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Teratozoospermia is a common factor associated with male infertility. However, teratozoospermia characterized by bubble-shaped acrosomes (BSAs) has not yet been identified in men and the causative genes are unknown. The present study is of a patient with severe teratozoospermia characterized by BSA and carrying a variant (c.1204G>A, p.Gly402Ser) of actin-like 7A (ACTL7A). For further verification, we generated an Actl7a-mutated mouse model (p.Gly407Ser) carrying an equivalent variant to that in the patient. We found that homozygous Actl7a-mutated (Actl7aMut/Mut) male mice were sterile, and all their sperm showed acrosomal abnormalities. We detected by transmission electron microscopy that during acrosomal biogenesis, the acrosome detaches from the nuclear membrane in Actl7aMut/Mut mice. Furthermore, mutant ACTL7A failed to attach to the acroplaxome and was discharged by cytoplasmic droplets, which led to the absence of ACTL7A in epididymal spermatozoa in mice. The mutant sperm failed to activate the oocyte, and sperm-borne oocyte activation factor phospholipase C zeta (PLCζ) discharge accompanied by ACTL7A was observed, leading to total fertilization failure (TFF). Immunoprecipitation followed by liquid chromatography-mass spectrometry showed that several differentially expressed proteins participate in acrosome assembly and actin filament organization. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF in the couple with an ACTL7A pathogenic variant. Our study defined a novel phenotype of an acrosomal abnormality characterized by BSA, revealed the underlying mechanism of a pathogenic variant in ACTL7A and provided a genetic marker and potential therapeutic option for male infertility., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Internet, magazine articles, and even biomedical journal articles, are full of cartoons of spermatozoa that bear minimal resemblance to real spermatozoa, especially human spermatozoa, and this had led to many misconceptions about what spermatozoa look like and how they are constituted. This review summarizes the historical and current state of knowledge of mammalian sperm ultrastructure, with particular emphasis on and relevance to human spermatozoa, combining information obtained from a variety of electron microscopic (EM) techniques. Available information on the composition and configuration of the various ultrastructural components of the spermatozoon has been related to their mechanistic purpose and roles in the primary aspects of sperm function and fertilization: motility, hyperactivation, capacitation, the acrosome reaction and sperm-oocyte fusion.

Study Question: What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility?, Summary Answer: While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function., What Is Known Already: CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4., Study Design, Size, Duration: In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization., Participants/materials, Setting, Methods: Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype., Main Results and the Role of Chance: While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis., Limitations Reasons for Caution: Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated., Wider Implications of the Findings: The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility., Large Scale Data: Not applicable., Study Funding and Competing Interest(s): This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. There is mounting evidence that the NL plays a role in spermatid differentiation during spermiogenesis. The mouse spermatid NL is composed of the ubiquitous lamin B1 and the spermatid-specific lamin B3, an N-terminally truncated isoform of lamin B2. However, nothing is known about the NL in human spermatids. We therefore investigated the expression pattern and localization of A-type lamins (A, C and C2) and B-type lamins (B1, B2 and B3) during human spermiogenesis. Here, we show that a lamin B3 transcript is present in human spermatids and that B-type lamins are the only lamins detectable in human spermatids. We determine that, as shown for their mouse counterparts, human lamin B3, but not lamin B2, induces strong nuclear deformation, when ectopically expressed in HeLa cells. Co-immunofluorescence revealed that, in human spermatids, B-type lamins are present at the nuclear periphery, except in the region covered by the acrosome, and that as the spermatid matures the B-type lamins recede towards the posterior pole. Only lamin B1 remains detectable on 33-47% of ejaculated spermatozoa. On spermatozoa selected for normal head density, however, this fell to <6%, suggesting that loss of the NL signal may be linked to complete sperm nucleus compaction. The similarities revealed between lamin expression during human and rodent spermiogenesis, strengthen evidence that the NL and lamin B3 have conserved functions during the intense remodelling of the mammalian spermatid nucleus., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca(2+) oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Proprotein convertase 4 (PCSK4) is a member of a family of proprotein convertases that convert inactive precursor proteins into their mature and active forms. PCSK4 is expressed by testicular germ cells and localizes to the sperm acrosome, suggesting roles in fertilization. Mice lacking PCSK4 exhibit a profound fertility defect; yet, to date, few substrates for PCSK4 are known. In this study, two-dimensional differential in-gel electrophoresis analysis was carried out in order to identify proteins that are altered in spermatozoa from PCSK4 null mice. Herein, we report that the sperm fertilization molecule acrosin-binding protein (ACRBP)/sp32, which normally undergoes processing from a 58.5 kDa precursor to a 27.5 kDa mature form, is not proteolytically processed in PCSK4 null mice and thus may be a substrate for PCSK4. However, analysis of the ACRBP sequence did not show a strong consensus site for convertase cleavage, suggesting that ACRBP processing may require the activity of a yet unknown enzyme that itself may be a PCSK4 substrate. Further analysis of spermatozoa from the PCSK4 null mice showed that proacrosin did not undergo autoactivation, supporting a role for the mature form of ACRBP in the regulation of proacrosin conversion into different acrosin isoforms. Finally, examination of ACRBP localization revealed a previously undetected morphological defect in the head/acrosomes of spermatozoa from PCSK4 null mice. Taken together, these results demonstrate that the fertility defect in the PCSK4 null mice may in part be due to altered ACRBP protein processing as well as abnormalities in the sperm head/acrosome.

Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ(2) = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.

Mammalian sperm must hyperactivate in order to fertilize oocytes. Hyperactivation is characterized by highly asymmetrical flagellar bending. It serves to move sperm out of the oviductal reservoir and to penetrate viscoelastic fluids, such as the cumulus matrix. It is absolutely required for sperm penetration of the oocyte zona pellucida. In order for sperm to hyperactivate, cytoplasmic Ca(2+) levels in the flagellum must increase. The major mechanism for providing Ca(2+) to the flagellum, at least in mice, are CatSper channels in the plasma membrane of the principal piece of the flagellum, because sperm from CatSper null males are unable to hyperactivate. There is some evidence for the existence of other types of Ca(2+) channels in sperm, but their roles in hyperactivation have not been clearly established. Another Ca(2+) source for hyperactivation is the store in the redundant nuclear envelope of sperm. To stabilize levels of cytoplasmic Ca(2+), sperm contain Ca(2+) ATPase and exchangers. The interactions between channels, Ca(2+) ATPases, and exchangers are poorly understood; however, mathematical modeling can help to elucidate how they work together to produce the patterns of changes in Ca(2+) levels that have been observed in sperm. Mathematical models can reveal interesting and unexpected relationships, suggesting experiments to be performed in the laboratory. Mathematical analysis of Ca(2+) dynamics has been used to develop a model for Ca(2+) clearance and for CatSper-mediated Ca(2+) dynamics. Models may also be used to understand how Ca(2+) patterns produce flagellar bending patterns of sperm in fluids of low and high viscosity and elasticity.

SUN domain proteins are integral proteins of the inner nuclear membrane and functions in linkage of the nuclear lamina to the cytoskeleton. Moreover, SUN domain proteins seem to mediate the tethering of the centrosome to the nuclear membrane, and they are involved in telomere attachment to the nuclear envelope in meiotic cells, and in germ cell development in invertebrates. In contrast to the widely expressed SUN domain proteins in mammals, SUN1 and SUN2, which have been analysed in great detail, there is virtually nothing known about testicular SUN domain proteins. Since a hallmark of male germ cell development is the profound remodelling of the nuclear envelope, emphasized, for example, by the reshaping of the nucleus during spermiogenesis, and the biogenesis of its tightly associated acrosome, SUN domain proteins might be engaged in these processes. We have isolated a novel SUN domain protein, SPAG4L-2, that differs from SPAG4L by an N-terminal insertion of 25 amino acids. Spag4l and Spag4l-2 are exclusively expressed in testis at about equimolar amounts, and show elevated transcription during ongoing spermiogenesis coincident with the appearance of round spermatids. Molecular dissection of the protein followed by cytological and biochemical investigations revealed that SPAG4L-2 and SPAG4L are transmembrane proteins that localize to the nuclear envelope. SPAG4L/4L-2 are restricted to the apical nuclear region of round spermatids that face the acrosomic vesicle, and thus are most probably involved in linkage of the acrosomic vesicle to the spermatid nucleus, and in acrosome biogenesis.

