1: Effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm.

Anim Reprod Sci 2002 Jan 23;69(1-2):91-7

Yi YJ, Cheon YM, Park CS.

A series of experiments were conducted to determine the effect of N-acetyl-D-glucosamine, glycerol concentration and equilibration time for the freezing of boar spermatozoa in 5 ml maxi-straws. The optimum final glycerol concentration in the diluent with 0.05% N-acetyl-D-glucosamine in the first diluent was 2-3% and the optimum glycerol equilibration time was 2-3h. In conclusion, we recommend the first diluent containing 11% lactose hydrate, 20% egg yolk and 0.05% N-acetyl-D-glucosamine in 100ml distilled water, and the second diluent containing 11% lactose hydrate, 20% egg yolk, 4% glycerol and 1% orvus es paste for the diluents of boar sperm freezing. Also, we found out that 0.05% soluble N-acetyl-D-glucosamine was the optimum concentration in the first diluent and a concentration of 0.05% soluble N-acetyl-D-glucosamine significantly enhanced the cryopreservation of boar spermatozoa.

2. 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.

3. 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.

4. Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl.

Glycoconj J 1999 Oct;16(10):639-48

Saez FJ, Madrid JF, Aparicio R, Leis O, Oporto B.

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal beta1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac alpha2,3Gal beta1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.

5. Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro.

Mol Hum Reprod 2000 Aug;6(8):699-706

Miranda PV, Gonzalez-Echeverria F, Blaquier JA, Mahuran DJ, Tezon JG.

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.

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. Heparin- and Zn2+-binding proteins from boar seminal plasma.

Acta Biochim Pol 1999;46(4):935-9

Holody D, Strzezek J.

Low molecular mass, heparin-binding proteins from seminal plasma play an important role in gametes interaction whereas plasmatic Zn2+-binding proteins stabilize chromatin and plasmalemma structures and protect spermatozoa in the female reproductive tract. By means of affinity chromatography the heparin- and Zn2+-binding proteins were isolated from boar seminal plasma and both preparations were analyzed by reverse HPLC. Most of the proteins bound to heparine and Zn2+-ions were classified as spermadhesins. Three fractions binding exclusively Zn2+ were isolated. They differ in amino-acid composition, content of glucosamine and content of protein components revealed by SDS/PAGE.

8. Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA).

FEBS Lett 1999 Sep 17;458(2):141-4

Montag M, van der Ven K, Dorbecker C, van der Ven H.

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.

9. The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs.

Biochem J 1999 Jul 1;341 ( Pt 1):1-4

Parrington J, Jones KT, Lai A, Swann K.

Cytosolic extracts of boar sperm contain a soluble phospholipase C (PLC) activity that induces Ca2+ release in sea-urchin (Lytechinus pictus) egg homogenates and an uncharacterized protein factor that causes Ca2+ oscillations when injected into mammalian eggs. In the present study we fractionated boar sperm extracts on three different FPLC chromatographic columns and found that the fractions that caused maximal Ca2+ release in sea-urchin egg homogenates were also the ones that triggered Ca2+ oscillations in mouse eggs. Our data suggests that the sperm factor which triggers Ca2+ oscillations in eggs contains a PLC and not the 33 kDa glucosamine deaminase previously suggested to be one its components.

10. Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein.

Braz J Med Biol Res 1999 Jan;32(1):59-65

Passos EP, Brugnara L, Facin AC, Riffel A, Lima GR, Freitas V, Brandelli A.

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control. NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

11. 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.

12. The role of carbohydrates in the induction of the acrosome reaction in mouse spermatozoa.

Biol Reprod 1999 Jan;60(1):94-101

Loeser CR, Tulsiani DR.

Center for Reproductive Biology Research and Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633, USA.

Capacitated acrosome-intact mouse spermatozoa bind to the egg's zona pellucida in a receptor-ligand-mediated manner. Mouse zona pellucida 3 (mZP3) is a glycoprotein that functions as a primary ligand and inducer of the acrosome reaction (AR). Multiple sugar residues on mZP3 are thought to be recognized by complementary sugar binding enzymes (glycosidases or glycosyltransferases) or sugar binding lectin-like proteins on the sperm surface. To elucidate the nature of the sugar residues involved in sperm-egg recognition, several neoglycoproteins (ngps) were tested for their ability to induce the AR. Ngps are synthetic glycoproteins with a known monosaccharide conjugated to BSA. Capacitated mouse spermatozoa were treated in the absence or presence of several concentrations of ngps. A significantly greater number of spermatozoa underwent the AR in the presence of mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA than in their absence. Glucose-BSA or galactose-BSA had no effect on the AR. Inclusion of millimolar concentrations of unconjugated sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) neither induced the AR nor blocked induction of the AR by ngps. These results demonstrate that some sugar residues can induce the AR, but only when conjugated to a protein backbone. Glucosaminyl-BSA (but not mannosyl-BSA or galactosaminyl-BSA) was a substrate for sperm-surface galactosyltransferase (GT), an enzyme thought to function as a receptor by binding to complementary glucosaminyl residues on mZP3. These data suggest a possible interaction between protein-conjugated glucosaminyl residues and sperm GT in the induction of the AR.

13. 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.

14. The human glucosamine-6-phosphate deaminase gene: cDNA cloning and expression, genomic organization and chromosomal localization.

Gene 1998 Aug 17;216(1):31-8

Shevchenko V, Hogben M, Ekong R, Parrington J, Lai FA.

