C-kit receptor and its possible function in human spermatozoa

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility.     

Appearance of an intra-acrosomal antigen during the terminal step of spermiogenesis in the rat

Summary In a survey of sperm antigens in the rat, a new intra-acrosomal antigen was found using a monoclonal antibody MC41 raised against rat epididymal spermatozoa. The MC41 was immunoglobulin G₁ and recognized spermatozoa from rat, mouse and hamster. Indirect immunofluorescence with MC41 specifically stained the crescent region of the anterior acrosome of the sperm head. Immuno-gold electron microscopy demonstrated that the antigen was localized within the acrosomal matrix. Immunoblot study showed that MC41 recognized a band of approximately 165000 dalton in the extract of rat sperm from the cauda epididymidis. Immunohistochemistry with MC41 demonstrated that the antigen was first detected in approximately step-2 spermatids, and distributed over the entire cytoplasmic region of spermatids from step 2 to early step 19. The head region became strongly stained in late step-19 spermatids and then in mature spermatozoa. Distinct immunostaining was not found in the developing acrosome of spermatids throughout spermiogenesis. These results suggest that the MC41 antigen is a unique intra-acrosomal antigen which is accumulated into the acrosome during the terminal step of spermiogenesis.     

Capacité des spermatozoïdes à se fixer sur la zone pellucide

After liberation from the seminiferous epithelium, the spermatozoa (SPZ), undergo in the epididymis a serie of functional and metabolic modifications resulting the capacity to ensure fertilization. Fertilization is the fundamental process in sexual reproduction as it permits the initiation and the formation of a new being by the fusion of two germinal cells: the male gamete (spermatozoa) and the female gamete (oocyte). For fertilization to occur the SPZ must recognize the zona pellucida (ZP), bind to it, penetrate it and fuse with the oocyte plasma membrane. Sperm binding to the ZP is an early, crucial event leading to fertilization and pre-embryo development. In mammals, sperm-ZP binding follows a serie of steps that occur in a well-defined chronological order: a) A loose association between SPZ and ZP referred to as «attachment». This shortlived interaction is heterospecific. b) Attachment is followed by a more distinct and persistent association of SPZ with ZP, thus called «binding». This sperm-zona interaction is species-specific, irreversible and mediated by complementary receptors present on the SPZ head and the ZP. c) The bound SPZ then undergoes the acrosome reaction (AR). Which involves fusion and vesiculation of the SPZ outer acrosomal membrane and plasma membrane leading to the release of acrosomal contents and the exposure of the inner acrosomal membrane. This AR is essential for SPZ passage through the ZP and to access to the oocyte plasma membrane where gamete fusion occurs.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight ?T20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization. © 1993 Wiley-Liss, Inc.     

Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa

Our laboratory has recently shown that in vitro-cultured oviductal cells secrete sperm motility maintaining factor(s). Since the binding of oviductal proteins to spermatozoa (SPZ) has been demonstrated in many species, the motility factor was postulated to bind the membranes of SPZ. Therefore, the current study was performed to evaluate which proteins from in vivo oviductal secretions bind to sperm membranes, to characterize binding conditions, and to evaluate the effect of this binding on sperm survival. Bovine oviducts were dissected, and oviductal cells and fluid were collected by pressing the oviductal tube with a glass slide. This mixture was incubated in Tris-EDTA buffer at 37°C for 30 min, and the cells were washed twice by centrifugation. The supernatant containing oviductal fluid proteins (OFP) was reserved, filtered, frozen (for later motility tests), or lyophilized and labeled with ¹²⁵|. Frozen-thawed SPZ were incubated either immediately, following capacitation, ionophore-induced acrosome reaction, death by heating, or flagellar removal with labeled OFP for 30 min. The resulting pellet after three washes was dissolved in SDS and submitted to 10% SDS-PAGE. An autoradiogram showed that 72, 66, 39, 38, and 36 kDa proteins bind strongly to the five types of SPZ used, and that this binding is very specific, since unlabeled OFP inhibited binding while serum proteins did not. Furthermore, for 39, 38, and 36 kDa proteins, the presence of calcium in the incubation medium was essential for dose-dependent binding, whereas magnesium was not. Preincubation of SPZ for 30 min at 37°C with oviductal fluid, followed by one wash and 6 hr of incubation in control media, showed that the percentage of motile SPZ is significantly higher (52 ± 6%) compared with SPZ not preincubated with oviductal fluid (24 ± 6%: P < 0.01). In summary, a limited number of proteins from oviductal secretions bind to the surface of bovine SPZ only in the presence of calcium, and this binding appears to be important for subsequent sperm viability. © 1996 Wiley-Liss, Inc.     

Liprin α3: a putative estrogen regulated acrosomal protein

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.     

Galactosyl receptor in human testis and sperm is antigenically related to the minor C-type (Ca2+-dependent) lectin variant of human and rat liver

Galactosyl receptor, a cell surface Ca²⁺-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca²⁺-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization. © 1995 Wiley-Liss, Inc.     

The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.     

Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa

Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.     

Calpactin-like proteins in human spermatozoa

Polyclonal antibodies directed against human calpactin I (p36) and calpactin II (p35) have been employed to investigate the distribution of calpactin-like proteins in human spermatozoa. Calpactins are a family of Ca2+-regulated cytoskeletal proteins that are major substrates of oncogene and growth factor receptor protein tyrosine kinases. The existence of a Triton-soluble 37-kDa protein antigenically related to calpactin II from somatic cells was revealed by Western blot analysis of human sperm extracts. The 37-kDa protein was not released from spermatozoa after experimental induction of the acrosome reaction by A23187 and Ca2+. Treatment of sperm homogenates with an EGTA-containing buffer partially solubilized the 37-kDa protein from the corpuscolate matter. Indirect immunofluorescence microscopy showed that anticalpactin II binds specifically to the sperm tail and to a band-like structure encircling the sperm head at the equatorial segment. In contrast, antibodies to calpactin I were found to bind to the tail midpiece, but failed to bind to Western blots of sperm proteins. This is the first immunological and biochemical report on the presence of calpactin proteins in a germ cell, the human spermatozoon.     

Brain synaptic junctional proteins at the acrosome of rat testicular germ cells.

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.     

受精蛋白β在人精子表面的免疫组织化学定位

Fertilin is a kind of sperm plasma membrane protein that mimics snake venom protein. It belongs to the ADAMs family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in the sperm-egg binding process by connecting the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta (HF93). Its localization on the sperm is in the change. In this study, the monoclonal antibody against human fertilin beta was prepared and used to analyze the localization of fertilin beta on capacitated and acrosome-reacted sperm by immunofluorescence and immunoelectron microscopy techniques. The results were as follows: (1) fertilin beta became restricted to the anterior head during the course of capacitation. (2) During the course of acrosome reaction, the expression and localization of fertilin beta changed immensely on the anterior head and restricted to the lateral of posterior head at last. The restrictions of fertilin beta to the anterior head of capacitated sperm of human beings indicated that fertilin beta may be involved in the binding the sperm to the epithelial cells of the oviduct; the restrictions of fertilin beta to the posterior head domain of acrosome-reacted sperm implied its function in sperm-egg binding and fusion.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.     

Sperm plasma membrane breakdown during Drosophila fertilization requires sneaky, an acrosomal membrane protein

Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. We previously proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. Here we support this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. We further show that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), we show that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed us to follow the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, we find that the Drosophila acrosome is a paternally inherited structure. We provide evidence that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. We relate the characteristics of Drosophila Snky, acrosome function and sperm PMBD to membrane fusion events that occur in other systems.     

Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.     

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.     

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.