Epithelial cadherin (E-cadherin) has been involved in several calcium-dependent cell-cell adhesion events; however, its participation in gamete interaction has not been fully investigated. Our results have demonstrated expression of E-cadherin mRNA in the human male reproductive tract showing higher levels in the caput, corpus and cauda epididymis than in the testis. The mature 122 kDa E-cadherin was detected in epididymal protein extracts and was localized in the epithelial cells from the three epididymal regions. Moreover, the 86 kDa E-cadherin ectodomain was found in cauda epididymal and seminal plasma. Western immunoblotting of human sperm protein extracts allowed the identification of four E-cadherin forms (122, 105, 97 and 86 kDa). The protein was localized in the acrosomal region of intact spermatozoa, remained associated with the head of acrosome-reacted cells and was also detected on the oocyte surface. A similar localization was determined for other proteins of the adhesion complex (beta-catenin and actin). Spermatozoa incubated with anti-E-cadherin antibodies showed impaired binding to homologous zona pellucida (ZP); in addition, presence of these antibodies inhibited the penetration of human spermatozoa to ZP-free hamster oocytes. The results presented here describe the expression of E-cadherin in the male reproductive tract and gametes and strongly suggest its involvement in adhesion events during human fertilization. The identification of proteins involved in gamete interaction will contribute to the understanding of the molecular basis of fertilization and help in the diagnosis and treatment of infertility.

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

A novel profilin, named profilin IV, was cloned and characterized as a testicular isoform, distinct from the previously described testis-specific profilin III. Profilin IV showed only 30% amino acid identity with the other mammalian profilins; nevertheless, database searches produced significant alignments with the conserved profilin domain. Northern blot analysis and in situ transcript hybridization suggested that profilin IV, like profilin III, is transcribed in the germ cells. However, the timing of their expression during post-natal development of rat testis and in the rat spermatogenetic cycle was distinct. In the human testis, profilin IV mRNA expression correlates with the presence of germ cells suggesting that it may be a suitable molecular diagnostic parameter to supplement conventional histopathological diagnostics in the assessment of testicular biopsies. The predicted profilin IV protein was verified employing an anti-oligopeptide antibody. Western blot analysis detected an immunorelated testicular protein of approximately 14 kDa. Immunohistochemistry revealed an intracellular protein of the rat, the mouse and the human testis accumulating asymmetrically in the cytoplasm of round and elongating spermatids with its perinuclear location coinciding with the position of the developing acrosome-acroplaxome and the manchette. Profilin IV thus may regulate testicular actin cytoskeleton dynamics and play a role in acrosome generation and spermatid nuclear shaping.

