Dall'Aglio C, Verini-Supplizi A.

The objective of this study was to characterize the glycoconjugates present in the zona pellucida of the follicular oocytes in sheep, goats and pigs. The zona pellucida was stained with periodic acid-Schiff, low iron diamine, high iron diamine, and nine different lectin horseradish conjugates: Con-A, SBA, DBA, PNA, RCA-I, GSA-II, WGA, LTA and UEA-I. Staining with DBA, PNA, SBA and RCA-I was performed with and without saponification with KOH and sialidase digestion. The results showed the presence of neutral and acidic glycoconjugates with different terminal sugars and also sialic acid radicals in the zona pellucida of all the animal studied. In particular, the positive staining with WGA, SBA, PNA and RCA-I suggests the presence of oligosaccharides with N-acetyl-D-glucosamine and sialic acid linked to the penultimate beta-N-acetyl-D-galactosamine and to the disaccharide galactosyl-(beta 1-3)-N-acetyl-D-galactosamine. The terminal trisaccharide sialic acid galactosyl-(beta 1-4)-N-acetyl-D-glucosamine was identified only in the zona pellucida of ovine and porcine oocytes. Thus, the zona pellucida exhibited species-specific variations in the content and distribution of lectin-binding patterns that may reflect the species specificity of gamete interaction.

11: Interaction of lectins with Cryptosporidium parvum.

J Infect Dis 1993 Jun;167(6):1477-80

Llovo J, Lopez A, Fabregas J, Munoz A.

Cell surface carbohydrates from four clinical isolates of Cryptosporidium parvum were analyzed by agglutination assays using a battery of 20 highly purified lectins with affinity for receptor molecules containing N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine, galactose, mannose, glucose, fucose, and N-acetyl-neuraminic acid. Tomentine, a lectin from the green seaweed Codium tomentosum, and UEA-II lectin, from Ulex europeus, both of them GlcNAc-specific lectins, agglutinated the oocysts. Subsequent inhibition assay confirmed the presence of this sugar on the surface of Cryptosporidium parvum oocysts. Codium fragile lectin, from another green seaweed, also exhibited agglutination activity against the oocysts. This is the first published demonstration of such an interaction between a human coccidian and lectins from seaweeds.

12: Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass.

Mol Reprod Dev 1993 Jan;34(1):87-93

Chen L, Russell PT, Larsen WJ.

Gonadotropin-stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2-cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation.

13: Artificial activation of porcine oocytes matured in vitro.

Mol Reprod Dev 1991 Apr;28(4):405-9

Prather RS, Eichen PA, Nicks DK, Peters MS.

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evaluated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29 degrees C activated at lower rates (45.5%) than those returning at 36 degrees C (78.9%). An experiment was designed to evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed to TL-Hepes or allowed an additional 2 minutes in EM prior to rinsing in TL-Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61% and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.

14: Timing of sequential changes in chromosome configurations during the 1st meiotic division of pig oocytes cultured in vitro.

Jpn J Vet Res 1990 Dec;38(3-4):127-37

Ocampo MB, Ocampo LT, Kanagawa H.

This study examines the timing of changes in chromosome configurations of pig oocytes derived from small antral follicles of follicular and/or inactive stage donors using modified 199 medium supplemented with gonadotropins (Follicle Stimulating Hormone (FSH), 10 IU/ml; Human Chorionic Gonadotropin (hCG), 10 IU/ml) and Glucosamine (0.539 mg/ml). Oocytes (n = 1,215) were fixed at the end of 3 hourly intervals from 0-48 hr of culture. Results were expressed as the percentage of oocytes at each stage of maturation for each time point. The germinal vesicle (GV) stage was observed for the first 17.6 hr; germinal vesicle breakdown (GVBD) stage between 17.6-26.4 hr; metaphase I (M-I) from 26.4-30.9 hr; anaphase I (A-I) ranged from 30.9-33.4 hr; telophase I (T-I) at 33.4-34.4 hr; and metaphase II (M-II) at 34.4-48 hr.

15: Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process.

J Cell Biol 1990 Oct;111(4):1571-82

Bataille N, Helser T, Fried HM.

To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.

16: Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass.

Mol Reprod Dev 1990 Jul;26(3):236-47

Chen L, Wert SE, Hendrix EM, Russell PT, Cannon M, Larsen WJ.

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

17: Selective activation of the N-glycosylation apparatus in uteri by estrogen.

J Biol Chem 1990 Feb 15;265(5):2947-55

Carson DD, Farrar JD, Laidlaw J, Wright DA.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional

18: Protein import through the nuclear pore complex is a multistep process.

J Cell Biol 1989 Sep;109(3):971-82

Akey CW, Goldfarb DS.

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.

19: Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification.

J Biol Chem 1989 Aug 15;264(23):13840-7

Salustri A, Yanagishita M, Hascall VC.

In most mammalian ovaries, the cumulus cell-oocyte complex (COC) expands at the time of ovulation by depositing an extensive extracellular matrix between the cumulus cells. This phenomenon can be reproduced in vitro by culturing COCs with follicle-stimulating hormone (FSH) and serum. Biosynthesis of hyaluronic acid (HA) and proteoglycans by mouse COCs in vitro was studied using [3H]glucosamine and [35S]sulfate as metabolic precursors. Radiolabeled complex carbohydrates were analyzed by ion exchange chromatography, specific enzyme digestion followed by high performance liquid chromatography, and gel filtration. The specific activities of [3H]hexosamines in the labeled molecules were determined by measuring the incorporation of 3H and 35S into chondroitin 4-sulfate disaccharides. When COCs were stimulated with FSH, HA biosynthesis increased 20-30-fold between 3-12 h later when expansion occurs, reaching a maximum rate of approximately 780 pmol (as glucosamine)/COC/h compared with the unstimulated rate of approximately 26 pmol/COC/h. The final concentration of HA in the expanded COC was calculated to be approximately 250 micrograms/ml. The effects of dibutyryl cyclic AMP (Bt2cAMP) on COC expansion and HA synthesis were similar to those of FSH, suggesting that the effects of FSH are mediated by cAMP. However, FSH significantly decreased the specific activity of the incorporated hexosamines while Bt2cAMP did not. Serum is necessary for the accumulation of HA in the COC matrix. HA synthesis in FSH-stimulated COCs was as high or higher in the absence of serum, but most was recovered in the medium and not in the COC matrix. The molecular size of the HA was greater than 2 million dalton in either case, suggesting that the serum did not alter physical properties of HA. Stimulation of proteoglycan biosynthesis by either FSH or Bt2cAMP was less pronounced (three to four times control) than for HA and was sustained throughout an 18-h culture period. A reduction of 80% in the deposition of newly synthesized PGs in the COC matrix by 0.5 mM beta-xyloside treatment did not affect the expansion of the cumulus.

20: Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization.

Am J Obstet Gynecol 1989 Jul;161(1):207-11

Mori K, Daitoh T, Irahara M, Kamada M, Aono T.

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.

21: Histochemistry of glycoconjugates in ovarian follicles of the adult house musk shrew, Suncus murinus.

Acta Anat (Basel) 1989;136(4):269-78

Suprasert A, Hirunagi K, Fujioka T, Yokoyama A.

In ovarian follicles of the adult house musk shrew, Suncus murinus, glycoconjugates have been studied by means of light- and electron-microscopic histochemistry. The results obtained are that: (1) glycoconjugates of the zona pellucida of oocytes are provided with vicinal diol and acidic groupings, and sialic acid-galactose dimer, alpha-D-mannose, alpha-D-glucose, beta-D-galactose, N-acetyl-D-glucosamine and alpha-L-fucose residues; (2) glycoconjugates of the intercellular matrix of the granulosa and theca folliculi are comparable in histochemical properties to those of the zona pellucida, except for the relatively smaller amount of vicinal diol and acidic groupings, and (3) the zona pellucida can be divided into outer and inner layers by the binding degrees of the lectins used. The possible histophysiological significances of all these glycoconjugates are discussed with special reference to the particular ovarian follicular structures.

22: 1-Deoxymannojirimycin inhibits Golgi-mediated processing of glycoprotein in Xenopus oocytes.

FEBS Lett 1988 Jul 18;234(2):489-92

Erratum in: FEBS Lett 1988 Oct 24;239(1):159

Fabbrini MS, Zoppe M, Bollini R, Vitale A.

We prepared in vitro an mRNA transcript coding for the erythroagglutinating subunit of the kidney bean glycoprotein phytohemagglutinin, E-PHA. The mRNA, injected into Xenopus oocytes, synthesized E-PHA carrying two Asn-linked carbohydrate chains, one of which was processed and acquired resistance to endo-beta-N-acetylglucosaminidase H, as occurs in the native bean cells. When the mannose analog 1-deoxymannojirimycin, an inhibitor of mammalian Golgi mannosidase I, was included in the oocyte culture medium, the acquisition of endo-beta-N-acetylglucosaminidase H resistance was abolished, indicating that also in an amphibian cell the inhibitor blocks a key reaction in Golgi-mediated processing.

23: An autoradiographic study of gonadotrophin regulation of labelled glycoconjugates within preovulatory mouse follicles during the final stages of oocyte maturation, using [3H]glucosamine as the radioactive precursor.

J Reprod Fertil 1988 Jul;83(2):759-72

Fowler RE.

Immature mice were treated with PMSG and hCG to induce follicular development and ovulation. [3H]Glucosamine was injected at the same time as the PMSG or 2 h before autopsy. The synthesis and distribution of labelled glycoconjugates within the preovulatory follicles was hormonally dependent. PMSG stimulated a rapid uptake of [3H]glucosamine into the zona pellucida and follicular fluid of the largest antral follicles although labelled macromolecules could not be demonstrated in any of the cellular components of these follicles. The injection of hCG stimulated a rapid incorporation of labelled macromolecules into the cellular components of the preovulatory follicle, namely into thecal, granulosa and especially the cumulus cells surrounding the oocyte. The density of labelled macromolecules within the follicular fluid also increased, while the specific and uniform labelling of the zona pellucida which was so characteristic of the period of PMSG stimulation changed. Between 4 and 8 h after the injection of hCG, labelled glycoconjugates containing [3H]glucosamine, became increasingly associated with the outer surface of the zona pellucida and with the region of the egg plasma membrane, even in Graafian follicles not destined to ovulate. The change in distribution of labelled macromolecules on the zona surface may be a prerequisite for successful sperm-zona binding and the specific association of labelled glycoconjugates in the region of the egg plasma membrane may be involved in the preparation of the egg surface for sperm-egg interactions involving cortical granule exocytosis and the block to polyspermy.

24: An autoradiographic study using [3H]glucosamine of gonadotrophin regulation of proteoglycan and glycoprotein synthesis in developing mouse follicles.

J Reprod Fertil 1987 Nov;81(2):415-26

Fowler RE, Guttridge K.

