Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.     

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.     

Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation.

Rat epididymal glycoprotein DE (37 kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in vitro and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibited the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus-associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.     

Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.     

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.     

Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg

Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Expression and structure-function analysis of de,a sperm cysteine-rich secretory protein that mediates gamete fusion

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.     

Immunochemical localization of epididymal protein DE on rat spermatozoa: Its fate after induced acrosome reaction

The localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto-chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.     

Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.     

Evidence for the involvement of zinc in the association of CRISP1 with rat sperm during epididymal maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.     

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.     

Effect of progesterone on bovine sperm capacitation and acrosome reaction

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0-6 h) with different concentrations of P (0.01-10 microg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 microg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 microg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

Background  

Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.     

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.     

S100 A9 is expressed and secreted by the oviduct epithelium,interacts with gametes and affects parameters of human sperm capacitation in vitro

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [³⁵S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [³⁵S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.     

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).     

Sperm protein "DE" mediates gamete fusion through an evolutionarily conserved site of the CRISP family

The first member of the cysteine-rich secretory protein (CRISP) family was described by our laboratory in the rat epididymis, and it is known as DE or CRISP-1. Since then, numerous CRISPs exhibiting a high amino acid sequence similarity have been identified in animals, plants and fungi, although their functions remain largely unknown. CRISP-1 proteins are candidates to mediate gamete fusion in the rat, mouse and human through their binding to complementary sites on the egg surface. To elucidate the molecular mechanisms underlying CRISP-1 function, in the present work, deletion mutants of protein DE were generated and examined for their ability to bind to the rat egg and interfere with gamete fusion. Results revealed that the egg-binding ability of DE resides within a 45-amino acid N-terminal region containing the two motifs of the CRISP family named Signature 1 and Signature 2. Subsequent assays using synthetic peptides and other CRISPs support that the egg-binding site of DE falls in the 12-amino-acid region corresponding to Signature 2. The interesting finding that the binding site of DE resides in an evolutionarily conserved region of the molecule provides novel information on the molecular mechanisms underlying CRISP-1 function in gamete fusion with important implications on the structure-function relationship of other members of the widely distributed CRISP family.     

Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation

Sperm capacitation is a maturation step that is deemed to be essential for sperm to fertilize an oocyte. A family of proteins, the binder of sperm (BSP), are known to bind choline phospholipids on sperm membranes and promote capacitation in bulls and boars. Recently, BSP-homologous genes have been identified in the epididymal tissues of human (BSPH1) and mouse (Bsph1, Bsph2). The aim of this study was to determine the binding characteristics of the murine binder of sperm protein homolog 1 (BSPH1) and evaluate its effects on sperm capacitation. Since it is not possible to purify the native BSP proteins from human and mouse in sufficient quantity, a murine recombinant BSPH1 (rec-BSPH1) was produced and used for the functional studies. Similarly to BSP proteins from other species, rec-BSPH1 bound to gelatin, heparin, phosphatidylcholine liposomes, and sperm. Both native BSPH1 and rec-BSPH1 were detected on the head and the midpiece region of sperm, although a stronger signal was detected on the midpiece region when sperm were incubated in a capacitating media containing bovine serum albumin. More importantly, murine rec-BSPH1 was able to capacitate sperm, but was unable to induce the acrosome reaction. These results show that murine epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and suggest that it might play a similar role in sperm functions.