ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

STUDY QUESTION: Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? SUMMARY ANSWER: A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. WHAT IS KNOWN ALREADY: ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. LIMITATIONS, REASONS FOR CAUTION: The absence of another independent pedigree to support our argument is a limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS: The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Effects of Cryopreservation on Acrosin Activity and DNA Damage of Russian Sturgeon (Acipenser gueldenstaedtii) Semen.

BACKGROUND: Cryopreservation of sturgeon sperm can be successful, but there can be a decrease in sperm viability and the reasons are not clear. OBJECTIVE: To investigate variations in the acrosin activity and the DNA integrity of Acipenser gueldenstaedtii semen during cryopreservation at -196ºC. MATERIALS AND METHODS: Fish semen samples were randomly divided into three groups: [1] fresh control; [2] native semen diluted 1:1 with 23.4 mM sucrose + 0.25 mM KCl + 30 mM Tris (pH 8.0) and the addition of 10% methanol as cryoprotectant; and [3] semen without any diluents or cryoprotectants. Acrosin activity and DNA damage (COMET assay) were assessed. RESULTS: The average acrosin activity fell to 61% and 27% of the control for cryoprotected and non-cryoprotected semen after cryopreservation. The differences among the three groups were significant (P<0.05). We also observed that various indexes of DNA damage (L-tail; tail DNA, tail momentum, olive tail momentum) were higher in semen that had been frozen. CONCLUSION: Although cryopreservation of semen induces decreased acrosin activity and increased DNA damage, cryoprotectants can protect the semen during cryopreservation.

Limited proteolysis by acrosin affects sperm-binding and mechanical resilience of the mouse zona pellucida.

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

Acrosin is essential for sperm penetration through the zona pellucida in hamsters.

During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

No evidence for phylosymbiosis in western chipmunk species.

Phylosymbiosis refers to a congruent pattern between the similarity of microbiomes of different species and the branching pattern of the host phylogeny. Phylosymbiosis has been detected in a variety of vertebrate and invertebrate hosts, but has only been assessed in geographically isolated populations. We tested for phylosymbiosis in eight (sub)species of western chipmunks with overlapping ranges and ecological niches; we used a nuclear (Acrosin) and a mitochondrial (CYTB) phylogenetic marker because there are many instances of mitochondrial introgression in chipmunks. We predicted that similarity among microbiomes increases with: (1) increasing host mitochondrial relatedness, (2) increasing host nuclear genome relatedness and (3) decreasing geographic distance among hosts. We did not find statistical evidence supporting phylosymbiosis in western chipmunks. Furthermore, in contrast to studies of other mammalian microbiomes, similarity of chipmunk microbiomes is not predominantly determined by host species. Sampling site explained most variation in microbiome composition, indicating an important role of local environment in shaping microbiomes. Fecal microbiomes of chipmunks were dominated by Bacteroidetes (72.2%), followed by Firmicutes (24.5%), which is one of the highest abundances of Bacteroidetes detected in wild mammals. Future work will need to elucidate the effects of habitat, ecology and host genomics on chipmunk microbiomes.

Photobiomodulation and gametogenic potential of human Wharton's jelly-derived mesenchymal cells.

Recently, light emitting diode (LED) irradiation has been introduced as a new strategy to enhance proliferation and affect differentiation of stem cells. Human Wharton's jelly-derived mesenchymal (hWJM) cells have unique characteristics that make them an appropriate source of stem cells for use in basic and clinical applications. In this study, we aimed to evaluate the effect of polarized (PL) and non-polarized (NPL) red light irradiation on gametogenic differentiation of hWJM cells in the presence or absence of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). Exposure of hWJM cells to PL and NPL red LED (625 nm, 1.9 J/cm2) with or without BMP4+RA pre-treatment effectively differentiated them into germ lineage when the gene expression pattern (Fragilis, DAZL, VASA, SCP3 and Acrosin) and protein synthesis (anti-DAZL, anti-VASA, anti-SCP3 and anti-Acrosin antibodies) of the induced cells was evaluated. These data demonstrated that photobiomodulation may be applied for gametogenic differentiation in-vitro.