Autoradiography of serial sections of ovaries of immature mice was used, after an injection of [3H]glucosamine, to follow the migration of newly synthesized macromolecules into preantral follicles during the period of treatment with PMSG and hCG. [3H]Glucosamine was injected at the same time as the PMSG or 2-h before the time of autopsy. PMSG stimulated a modest uptake of [3H]glucosamine into the zona pellucida of preantral follicles. However, the in-vivo synthesis of labelled macromolecules increased substantially during the period of hCG stimulation, especially in those mice in which the label was injected at the same time as the PMSG. After both short and longer term exposure to [3H]glucosamine, the maximum uptake of label in preantral follicles occurred 4-8 h after the injection of hCG, indicating that hCG rather than PMSG probably exerts the greatest control over the uptake and incorporation of [3H]glucosamine into the zona pellucida and oocyte of preantral follicles. It is suggested that [3H]glucosamine is largely incorporated into non-sulphated glycosaminoglycans.

25: Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices.

Histochemistry 1986;85(6):523-8

Tesarik J, Kopecny V.

Light- and electron-microscope autoradiography using 3H-glucosamine and 3H-fucose as precursors was employed to investigate proteoglycan synthesis and secretion by late preovulatory human oocytes and cumulus cells. Both the oocyte and cumulus cells were found to be important cellular sources supplying proteoglycans to the oocyte-cumulus-complex extracellular matrices, i.e., the zona pellucida and the cumulus intercellular matrix. Both the oocyte and cumulus cells were shown to secrete labelled proteoglycans into the zona pellucida. Labelled proteoglycans were also detected in the cumulus intercellular matrix. Chase experiments revealed the labelled molecules to be relatively closely associated with both the zona pellucida and the cumulus intercellular matrix. Staining with chromic acid and phosphotungstic acid showed proteoglycan material to penetrate from the cumulus intercellular matrix into pores of the zona pellucida. This material is thought to be a structural equivalent of the newly synthesized proteoglycans secreted by cumulus cells and migrating into the zona pellucida (as detected by autoradiography). It is concluded that newly synthesized proteoglycans secreted by the oocyte and cumulus cells in the late preovulatory period are a component of the microenvironment in which fertilization takes place.

26: Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs.

J Exp Zool 1985 Sep;235(3):335-40

Prody GA, Greve LC, Hedrick JL.

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.

27: In vitro maturation and fertilization of bovine oocytes are temperature-dependent processes.

Biol Reprod 1983 Aug;29(1):173-9

Lenz RW, Ball GD, Leibfried ML, Ax RL, First NL.

Effects of temperature on bovine sperm acrosome reaction, oocyte maturation, hyaluronic acid production by cumulus cells and in vitro fertilization were studied. Viability and a true acrosome reaction of bovine spermatozoa were impaired at 40 degrees C. Temperatures lower than 35 degrees C did not enhance the acrosome reaction. However, viability between 30 degrees C-38 degrees C was not altered after 22 h of incubation. The optimal temperature for the acrosome reaction was 38 degrees C. Labeled glucosamine incorporation into glycosaminoglycans was not different among temperatures of 35 degrees C, 37 degrees C or 39 degrees C, whereas 41 degrees C caused a significant reduction (P less than 0.02). Temperatures ranging between 35 degrees C-39 degrees C had no deleterious effects on resumption and completion of meiosis, but at 41 degrees C the frequency of oocytes that progressed to Metaphase II was significantly reduced (P less than 0.0001). Ova matured at 39 degrees C had significantly higher rates of fertilization than at 35 degrees C, 37 degrees C, or 41 degrees C. Killed spermatozoa (control) had no effect on ovum activation at 39 degrees C. From these results it was concluded that events occurring prior to and during fertilization are temperature sensitive.

28: Glycosaminoglycans in bovine cumulus-oocyte complexes: morphology and chemistry.

Mol Cell Endocrinol 1982 Sep;28(1):113-122

Ball GD, Bellin ME, Ax RL, First NL.

Bovine cumulus-oocyte complexes from small (1-5 mm) follicles were cultured for 24 h in 0.25 ml minimum essential medium supplemented with 10% fetal bovine serum and 20 microCi [3H]glucosamine. Treatment groups consisted of supplementing the culture medium with no hormone (control), 0.5 IU/ml follicle-stimulating hormone (FSH) or 10 mM 8-Br-adenosine cyclic monophosphate (cAMP). After culture, the complexes were fixed for light and scanning electron microscopy. Electron photomicrographs revealed that complexes induced to expand with FSH or cAMP contained a copious glycosaminoglycan (GAG) matrix extending between and around the cumulus cells. Control complexes did not exhibit expansion or an extracellular matrix. The radiolabeled GAG material was isolated for chemical identification. Chemical analyses included: (1) electrophoresis of GAG material, (2) electrophoresis of GAG material after enzyme or nitrous acid treatment, (3) thin-layer chromatography of GAG hydrolysates. The results from electrophoresis showed that the radiolabeled GAG co- migrated with hyaluronic acid. The GAG material was resistant to chondroitinase ABC and nitrous acid degradation but was digested by hyaluronidase. Complexes treated with FSH and cAMP incorporated higher (P less than 0.1 and P less than 0.025 respectively) amounts of [3H]glucosamine into hyaluronic acid than control cultures. Thin-layer chromatography identified the primary amino sugar of the GAG to be glucosamine. These data collectively showed that the radioactive GAG produced by bovine cumulus-oocyte complexes was hyaluronic acid.

29: Effect of removal of carbohydrate residues upon the half life and in vivo biological activity of human chorionic gonadotropin.

Adv Exp Med Biol 1979;112:749-56

Batta SK, Rabovsky MA, Channing CP, Bahl OP.

The effect of progressive enzymatic removal of various carbohydrates on the half life (T 1/2) and biologic activity in vivo of hCG was studied in rats. The cleavage of carbohydrates such as galactose, glucosamine and mannose, in addition to sialic acid, reduced the T 1/2 from 60--90 for native hCG to less than 3 minutes. The hCG derivatives had no inherent ability to cause ovulation, but in PMSG, Nembutal-treated immature rats, the hCG derivatives with carbohydrate residues internal to sialic acid removed blocked the stimulatory effect of native hCG upon ovulation.

30: Studies of a glycoprotein in the oocysts of Eimeria tenella.

J Biol Chem 1976 Jan 25;251(2):302-7

Stotish RL, Wang CC, Hichens M, VandenHeuvel WJ, Gale P.

A glycoprotein unique to the cytoplasm of the unsporulated oocyst of Eimeria tenella has been purified and partially characterized. The protein has a molecular weight of 30,000, of which approximately 40% is carbohydrate. The carbohydrate portion of the molecule consists of glucose, galactose, mannose, xylose, glucosamine, and galactosamine, with no detectable sialic acid. The protein portion contains approximately 141 residues, being rich in hydrophilic amino acids with very few aromatic amino acids and no cystine. The protein comprises 14% of the total soluble protein of the unsporulated oocyst but has not been identified in the cytoplasm of any other developmental stage of the organism. Using polyacrylamide gel electrophoresis and a radioimmunoassay specific for the protein, it has been shown to disappear from the cytoplasm between the 15th and 20th hour of the 20-hour sporulation process. Subsequent immunofluorescence experiments have shown a reactive material as a component of the sporozoite membrane. These results indicate that the glycoprotein is a structural protein of the sporozoite membrane, apparently synthesized by the unsporulated oocyst and incorporated into the sporozoite membrane as one of the last steps involved in the sporulation process.

31: Oogenesis in the bluefin tuna, Thunnus thynnus L.: a histological and histochemical study.

Histol Histopathol 2002;17(3):775-88

Sarasquete C, Cardenas S, de Gonzalez CM, Pascual E.

Histology and histochemistry are useful tools to study reproductive mechanisms in fish and they have been applied in this study. In the bluefin tuna, Thunnus thymus L., oocyte development can be divided into 4 principal phases based on the morphological features of developing oocytes and follicles. The primary growth phase includes oogonia and basophilic or previtellogenic oocytes classified as chromatin-nucleolus and perinucleolus stages. The secondary growth phase is represented by vitellogenic oocytes at early (lipid globule and yolk granule 1), mid (yolk granule 2) and late (yolk granule 3) vitellogenesis stages. The maturation phase involves postvitellogenic oocytes undergoing maturation process. During the spawning period, both postovulatory follicles, which indicate spawning, and atretic follicles can be distinguished in the ovary. Carbohydrates, lipids, proteins and specially those rich in tyrosine, tryptophan, cystine, arginine, lysine and cysteine, as well phospholipids and/or glycolipids and neutral glycoproteins were detected in yolk granules. Moreover, affinity for different lectins (ConA, WGA, DBA and UEA) was detected in vitellogenic oocytes (yolk granules, cortical alveoli, follicular layer and zona radiata), indicating the presence of glycoconjugates with different sugar residues (Mannose- Man- and/or Glucose -Glc-; N-acetyl-D-glucosamine- GlcNAc- and/or sialic acid- NANA-; N-acetyl-D-galactosamine- GalNAc-; L-Fucose -Fuc-). Histochemical techniques also demonstrated the presence of neutral lipids in globules (vacuoles in paraffin sections) and neutral and carboxylated mucosubstances in cortical alveoli. By using anti-vitellogenin (VTG) serum, immunohistochemical positive results were demonstrated in yolk granules, granular cytoplasm and follicular cells of vitellogenic oocytes. Calcium was also detected in yolk granules and weakly in follicular envelope. In females, the gonadosomatic index (GSI) increased progressively from May, during early vitellogenesis, until June during mid and late vitellogenesis, where the highest values were reached. Subsequently, throughout the maturation-spawning phases (July), GSI decreased progressively reaching the minimal values during recovering-resting period (October).

32: EP(4) receptors mediate prostaglandin E(2)-stimulated glycosaminoglycan synthesis in human cervical fibroblasts in culture.

Mol Hum Reprod 2001 Apr;7(4):397-402

Schmitz T, Dallot E, Leroy MJ, Breuiller-Fouche M, Ferre F, Cabrol D.