When mammalian eggs are fertilized by sperm, a distinct series of calcium oscillations are generated which serve as the essential trigger for egg activation and early embryo development. The identification of a soluble hamster sperm 33-kDa protein that co-migrated with calcium oscillation-inducing activity was recently described by Parrington et al. (Parrington, J., Swann, K., Shevchenko, V.I., Sesay, A.K. and Lai, F.A., 1996. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379, 364-368). The hamster sperm 33 kDa protein was termed oscillin because it correlated with calcium oscillation-inducing activity in mammalian eggs. Sequence analysis of the hamster sperm 33 kDa protein indicated no similarity to any known cell signalling molecule, however, it displayed extensive homology with a bacterial glucosamine-6-phosphate deaminase. We have isolated the corresponding human testis homologue of the hamster sperm 33 kDa cDNA. Nucleotide sequence analysis reveals a high level of sequence identity between the hamster and human genes. The deduced protein sequence of the human gene also shares extensive amino acid identity with the bacterial glucosamine-6-phosphate deaminase enzyme. Heterologous expression of the human testis 33 kDa protein produced a glucosamine-6-phosphate deaminase activity. The genomic structure of the human glucosamine-6-phosphate deaminase has been mapped and the gene was localized by fluorescence in situ hybridization (FISH) to chromosome 5q31.

15. Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes Lewis-a trisaccharide.

Biol Reprod 1998 Jul;59(1):39-44

Suarez SS, Revah I, Lo M, Kolle S.

Sperm binding to oviductal epithelium produces a reservoir in vivo that may serve to maintain sperm fertility and provide sperm for fertilization when ovulation occurs. Previously, it was determined that bull sperm binding could be blocked by fucoidan and its component fucose; furthermore, treatment of epithelium with fucosidase prevented binding. The present study was conducted to further characterize binding. Because fucose would probably be present on the epithelium as part of oligosaccharide moieties of glycoproteins and/or glycolipids, competitive binding inhibition activity was tested for fucose in five linkages commonly found in oligosaccharides. Binding inhibition was assayed by determining the concentration of motile, frozen/thawed sperm bound to fresh epithelial explants in the presence of test inhibitors. Initially, 5 monosaccharides were tested at 30 mM (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose), and only fucose significantly reduced sperm binding compared to vehicle control (p = 0.03). Of the oligosaccharides tested (lacto-N-fucopentaose I, 3-fucosyllactose, Lewis-X, Lewis-a, and GlcNAcbeta1-4[Fucalpha1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.

16. Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.

FASEB J 1998 Jan;12(1):91-9

Wolosker H, Kline D, Bian Y, Blackshaw S, Cameron AM, Fralich TJ, Schnaar RL,
Snyder SH.

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.

17. The metabolic properties of spermatozoa from the epididymis of the tammar wallaby, Macropus eugenii.

Mol Reprod Dev 1998 Jan;49(1):92-9

Murdoch RN, Jones RC.

The utilization of various substrates by sperm from the cauda epididymidis of the tammar was examined because the major naturally occurring sugar in the semen of this species is N-acetyl-D-glucosamine (NAG) and not furctose, as in eutherian mammals. The sperm displayed a high level of endogenous respiration that supported motility for relatively prolonged periods of time in vitro. They also metabolised exogenous 14C-labelled glucose, NAG, sucrose, and acetate through glycolytic and/or oxidative processes to produce lactate and 14CO2 at varying rates. The rate of uptake of NAG by tammar sperm was about four times greater than that of other substrates. Glucose and/or NAG stimulated the rate of oxygen consumption by about 20%, but acetate stimulated oxygen consumption by more than 40%. The most striking findings were that NAG almost completely inhibited the oxidation of glucose and sucrose by the sperm and depressed the uptake of glucose, 3-O-methylglucose, and sucrose. Acetate oxidation also was inhibited by NAG, but only by about 50%. Tammar sperm generated substantial amounts of free glucose during incubation with NAG, but this and the inhibitory effects of NAG on glucose oxidation were not mimicked by rat sperm. It is proposed that tammar sperm fail to oxidise glucose in the presence of NAG because of the rapid cellular uptake of NAG relative to glucose. Also, the intracellular glucose and acetate liberated from NAG would compete with exogenous glucose for processing in the Embden-meyerhof and tricarboxylic acid (TCA) cycle pathways. It is also suggested that tammar sperm oxidise sucrose after extracellular hydrolysis into its glucose and fructose components. The biological implications of these metabolic and transport properties of tammar sperm have as yet to be determined.

18. 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.

19. Major goat sperm 105 kDa maturation antigen: purification, characterization, and effect of its antiserum on acrosin activity.

Am J Reprod Immunol 1997 May;37(5):399-407

Erratum in: Am J Reprod Immunol 1999 Mar;41(3):232

Sarkar M, Das T, Chatterjee T.

A ConA binding membrane glycoantigen of 105 kDa molecular mass was purified from mature goat sperm by ion exchange and gel filtration chromatography. Of the detergents examined, the anionic deoxycholate was found to be highly effective in maximum solubilization of this sperm membrane antigen (SMA2). The analysis of the saccharide components by gas liquid chromatography revealed that the 105 kDa antigen (SMA2) contained the highest amount of mannose, followed by galactose and glucose in a ratio of 4:3:1. One amino sugar, N-acetylglucosamine, was also found to be present in the polysaccharide branching of the SMA2 antigen. The internal sulfydryl linkage is essential for the maintenance of the protein backbone of 105 kDa antigen. The antigen selectively resides on the anterior head of goat sperm. The binding of anti-SMA2 antibody to the integrated mature goat spermatozoa inhibited the release of acrosin after the induction of spermatozoa with Ca-ionophore.

