Biology of male fertility control: an overview of various male contraceptive approaches.

The population of our planet continues to grow at an alarming rate. If the growth continues at the present rate, the estimated current world population of about seven billion is expected to double in the next forty years. Accumulated data from surveys by the United Nations Population Control Division suggest that a majority of today's young men in many countries are willing to have fewer children than their parents did. However, the contraceptive options available to them have not changed in several decades. In spite of the general agreement that men, like women, must take full responsibility of their fertility, the availability of safe, reversible and affordable contraceptives for men have lagged behind because of the complexity of the science of the male reproductive system. Thus, the contraceptive needs of millions of men/couples go unmet every single day and results in millions of unwanted pregnancies. In this article, we intend to discuss new hormonal and non-hormonal contraceptive approaches that are at various stages of research and development and may someday provide new contraceptives for men. In addition, we intend to discuss many details of three safe, effective, affordable and reversible vas-based approaches that are inching closer to being approved for use by millions of men in multiple countries. Finally, our intention is to discuss the male contraceptive pill that will soon be available to men only in Indonesia. The availability of these male contraceptives will allow both men and women to take full control of their fertility and participate in slowing down the growth of world population.

Significance of egg's zona pellucida glycoproteins in sperm-egg interaction and fertilization.

Mammalian fertilization is a highly programmed process by which sperm and egg unite to form a zygote, a cell with somatic chromosome numbers. To fertilize an egg, the capacitated (acrosome-intact) spermatozoa recognize and bind to the egg's extracellular glycocalyx coat, the zona pellucida (ZP). The tight and irreversible binding of the opposite gametes in the mouse and many other species studied, including man, results in the opening of Ca2+ channels on sperm plasma membrane (PM) and influx of Ca2+. The transient rise in Ca2+ and other second messengers, such as cAMP and IP3, initiates a cascade of signaling events that elevate sperm pH and triggers the fusion of the sperm PM and underlying outer acrosomal membrane at multiple sites (induction of the acrosomal reaction). The fusion of the two membranes results in the exocytosis of acrosomal contents at the site of sperm-egg adhesion. The hydrolytic action of the acrosomal enzymes (glycohydrolases, proteinases, esterases, sulfatases etc), released at the site of sperm-egg adhesion, along with the enhanced thrust generated by the hyperactivated spermatozoon, are important factors that regulate the penetration of the ZP and the fusion of the acrosome-reacted spermatozoon with the egg. Evidence accumulated over the past two decades strongly suggests that glycan units of the ZP have a significant role in the recognition and adhesion of the opposite gametes and induction of the AR. In this review article, we intend to highlight well programmed molecular events that results in the sperm-egg adhesion and fertilization. Our intention is also to discuss the increasing controversy on the role of ZP glycan chains in sperm-egg interactions.

Molecular events that regulate mammalian fertilization.

Mammalian fertilization is the net result of a highly programmed sequence of molecular events that collectively result in the union of two radically different looking haploid cells, sperm and egg, to form a diploid zygote. For successful fertilization, sperm cells undergo continuous modifications during their formation in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only capacitated acrosome-intact spermatozoa are capable of binding to the egg's extracellular coat, the zona pellucida (ZP) in a receptor-ligand manner. The species-specific irreversible binding of the opposite gametes elevates intrasperm Ca2+ and triggers a signal transduction cascade that results in the fusion of the sperm plasma membrane and outer acrosomal membrane at multiple sites (i.e., induction of the acrosomal reaction) and the secretion of acrosomal contents. The hydrolytic action of the acrosomal enzymes (i.e., glycohydrolases, proteinases etc.) released at the site of sperm-egg binding along with the hyperactivated beat pattern of the bound spermatozoon, are important factors that regulate its penetration of the ZP and fertilization of the egg. In this article, we intend to discuss data from this and other laboratories that provide useful insights into biology underlying sperm development in the testis, maturation in the epididymis, capacitation in the female genital tract, sperm-egg interaction, and induction of the acrosome reaction (AR) before the acrosome reacted sperm can fertilize an egg. Our intention is also to discuss how Ca2+ signaling cascades regulate sperm functions and male fertility. Finally, we will discuss sperm molecules that are under intensive research to regulate male fertility.

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa.

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).

Mammalian sperm molecules that are potentially important in interaction with female genital tract and egg vestments.