The aim of this study was to determine the prostaglandin E (EP) receptors and second messengers implicated in glycosaminoglycan (GAG) synthesis by human cervical fibroblasts in culture. Human cervical fibroblasts were obtained from cervical biopsies in pre-menopausal, cycling women. Cultured cells were incubated with prostaglandin E(2) (PGE(2)) and an array of agonists and antagonists. Glycosaminoglycan synthesis was assayed after extraction by measuring the [(3)H]glucosamine and [(35)S]sulphate incorporated into GAG and cAMP production was determined by radioimmunoassay. PGE(2) significantly stimulated GAG synthesis. Neither 17-phenyl-trinor-PGE(2), the EP(1) selective agonist, nor sulprostone, an EP(3) agonist, had any effect on GAG production. Butaprost, the EP(2) selective agonist, also failed to increase GAG synthesis. AH6809, an EP(2) antagonist, had no effect on PGE(2)-stimulated GAG production. AH23848, an EP(4) antagonist, inhibited the GAG synthesis provoked by PGE(2). PGE(2) and butaprost significantly increased cAMP production. Both AH6809 and AH23848 inhibited the PGE(2)-stimulated cAMP production. H89, a cAMP-dependent protein kinase (PKA) inhibitor, did not inhibit PGE(2)-stimulated GAG synthesis and Sp-cAMPS, a selective PKA activator, failed to increase GAG production. In conclusion, both EP(4) and EP(2) receptors are present and functional in human cervical fibroblasts. Only EP(4) receptors mediate PGE(2) stimulated GAG synthesis in a PKA-independent pathway.

33: Induction of acrosomal exocytosis in chicken spermatozoa by inner perivitelline-derived N-linked glycans.

Biochem Biophys Res Commun 2000 Nov 11;278(1):84-9

Horrocks AJ, Stewart S, Jackson L, Wishart GJ.

In birds, the ovum is surrounded by a glycoprotein coat known as the inner perivitelline layer (IPVL), which is analogous to the mammalian zona pellucida and, as such, is the site of initial sperm binding and induction of acrosomal exocytosis (the acrosome reaction). In this study, we demonstrate that oligosaccharides isolated from chicken-IPVL glycoproteins are capable of inducing the acrosome reaction in chicken spermatozoa. Preparations containing only O-linked glycans were unable to induce the acrosome reaction whereas N-linked oligosaccharides released from the IPVL by PNGaseF treatment could induce the acrosome reaction. Addition of galactose to terminal N-acetyglucosamine residues suppressed the acrosome reaction-inducing capacity of the oligosaccharide preparation; however, this capacity could be restored by co-incubation with beta-galactosidase. This evidence suggests that the acrosome reaction-inducing factor is probably an N-linked oligosaccharide with terminal N-acetyl-glucosamine residues. Copyright 2000 Academic Press.

34: Identification of perivitelline N-linked glycans as mediators of sperm-egg interaction in chickens.

J Reprod Fertil 2000 Nov;120(2):397-403

Robertson L, Wishart GJ, Horrocks AJ.

This study demonstrates that carbohydrates play an essential role in sperm-egg interactions in birds. Sperm-egg interaction was measured in vitro as the ability of spermatozoa to hydrolyse a small hole in the inner perivitelline layer, the equivalent of the mammalian zona pellucida. Preincubation with Triticum vulgaris lectin (WGA) and succinyl-WGA (S-WGA) at 10 microgram ml(-1) resulted in complete inhibition of sperm-egg interaction, whereas at the same concentration a range of other lectins (Canavalia ensiformis (Con A), Arachis hypogea (PNA), Ulex europaeus II (UEA II), Solanum tuberosum (STA), Tetragonolobus purpureas (LTA) and Pisum sativum (PSA)) were unable to inhibit sperm egg interaction significantly, although fluorescein-labelled derivatives of these lectins were found to stain the inner perivitelline layer. Significant inhibition of sperm-egg interaction was achieved by the addition of N-acetyl-D-glucosamine and fucoidin to the assay mixture; however, D-glucose, D-galactose, D-fucose and L-fucose had no significant effect on sperm-egg interaction. Pretreatment of the inner perivitelline layer with N-glycanase significantly reduced sperm-egg interaction, whereas treatment with O-glycanase had no effect. These results demonstrate that N-linked glycans play an essential role in sperm-egg interaction in chickens.

35: Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate. II. Combination with ovarian hormones.

Acta Histochem 2000 Aug;102(3):309-21

Hosie M, Terry V, Shaw T, Dwarte D, Murphy CR.

We used a single administration of clomiphene citrate (CC), a synthetic oestrogen that is prescribed for infertility treatment, in combination with either a single administration of oestradiol 17beta (E2) or progesterone (P4) to assess the combined effects of these hormones on the uterine surface. The aim of these experiments was to investigate how CC in combination with these hormones affected both expression of oligosaccharides on the uterine surface and membrane architecture further elucidating CC's agonistic/antagonistic properties. Ovariectomized sexually mature rats were given combinations of E2 and CC (E2 + CC) or P4 and CC (P4 + CC) or P4 and E2 (P4 + E2) and were killed 24 h later. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin and subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results of the administration of these hormone combinations indicate that CC, when administered in conjunction with E2, had the ability to downregulate expression of oligosaccharides on the membrane surface caused by E2. When administered with P4, CC had the ability to upregulate the effects of P4. Thus, when combined with E2, CC has an antagonistic effect but when combined with P4, CC has an agonistic effect.

36: Effects of progesterone on prostaglandin E(2)-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture.

Mol Hum Reprod 2000 Jul;6(7):661-4

Carbonne B, Dallot E, Haddad B, Ferre F, Cabrol D.

Prostaglandins are known to induce cervical ripening and this effect may be mediated by an increase in glycosaminoglycan (GAG) concentration. The aim of this study was to assess the effects of progesterone on prostaglandin E(2) (PGE(2))-induced changes in GAG synthesis by human cervical cells in culture. Human cervical fibroblasts were obtained by cervical biopsies in hormonally active women and cultured. Cells were submitted to an incubation with progesterone or control medium. A second incubation was then performed with increasing concentrations of PGE(2). GAG synthesis by the cervical cells was assayed after extraction, by incorporation of [(3)H]-glucosamine and [(35)S]-sulphate into GAGs. It was found that progesterone alone induced a dose-dependent increase in GAG synthesis. After pre-incubation with progesterone, PGE(2) further increased [(3)H]-glucosamine and [(35)S]-sulphate uptake. However, when expressed as percentage of stimulation, the stimulatory effect of PGE(2) on GAG synthesis was inhibited at high progesterone concentrations. Therefore we concluded that, although high concentrations of progesterone increase the overall synthesis of GAG, they may also play a preventative role against PGE(2)-induced changes in GAG production during pregnancy.

37: Use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro.

Am J Vet Res 2000 Apr;61(4):446-9

King SS, Young DA, Nequin LG, Carnevale EM.

OBJECTIVE: To determine whether specific sugars inhibit adhesion of Streptococcus zooepidemicus, Pseudomonas aeruginosa, and Escherichia coli to equine endometrial epithelial cells in vitro. SAMPLE POPULATION: Endometrial biopsy specimens collected during estrus from 7 healthy mares. PROCEDURE: Endometrial specimens on glass slides were incubated for 30 minutes at 4 C with suspensions of S. zooepidemicus, P. aeruginosa, or E. coli in phosphate-buffered saline solution (PBSS) alone or with various concentrations of D-(+)-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-(+)-glucose, galactose, or N-acetyl-neuraminic acid. Inhibition of bacterial adherence was determined by comparing adhesion of bacteria (i.e., percentage of glandular epithelial cells with adherent bacteria) suspended in each sugar solution with that of bacteria suspended in PBSS. RESULTS: Mannose and N-acetyl-D-galactosamine inhibited adhesion of E. coli and P. aeruginosa to epithelial cells, whereas only mannose inhibited adhesion of S. zooepidemicus. The other sugars did not affect bacterial adherence. CONCLUSIONS AND CLINICAL RELEVANCE: Mannose and N-acetyl-D-galactosamine appear to play a role in adhesion of S. zooepidemicus, P. aeruginosa, and E. coli to equine endometrium. In horses with uterine infections, use of sugars to competitively displace bacteria from attachment sites on cells may provide an adjunct to antibiotic treatment.

38: Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate.

Acta Histochem 1999 Nov;101(4):383-96

Hosie MJ, Shaw TJ, Dwarte DM, Murphy CR.

We have studied histochemically the effects of clomiphene citrate on the expression of oligosacchrides on the apical plasma membrane of uterine epithelial cells using the lectin Phytolacca americana. Ovariectomized sexually mature rats were given a single injection of either clomiphene in two concentrations or estradiol 17 beta or progesterone and were killed 24 hr later. Uterine tissue was labeled with Phytolacca americana conjugated with avidin and subsequently labeled with biotinalyted ferritin and prepared for transmission electron microscopy. Our results indicate that clomiphene and to a lesser degree progesterone significantly increased lectin binding. However, the increase was not as large as that observed with a single dose of estrodiol 17 beta. When the proportion of lectin positivity in relation to total membrane length was analyzed, treatment with clomiphene and progesterone did not have significantly different effects. Low dose clomiphene did not have a significant effect as compared with controls. Our data show that clomiphene has a dose-dependent adverse effect on lectin binding as compared with ovarian hormones. We suggest that these effects contribute to low pregnancy rates with clomiphene use.

39: Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma.

J Reprod Fertil 1998 Sep;114(1):25-34

Jonakova V, Kraus M, Veselsky L, Cechova D, Bezouska K, Ticha M.

Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.

40: Purification and multimeric structure of bovine N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

J Biol Chem 1998 Sep 4;273(36):23203-10

Kornfeld R, Bao M, Brewer K, Noll C, Canfield WM.

N-Acetylglucosamine-1-phosphodiester alpha-N-Acetylglucosaminidase (EC; phosphodiester alpha-GlcNAcase) catalyzes the second step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. A partially purified preparation of phosphodiester alpha-GlcNAcase from bovine pancreas was used to generate a panel of murine monoclonal antibodies. The anti-phosphodiester alpha-GlcNAcase monoclonal antibody UC1 was coupled to a solid support and used to immunopurify the bovine liver enzyme 670,000-fold in two steps to apparent homogeneity with an overall yield of 14%. The purified phosphodiester alpha-GlcNAcase has a specific activity of 498 micromol of [3H]GlcNAc-alpha-phosphomannose-alpha-methyl cleaved per h per mg of protein using 0.5 mM [3H]GlcNAc-alpha-phosphomannose-alpha-methyl as substrate. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine phosphodiester alpha-GlcNAcase is a 272,000-Da complex of four identical 68,000-Da glycoprotein subunits arranged as two disulfide-linked homodimers. A soluble form of the enzyme, isolated from fetal bovine serum, showed the same subunit structure. Both forms of the enzyme reacted with a rabbit antibody raised to the amino-terminal peptide of the liver enzyme, suggesting that phosphodiester alpha-GlcNAcase is a type I membrane-spanning glycoprotein with its amino terminus in the lumen of the Golgi apparatus.

41: Glycosidic residues involved in human sperm-zona pellucida binding in vitro.

Mol Hum Reprod 1997 May;3(5):399-404

Miranda PV, Gonzalez-Echeverria F, Marin-Briggiler CI, Brandelli A, Blaquier JA, Tezon JG.