20. Modulation of sperm acrosomal exocytosis by guanyl nucleotides and G-protein-modifier agents.

Biochem Mol Biol Int 1997 May;41(6):1217-25

Brandelli A.

Mammalian sperm must undergo an exocytotic event during fertilization, the acrosome reaction (AR). This process is specifically induced by egg-surface glycoproteins and it involves guanine nucleotide binding proteins (G-proteins). Neoglycoproteins (NGP) with mannose or N-acetylglucosamine residues has been demonstrated to induce the AR in human sperm. Activators of G-proteins, like GTP gamma S, GppNHp, mastoparan and AlF4- were capable of inducing the AR, while other nucleotides or analogues did not. When sperm were preincubated with these agents and then with NGPs, only the G-protein inhibitor GDP beta S decreased the AR rate. The preincubation of sperm with Pertussis toxin resulted in the inhibition of NGP-induced AR, while no effect was observed with cholera toxin. Results indicate that direct activation of G-proteins is sufficient to elicit the AR, and the induction of the AR in human sperm is mediated by N-acetylglucosaminyl and mannosyl binding sites involving PTx-sensitive G-proteins similar to the induction by zona pellucida glycoproteins.

21. Binding of human spermatozoa to lectin-coated agarose microbeads.

Arch Androl 1997 Mar-Apr;38(2):133-41

Gabriel LK, Franken DR.

An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP) may be at the origin of many cases of poorly explained or idiopathic infertility. It would be clinically useful to be able to distinguish this condition from other causes of infertility. A major problem in testing the sperm-ZP binding ability is the paucity of biological ZP. Examination of whether sperm binding to PNA-, UEA-1-, WGA-, Con A-, or PSA-coated agarose microbeads reflected sperm binding to biological ZP and correlated with in vitro fertilization rates showed that only binding to WGA-coated microbeads showed significant positive and negative predictive values when compared to IVF rates in 2 x 2 contingency. Sperm binding to PNA, Con A, and PSA was indiscriminately high, irrespective of IVF rate. Human spermatozoa did not bind to UEA-1-coated agarose microbeads. Furthermore, sperm binding to WGA-coated microbeads correlated with sperm morphology ratings. These results implicate terminal N-acetyl-D-glucosamine and/or sialic acid (specific saccharides for WGA) in sperm-ZP interaction and also suggest that the use of lectin-coated microbeads may represent an initial step in the development of a synthetic sperm binding assay.

22. Fractionation and characterization of boar seminal plasma spermadhesion PSP-II glycoforms reveal the presence of uncommon N-acetylgalactosamine-containing N-linked oligosaccharides.

Glycoconj J 1997 Feb;14(2):275-80

Solis D, Calvete JJ, Sanz L, Hettel C, Raida M, Diaz-Maurino T, Topfer-Petersen E.

Lectin mapping, carbohydrate analysis and electrospray mass spectrometry of boar seminal plasma PSP-II glycoforms show that its single N-glycosylation site displays a repertoire of carbohydrate structures consisting of the basic pentasaccharide core Man alpha 1-6[Man alpha 1-3]Man beta1-4GlcNAc beta1-4GlcNAc with a fucosyl residue alpha1-6-linked to the innermost N-acetylglucosamine residue. Other glycoforms display fucosylated hybrid-type or monoantennary complex-type chains, some of which contain alpha2-6-linked sialic acid. N-acetylgalactosamine, possibly in Gal beta1-3GalNAc sequence, is present in most of the PSP-II glycoforms.

23. Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse.

Mol Reprod Dev 1996 Dec;45(4):535-46

Thaler CD, Cardullo RA.

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized 125I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 x 10 (7)M-1 min-1. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble 125I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (approximately 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the gal beta 1, 3-GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor.

24. 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.

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

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.

25. Voltage-dependent calcium channels and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosaminyl/mannosyl neoglycoproteins.

J Androl 1996 Sep-Oct;17(5):522-9

Brandelli A, Miranda PV, Tezon JG.

Mammalian spermatozoa must undergo an exocytotic event during fertilization, the acrosome reaction (AR). In most species studied this process is induced by specific glycoproteins of the oocyte extracellular matrix, the zona pellucida (ZP), and it involves guanine nucleotide-binding regulatory proteins (G-proteins), resulting in an uptake of extracellular calcium by the sperm. In the bull, this event has been reported to be mediated by voltage-dependent calcium channels (VDCC). Previous observations showed that neoglycoproteins (NGPs) with N-acetylglucosamine or mannose (GlcNAc-BSA or Man-BSA) residues induce the AR in capacitated human spermatozoa. We report here that the pretreatment of spermatozoa with 125 ng/ml pertussis toxin (PTx) inhibited GlcNAc-BSA- or Man-BSA-induced AR, whereas 1 microgram/ml cholera toxin had no effect. These data indicate that the transduction mechanism for GlcNAc-BSA- and Man-BSA-induced AR involves G-proteins of the inhibitory type (GI). An increase in the AR rate was observed when capacitated spermatozoa were incubated with increasing concentrations of potassium ions (K+) in Biggers-Whitten-Whittingham (BWW) modified medium (2.6 +/- 0.3-fold at 80 mM K+). This induction was observed only when the pH was raised to 8.5, and it was inhibited by verapamil, nitrendipine, omega-conotoxin, nickel ions (Ni2+), lanthanum ions (La3+), or cadmium ions (Cd2+) in a concentration-dependent manner, indicating the participation of VDCC activated by membrane depolarization. The GlcNAc-BSA- or Man-BSA-induced AR was completely inhibited by preincubation of spermatozoa with VDCC blockers and calcium antagonists, indicating a link between the binding of sugar residues of the NGPs and channel activation. The AR induced by membrane depolarization with high K+ medium was not inhibited by PTx, suggesting that Ca2+ entry is downstream to GI-protein activation. These data show that the induction of the AR in human spermatozoa by GlcNAc- or Man-NGPs involves VDCC and GI-like regulatory proteins similar to the induction described for ZP in other mammalian species.

26. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein.

Nature 1996 Jan 25;379(6563):364-8

Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA.

At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo. It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated. Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion. Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs. Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head. Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes. This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

27. Distribution of lectin binding in the testes of the musk shrew, Suncus murinus.

J Anat 1995 Oct;187 ( Pt 2):323-9

Kurohmaru M, Kobayashi H, Kanai Y, Hattori S, Nishida T, Hayashi Y.

Distribution of lectin binding in the testis of the musk shrew, Suncus murinus, was investigated by light and transmission electron microscopy. Not only spermatogenic cells but also Sertoli cells bound some lectins. Canavalia ensiformis agglutinin (Con A) and wheat germ agglutinin (WGA, Triticum vulgaris), indicating the presence of D-mannose and N-acetyl-D-glucosamine respectively, showed an intense reaction in the acrosomal region of early to late spermatids. Ricinus communis agglutinin I (RCA-I), peanut agglutinin (PNA, Arachis hypogaea), Bauhinia purpurea agglutinin (BPA), soybean agglutinin (SBA, Glycine max), revealing the presence of D-galactose and/or N-acetyl-D-galactosamine, bound to the acrosomal region from Golgi to acrosome-phase spermatids and abruptly decreased in intensity in maturation-phase spermatids. GS-II, indicating the presence of N-acetyl-D-glucosamine, gave an intense reaction only in the acrosome of acrosome-phase spermatids. These findings demonstrate that the appearance/disappearance of some glycoconjugates in the spermatid acrosome occurs in the musk shrew during acrosomal formation. Additionally, RCA-I, PNA and BPA revealed a strong reaction in the cytoplasm of Sertoli cells. The reaction that was observed in the intramembranous region of Sertoli cell cytoplasm at the electron microscope level appeared from stages VIII to XIII but not from stages I to VII. This finding suggests that glycoconjugates containing D-galactose may change stage-dependently in the musk shrew Sertoli cell.

28. A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein.

Hum Reprod 1995 Jul;10(7):1751-6

Brandelli A, Miranda PV, Anon-Vazquez MG, Marin-Briggiler CI, Sanjurjo C, Gonzalez-Echeverria F, Blaquier JA, Tezon JG.

Neoglycoproteins with N-acetylglucosamine residues (BSA-GlcNAc) induced specifically the acrosome reaction (AR) in human spermatozoa. Our objective was to investigate the relationship between this phenomenon and the invitro fertilization (IVF) rate. Sperm suspensions from IVF protocols were incubated with BSA-GlcNAc (t), using calcium ionophore (i) or medium alone (c) as positive or negative controls. When the normalized AR percentage ratio (STIM) (% ARt-%ARc):(%ARi-%ARc) was compared with fertilization rate in 31 couples from our IVF programme, a positive correlation was found (r = 0.46, P < 0.01). The fertilization rate in patients with STIM > or = 0.2 was higher than in non-responders (STIM < 0.2); 72 +/- 7% compared with 5 +/- 3%. The overall predictive value of this test for adequate fertilization rate (> 30%) was 87%, sensitivity 91% and specificity 78%. False positives were 9% and false negatives 22%. For successful fertilization rates (> 60%), the results were: overall predictive value, 84%; sensitivity 100%; specificity 64%. False positives were 23% and no false negatives were found. The results indicated that the induction of AR in human spermatozoa by GlcNAc-neoglycoproteins could be used to predict their fertilizing ability in vitro.

29. Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma.

Biol Reprod 1995 Jul;53(1):201-8

Drisdel RC, Mack SR, Anderson RA, Zaneveld LJ.

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.

30. Biochemical characterization of a bovine oviduct-specific sialo-glycoprotein that sustains sperm viability in vitro.

Biochim Biophys Acta 1995 Apr 28;1266(2):117-23

Satoh T, Abe H, Sendai Y, Iwata H, Hoshi H.

A bovine oviduct-specific glycoprotein (BOGP) that sustained the viability of bovine spermatozoa in vitro was purified from an extract of bovine oviducts. The amino-terminal amino acid sequence of BOGP was found to be a homologous with that of oviductin, a protein from hamster that was recently characterized by Mallete and Bleau (1993: Biochem. J. 295, 437-445). Purified BOGP was characterized as a sialo-glycoprotein containing N-linked and O-linked sialo-oligosaccharides side chains with galactose, mannose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, fucose and sialic acids in its core protein (57 kDa). Intact BOGP has a wide range of isoelectric points (pIs) from 6.5 to 3.0 but a narrow range of molecular masses around 95 kDa. On isoelectric focusing of neuraminidase-treated BOGP (AS-BOGP), a narrow band with a pI of 9.3 was observed, and the ability of AS-BOGP to maintain sperm viability was negligible. We propose that BOGP is a mucin-type sialo-glycoprotein with a molecular mass of 72 kDa that contains one N-linked and approx. 15 O-linked sialo-oligosaccharide chains. These side chains appear to be important for the maintenance of sperm viability.