Previous studies have implicated receptor tyrosine kinases in progesterone-induced [Ca2+]i signalling, and consequent induction of the acrosome reaction, in human spermatozoa. We have investigated the effects of tyrosine kinase inhibition on [Ca2+]i responses in large numbers of individual human spermatozoa. Genistein (5, 50 and 250 micromol/l), an inhibitor of receptor-linked tyrosine kinases, significantly inhibited the progesterone-induced acrosome reaction (P < 0.05). However, we could detect no effect of genistein on progesterone-induced [Ca2+]i signalling. In control experiments, application of progesterone induced a significant transient [Ca2+]i response in approximately 77% of cells and a sustained [Ca2+]i ramp/plateau in approximately 48% of cells (n = 26; 5411 cells). In preparations pretreated with genistein (50 micromol/l), significant transient and sustained responses were detected in 69.5 and 39.1% of cells respectively (n = 5; 1109 cells). The amplitudes of both transient and sustained [Ca2+]i responses were similar in control and genistein-pretreated preparations. Tyrphostin A47 (100 micromol/l), another receptor tyrosine kinase inhibitor, also failed to inhibit either the transient or sustained [Ca2+]i response (n = 3; 468 cells). Assessment of tyrosine phosphorylation of two sperm proteins (p105/81) showed greatly increased levels of phosphotyrosine in response to capacitation but a negligible increase in response to progesterone stimulation. Pretreatment with genistein (50 and 250 micromol/l) decreased capacitation-induced tyrosine phosphorylation and resulted in a loss of phosphorylation in response to progesterone treatment. We conclude that neither the transient nor sustained phases of the progesterone-induced [Ca2+]i response require receptor tyrosine kinase signalling. Previous reports of modulation of the progesterone-induced [Ca2+]i signal by tyrosine kinase inhibition probably reflect inhibition of the acrosome reaction.

Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.

Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm-zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm-ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.

High concentrations of FSH have been shown to boost in-vitro differentiation of germ cells from men with normal spermatogenesis and from some patients with in-vivo maturation arrest. This study shows that the differentiation-promoting effect of FSH is connected to protection against germ cell apoptosis and that both effects can be mimicked by the intracellular cyclic AMP (cAMP)-elevating drug pentoxifylline. On the other hand, a high concentration of insulin, supposed to act at the insulin-like growth factor I receptor, did not exert any effect either on differentiation or apoptosis of germ cells in vitro. These data show that the in-vitro effects of supraphysiological concentrations of FSH on human spermatogenesis are mediated by the classical FSH signal transduction pathway involving cAMP as a second messenger. Pentoxifylline may thus be useful as an alternative means for intracellular cAMP elevation in men with high circulating FSH concentrations leading to desensitization of the FSH receptor.

A predictive test for determining whether motile populations of human spermatozoa will fertilize eggs in vitro has been an elusive goal of clinical research. We have developed an assay for the ability of motile human spermatozoa to bind fluorescein isothiocyanate-labelled mannosylated bovine serum albumin (Man-FITC-BSA) as a test for the presence of sperm surface receptors (lectins) for mannose ligands. Mannosylated ligands are present on the human zona pellucida and are involved in the species-specific binding of human spermatozoa to the zona pellucida. We now demonstrate in prospective blinded analysis that the fractional increase in acrosome loss following a mannose ligand challenge is highly correlated with the rate of fertilization in vitro. Using an incremental increase of acrosome exocytosis of >0.1 as a threshold to predict which specimens will yield normal fertilization, the assay has a sensitivity of 97.8%, a specificity of 83.3%, a positive predictive value of 95.7% and a negative predictive value of 90.7%. These data indicate that testing for a mannose-induced acrosome reaction may be useful in assessment of sperm function prior to in-vitro fertilization in order to assign males to conventional insemination or intracytoplasmic sperm injection protocols.

In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar ligand binding properties of the human sperm surface and the functional capacity of the acrosome for exocytosis--key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man-FITC-BSA). We now report that Man-FITC-BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding of Man-FITC-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose monosaccharide to Man-FITC-BSA increases the number of polyvalent mannose ligands bound by individual spermatozoa and increases the rate of the acrosome reactions induced by Man-FITC-BSA, thereby increasing specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose ligands + D-mannose monosaccharide offers a new, convenient and readily available system to study sperm capacity for induced acrosome loss.