Glycosidic residues of the mammalian zona pellucida (ZP) are known to be involved in sperm binding, suggesting the presence of complementary carbohydrate binding sites on spermatozoa. However, in previous studies, in which sperm suspensions were incubated with monosaccharides, no inhibitory effect was observed. Results of studies in which sperm were treated shortly after swim-up suggest that the use of non-capacitated cells may explain the apparently conflicting results. In the present report, we studied the effect of preincubation of capacitated spermatozoa with different monosaccharides on their ability to bind to ZP. After 5 h under capacitating conditions, spermatozoa were incubated in medium with or without a monosaccharide, resuspended in fresh medium and used for hemizona (HZ) binding assay. When ZH were incubated with spermatozoa treated with N-acetyl-D-glucosamine, D-mannose, D-fucose, L-fucose or D-galactose, a significant decrease in the number of spermatozoa bound was observed (level of inhibition: 62, 58, 82, 68 and 48% respectively) while treatment of spermatozoa with D-glucose produced no inhibition. Sugar treatment neither altered sperm motility nor the rate of acrosome reaction. These results suggest that N-acetylglucosamine, mannose, fucose and galactose residues are involved in human sperm-zona pellucida binding in vitro.

42: Regulation of hyaluronate metabolism by progesterone in cultured fibroblasts from the human uterine cervix.

FEBS Lett 1997 Feb 3;402(2-3):223-6

Tanaka K, Nakamura T, Takagaki K, Funahashi M, Saito Y, Endo M.

The effects of progesterone, dehydroepiandrosterone sulfate and 17beta-estradiol on the synthesis and degradation of hyaluronate were investigated using human uterine cervix fibroblasts. The cells were incubated with [3H]glucosamine in the presence of the hormones and then [3H]hyaluronate was isolated from the medium. The changes in the radioactivity of [3H]hyaluronate showed that progesterone suppressed hyaluronate synthesis by 22% of the control levels, while dehydroepiandrosterone sulfate and 17beta-estradiol enhanced it by 22% and 12% of the control levels, respectively. Furthermore, progesterone induced degradation of high-molecular-weight [14C]hyaluronate into low-molecular-weight hyaluronate (Mr approximately 40000). These results suggest that in cultured fibroblasts from the human uterine cervix progesterone converts hyaluronate metabolism from the synthesis phase to the degradation phase.

43: Synthesis of glycosaminoglycans by human cervical fibroblasts in culture: effects of prostaglandin E2 and cyclic AMP.

Eur J Obstet Gynecol Reprod Biol 1996 Dec;70(1):101-5

Carbonne B, Jannet D, Dallot E, Pannier E, Ferre F, Cabrol D.

OBJECTIVE: To study the mechanism of action of prostaglandin E2 (PGE2) and its analogue sulprostone leading to production of glycosaminoglycans (GAGs) in the human uterine cervix. STUDY DESIGN: We analysed the effects of PGE2 and its analogue sulprostone upon production of adenosine 3',5'-monophosphate (cAMP), in human cultured fibroblasts. We also studied the effects of PGE2, sulprostone and a cAMP analogue (8-Bromo-cAMP), on the incorporation of [3H]glucosamine into GAGs in human cervical fibroblasts in culture. RESULTS: Following treatment with PGE2 (10(-4)-10(-6) M), we observed a significant increase in the production of cAMP from 96.3 +/- 8.4 pmol/10(6) cells without phosphodiesterase inhibitor 3-isobutyl-methylxanthine (IBMX) to 325 +/- 63 pmol/10(6) cells with 10(-4) M IBMX (Spearman correlation test; P < 0.05). Under the same conditions, the effects of sulprostone (10(-6) M) were limited (from 8.1 +/- 1.5 to 51.3 +/- 14.1 pmol/10(6) cells without and with IBMX, respectively; not significant). Both PGE2 and 8-bromo-cAMP (from 10(-12) to 10(-4) M) increased [3H]glucosamine uptake into GAGs (Spearman correlation test; P < 0.05). Sulprostone (10(-12)-10(-4) M) was unable to reproduce such an effect even after a 24 or 48 h treatment. CONCLUSION: Since firstly, PGE2 acts through EP1, EP2 and EP3 specific receptors, whereas the action of sulprostone is only mediated by EP1 and EP3, and secondly EP2 receptor is coupled with cAMP production, we conclude that cAMP is involved in mediating the action of PGE2 upon GAG synthesis by human cultured cervical fibroblasts.

44: Synthesis and intracellular trafficking of Muc-1 and mucins by polarized mouse uterine epithelial cells.

J Biol Chem 1996 Nov 8;271(45):28128-37

Pimental RA, Julian J, Gendler SJ, Carson DD.

Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 +/- 1 and 16.5 +/- 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 +/- 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 +/- 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.

45: Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro.

Am J Vet Res 1996 Nov;57(11):1635-9

Comment in: Am J Vet Res. 1997 Jan;58(1):4.

Dobrinski I, Ignotz GG, Thomas PG, Ball BA.

OBJECTIVE: To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. ANIMALS: 4 reproductively sound stallions, and 1 mare in estrus. PROCEDURES: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fetuin or asialofetuin/ml in modified Tyrode's solution (TALP), or in TALP alone. After 2 hours of coculture, numbers of attached spermatozoa were counted by fluorescence microscopy and analysis of digitized images. In experiment 1b, progressive motility, viability, acrosomal integrity, and capacitation status were determined in spermatozoa incubated for 2 hours in the presence of the respective monosaccharides and glycoproteins or in TALP alone. In experiment 2, proteins isolated from the periacrosomal plasma membrane of equine spermatozoa were subjected to galactose affinity chromatography and subsequent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. RESULTS: Numbers of spermatozoa attached to OEC were reduced (P < 0.05) after all treatments except N-acetyl glucosamine, compared with incubation in TALP alone. The lowest numbers of spermatozoa were bound in cultures incubated in the presence of galactose and asialofetuin. Spermatozoal motility was lower (P < 0.05) after incubation for 2 hours in the presence of fetuin, compared with control, and incubation in the presence of fetuin or asialofetuin caused a significant (P < 0.05) increase in the percentage of capacitated spermatozoa, compared with control. Affinity chromatography of periacrosomal plasma membrane proteins revealed a galactose-binding protein of about 66 kd. CONCLUSION: Recognition of glycoconjugates with exposed galactosyl residues on OEC by galactose-binding proteins on the periacrosomal plasma membrane of equine spermatozoa could mediate the attachment of equine spermatozoa to OEC in vitro.

46: Sialyl-Lewis x and Sialyl-Lewis a are associated with MUC1 in human endometrium.

Glycoconj J 1996 Oct;13(5):769-79

Hey NA, Aplin JD.

Endometrial epithelial cells express MUC1 with increased abundance in the secretory phase of the menstrual cycle, when embryo implantation occurs. MUC1 is associated with the apical surface of epithelial cells and is also secreted, being detectable in uterine fluid at elevated levels in the implantation phase. However, its physiological role is uncertain; it may either inhibit intercellular adhesion by steric hindrance or carry carbohydrate recognition structures capable of mediating cell-cell interaction. Here we show that endometrial epithelium expresses both Sialyl-Lewis x (SLex) and Sialyl-Lewis a (SLea), with a distribution and pattern of menstrual cycle regulation similar to that of MUC1. Using Western blotting and double determinant ELISA of uterine flushings, we demonstrate that SLex is associated with MUC1 core protein. The endometrial carcinoma cell lines HEC1A and HEC1B are shown to express MUC1 in a mosaic pattern, while three other cell lines express much lower amounts. HEC1A expresses both SLex and SLea while HEC1B expresses SLea only. Immunoprecipitation has been used to demonstrate that SLea is associated with MUC1 in HEC1B cells, and both SLex and SLea are associated with MUC1 in HEC1A cells.

47: Ultrastructural localization of lectin receptors in the preimplantation ovine embryo.

Anat Rec 1994 Dec;240(4):537-44

De Paz P, Sanchez AJ, Fernandez JG, Garcia C, Chamorro CA, Anel L.

BACKGROUND: Preimplantation development of mammalia is characterized by cell surface changes functioning in intercellular communication and adhesion. The glycoconjugate role in cellular interactions has been analysed for several groups but not in sheep embryos. The binding patterns of eleven lectins during sheep preimplantation development were investigated and the role of glycoconjugates in early development was discussed. METHODS: Ultrathin sections from preimplantation ovine embryos (3-7 days) were incubated with different colloidal gold conjugated lectins and the frequency of gold particles on the cell membrane, some organelles, and the zona pellucida was evaluated. RESULTS AND CONCLUSIONS: We observed a higher staining of WGA, DBA, and SBA lectins in the intercellular contact zone with respect to the free cell surface of blastomeres during cleavage. This indicates that the N-acetyl galactosamine and N-acetyl glucosamine residues may be involved in sheep morula compaction. In contrast, the trophoblast cell displays an increase of staining of some lectins previously identified during cleavage (LcH, WGA, SBA, MPA, and PNA) on the free membrane, and a lack of sugar residues in the intercellular surface. This polarization of the trophoblast cell surface is not observed in the inner cell mass and could provide a mechanism for differentiation within the blastocyst. Intracytoplasmic vesicles show a cytochemical identity with lysosomes in the blastocyst (abundant GlcNAc and Man/Glc residues) that may reflect a functional relationship between both organelles in an intracellular cycle. The zona pellucida presents abundant GalNAc, GlcNAc, and Gal residues during preimplantation ovine development.

48: Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein.

Mol Cell Biochem 1994 Aug 31;137(2):91-9

Das M, Mukhopadhyay PK, Chowdhury M.

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well. In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors. SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64 kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents. UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose. De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (125I)-labelled lectin is bound to Mannan-Sepharose affinity matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

49: Inhibition of sperm-egg fusion in the hamster and mouse by carbohydrates.

Zygote 1994 Aug;2(3):253-62

Ponce RH, Urch UA, Yanagimachi R.

After spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm-egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm-egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm-egg fusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and -galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.

50: Human uterine cervical epithelial cells grown on permeable support--a new model for the study of differentiation.

Differentiation 1994 Apr;56(1-2):107-18

Gorodeski GI, Romero MF, Hopfer U, Rorke E, Utian WH, Eckert RL.

The purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5-12 cell layers compared to 1-3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support. Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cysteine-, [3H]fucose- and [14C]glucosamine-labeled molecules, and positive short-circuit current (Isc) under voltage-clamp conditions. Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7-22.10(-6) cm.sec-1, which was 5-100 fold smaller compared to blank inserts, with a cut-off MW of 40-70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5, indicating a leaky but confluent epithelia. Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium respectively.

51: Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain.

Eur J Biochem 1994 Jan 15;219(1-2):331-48

Pfeiffer G, Strube KH, Schmidt M, Geyer R.