31. 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.

32. Developmentally regulated oligosaccharides in mouse spermatogenic cells.

Arch Biochem Biophys 1994 Jun;311(2):469-79

Maylie-Pfenninger MF.

Mouse spermatogenic cells were separated into four populations, pachytene spermatocytes, round spermatids, elongated spermatids, and residual bodies. Each cell population was metabolically labeled with [3H]galactose, [3H]glucosamine, or [3H]fucose. Glycopeptides were prepared from the radiolabeled glycoproteins by pronase digestion and fractionated by serial lectin affinity chromatography and gel filtration. The presence of O-linked oligosaccharides was assessed by pronase digestion of [3H]galactose-labeled glycoproteins, exclusion of the radiolabeled glycopeptides from a ConA-Sepharose column, gel filtration, and treatment with alkaline borohydride. This analysis reveals that a large proportion of [3H]galactose-labeled oligosaccharides (47-52%) are O-linked structures, while the majority (80-90%) of [3H]fucose-labeled oligosaccharides are N-linked. The proportions of triantennary, biantennary, oligomannose, and hybrid oligosaccharides linked to asparagine vary with the cell populations. Furthermore, in round spermatids, but not in the other cell populations, a relatively large proportion (15%) of [3H]glucosamine-labeled oligosaccharides consists of terminal O-linked N-acetylglucosamine. Taken together these data show that each spermatogenic cell population contains a unique complement of oligosaccharide structures that could play an important role as differentiation signals in the interactions among these cells and/or with Sertoli cells.

33. pH-sensitive dissociation and association of beta-N-acetylhexosaminidase from boar sperm acrosome.

Biol Reprod 1994 Apr;50(4):860-8

Takada M, Yonezawa N, Yoshizawa M, Noguchi S, Hatanaka Y, Nagai T, Kikuchi K, Aoki H, Nakano M.

beta-N-Acetylhexosaminidase (beta-Hex, EC, 3.2.1.52) was released from cauda epididymal boar sperm by treatment with ionophore A23187, indicating that this enzyme is localized in the acrosome. beta-Hex was extracted on a large scale, with 2% acetic acid containing 0.2% Brij 35, from washed ejaculated sperm. By gel filtration chromatography, beta-Hex was separated into a high-molecular-weight fraction (beta-Hex I) and a low-molecular-weight fraction (beta-Hex I). beta-Hex I, which is predominant under acidic conditions (pH 6.5), dissociated into beta-Hex II under alkaline conditions (pH 7.4). beta-Hex II, converted from beta-Hex I, associated again to form beta-Hex I under acidic conditions. By sequential chromatography on ion-exchange, lectin, gel filtration, and ion-exchange HPLC columns, beta-Hex I and II were purified 1200-fold and 4000-fold, respectively, with a combined recovery of 23% as measured with synthetic substrate. An inhibitor of beta-Hex, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl-carbamate (PUGNAC), reduced the in vitro fertilization rate in porcine cumulus-enclosed eggs, but barely changed the rate when cumulus-free eggs were used. beta-Hex I was shown to possess cumulus dispersion activity, suggesting that beta-Hex plays a role in the passing by sperm through cumulus cells before they bind to the zona pellucida.

34. Ultrastructural and cytochemical studies of the spermatid and spermatozoon of Culex quinquefasciatus (Culicidae).

J Submicrosc Cytol Pathol 1993 Apr;25(2):213-22

Bao SN, de Souza W.

Ultrastructural and cytochemical studies were carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. The spermatozoon appeared threadlike, lacking distinct head and tail regions. The nucleus development involved nuclear shape change and chromatin condensation. The acrosome showed two distinct zones and a subacrosomal space. In the sperm tail an axoneme and two mitochondrial derivatives containing crystalline matrix were observed. The axoneme presented a 9+9+'1' microtubule pattern and the accessory microtubules were composed of fifteen protofilaments. The plasma membrane of the spermatozoon was asymmetric, presenting a 30 nm thick surface coat which was not sensitive to the action of pepsin. Carbohydrates were located in the plasma membrane and in the interface basal layer of the coat, by using the periodic acid-thiosemicarbazide-silver proteinate technique. Lectin gold studies revealed mannose, glucose, galactose, N-acetylglucosamine and sialic acid residues on the cell surface. These carbohydrate residues groups randomly occurred on the surface at the head and tail regions. The surface coat disappeared during the sperm storage in the spermatheca before fertilization.

35. Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction.

Dev Biol 1991 Oct;147(2):440-4

Macek MB, Lopez LC, Shur BD.

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.

36. Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains.

Mol Reprod Dev 1991 Jun;29(2):172-9

Anakwe OO, Sharma S, Hardy DM, Gerton GL.

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pulse-chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of proacrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N-glycosidase or O-glycosidase reduced the Mr by 3-7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N- and O-linked oligosaccharides.

37. 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.

38. The vitelline coat spikes: a new peculiar structure of Mytilus galloprovincialis eggs with a role in sperm-egg interaction.

Mol Reprod Dev 1991 Feb;28(2):143-9

Focarelli R, Rosa D, Rosati F.