The involvement of protein kinase C (PKC) in exocytosis of the mammalian sperm acrosome is still a controversial issue. Work carried out thus far has failed to provide direct evidence for the activation of this enzyme upon stimulation with natural agonists of acrosomal exocytosis. We have therefore used progesterone stimulation of the acrosome reaction in human spermatozoa to clarify this issue. In spermatozoa preincubated under conditions known to support capacitation and fertilization in vitro, treatment with progesterone caused a time-dependent stimulation of phosphorylation of at least eight proteins ranging in size from approximately 20-220 kDa. The inclusion of the PKC inhibitors chelerythrine chloride or calphostin C reduced the observed phosphorylation in a concentration-dependent manner. Exogenously supplied phorbol 12-myristate-13-acetate (PMA) or the permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG), synthetic activators of PKC, also stimulated phosphorylation in preincubated spermatozoa, but inclusion of calphostin C diminished the response. Furthermore, the prior inclusion of the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine also inhibited phosphorylation, suggesting that PKC is activated downstream of Ca2+ channel opening. Exocytosis triggered by progesterone was significantly inhibited by chelerythrine chloride or calphostin C. Both PMA and OAG triggered exocytosis, but the inclusion of chelerythrine chloride significantly inhibited the response; exocytotic responses were seen only in capacitated cells. These results provide the first direct evidence that PKC activation plays a role in the signal transduction pathway underlying acrosomal exocytosis in progesterone-stimulated capacitated spermatozoa.

Selectins are a family of adhesive molecules, involved in the interactions between leukocytes and endothelium and in platelet adhesion. P-selectin, one of the members of this family, is stored in alpha-granules and dense granules of platelets as well as in Weibel-Palade bodies of endothelial cells, and it is rapidly redistributed to the cell surface after activation. It recognizes carbohydrate structures as ligands, in particular sialyl-Lewis(x), which is part of the CD15 antigen. In this work we studied P-selectin expression on gametes. While zona-free human and hamster oocytes did not react with a monoclonal antibody directed against P-selectin, oocytes from both species displayed a reactivity with this antibody following their contact with human spermatozoa, as demonstrated both by covasphere binding and indirect immunofluorescence. Artificial activation of zona-intact human oocytes by means of the calcium lonophore A23187 induced the expression on the oolemma of a moiety reacting with anti-P-selectin antibody as well. P-selectin also appeared to be expressed on the sperm surface following the acrosome reaction, as demonstrated by a flow cytometric study of reactivity of spermatozoa with the anti-P-selectin antibody, using the expression of CD46 as a marker of the acrosome reaction. The localization of the P-selectin moiety on the equatorial region of the plasma membrane of acrosome reacted spermatozoa was confirmed by transmission electron microscopy using immunogold labelling. We suggest that P-selectin might be involved in gamete interactions.

Human preimplantation embryos, gametes and cumulus cells were studied for expression of the complement-binding proteins CD46 (membrane cofactor protein), CD55 (decay accelerating factor) and CD59 (membrane attack complex inhibitory factor) as well as complement receptors type 1 (CRI), type 2 (CR2) and type 3 (CR3). Both the CD55 and CD59 glycosyl phosphatidylinositol (GPI)-anchored proteins were expressed by the plasma membrane and zona pellucida of oocytes, early embryos and expanded preimplantation blastocysts; in contrast, CD46 was expressed only on the plasma membrane. Cumulus cells consistently expressed CD46 and, most strongly, CD59 whereas CD55 expression was variable. Monoclonal antibodies (mAbs) to CRI, CR2 and CR3 epitopes gave only occasional reactivity on oocytes and were unreactive with blastocysts, spermatozoa and cumulus cells. CD46 is expressed only on the spermatozoal inner acrosomal membrane, and CD59 on the plasma membrane; CD55 expression was confirmed on the plasma membrane as well as the inner acrosomal membrane. Control mAbs specific for factor H were usually unreactive with gametes, blastocysts and cumulus cells. These data support the concept that gametes and early embryonic cells are protected from complement-mediated attack by expression of CD46, CD55 and CD59, although these complement-binding proteins may have additional roles in reproductive events.