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glucans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing. The results revealed that Asn117 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273-286]. In contrast, Asn117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Asn67 (YN-tPA) or Asn58 (GS-tPA) as well as those at Asn184 and Asn448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gal alpha 3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats. Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex-type glycans at Asn117 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at the position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.

52: Polypeptides synthesized and released by rat ectopic uterine implants differ from those of the uterus in culture.

Biol Reprod 1993 Jun;48(6):1334-40

Sharpe KL, Vernon MW.

Unique endometriosis-specific secretory proteins would be of paramount importance as noninvasive markers for diagnosis and evaluation of therapeutic approaches for endometriosis. Furthermore, identification of endometriosis-specific secretory proteins may be an important step towards understanding the pathophysiology of endometriosis-associated pain and infertility. Therefore this study was designed to assess protein synthesis and secretion by ectopic uterine implants from steroid-treated and reproductively cyclic rats with surgically induced endometriosis. Uteri, ectopic uterine implants, and control tissues were incubated in L-[35S]methionine or D-[6-3H]glucosamine for 0-24 h and 24-48 h. De novo-synthesized proteins released into the culture media were identified using two-dimensional SDS-PAGE, fluorography, and computer-assisted image analysis. Two distinct groups of ectopic uterine implant proteins were identified: ENDO I (M(r) 40,000-50,000; pI 4.0-5.2) and ENDO II (M(r) 28,000-32,000; pI 7.5-9.0) were produced by ectopic uterine implants and not the uteri. A third group of proteins, previously identified in culture media of the uteri from progesterone-treated rats and called PUP-1 (M(r) 70,000; pI 5.7), was synthesized and secreted by ectopic uterine implants 24-48 h later than in parallel uterine cultures. The detection of ectopic uterine implant proteins suggests biochemical characteristics of the ectopic tissue that may be used to develop unique markers for endometriosis. Furthermore, the delayed synthesis and secretion of the uterine protein PUP-1 by the ectopic uterine implants illustrates yet another example of the asynchronous behavior of these two tissues, which may be related to the etiology or pathophysiology of the disease.

53: Neonatal porcine endometrial development involves coordinated changes in DNA synthesis, glycosaminoglycan distribution, and 3H-glucosamine labeling.

Biol Reprod 1993 Apr;48(4):729-40

Spencer TE, Bartol FF, Wiley AA, Coleman DA, Wolfe DF.

To determine whether neonatal porcine endometrial development involved alterations in endometrial DNA synthesis, glycosaminoglycan (GAG) distribution, and/or 3H-glucosamine labeling, gilts were assigned randomly at birth (Day 0) to be hysterectomized on either Day 0, 7, 14, 28, 42, or 56. Uteri were processed for histology and Alcian blue-8GX (AB) histochemistry or cultured as explants in the presence of [methyl-3H]thymidine (3H-Thd) or D-[6-3H]glucosamine (3H-GlcN) and processed for subsequent autoradiography. For 3H-Thd-labeled tissues, labeling index (LI; % of nuclei labeled) was determined for two endometrial tissues (epithelium and stroma); two epithelial areas (luminal and glandular); and, for glandular epithelium, in three endometrial zones (zone 1 = shallow, zone 2 = intermediate, zone 3 = deep). For 3H-GlcN-labeled tissues, LI (grains/100 microns2) was determined for two stromal zones (shallow and deep). Endometrial glands were absent on Day 0, present in shallow stroma on Days 7 and 14, and extended to the myometrium in tissues from Day 28 through Day 56. Appearance of endometrial glands was associated with a dramatic increase in glandular epithelial 3H-Thd LI, which was maximal on Days 7 and 14 and declined thereafter. When glands were present in all three endometrial zones (Days 28-56), glandular epithelial 3H-Thd LI was consistently greatest in zone 2. Stromal 3H-Thd LI decreased after Day 0. In tissues obtained after Day 0, a distinct zone of alcianophilia was observed in shallow stroma adjacent to luminal epithelium and surrounding the necks of newly developed endometrial glands. This staining pattern was marked in tissues from Days 7, 14, and 28. Generally, stromal 3H-GlcN LI was greater in shallow than in deep stromal zones; it decreased after Day 0 to minimum values on Days 28 and 42 in both zones, and increased slightly in shallow stroma on Day 56. Data indicate that development of the neonatal porcine endometrium between birth and Day 56 involves coordinated alterations in patterns of DNA synthesis, GAG distribution, and glycoconjugate biosynthesis. The morphogenetic processes characterized here are likely to be regulated locally via changes in tissue microenvironment.

54: Prolactin suppression during pre and post-implantation periods on rat uterine glucosamine synthase activity.

Indian J Exp Biol 1993 Apr;31(4):386-8

Vijayan E, Jayashree J.

Administration of bromocriptine (Bc), an ergot derivative having dopamine receptor agonist activity, to rats on day 1-5 of pregnancy prevented implantation of blastocysts and significantly suppressed uterine glucosamine 6-phosphate synthase activity. There was no effect on implantation or the enzyme activity when Bc was injected on day 7 or later of pregnancy. Injection of prolactin following Bc partially restored the enzyme activity and increased number of implantation sites. These results indicate that suppression of prolactin on day 1 to 5 of pregnancy causes failure of implantation. Bc on day 9 or later had no effect possibly due to the availability of placental LH/hCG to support the luteal cells.

55: Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep.

Biol Reprod 1992 Dec;47(6):917-24

Murray MK, Sower SA.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.

56: WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

J Cell Physiol 1992 Jun;151(3):451-65

Valdizan MC, Julian J, Carson DD.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)

57: Purification and characterization of rat uterine glycerylphosphorylcholine diesterase and its tissue-specific induction by 17 beta-estradiol.

Endocrinology 1991 Sep;129(3):1147-54

Mitra J, Chowdhury M.

The rat uterine enzyme glycerylphosphorylcholine (GPC) diesterase found in the proestrous secretions was purified and characterized biochemically with respect to its subunit mol wt, native size, pI, and amino acid and carbohydrate composition. The 30-kDa protein was assessed to have a native mol wt of 105 kDa, as determined by analytical gel filtration. It had a basic pI with an unusually high carbohydrate/protein ratio (0.83:1). The estrogen inducibility of the protein was identified by its de novo synthesis in vitro after in vivo 17 beta-estradiol administration. For this experiment, uteri from estradiol-treated immature rats were incubated in the presence of [14C]glucosamine, and the 14C-labeled total glycoconjugates were then subjected to the enzyme purification procedure. The peak enzyme fraction was observed to correspond to one of the 14C-labeled protein peaks in the elution profile of the glycoconjugates. Assay of GPC diesterase activity showed significant enhancement only in the uterus, not in other organ systems, after in vivo estradiol treatment, and this hormone-stimulated increase was inhibited by nafoxidine, a potent estrogen antagonist. The enzyme activity profile of the three main separated uterine cellular types showed that the enzyme was secreted by epithelial cells. No antigenic homology of the enzyme was observed with GPC diesterase from nonmammalian sources. The results suggested that GPC diesterase was one of the estrogen-regulated proteins of the preovulatory uterine fluid of rats, and the availability of a purified preparation of the enzyme could serve as a useful tool to study the mechanism of estrogen action.

58: Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation.

Biol Reprod 1991 Jun;44(6):1063-79

Blair RM, Geisert RD, Zavy MT, Yellin T, Fulton RW, Short EC.

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.

59: Studies on mouse sperm lectin; the existence and the participation in sperm-egg binding.

J Pharmacobiodyn 1991 May;14(5):244-9

Kawai Y, Shimaji H, Hama T, Mayumi T.

Mouse sperm bound to egg zona pellucida and attached to unfixed rabbit erythrocytes specifically, but not to fixed-erythrocytes. When mouse sperm were treated with ionophore A23187, the sperm lost both the binding ability to the zona pellucida and the attachment ability to rabbit erythrocytes. The membrane vesicles released from A23187-treated sperm agglutinated rabbit unfixed erythrocytes. Sperm binding to egg zona pellucida was also inhibited by the membrane vesicles. The inhibition spectrum of a hemagglutination and of an inhibitory activity of sperm-egg binding by various carbohydrates was studied. The hemagglutination was inhibited by N-acetyl-glucosamine, N-acetylgalactosamine and glyco-proteins, such as fetuin and orosomucoid, but not by N-acetylneuraminic acid (NeuNAc) in saccharides and asialo-transferrin in glycoproteins. On the other hand, the sperm-egg binding was inhibited most effectively by NeuNAc and also by asialo-transferrin. The inhibition spectrum of hemagglutination and sperm-egg binding didn't agree with each other. These results suggest that a lectin-like molecule is expressed on mouse sperm surface but is not directly concerned in sperm-egg binding.

60: Identification and characterization of de novo-synthesized porcine oviductal secretory proteins.

Biol Reprod 1990 Dec;43(6):929-38

Buhi WC, Alvarez IM, Sudhipong V, Dones-Smith MM.

Oviductal secretory products provide a biochemical environment important for establishment of pregnancy. A previous study identified three de novo-synthesized glycoproteins by one-dimensional SDS-PAGE as well as increased incorporation of [3H]Leu into secretory protein by whole oviduct and ampulla associated with proestrus, estrus, and metestrus only. Here, our objective was to further identify and characterize oviductal secretory proteins, specifically 115,000- and 85,000-Mr estrus-associated proteins (EAP). Two-dimensional SDS-PAGE resolved the 115,000-Mr protein into two proteins of 100,000 Mr, one basic and one acidic, and the 85,000-Mr protein into 75,000- and 85,000-Mr species (pI less than 4.0). Differential secretion of proteins between ampulla and isthmus was indicated. The 100,000-, 75,000-, and 85,000-Mr proteins were synthesized by ampulla during estrus but not by isthmus nor by uterine endometrium. De novo-synthesized EAP were labeled with glucosamine, Leu, and Met, and the 75,000-85,000-Mr proteins from ampulla and a 30,000-Mr family from isthmus were labeled with fucose. Inorganic [35S]sulfate labeled three EAP. Fractionation of culture medium by gel filtration demonstrated differences between products secreted by ampulla and isthmus and suggested that some EAP may be found as high-molecular weight forms in the native state. Results indicate that porcine oviductal tissue synthesizes specific EAP at the time of fertilization and early cleavage-stage embryonic development, that there are differences in the type and distribution of glycoproteins from ampulla and isthmus, and that post-translational modifications occur with the addition of glucosamine, fucose, and inorganic sulfate.

61: The involvement of surface sugars of mammalian spermatozoa in epididymal maturation and in vitro sperm-zona recognition.

Andrologia 1990 Mar-Apr;22(2):184-94

Kumar GP, Laloraya M, Agrawal P, Laloraya MM.