In the course of this study we found that in Mytilus galloprovincialis eggs long filamentous protrusions never described before, which we have termed "vitelline coat spikes," could be clearly detected using the lectin from Dolichos biflorus, which recognizes the GalNAc residues. The spikes could be also observed by transmission electron microscope but only in some fortuitous sections could their origin in the vitelline coat be clearly observed. The spikes were also clearly visible using the scanning electron microscope. Observations of the sperm-egg interaction very few seconds after insemination or using fixed eggs suggested that the spikes could play a role in a primary binding to the unreacted sperm. Experiments have been done to test the effect of GalNAc on the sperm-egg binding and on the fertilization process which seem to confirm this hypothesis.

39. Identification of two maturation-related, wheat-germ-lectin-binding proteins on the surface of mouse sperm.

Acta Anat (Basel) 1991;142(2):165-70

Liu HW, Wang JJ, Chao CF, Muller C.

Epididymal maturation is essential for mammalian sperm to develop fertility. Wheat germ agglutinin (WGA), a lectin which specifically recognizes N-acetyl-glucosamine and sialic acid, was used to investigate membrane characteristics of epididymal sperm in the mouse. Histochemical and cytochemical localization revealed that WGA-binding sites were increased as sperm became mature. The binding sites were mainly localized in the plasma membrane of the anterior acrosome and tails. On Western blots of NP-40 extracts, two WGA-binding glycoproteins, GP-49 and GP-83, were identified on the sperm recovered from both corpus and cauda epididymidis. GP-83 was removed from the sperm surface by centrifugation and resuspension in phosphate-buffered saline (PBS) three times. GP-49 was resistant to centrifuging at 400 g for 5 min up to seven times and the treatment with 1M NaCl in PBS. These observations suggest that GP-49 is very likely an intrinsic membrane protein of the sperm, whereas GP-83 is an extrinsic protein.

40. 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.

41. 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.

42. 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.

43. 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.

44. Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse.

Biol Reprod 1988 May;38(4):955-67

O'Brien DA, Gerton GL, Eddy EM.

Monoclonal antibody 1D4, a mouse immunoglobulin M raised against CD-1 mouse spermatogenic cell membranes, recognizes acrosomal constituents in the mouse, rabbit, and guinea pig. In the mouse, acrosomes of round and condensing spermatids were labeled with 1D4 by indirect immunofluorescence on isolated cells and by immunohistochemistry on paraffin sections. During the terminal steps of spermiogenesis, however, acrosomal labeling in mouse germ cells was lost. Little or no 1D4 immunoreactivity was detected by enzyme-linked immunosorbent assays in prepubertal testes, Sertoli cells, or several somatic tissues. To identify antigens recognized by 1D4, mouse spermatogenic cell proteins were separated by one- (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. Multiple antigens larger than 200,000 relative molecular weight (Mr) were resolved on 1D immunoblots from round and condensing spermatids isolated by sedimentation velocity at unit gravity. A smaller antigen (Mr 85,000 isoelectric point approximately 5.7) was also detected on 1D and 2D immunoblots of round spermatid proteins. These antigens can be labeled biosynthetically with [3H] glucosamine and immunoprecipitated, suggesting that they are a set of glycoconjugates that share a common epitope recognized by 1D4. This determinant is no longer detectable in late spermatids, indicating that biochemical modifications of acrosomal constituents occur during the terminal steps of germ cell differentiation.

45. Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit.

Gamete Res 1987 May;17(1):35-42

Kopecny V, Flechon JE.

Rabbit spermatozoa were labeled predominantly in their acrosomal glycoproteins by 1-3H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.

46. Treatment of cervical mucus with lectins: effect on sperm migration.

Fertil Steril 1985 Nov;44(5):633-7

Snyder MG, Zaneveld LJ.

Treatment of midcycle human cervical mucus with the lectins Ulex europaeus agglutinin I (UE), wheat germ agglutinin (WGA), and peanut agglutinin (PA) did not alter the ability of spermatozoa to enter and migrate through the mucus. These lectins form glycoconjugates by binding to L-fucose (UE), N-acetylglucosamine and sialic acid (WGA), and D-galactose and D-galactose-(1-3)-D-N-acetylgalactosamine (PA), sugars that are present in the carbohydrate side chains of the mucus glycoproteins (mucins). Ricinus communis, with high affinity for D-galactose and N-acetylgalactosamine, did cause a significant decrease in sperm migration but only within the mucus (not at the sperm-mucus interface); this may have been because of an effect on sperm motility. These sugars are thought to be important for the secondary structure of the glycoproteins and for the cross linking between the mucins that produce the rheologic properties of cervical mucus. However, it appears that interference with the sugars by lectin binding does not significantly alter the ability of spermatozoa to migrate through cervical mucus.

47. Lactosaminoglycans synthesized by mouse male germ cells are fucosylated by an epididymal fucosyltransferase.

Dev Biol 1984 Apr;102(2):402-8

Cossu G, Boitani C.

We have studied the synthesis of protein-bound carbohydrates in differentiating male germ cells in the mouse. Spermatocytes and spermatids synthesize asparagine-linked and high-molecular-weight glycopeptides as the major classes of protein bound carbohydrates. Asparagine-linked glycopeptides were found to be mainly composed of the complex bi-antennary type as shown by affinity chromatography on concanavalin-A Sepharose; high-molecular-weight glycopeptides were represented by nonfucosylated lactosaminoglycans since they were metabolically labeled with [14C]glucosamine but not with [3H]fucose, did not bind to DEAE-cellulose, and were susceptible to endo-beta-galactosidase. Labeling with galactose oxidase/Na B3H4 technique demonstrated that lactosaminoglycans were present on the surface of differentiating germ cells and of testicular and epididymal spermatozoa. Since lactosaminoglycans from germ cells and testicular spermatozoa were not retained on a column of fucose-binding lectin, it was concluded that these molecules do not contain fucose. On the other hand, epididymal spermatozoa lactosaminoglycans bound to the lectin and therefore contained fucose. A soluble fucosyltransferase, capable of transferring fucose to germ cell lactosaminoglycans, was found to be present in the epididymis but not in the testis. These data show that developing germ cells synthesize nonfucosylated lactosaminoglycans which are probably preserved throughout spermiogenesis. We suggest that these molecules are fucosylated in vivo by a fucosyltransferase secreted by the epididymal epithelium.