Fluoride Interferes with the Sperm Fertilizing Ability via Downregulated SPAM1, ACR, and PRSS21 Expression in Rat Epididymis.

Fluoride is a widespread environmental pollutant that can induce low sperm quality and fertilizing ability; however, the underlying mechanism still remains unclear. Hence, we aimed to investigate the influence of fluoride on the sperm fertilizing ability via some key proteins in the epididymis. For this, 40 adult rats were assigned randomly into four groups. The control group was given distilled water, while the other three groups were given 25, 50, and 100 mg of NaF/L via drinking water for 56 days, respectively. After 1 day, epididymides were processed for sperm-egg binding, RNA extraction, western blot, and immunofluorescence analysis. Fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer. A further study revealed that fluoride altered the expression levels of genes and proteins related to acrosome reaction in vivo, including SPAM1, ACR, and PRSS21. However, fluoride only affected the expression of the ACR protein only in the epididymis but not in the testis. Fluoride also affected the expression levels of the membrane proteins CD9 and CD81 of epididymosomes in the epididymis. From the results, it can be concluded that fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer, which could be one of the reasons for decreased fertility ability in males treated with fluoride. These results provide some theoretical guidance and new ideas for treatments of low fertility, infertility, and other reproductive diseases.

A delayed sperm penetration of cumulus layers by disruption of acrosin gene in rats.

Acrosin, the trypsin-like serine protease in the sperm acrosome, was long viewed as a key enzyme required for zona pellucida penetration to fertilize eggs. However, gene disruption experiments in mice surprisingly showed that acrosin-disrupted males were fertile. Thus, the acrosin was considered to be not an essential enzyme for fertilization in mice. However, the involvement of acrosin in fertilization has been suggested in various species such as rat, bull, and pig. Moreover, it has been reported that serine protease (including acrosin) activity in mice is significantly weaker compared to other species, including rats. We analyzed the role of acrosin by disrupting the rat acrosin gene. It was found that, unlike in mice, acrosin was almost the sole source of serine protease in rat spermatozoa. Nevertheless, the acrosin-disrupted males were not infertile. However, the litter size from acrosin-disrupted males was decreased compared to heterozygous mutant rats. Further investigation using an in vitro fertilization system revealed that the acrosin-disrupted spermatozoa possessed an equal ability to penetrate the zona pellucida with wild-type spermatozoa, but the cumulus cell dispersal was slower compared to wild-type and heterozygous spermatozoa. This delay was presumed to be the cause of the small litter size of acrosin-disrupted male rats.

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm.

STUDY QUESTION: Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? SUMMARY ANSWER: Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. WHAT IS KNOWN ALREADY: Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCζ antibody specificity and detection protocols. STUDY DESIGN, SIZE DURATION: Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1-0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. MAIN RESULTS AND THE ROLE OF CHANCE: Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCζ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCζ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males. LIMITATIONS, REASONS FOR CAUTION: Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status. WIDER IMPLICATIONS OF THE FINDINGS: We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCζ appears to be kept in an inactive form in the sperm. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTERESTS: J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-European Research Fellowship award. This work was also partly funded by a research grant from Cook Medical Technologies LLC. There are no competing financial interests to declare.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin.

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Acrosome Reaction as a Preparation for Gamete Fusion.

The acrosome reaction (AR) is a universal requisite for sperm-egg fusion. However, whereas through the animal kingdom fusion of spermatozoa with the egg plasma membrane occurs via the inner acrosomal membrane exposed after the AR, in eutherian mammals, gamete fusion takes place through a specialized region of the acrosome known as the equatorial segment (ES) which becomes fusogenic only after the AR is completed. This chapter focuses on the different molecular mechanisms involved in the acquisition of the fusogenicity of the ES after the AR. We provide an update of the knowledge about the proteins proposed to have a role in this process either by modifying cytoskeletal and/or membrane molecules or by relocalizing to the ES after the AR to subsequently participate in gamete fusion.