The distribution of various simple sugar residues over the spermatozoa surfaces of five different mammalian species is characterized and compared. Epididymal maturation of the spermatozoa of all the five species studied exhibited an increase in the amount of N-acetyl-D-glucosamine residues over their acrosomal domains. A complete blockade of sperm-zona pellucida attachment of hamster gametes could be brought about when spermatozoa were treated previously with 0.1 M of N-acetyl-D-glucosamine. This sugar seems to be specifically involved in sperm-zona pellucida attachment in hamsters. The inter-specific cross-reactivity of gametes of laboratory mammals like rat, mouse, rabbit and hamster could, quite likely, be because of the involvement of N-acetyl-D-glucosamine as a common factor in this reaction in these animals.

62: Lactosaminoglycan assembly, cell surface expression, and release by mouse uterine epithelial cells.

J Biol Chem 1990 Jan 5;265(1):430-8

Dutt A, Carson DD.

The kinetics of assembly, cell surface expression, secretion, and degradation of the major lactosaminoglycan (LAG)-bearing glycoproteins in mouse uterine epithelial cells have been studied. LAGs have been shown previously to be synthesized preferentially by these cells in the uterus and are expressed at the cell surface, where they participate in cell adhesion processes (Dutt, A., Tang., J.-P., and Carson, D. D. (1987) Dev. Biol. 119, 27-37). We utilized selection on pokeweed mitogen-Sepharose, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent electroelution to isolate the major LAG-bearing glycoproteins. The intact LAG-bearing glycoproteins exhibited very high apparent Mr (greater than 500,000) both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography under dissociative conditions. The subset of LAGs at the cell surface exhibited a half-life of approximately 11 h, whereas total cell-associated LAGs had a half-life of 6 +/- 1 h. Pulse-chase experiments indicated that the transit time of LAG core proteins from the rough endoplasmic reticulum to the site of LAG addition in the Golgi was 30-45 min. LAG glycoprotein transit from the Golgi to the cell surface required at least an additional 30-45 min. The major metabolic fate of the cell-associated LAGs was secretion to the medium with no evidence of lysosomal degradation. Some (30%) of the LAGs appeared to be released to the medium via the action of cell surface proteases. Epithelial cell surfaces bound fluoresceinated pokeweed mitogen, indicating the constitutive presence of LAG-bearing molecules at the cell surface; pokeweed mitogen binding to the cell surface was completely blocked by 10 mM chitotriose. These observations provide the first comprehensive description of the intracellular transport and metabolism of this interesting class of glycoproteins of the uterine epithelial cell surface.

63: Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro.

Am Rev Respir Dis 1990 Jan;141(1):72-8

Goswami SK, Kivity S, Marom Z.

Erythromycin and other antibiotics have been used empirically in the treatment of patients with chronic obstructive pulmonary disease (COPD). We studied whether this empirical role of antibiotics might not be related to a possible direct effect on respiratory glycoconjugate (RGC) secretion. The effect of erythromycin on RGC secretion and hypersecretion was studied in an in vitro preparation of human airways that were secreting [3H]glucosamine respiratory glycoconjugate (RGC), and on a human endometrial adenocarcinoma cell line secreting a glycoconjugate (tumor glycoconjugate = TGC) chemically similar to the RGC secreted by the airways. Erythromycin at 10(-5) M reduced RGC secretion by 35 +/- 4% (n = 9, p less than 0.001) in both human airways and the adenocarcinoma cells, and was increasingly active in the pharmacologic range of 10(-7) to 10(-4) M. The inhibitory effect of erythromycin was maximal within 16 h and was still evident 34 h after incubation. Erythromycin was noted to reduce both spontaneous (baseline) and stimulated RGC secretion (by histamine and methacholine) from airways in culture. The blocking effect appeared to be more selective for histamine than methacholine. These effects were not associated with any toxicity to the tissues and were not associated with the inhibition of protein synthesis. Dexamethasone also inhibited RGC release in both assay systems and exhibited dose-related effects in the physiologic ranges (10(-9) to 10(-5) M). When administered together, erythromycin and dexamethasone had an additive inhibitory effect on RGC secretion (68.0 +/- 3.0%, n = 7, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

64: Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells.

Eur J Biochem 1989 Dec 8;186(1-2):273-86

Pfeiffer G, Schmidt M, Strube KH, Geyer R.

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.

65: The uptake of [3H] glucosamine-labelled glycoconjugates into the perivitelline space of preimplantation mouse embryos.

Hum Reprod 1989 Oct;4(7):821-5

Fowler RE, Barratt E.

Autoradiography has been used to follow the synthesis and migration of labelled glycoconjugates into fertilized and unfertilized mouse eggs. Adult female mice were paired individually with males and injected with 100 microCi [3H] glucosamine, either when they were paired with males or at the time a vaginal plug was first detected. The mice were killed at intervals after the injection of label and 5-microns and semi-thin sections of the oviducts were processed for autoradiography. Labelled glycoconjugates passed rapidly into the perivitelline space of fertilized and unfertilized mouse eggs. There was a positive correlation between the density of label within the zona pellucida and the perivitelline space. Labelled glycoconjugates were maintained within the perivitelline space of many developing embryos for at least 48 h despite very low levels of label within the oviduct by this time. Other 2-cell embryos were only lightly labelled. It is suggested that labelled glycoconjugates may be derived from secretions of the cumulus cells which surround the egg at ovulation rather than from oviductal secretions.

66: O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells.

Endocrinology 1989 Apr;124(4):1602-12

Peters BP, Krzesicki RF, Perini F, Ruddon RW.

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.

67: Glucosamine enhanced sperm-egg binding but inhibited sperm-egg fusion in mouse.

Experientia 1989 Feb 15;45(2):193-4

Okabe M, Yagasaki M, Matzno S, nagira M, Kohama Y, Mimura T.

In order to study the sperm-egg recognition mechanism on the surface of the plasma membrane, zonae were removed from mouse eggs by exposure to acidic conditions. Sperm binding to denuded eggs was then observed in the presence of various sugars. Among several carbohydrates tested, only glucosamine (GlcN) was found to increase the number of sperm bound to eggs while inhibiting sperm-egg fusion. The inhibition was reversible; when denuded eggs were transferred to a GlcN free medium, a high rate of polyspermy was observed.

68: Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin.

Endocrinology 1989 Feb;124(2):862-9

Ruddon RW, Krzesicki RF, Beebe JS, Loesel L, Perini F, Peters BP.

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.

69: Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts.

Biol Reprod 1988 Dec;39(5):1171-82

Baumbach GA, Climer AH, Bartley NG, Kattesh HG, Godkin JD.

Department of Animal Science, College of Veterinary Medicine, University of Tennessee, Knoxville 37901.

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.

70: Characterization of a high molecular weight glycoprotein secreted by the peri-implantation bovine conceptus.

Biol Reprod 1988 Oct;39(3):553-60

Newton GR, Hansen PJ, Low BG.

Cow conceptuses were flushed from uteri on Day 17 of pregnancy and cultured with [3H]glucosamine and [14C]leucine. A high molecular weight glycoprotein (HMWG) having an Mr = 765,000 was isolated by a combination of anion-exchange and gel-filtration chromatography. Selective chemical and enzymatic degradations were performed. The HMWG was resistant to Pronase and peptide: N-glycanase F. Only endo-beta-galactosidase and harsh alkaline reducing conditions were successful in dissociating carbohydrate from the protein core, suggesting that carbohydrate chains are N-linked to Asn and contain beta-galactosidic linkages. The intact molecule could bind to an affinity column of Datura stramoniom lectin, suggesting the presence of beta(1-4)-linked oligomers of N-acetylglucosamine. The susceptibility of HMWG to endo-beta-galactosidase suggests that at least some of these oligomers are substituted with galactose to form N-acetyllactosamine. Binding of HMWG to lectin could be inhibited partially with N-acetyllactosamine or completely with a mixture of N, N'-diacetylchitobiose and N, N', N"-triacetylchitotriose. In summary, properties of the HMWG suggest it contains lactosaminoglycan components and is almost identical to an HMWG secreted by the Day 16 ovine conceptus. Thus, embryos of these two ruminant species secrete similar molecules during early pregnancy.

71: Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring.

J Biol Chem 1988 Feb 25;263(6):3016-21

Takami N, Ogata S, Oda K, Misumi Y, Ikehara Y.

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.

72: Hormonal influence on glycosaminoglycan synthesis in uterine connective tissue of term pregnant women.

Hum Reprod 1987 Apr;2(3):177-82

Wiqvist I, Linde A.

Adaptation of the uterus to the growing fetus necessitates remodelling of the uterine connective tissue. Proteoglycans, being a main constituent of the extracellular matrix, influence the physical properties of the tissue and play an important regulatory role for a number of functional events. The synthesis of glycosaminoglycans (GAGs), the carboxyhydrate side chains of proteoglycans, in tissue from the lower uterine segment of term pregnant women was investigated in vitro by measurement of 35SO4 and [14C]glucosamine incorporation. Prostaglandin E2 and oestradiol-17 beta significantly increased the synthesis of sulphated GAGs but decreased the incorporation of [14C]glucosamine, while relaxin, prostaglandin F2 alpha and oxytocin had no significant effect. To further explore the influence of prostaglandin E2, tissue specimens were incubated with [14C]glucosamine and GAGs separated into three fractions on cetylpyridinium chloride cellulose micro columns. Prostaglandin E2 was found to significantly reduce the synthesis of components recovered in the glycoprotein and hyaluronate fractions, whereas synthesis of components in the sulphated GAG fraction was increased. The results indicate that prostaglandin E2 and oestradiol-17 beta have differential effects on different GAGs whereas relaxin, oxytocin and prostaglandin F2 alpha have no effect.

73: Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri.

Biochemistry 1987 Mar 24;26(6):1598-606

Carson DD, Tang JP, Hu G.

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

74: Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function.

Dev Biol 1987 Mar;120(1):228-35

Carson DD, Dutt A, Tang JP.

The synthesis of various glycoconjugate classes by mouse uteri during the pre- and peri-implantation period has been examined using [3H]glucosamine as a metabolic precursor. A unique and dramatic (five- to sixfold) increase was observed in the synthesis of hyaluronate on the day upon which embryo implantation normally occurs. Mated, but nonpregnant mice did not display increased hyaluronate biosynthesis. In contrast to hyaluronate, the synthesis of most other types of glycoconjugates remained fairly constant during the first 5 days of pregnancy. Low (1500-5000)-molecular-weight N-linked oligosaccharides constituted the major class of oligosaccharides synthesized under all conditions. High (greater than 10,000)-molecular-weight glycoconjugates constituted the second most abundant class of glycoconjugates synthesized (20-30%). Most (85%) of the newly synthesized hyaluronate was associated with the nonepithelial cell types of the uterus. Experiments using ovariectomized mice receiving steroid hormones demonstrated that uterine hyaluronate synthesis was induced preferentially by an artificial decidual stimulus and implicated stromal cells as the site of hyaluronate synthesis. In addition, it was demonstrated that tissue culture plates coated with hyaluronate, but not other polysaccharides, support attachment and spreading of a large fraction (60 to 70%) of embryos cultured in serum-free medium. Collectively, these studies indicate that increased hyaluronate biosynthesis accompanies decidual responses in the endometrium and may promote embryo implantation following initial penetration of the uterine epithelium.