Fertilisation is a highly programmed process by which two radically different cells, sperm and egg, unite to form a zygote, a cell with somatic chromosome numbers. Development of the zygote begins immediately after sperm and egg haploid pronuclei come together, pooling their chromosomes to form a single diploid nucleus with the parental genes. Mammalian fertilisation is the net result of a complex set of molecular events which allow the capacitated spermatozoa to recognise and bind to the egg's extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fuse with the egg plasma membrane. Sperm-zona (egg) interaction leading to fertilisation is a species-specific carbohydrate-mediated event which depends on glycan-recognising proteins (glycosyltransferases/glycosidases/lectin-like molecules) on sperm plasma membrane (receptors) and their complementary glycan units (ligands) on ZP. The receptor-ligand interaction event initiates a signal transduction pathway resulting in the exocytosis of acrosomal contents. The hydrolytic action of the sperm glycohydrolases and proteases released at the site of sperm-egg interaction, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of egg investments. This review focuses on sperm molecules believed to be important for the interaction with the female genital tract, passage through cumulus oophorus and attachment to ZP, induction of the acrosome reaction, secondary binding events, and passage through the ZP. An understanding of the expression and modifications of molecules thought to be important in multiple events leading to fertilisation will allow new strategies to block these modifications and alter sperm function.

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida.

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/ exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization.

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase.

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

Mammalian sperm acrosome: formation, contents, and function.

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.

Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction.

The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of the opposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during sperm development in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum's extracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed to take place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-intact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence of glycan-binding proteins (receptors) on the sperm plasma membrane and their complementary bioactive glycan units (ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivated acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohydrate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensive progress that has been made to enhance our understanding of the well programmed multiple molecular events necessary for successful fertilization. This review will identify these events, and discuss the functional significance of carbohydrates in these events.

Characterization of the pharmacological-sensitivity profile of neoglycoprotein-induced acrosome reaction in mouse spermatozoa.

Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.

Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa.

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.

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

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.

Mannose-binding molecules of rat spermatozoa and sperm-egg interaction.

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.

Rat sperm surface mannosidase is first expressed on the plasma membrane of testicular germ cells.

In previous publications (Tulsiani et al., Biochem J 1993; 290:427-436 and Tulsiani et al., Dev Biol 1995; 167:584-595), we reported that sperm surface mannosidase is present in rat testis and is modified during spermatogenesis and sperm maturation. The present studies were directed towards examining the origin of alpha-D-mannosidase activity present on fertile spermatozoa. Mixed germ cells prepared after sequential enzymatic digestions of rat testis were separated by unit gravity sedimentation using 2-4% linear bovine serum albumin gradient. Fractions enriched in spermatocytes, round spermatids, and condensed/elongated spermatids (> 95% pure cells) were separately pooled and assayed for [3H]Man9-mannosidase activity before (intact) and after lysis with Triton X-100. Interestingly, the cells contained a significant level of alpha-D-mannosidase activity. Approximately 70% of the total [3H]Man9-mannosidase activity present in the detergent-solubilized germ cell extract cross-reacted with anti-rat sperm mannosidase, and 25% of the activity cross-reacted with anti-Golgi mannosidase I. This result indicates that most of the mannosidase activity present in the germ cell extract is antigenically similar to the enzyme present on the cauda spermatozoa. Using cell fractionation techniques, we obtained evidence suggesting that the germ cell-associated mannosidase activity is an integral component of the plasma membranes. Taken together, these results indicate that sperm surface mannosidase is first expressed on the testicular germ cells.

Rat zona pellucida glycoproteins: molecular cloning and characterization of the three major components.

The zona pellucida (ZP), the extracellular glycocalyx that surrounds the oocyte, is well known to mediate homologous gamete interaction. In a previous study from our laboratories, we reported the qualitative characterization of the rat ZP. The ZP in this species, like the mouse, hamster, and human, was found to contain three glycoproteins, namely rZP1, rZP2, and rZP3 (Araki et al. [1992] Biol Reprod 46:912-919). In this study, cDNAs encoding whole rat ZP major components have been isolated and characterized. A rat ovary cDNA library was screened with the mouse ZP3 and ZP2 cDNA probes, respectively. For rZP1 cDNA cloning, cDNAs generated using reverse transcriptase-polymerase chain reaction and rapid amplification of 5' and 3' cDNA ends, were isolated and sequenced. The rZP3 cDNA showed 1338 bp with a coding region containing 1272 bp, that translates into 424 amino acids. The total translation of rZP3 peptide has a molecular weight of 45,820, containing six potential N-glycosylation sites and 75 Ser/Thr residues, possible O-glycosylation sites. The amino acid sequence derived from the cDNA sequence shares high sequence homologies to mouse (90%), hamster (78%), and human (65%) ZP3 (ZPC) glycoproteins, indicating that the rat and mouse ZP3 have quite a conserved amino acid sequence, including the potential glycosylation sites. The total transcript of the rZP2 was 2154 nucleotides and the largest open reading frame was 695 amino acids. This would translate into a protein of 78.4 kDa. In the case of rZP1, the cDNA clone consisted of 1960 bp, and the coding region contained 1851 bp translating into 617 amino acids. Significant homologies were observed between rZP2 and ZPA family from various mammalian species. The rZP1 also showed a sequence homology to mouse ZP1, known as a mouse orthologue of ZPB family, suggesting that the rZP2 and rZP1 are members of ZPA and ZPB families, respectively. The message distributions for each zona components were limited within the ovary and the signal was detectable in the growing oocytes. The present results will further our understanding of the structure of rat zona components and lead to a better understanding of species-specificity observed during sperm-egg interaction.

The biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization.

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm-egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.

Identification and androgen regulation of egasyn in the mouse epididymis.

The expression and androgen regulation of egasyn, the endoplasmic reticulum-targeting protein of beta-D-glucuronidase, was examined in the mouse-epididymis. The proximal (caput) and distal (corpus & cauda) epididymal tissue extracts were prepared by homogenization and sonication in buffered Triton X-100 solution, and high speed centrifugation. The supernatant when resolved by 2D-PAGE under non-denaturing conditions and stained for esterase activity showed that the distal (but not proximal) epididymis of the normal mouse contain several specific forms of esterases. These forms include a series of four variants (pI 5.2-5.75) with high mobility (HM) and esterase activity, and three faintly staining variants (beginning at pI 6.0) with low mobility (LM). Several lines of evidence indicate that the specific esterases seen in the corpus/cauda epididymidis are egasyn-esterases. Firstly, these molecular forms were not seen in the distal epididymal extracts from the egasyn-deficient mouse. Secondly, the HM forms can be immunoprecipitated with anti-egasyn antibody, suggesting the presence of free egasyn. Finally, the LM forms disappeared after heat treatment (56 degrees C for 8 min), a condition known to dissociate egasyn:beta-D-glucuronidase complex. This result indicates that a small amount of egasyn is complexed with beta-D-glucuronidase. Immunoblotting (Western blot) studies (using anti-egasyn antibody) following resolution of egasyn released from the egasyn:beta-D-glucuronidase complex revealed a single band of an apparent molecular weight 64 kDa in the distal (but not proximal) epididymis, indicating that the mouse epididymal egasyn is identical or very similar to the liver egasyn. Castration of mice lead to the appearance of free and complexed egasyn forms in the proximal epididymis. Testosterone supplementation to the castrated mice resulted in the disappearance of the induced egasyn forms from the caput epididymidis. Taken together, these results indicate that the expression of egasyn in the epididymis is region-specific and is differentially regulated by androgens.

Role of luminal fluid glycosyltransferases and glycosidases in the modification of rat sperm plasma membrane glycoproteins during epididymal maturation.

It is generally accepted that mammalian spermatozoa undergo biochemical and morphological changes during epididymal transit, collectively termed epididymal maturation. Although many details of the biochemical modification are not fully understood, lectin binding studies from several laboratories strongly suggest that glycan moieties of sperm plasma membrane glycoproteins are extensively modified as spermatozoa transit from the proximal to the distal epididymis. In the present article, we summarize our studies with two sets of glycan modifying enzymes, namely glycosyltransferases (synthetic enzymes) and glycosidases (hydrolytic enzymes) in rat spermatozoa collected from different regions of the epididymis, and similar enzyme activities present in the epididymal luminal fluid. Our data show that the activities of these enzyme are high in the epididymal luminal fluid (> 80% of the total enzyme activities was in the plasma). Evidence presented in this report also demonstrates that: (1) at least one sperm surface glycoprotein (apparent molecular mass of 86 kDa) is fucosylated in vitro when caput spermatozoa are incubated with GDP [14C]fucose; and (2) a peanut agglutinin (PNA)-positive glycoprotein of 135-150 kDa present on plasma membrane of sperm from the caput (but not cauda) epididymidis is degalactosylated by digestion with purified luminal fluid beta-D-galactosidase. Taken together, these results strongly suggest a role for glycoprotein modifying enzymes in the modification of sperm surface glycoproteins during epididymal maturation.