48. The structure and epididymal maturation of the spermatozoon of the common marmoset (Callithrix jacchus).

J Anat 1984 Mar;138 ( Pt 2):227-35

Moore HD, Hartman TD, Holt WV.

Maturation of the spermatozoon of the common marmoset (Callithrix jacchus) during epididymal transit was investigated using light and electron microscopy. Except for the caudal migration of the cytoplasmic droplet there were no apparent ultrastructural modifications, although interesting morphological features of the sperm head were observed such as the substructure of the acrosome and a waist at the equatorial region. Alteration to the plasmalemma during maturation was reflected by an increasing capacity of the sperm surface to bind wheat germ agglutinin (recognising n-acetyl-d-glucosamine) but not ricin communis or concanavalin agglutinins. This change was concomitant with the development of sperm fertilising capacity in this species.

49. Binding of secreted glycoproteins to spermatozoa in the mammalian epididymis: a fine-structure autoradiographic study.

Anat Rec 1984 Feb;208(2):197-206

Kopecny V, Flechon JE, Pivko J.

Mouse and guinea pig epididymal tissues have been investigated by light and electron microscopic autoradiography after long intervals ranging from 24 h to 5 days postinjection (p.i.) of the glycoprotein precursors, L-fucose-6-3H or D-glucosamine-1-3H. Using modified fixations to enhance glycoprotein preservation in situ, we found intense labelling of luminal contents in at least some of the epididymal segments after all the intervals investigated. At 24 h p.i., the label in guinea pig was associated with spermatozoa during remodelling of the acrosome in segment II, and at 3 days p.i., radioactivity was trapped within sperm head associations ("rouleaux") in segment IV of the epididymis. At this time, similar rouleau labelling extended from segment IV to segment VIII. In mouse, the luminal contents of the cauda epididymis were still intensely labelled at 5 days p.i.; analysis of the electron microscopic autoradiograms showed that relative grain concentration over the spermatozoa was twice that of the epididymal plasma. This concentration was especially elevated in the region of the sperm head. These findings taken together were interpreted as the binding of secreted epididymal glycoproteins to spermatozoa during sperm transit through the epididymis. In contrast to luminal contents, the labelling of the epididymal epithelium was generally lower, except on the clear cells which showed more pronounced labelling than the neighboring principal cells in mouse cauda epididymis at 5 days p.i. This label probably originated from the resorption of luminal glycoproteins.

50. Studies on acrosome labelling of mammalian spermatozoa by radioactive sugars.

Reprod Nutr Dev 1984;24(4):419-29

Kopecny V, Cechova D, Zelezna B, Flechon JE, Motlik J, Pech V.

The localization of glycoprotein synthesis and storage was studied during acrosome formation in guinea-pig using fine-structure autoradiography after (3H)-fucose incorporation. Three days after (3H)-fucose injection, labelling in spermatids was concentrated in the matrix of developing acrosomes, and it was evident that the fucosylation of acrosomal glycoproteins largely overshadowed the fucosylation of other spermatid glycoproteins. Acrosin labelling and its quantitative relation to labelling of other glycoproteins was examined in mature rabbit spermatozoa after incorporation of (14C)-fucose or (14C)-glucosamine during spermatogenesis. Cauda epididymis spermatozoa recovered 21 days after intratesticular application of (14C)-fucose or (14C)-glucosamine were analysed for acrosin specific labelling after acid extraction and gel filtration. In all the material examined, radioactivity was detected in the proacrosine fractions; radioactivity in purified proacrosin amounted to at least 2% of the total radioactivity in the epididymal sperm population. In addition to the peak with radioactive proacrosin, another radioactive peak in (14C)-glucosamine-labelled material was attributed to a glycoprotein intraacrosomal inhibitor of acrosin. It is concluded that (pro)acrosin (acrosin-inhibitor) complexes seem to contribute significantly to acrosomal glycoprotein labelling by radioactive sugars and that the distribution of these complexes may at least correspond to their cytochemically detectable component, acrosin. The superposition of the distribution of acrosin and of other acrosomal glycoproteins during acrosome reaction can be explained by the fact that the dispersal of most of the acrosomal content is linked to proacrosin activation.

51. Hexosamine content of normal and pathological human sperm.

Andrologia 1983 Nov-Dec;15(6):655-8

[Original Article in German]

Nadermann E, Nissen HP, Kreysel HW.

Glucosamine and galactosamin were determined in 96 human ejaculates. The principal hexosamine of spermatozoa was galactosamin on the other hand glucosamin was the principal aminosugar of seminal plasma. The results were compared with the fertility parameters. Our results show no definity relation between the hexosamine concentration and the andrological conditions.

52. Purification of rabbit sperm autoantigens by preparative SDS gel electrophoresis: amino acid and carbohydrate content of RSA-1.

Biol Reprod 1982 Oct;27(3):713-21

O'Rand MG, Porter JP.