75: Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus.

Biol Reprod 1987 Mar;36(2):405-18

Hansen PJ, Ing NH, Moffatt RJ, Baumbach GA, Saunders PT, Bazer FW, Roberts RM.

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.

76: Physicochemical properties of apolipoprotein(a) and lipoprotein(a-) derived from the dissociation of human plasma lipoprotein (a).

J Biol Chem 1986 Jul 5;261(19):8712-8

Fless GM, ZumMallen ME, Scanu AM.

Chemical reduction of human plasma lipoprotein(a) (Lp(a)) yielded two water-soluble products which were separated by rate zonal ultracentrifugation. Apolipoprotein(a) (apo(a)) was completely recovered from the bottom of the gradient, whereas lipoprotein(a-) (Lp(a-)), which contained all of the lipids and apo-B100 of Lp(a), floated. By the techniques of circular dichroism and viscometry Lp(a-) was identical to low density lipoprotein (LDL). Lp(a-) was slightly larger in mass than autologous LDL and contained proportionally more triglyceride. The difference in mass between Lp(a) and Lp(a-) was accounted for by the loss of 2 molecules of apo(a) from the Lp(a) particle. The molecular weight of reduced and carboxymethylated apo(a) was 281,000 as determined by sedimentation equilibrium in 6 M guanidine HCl. By circular dichroism the structure of apo(a) was mostly random (71%) with the remainder representing 8% alpha-helix and 21% beta-sheet; its intrinsic viscosity, 28.3 cm3/g, was consistent with an extended flexible coil. The amino acid composition was characterized by an unusually high content of proline (11.4 mol %) as well as tryptophan, tyrosine, arginine, threonine, and a low amount of lysine, phenylalanine, and isoleucine. Apo(a) contained 28.1% carbohydrate by weight represented by mannose, galactose, galactosamine, glucosamine, and sialic acid in an approximate molar ratio of 3:7:5:4:7, respectively. Overall, the structure of Lp(a) appears to be consistent with a rigid spherical LDL-like core particle which, as a consequence of its association with a flexible glycoprotein such as apo(a), favors the entrapment of significant amounts of hydrodynamically associated solvent. Furthermore, the Lp(a-) remnant generated by the removal of apo(a) from Lp(a) was similar in structure but not identical to autologous LDL.

77: Glycoproteins in rabbit uterus during implantation. Differential localization visualized using 3H-N-acetyl-glucosamine labelling and FITC-conjugated lectins.

Histochemistry 1986;84(1):73-9

Thie M, Bochskanl R, Kirchner C.

The synthesis of glycoproteins in rabbit uterine epithelium during the late preimplantation period was studied using tritiated N-acetylglucosamine. In vivo labelling was achieved by the intra-uterine implantation of agar gel columns containing the precursor. Autoradiography showed the radioactivity to be predominantly localized in the apical cell surfaces, with single cells exhibiting an accumulation of silver grains in their supranuclear cytoplasm. After gel electrophoresis of uterine flushings, activity was mainly found in the beta-glycoprotein fraction. Fluorescein isothiocyanate (FITC)-conjugated wheat-germ agglutinin reacted with the apical cytoplasm and surfaces of the endometrial cells. However, FITC-conjugated concanavalin A exhibited a different binding pattern, reacting first with the basal cytoplasm, and later with the apical cytoplasm. After concanavalin-A staining, single cells exhibited positive vesicles in their lateral and apical parts. These cells may be released into the uterine lumen until 210 h post column. Neither of the lectins reacted with ciliated cells. Concanavalin A showed an affinity for the beta-glycoprotein fraction of the uterine secretion. The results indicate that, although all endometrial cells contain glycoproteins, only a few of these seem to be involved in the synthesis of secretory products.

78: Characterization of proteins produced in vitro by bovine endometrial explants.

Biol Reprod 1985 Oct;33(3):745-59

Bartol FF, Roberts RM, Bazer FW, Thatcher WW.

Endometrial tissues were obtained from 17 pregnant (P, estrus/mating = Day 0; Day 16, n = 4; Day 19, n = 6; Day 22, n = 3; Day 24, n = 4) and six nonpregnant (NP; Day 16, n = 4; Day 19, n = 2) cattle, as well as from one cyclic (nonbred) cow (Day 4), one later-pregnant cow (Day 69), and both ligated and pregnant uterine horns of three unilaterally pregnant cattle (UP; Day 270). Tissues (approximately equal to 500 mg wet tissue/explant) were cultured for 24 or 48 h in modified minimal essential medium (MEM), in the presence of radioactive amino acid and/or amino sugar substrate(s) (L-[3H] leucine, L-[14C] leucine, and D-[3H] glucosamine), in order to characterize substrate uptake and de novo synthesis and release of proteins and polypeptides in vitro. Endometrial explants from all cattle produced proteins de novo from radiolabeled substrates. Chromatographic (gel filtration, cation, and anion exchange) and two-dimensional polyacrylamide gel electrophoretic analyses revealed complex patterns of primarily acidic radiolabeled polypeptides in dialyzed MEM, which were absent from endometrial tissue homogenates. No qualitative differences were noted in the array of proteins released into MEM associated with either pregnancy status (P vs. NP, UP) or stage of gestation (Days 16, 19, 22, 24, 69, and 270). Medium from all endometrial explants was enriched in polypeptides in four Mr (X 10(3)/pH classes (I, approximately equal to 14/greater than 7.2; II, 19-24/5.4-6.3; III, 28-31/6.9-7.3; and IV, greater than or equal to 150/less than or equal to 5.1).

79: Suppression of placental alkaline phosphatase biosynthesis by tunicamycin.

J Biol Chem 1984 Dec 25;259(24):14997-9

Ito F, Chou JY.

Placental alkaline phosphatase activity and immunoreactivity were inhibited in a parallel fashion in choriocarcinoma cells by tunicamycin, a protein glycosylation inhibitor. Tunicamycin suppressed placental alkaline phosphatase biosynthesis in addition to inhibiting protein glycosylation in general. An anti-placental alkaline phosphatase-precipitable polypeptide of 58,000 daltons was formed in the presence of this antibiotic. The 58,000-dalton polypeptide had a degradation rate similar to that of the glycosylated phosphatase monomer from control cultures. Tunicamycin suppressed placental alkaline phosphatase mRNA activity leading to the observed decrease in biosynthesis.

80: Lectin binding in endometrial adenocarcinoma.

Am J Clin Pathol 1984 Sep;82(3):259-66

Kluskens LF, Kluskens JL, Bibbo M.

The relative increase in endometrial adenocarcinoma in women has increased the need for more objective criteria in the distinction of hyperplastic and neoplastic endometrium. The authors have used the ability of lectins to detect changes in surface glycoproteins to probe the differences among proliferative endometrium, endometrial adenomatous hyperplasia and adenocarcinomas. Paraffin-embedded sections of tissue obtained by Vakutage endometrial sampling technics were stained with each of 7 FITC lectin conjugates. Thirty-four specimens were examined (7 proliferative, 12 hyperplastic, and 15 adenocarcinomatous). Wheat germ agglutinin binding was detectable in all specimens with a distribution at the cell luminal border of glandular formations irrespective of diagnosis. However, adenocarcinoma cases showed distribution along the lumenal border and the cell periphery with loss of orientation of the lectin binding. Similar alterations and increased binding were noted for Concanavalin A. The WGA binding to sections was specifically inhibitable by oligosaccharides of N-acetyl-glucosamine. The results provide an objective criterion for detection of loss of cell orientation useful in the diagnosis of endometrial adenocarcinoma in tissue fragments.

81: Acute effects of prostaglandins on the biosynthesis of connective tissue constituents in the non-pregnant human cervix uteri.

Acta Obstet Gynecol Scand 1984;63(2):169-73

Norstrom A.

Biochemical effects of prostaglandins (PGs) on the connective tissue of the human cervix uteri were studied by measuring the net incorporation of [3H] glycine and [3H] glucosamine into cervical specimens, incubated in vitro in the presence of PGE2 and PGF2 alpha. The net radiolabelling with these isotopes, precursors of collagen and proteoglycans, was influenced by PGE2 and PGF2 alpha in a similar manner. In the follicular phase of the menstrual cycle, both PGs induced a decreased radiolabelling with glycine but caused an increased labelling with glucosamine. In the luteal phase, the situation was reversed, i.e. the incorporation of glycine was augmented and that of glucosamine was reduced in tissue incubated with PGs. It is suggested that PGs can modulate the fibroblast biosynthesis activity in such a way that either the synthesis of fibrous structures (collagen) or that of ground substance constituents (proteoglycans) is favored.

82: Secretion of mucin by explants of rabbit and human cervix in organ culture.

Biol Reprod 1983 Oct;29(3):751-65

Adler KB, Alberghini TV, Counts DF, Auletta FJ.

Small explants (2-3 mm3) of endocervix from virgin, estrous rabbits, and from hospitalized patients undergoing hysterectomy for nonneoplastic disease, were placed in organ culture and maintained in serum-free media for 4 days at 35 degrees C in a humid environment of 95% air/5% CO2. Waymouth's MB 752/1 with 10-5 M hydrocortisone succinate, 10-7 M retinyl acetate, and 1 microgram/ml insulin proved to be an excellent medium for maintaining these tissues, as judged by examination with light and scanning electron microscopy after incubation for 5 days. The explants incorporated the radiolabeled glycoprotein precursor, tritiated glucosamine, and secreted labeled mucin glycoproteins in vitro. Mucin released into the culture medium contained sialic acid and hexosamine in a molar ratio of approximately 0.5-0.8:1.0. Although some alterations occur in the morphology of secretory cells and their products after maintenance in culture for several days, the system can be utilized for studying various aspects of the cell biology of cervical mucin secretion.

83: Glycosaminoglycans: their distribution and potential vasoactive action in the nonpregnant and pregnant ovine uterus.

Am J Obstet Gynecol 1983 Apr 15;145(8):1041-8

Greiss FC Jr, Wagner WD.

The effects of direct uterine artery infusions of glucosamine-hydrochloric acid, dextrose, and mannitol were evaluated in ewes that had undergone oophorectomy. Glucosamine-hydrochloric acid caused a prompt dose-related increase in uterine blood flow which approximated uterine blood flow rates induced by estradiol. Infusions of dextrose caused similar but transient vasodilatation, whereas infusions of mannitol had no effect. The glycosaminoglycans content of endometrium, myometrium, and caruncles/cotyledons (placentomes) was measured in nonpregnant and early pregnant ewes between 19 and 45 days' gestation. In endometrium and myometrium, total hexosamine levels were similar in nonpregnant and pregnant ewes. In the placentomes, total hexosamine levels increased steadily after 20 to 25 days' gestation. The hyaluronic acid fraction of total glycosaminoglycans hexosamine increased similarly and paralleled the total hexosamine levels. Galactosamine levels were unchanged in all tissues. It is postulated that a function of Wharton's jelly may be to provide the mechanism for shunting of uterine blood flow to the ovine placentomes.