Sperm proteases that may be involved in the initiation of sperm motility in the newt, Cynops pyrrhogaster.

A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR) is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS). Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR. In an in silico analysis of the testes, acrosins and 20S proteasome were identified as possible candidates of the acrosomal proteases. We also detected another AEBSF-sensitive protease activity on the sperm surface. Fluorescence staining with AlexaFluor 488-labeled AEBSF revealed a cysteine protease in the principal piece; it is localized in the joint region between the axial rod and undulating membrane, which includes an axoneme and produces powerful undulation of the membrane for forward sperm motility. These results indicate that AEBSF-sensitive proteases in the acrosome and principal piece may participate in the initiation of sperm motility on the surface of egg jelly.

Antisperm antibodies: invaluable tools toward the identification of sperm proteins involved in fertilization.

The identification of sperm proteins involved in fertilization has been the subject of numerous investigations. Much interest has been dedicated to naturally occurring antisperm antibodies (ASA) and their impact in fertility. Their presence in men and women has been associated with 2-50% of infertility cases. ASA may impair pre- and post-fertilization steps. Experimental models have been developed using sperm proteins as immunogens to evaluate their involvement in sperm function. Our team has pursued investigations to assess ASA presence in biological fluids from patients consulting for infertility and their effect on fertilization. We found ASA in follicular fluids with ability of inducing the acrosome reaction and blocking sperm-zona pellucida interaction and used them to identify sperm entities involved in these events. We generated and utilized antibodies against proacrosin/acrosin to characterize the sperm protease system. We implemented an ELISA to detect proacrosin/acrosin antibodies in human sera and evaluated their impact upon fertility by developing in vitro assays and a gene immunization model. This review presents a summary of ASA history, etiology, current approaches for detection and effects upon fertility. ASA (naturally occurring, generated by animal immunization and/or of commercial origin) are invaluable tools to understand the molecular basis of fertilization, better diagnose/treat immunoinfertility and develop immunocontraceptive methods.

Modes of acrosin functioning during fertilization.

Mammalian fertilization is a complex process that involves gamete recognition, penetration, and fusion. Biochemical studies that identified the role of acrosome components during sperm-ova interaction especially the zona pellucida (ZP) provided major advances in sperm cell biology. Acrosin (a typical serine protease) functions during fertilization in several significant ways which include: a) activation of acrosome components, b) secondary binding with the ZP, and c) hydrolysis of the ZP. However, studies using knockout (KO) acrosin-deficient mice cast doubt on the traditional role of acrosin in fertilization. The KO acrosin-deficient mice exhibit normal fecundity except for delayed fertilization. Despite the doubt cast on the traditional role of acrosin by the KO acrosin-deficient mouse studies, acrosin still remains a major protease involved in multiple processes of fertilization. In this review, we assess the functional profile of acrosin and briefly summarize recent findings on proteases involved in fertilization. We propose a refined scheme for the functional role of acrosin in fertilization. We particularly emphasize the role of acrosin in acrosome exocytosis and activation of other acrosome components based on advanced technology like structural X-ray analysis.

Acrosin activity is a suitable indicator of boar semen preservation at 17 °C when increasing environmental temperature and radiation.