This study has described a relatively simple preparative procedure for isolating sperm autoantigens from testis membrane pellets using an SDS-7%-15% polyacrylamide gradient prep-disc gel column. Using this system, the rabbit sperm membrane autoantigen, RSA-1, has been isolated without the use of an immunoadsorbent column, giving an almost 10-fold greater yield than with the immunoadsorbent column method. A second autoantigen (RSA-2) has also been isolated. Both RSA-1 and RSA-2 retain their antigenic activity after isolation from the prep-disc gel. The carbohydrate and amino acid content of RSA-1 was determined. RSA-1 is 9.1% carbohydrate and by gas liquid chromatographic analysis contains xylose, galactose, mannose, glucose and glucosamine. The average hydrophobicity of RSA-1 indicates that it is a relatively nonpolar, asymmetric protein in the general category of fibrous or tropomyosin-like proteins.

53. Origin of glycerylphosphorylcholine, inositol, N-acetylaminosugar, and prostaglandins in human seminal plasma and their effects on sperm metabolism.

Arch Androl 1980 Mar;4(2):149-55

Brown-Woodman PD, Marley PB, Morris S, Rodger JC, White IG.

The origin of glycerylphosphorylcholine (GPC), N-acetylaminosugar, inositol, and prostaglandins in human seminal plasma was investigated by correlating the concentration of these components in split ejaculates with known marker constituents. Fructose and acid phosphatase were selected as markers of the secretory activity of the seminal vesicles and prostate gland, respectively, and spermatozoa indicated epididymal origin. The concentration of fructose was lowest in the first fraction of the semen and increased to a maximum in the final portion. Prostaglandins E and F and N-acetylaminosugar values closely followed this pattern, indicating that these components originate in the seminal vesicles. The concentration of spermatozoa was high in the first two fractions, decreasing to a minimum in the final fraction. The distribution of GPC was similar to that of the spermatozoa, indicating that the epididymis secretes this compound. Inositol levels were similar in all fractions, indicating that it is probably present in epididymal, vesicular, and prostatic fluid. Human spermatozoa were unable to utilize N-acetylglucosamine or inositol. High concentrations of some prostaglandins (100 micrograms/ml of PGF1 alpha, 15S 15 met. F2 alpha, PGA1, and PGA2) depressed the endogenous oxygen uptake of human spermatozoa.

54. Protein composition of human sperm from oligozoo-, teratozoo-, asthenozoo- and azoospermia.

Andrologia 1980 Jan-Feb;12(1):61-5

[Original article in German]

Paulsen H, Nissen HP, Heinze I, Schirren C, Kreysel HW.

The amino acid analysis of seminalplasma and spermatozoa shows for the different andrological diagnoses that the predominant amino acids were aspartic--and glutamic acid, serine, glycine and lysine. Analytical data indicate that there were differences between plasma and spermatozoa for a lot of amino acids. No differences in the protein composition of the ejaculates were found between the various groups of andrological diagnoses.

55. Isolation and characterization of the vitelline layer of sea urchin eggs.

J Cell Biol 1977 Nov;75(2 Pt 1):410-21

Glabe CG, Vacquier VD.

The vitelline layers (VLs) of unfertilized sea urchin eggs were isolated by homogenization in a hypotonic medium containing Triton X-100 and EDTA. The surface topography of the VL is not changed by isolation. The thickness of the isolated VLs (300-400 A) is greater than that reported for VLs on intact eggs (100-200 A). Sperm adhere to the isolated VLs. When both internal and external VL surfaces are accessible to sperm, the sperm attach only to the external surface, suggesting that the external surface may carry sperm receptor proteins not present on the internal surface. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis shows that isolated VLs are composed of numerous proteins ranging from greater than 213,000 to 25,000 daltons. Lactoperoxidase-catalyzed 125I-iodination of unfertilized eggs labels two high molecular weight bands that stain faintly for carbohydrate. VLs are 90% protein and 3.5% carbohydrate. No predominance of a single amino acid or class of amino acids was found. Carbohydrate analysis yields fucose, mannose, galactose, glucose, xylose, glucosamine, galactosamine, and sialic acid. Controls for purity indicate that isolated VLs contain 2% protein of cytoplasmic origin and no more than 2.5% egg jelly.

56. Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. II. Autoradiographic observations on the sites of synthesis and mode of transport of galactose- and glucosamine-labelled materials.

J Cell Sci 1977 Jun;25:323-34

Johnson KE.

Pulse-chase labelling experiments and light- and electron-microscopic autoradiography were used to examine the sites of synthesis, mode of transport, and sites of deposition of galactose- and glucosamine-labelled materials in different developmental stages of normal developing Rana pipiens embryos and interspecific hybrid embryos formed by fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana. In both normal and hybrid embryos, after 15-min pulse, grains are closely associated with juxtanuclear and cytoplasmic collections of membrane-bound vesicles which resemble the Golgi apparatus. In normal embryos following a 15-30 min pulse and a 60-min chase, grains are largely cleared from the cytoplasmic vesicles and deposited in the extracellular spaces or along cell surfaces where the extracellular spaces are relatively large. In contrast, arrested hybrid embryos given a 15-30-min pulse and a 60-min chase show a marked accumulation of grains over cytoplasmic structures such as the Golgi apparatus and vesicular elements in the cell cortex. Finally, early gastrula stage normal embryos are most active in the synthesis of galactose-labelled materials in cells above the dorsal lip of the blastopore, where cell migration is initiated.