84: Biosynthesis and processing of placental alkaline phosphatase.

Biochem Biophys Res Commun 1983 Mar 16;111(2):611-8

Ito F, Chou JY.

Polypeptides of 61,500 and 64,500 apparent molecular weights were the precursor and fully processed forms of placental alkaline phosphatase monomer synthesized by choriocarcinoma cells in vivo. [3H] Mannose was incorporated into both polypeptides whereas [3H] glucosamine was incorporated mainly into the 64,500-dalton polypeptide, suggesting processing by the addition of glucosamine moieties. In the absence of microsomal membranes, choriocarcinoma mRNA directed the cell-free synthesis of the preprotein form of alkaline phosphatase monomer of apparent Mr = 60,000. The unglycosylated monomer had an apparent Mr = 58,000. In the presence of microsomal membranes, the 60,000-dalton polypeptide was processed to a polypeptide of apparent Mr = 61,500, comigrating with the precursor form of alkaline phosphatase monomer.

85: An improved gas chromatographic method for measuring glucosamine and muramic acid concentrations.

Anal Biochem 1983 Feb 1;128(2):438-45

Hicks RE, Newell SY.

A method of simultaneously measuring glucosamine and muramic acid concentrations in marsh grass litter was developed. Spartina alterniflora samples were preextracted with acetone to remove lipids containing amino sugars and then hydrolyzed in 6 N HCl (100 degrees C, 4.5 h). Amino sugars in the hydrolysates were isolated by ion-exchange chromatography, which gave good recoveries (greater than 90%) and reproducibility (CV less than 5%). Isolated amino sugars were converted to O-methyloxime acetates. beta-Phenylglucose and N-methylglucamine were added as internal standards. Sample derivatives were quantified by capillary column gas chromatography. OV-101 and SE-54 capillary columns completely separated glucosamine and muramic acid from other amino sugars. The detection limit of glucosamine and muramic acid during gas chromatographic analysis was below 30 pmol using splitless-mode injection (SE-54 column). Filamentous fungal and procaryotic biomasses may be estimated simultaneously by using glucosamine and muramic acid biomass conversion factors in conjunction with this method.

86: Experimental studies on the influence of prostaglandins on the connective tissue of the human cervix uteri.

Acta Obstet Gynecol Scand Suppl 1983;113:167-70

Norstrom A, Wilhelmsson L, Hamberger L.

The influence of prostaglandins (PGs) on the synthesis of collagen and proteoglycans in the human cervix was estimated by incubation in vitro of cervical biopsy samples in the presence of tritiated amino acids and glucosamine after addition of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. In the follicular phase of the menstrual cycle PGs increased the labelling with [3H] glucosamine but reduced the labelling with [3H] hydroxyproline, [3H] proline and [3H] glycine. Conditions were reversed in the luteal phase. The stable metabolite of PGI2, 6-keto-PGF1 alpha, had an effect similar to that of the classical PGs. In the early period of the first trimester the pattern of PG effects was the same as in the luteal phase, i.e. the labelling with [3H] proline was increased and the incorporation of [3H] glucosamine was reduced after treatment with PG. At the end of the first trimester, PG affected the radiolabelling in an inverse way. It is concluded that PGs are involved in the control of the biosynthetic activity of the cervical connective tissue. PGs may play a role of modulators, reinforcing or accelerating the current biosynthetic activity, and directed mainly by hormonal, neuronal and other factors.

87: D-Glucosamine-induced increase of the glycerol-containing lipids in growing cultures of human malignant epithelial cells.

J Natl Cancer Inst 1983 Jan;70(1):57-61

Krug E, Dussaulx E, Rozen R, Griglio S, de Gasquet P, Zweibaum A.

D-Glucosamine was found to inhibit the growth of human malignant epithelial cells SW-839, HT-29, RT-4, and SK-OV-3 in culture in a process that was associated with significant increments in glycerol-containing lipids. Each cell line had a different sensitivity to the drug, but all four cell lines shared the same features in their response, i.e., dose-dependent (at concentrations of 1, 5, and 10 mM), noncytotoxic reductions in growth (minimum 30%, maximum 70%), and simultaneous 1.5-fold to sevenfold increases in lipid contents. Cells regained their normal growth and lipid patterns when glucosamine was removed. Glucosamine did not modify the lipid contents of cells in the late phase of culture when growth was minimal.

88: Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin.

J Biol Chem 1982 Sep 10;257(17):10172-7

Hanover JA, Elting J, Mintz GR, Lennarz WJ.

The glycoprotein hormone, human chorionic gonadotropin (hCG), contains both N- and O-linked oligosaccharide chains linked to its beta-subunit. Using the human choriocarcinoma cell line, BeWo, we have examined the temporal relationship between N- and O-glycosylation of hCG and the subsequent processing of both types of oligosaccharide chains. The results indicate that, as observed in related cell lines, mature, completely glycosylated forms of the subunits of hCG cannot be detected intracellularly in BeWo cells during pulse-chase experiments with [35S]methionine. To more directly study the temporal relationship between N- and O-glycosylation of hCG in BeWo cells, 14C-amino acids and [3H]glucosamine (which also serves as a precursor to N-acetylgalactosamine) were used to label hCG. The results of these studies are consistent with a model for the N- and O-glycosylation of hCG in which 1) N-glycosylation of hCG occurs co-translationally or very shortly after translation, and 2) the addition of O-linked GalNAc residues to the polypeptide and the addition of peripheral GlcNAc residues to the N-linked oligosaccharide chains occur just prior to secretion, presumably in the Golgi complex.

89: Influence of prostaglandin D2 on the biosynthesis of connective tissue constituents in the pregnant human cervix.

Prostaglandins 1982 Mar;23(3):361-7

Norstrom A.

Uterine cervical tissue was obtained from pregnant women undergoing abortion or caesarean section. The tissue was incubated in Krebs-Ringer bicarbonate buffer containing prostaglandin (PG) E2 and radioactive precursors for collagen (3H proline) and proteoglycans (3H glucosamine). After incubation the tissue-bound radioactivity was determined and related to the tissue dry weight. The effect of PGE1 on the net tissue radiolabelling varied with the gestational age and with the cervical status at operation. In early 1st trimester PGE2 increased the labelling with 3H proline but decreased that with 3H glucosamine. From the 12th week of gestation until term pregnancy conditions were reversed, i.e. the incorporation of 3H proline was reduced and that of 3H glucosamine was augmented following treatment with PGE2. After start of labour and rupture of the membrane, however, PGE2 diminished the labelling with 3H proline as well as 3H glucosamine. It is suggested that PGE2 is a modulator of biochemical events which underlie cervical ripening.

90: Progesterone effect on the biosynthesis of glycoconjugates, specifically of sulfated glycoprotein, in the endometrium of rabbit uterus.

Tohoku J Exp Med 1980 Oct;132(2):147-52

Endo M, Yosizawa Z.

The endometrial scrapings obtained from the uteri of estrogen-progesterone-treated and estrogen-treated rabbits were incubated with N-acetyl-D-[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25 M sucrose, and the insoluble residue (R-Fr) was separated. The supernatant at 8,5000 X g for 10 min of the homogenate was subjected to subcellular fractionation by discontinuous sucrose gradient ultracentrifugation, and a Golgi-rich fraction (G-Fr) was obtained. Crude glycoconjugates (glycosaminoglycans and glycoproteins) were then separated from M-Fr, R-Fr and G-Fr after pronase digestion. The amounts of the radioactivities incorporated into these glycoconjugates suggested that progesterone markedly suppressed the estrogen effect within 6 hr in R-Fr and G-Fr, whereas the suppression slightly delayed in M-Fr. The amounts of the radioactivities incorporated into acidic GC, which obtained by CPC-precipitation of the crude GC after digestion with crude heparinase, indicated that the biosynthesis of the CPC-precipitable sulfated glycoproteins was almost completely ceased by progesterone, although other glycoconjugates were still actively synthesized under the progesteronic condition.

91: Hormonal effects on the biosynthesis of sulfated glycoprotein in a microsomal fraction of the endometrium of rabbit uterus.

J Biochem (Tokyo) 1978 Feb;83(2):537-42

Yamamoto R, Munakata H, Yamamoto M, Yosizawa Z.

A crude microsomal fraction (M-Fr) was separated from the endometrial scrapings of uteri of ovariectomized rabbits with or without hormonal treatment. The effects of estrogen and progesterone on the incorporation into M-Fr of L-[U-14C]-fucose and N-acetyl-D-[6-3H]-glucosamine from their nucleotides were investigated. Estrogen increased the incorporation of these sugars, whereas progesterone suppressed this effect. The results of fractionation on a DEAE-Sephadex A-25 (Cl- form) column of the isotope-labelled complex saccharide mixtures, obtained by pronase digestion of the incubation mixtures, indicated that biosynthesis of sulfated glycoprotein was most sensitive to the hormones among the complex saccharides in M-Fr. Thus, a hormonal effects on the biosynthesis of sulfated glycoprotein in the endometrium of ovariectomized rabbit has been unambiguously confirmed at the microsomal level.

92: Biosynthesis of sulfated glycoprotein in the endometrium of rabbit uterus.

J Biochem (Tokyo) 1976 Feb;79(2):293-8

Endo M, Yosizawa Z.

The endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-d[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25m sucrose, and the insoluble residue (R-Fr) was separate. The supernatant at 8,500Xg for 10 min of the homogenate was subjected to subcelular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (g-fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr. In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-ACETYL-D[1-3H]glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.

93: Metabolic rate of acidic complex saccharides in rabbit uterus under estrogenic condition.

J Biochem (Tokyo) 1976 Jan;79(1):1-4

Endo M, Yosizawa Z.

Uterine slices obtained from estrogen-treated rabbits were incubated in vitro with N-acetyl-D-[1-3H]glucosamine together with D-[U-14C]glucose. The isotope-labelled acidic complex saccharides were then isolated by pronase digestion, Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane, in succession. In this way, individual acidic complex saccharides (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide, and sialoglycopeptide) were separated into 2-5 subfractions. The specific radioactivity of hexosamine in the subfractions indicated that the metabolic rate of the uterine complex saccharides as follows: hyaluronic acid greater than sulfated glycopeptide greater than heparan sulfate greater than chondroitin sulfate C greater than dermatan sulfate. In addition, metabolic heterogeneity of heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate was suggested.

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