The effect of increasing environmental temperature and radiation on the sperm quality and the field fertility of refrigerated seminal doses from AI boars (N = 30) was analyzed throughout four experimental months (from March through June). In each experimental month, analyses of sperm quality were performed at days 0, 1, 3, 5, 7, and 9 of refrigeration of seminal doses; pregnancy rate and litter size were evaluated using double monospermic inseminations of multiparous female animals using seminal doses at Days 1 to 2 and Days 3 to 4 of refrigeration. Sperm quality was assessed from the evaluation of conventional parameters of sperm concentration, sperm motility, sperm morphology, and sperm viability, and capacitation parameters of membrane lipid disorder, intracellular calcium content, and acrosin activity. Results showed that sperm quality of boar seminal doses was negatively affected by increasing temperature and radiation, which resulted in significantly decreased sperm motility and viability, acrosin activity, pregnancy rate, and litter size, and significantly increased intracellular calcium levels in the trials performed in June. In any experimental month, aging of refrigerated doses was associated with the progressive increase of intracellular calcium levels and inactivation of acrosin, that began from Day 5 of storage in the trials performed in March and April, from Day 3 in those of May, and from Day 0 in those of June. Among the sperm parameters analyzed, only acrosin activity exhibited a clearly differentiated pattern in association with increasing temperature and radiation, and a significant correlation with pregnancy rate and litter size. These results highlighted the potential role of acrosin activity as an indicator of boar sperm preservation at 17 °C in boars.

Expression analysis of MND1/GAJ, SPATA22, GAPDHS and ACR genes in testicular biopsies from non-obstructive azoospermia (NOA) patients.

BACKGROUND: High-throughput studies provide a wide spectrum of genes for use as predictive markers during testicular sperm extraction (TESE) in combination with ICSI. In this work, we used the specimens from testicular biopsies of men with non-obstructive azoospermia who underwent TESE to investigate the expression of spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR. METHODS: Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis (HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO). The levels of expression of the spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR in the testes were compared among these three groups using the reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Analysis of the expression of spermatogenic genes in human testes with abnormal spermatogenesis showed different expression patterns in patients from different groups. Fertilization rate for studied set of patients was 66% and pregnancy rate 29%. For HS group fertilization rate was 72% and pregnancy rate 32%, while for MA group fertilization and pregnancy rates were 54% and 26%, respectively. Fertilization rates in relation to the studied genes were uniformly around 70%, pregnancy rates for ACR and GAPDHS genes were surprisingly low at 6% and 8% correspondingly. CONCLUSIONS: Analysis of the expression of genes involved in spermatogenesis can be a fast additional test for the level of spermatogenesis in testicular samples.

Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm.

This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.

DNA immunization against proacrosin impairs fertility in male mice.

PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.

Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization.

Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.

Acrosin activity regulation by protein kinase C and tyrosine kinase in bovine sperm acrosome exocytosis induced by lysophosphatidylcholine.

Acrosin is an important proteolytic enzyme that is capable of hydrolysing the zona pellucida in bovine oocyte. Lysophosphatydic acid (LPA) derivated from lysophosphatidylcholine (LPC) is known to trigger the acrosome exocytosis. The present study was aimed at examining the acrosin activity variations in LPC-induced acrosome exocytosis and its regulation by tyrosine kinase, protein kinase C (PKC) and voltage-dependent calcium channels (VDCC) in spermatozoa previously capacitated with heparin or quercetin. The enzyme activities were spectrophotometrically measured using N-α-benzoyl-DL-arginine p-nitroanilide as an acrosin-specific substrate. The capacitation and acrosomal reaction were evaluated by chlorotetracycline assay, and the viability and acrosome integrity were evaluated by the trypan blue stain/differential interference contrast. It was observed that LPC induced acrosome exocytosis and increased the activity of acrosin in spermatozoa previously capacitated with heparin. In heparin/LPC-treated samples, it was observed that the inhibition of tyrosine kinase and PKC blocked the acrosome exocytosis and the acrosin activity (p < 0.05). Under these conditions, in heparin-capacitated spermatozoa, the LPC provokes an acrosin activity increase that is independent of calcium influx through VDCC Type L. In cryopreserved bovine spermatozoa, LPC might require modulation, mainly tyrosine kinase participation with respect to PKC activity to induce acrosome exocytosis and increase acrosin activity.