The correlation between sperm percentage with a small acrosome and unexplained in vitro fertilization failure.

PURPOSE: Since the unexplained in vitro fertilization failure occurs frequently, it is of great importance and clinical value to identify potential underlying predictors. This study aimed to explore whether the percentage of sperm with a small acrosome was correlated with unexplained in vitro fertilization failure. METHODS: A new acrosomal function evaluation index (the percentage of sperm with a small acrosome) was introduced into the analysis of sperm morphology. The association between the index and acrosome function by acrosin activity detection test and acrosome reaction test was investigated. In addition, the correlation with unexplained in vitro fertilization failure was further explored. Finally, the ROC curve was used to analyze the diagnostic efficacy on the failure of in vitro fertilization and the cutoff value was calculated. RESULTS: As the increasing of the percentage of sperm with a small acrosome, the value of acrosin activity, acrosome reaction rate, and in vitro fertilization rate were reduced, with a statistically significant difference (P < 0.05). The index in the low fertilization rate group was significantly higher than that in the normal fertilization rate group (P < 0.05). Finally, the results of ROC curve found that when the index was 43.5%, the sensitivity and specificity were 74.2% and 95.3%, respectively. CONCLUSION: The percentage of sperm with a small acrosome was positively correlated with unexplained in vitro fertilization failure, which could be potentially used as a prognostic index for the failure of in vitro fertilization. TRIAL REGISTRATION: [Ethics review acceptance No IIT20210339B].

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells.

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10 mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20 mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

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.

Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

BACKGROUND: Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. OBJECTIVES: This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction. MATERIALS AND METHODS: The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2 in pig sperm. RESULTS: Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. DISCUSSION AND CONCLUSION: These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

Male haploid cells through direct spherification.

OBJECTIVE: To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system. DESIGN: Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells. MAIN OUTCOME MEASURES: Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging. RESULTS: On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively. CONCLUSION: Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.

Immature rat testis sustained long-term development using an integrative model.

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Insights into the influence of canine breed on proteomics of the spermatozoa and seminal plasma.

This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.

[Pricking-reinforcing -reducing therapy improves semen quality and seminal plasma biochemical indexes in patients with varicocele infertility].

OBJECTIVE: To investigate the effect of pricking-reinforcing -reducing therapy (PRRT) on the semen quality and seminal plasma biochemical indexes of varicocele (VC) infertility patients. METHODS: We randomly and equally assigned 160 patients with VC infertility into a PRRT and a control group, the former treated by PRRT and the latter with oral ShengjingCapsules. Before and after treatment, we obtained the semen parameters, sperm morphology, sperm survival rate, sperm acrosin activity, seminal plasma neutral α glucosidase and seminal plasma zinc in the patients and compared them between the two groups. RESULTS: Before treatment, there were no statistically significant differences between the PRRT and control groups in sperm concentration (ï¼»16.81 ± 7.83ï¼½ vs ï¼»16.80 ± 7.54ï¼½ ×106 /ml, P > 0.05), total sperm count (ï¼»42.01 ± 19.57ï¼½ vs ï¼»41.99 ± 18.84ï¼½ ×106, P > 0.05), percentages of progressively motile sperm (PMS) (ï¼»15.37 ± 11.03ï¼½% vs ï¼»14.68 ± 10.27ï¼½%, P > 0.05) and morphologically normal sperm ( MNS) (1.62 ± 1.51ï¼½% vs ï¼»1.62 ± 1.13ï¼½%, P > 0.05), sperm survival rate (ï¼»28.11 ± 18.95ï¼½% vs ï¼»28.23±18.38ï¼½%, P > 0.05) and sperm acrosin activity (ï¼»28.11 ± 14.64ï¼½ vs ï¼»27.19 ± 14.07ï¼½ U/L, P > 0.05). After three months of treatment, all the patients showed evident increases in the above parameters (P < 0.05), even higher in the PRRT than in the control group, more significantly in sperm concentration (ï¼»38.88 ± 30.54ï¼½ vs ï¼»25.60 ± 14.71ï¼½ ×106 /ml, P < 0.05), PMS (ï¼»32.60 ± 12.46ï¼½% vs ï¼»27.67 ± 12.27ï¼½%, P < 0.05) and sperm acrosin activity (ï¼»65.74±31.81ï¼½ vs ï¼»67.94±17.95ï¼½ U/L, P < 0.05), though not significantly in total sperm count (97.20 ± 76.35ï¼½ vs ï¼»88.19 ± 39.56ï¼½ ×106, P > 0.05), MNS (ï¼»2.35 ± 1.83ï¼½% vs ï¼»1.87 ± 1.20ï¼½%, P > 0.05) and sperm survival rate (ï¼»61.44 ± 20.02ï¼½% vs ï¼»59.12 ± 22.48ï¼½%, P > 0.05). Compared with the baseline, after treatment, the patients in the PRRT group also exhibited elevated levels of neutral α-glucosidase (ï¼»14.42 ± 5.90ï¼½ vs ï¼»28.43 ± 19.76ï¼½ U/L, P < 0.05) and seminal plasma zinc (ï¼»2.11 ± 1.22ï¼½ vs ï¼»2.89 ± 1.23ï¼½ mmol/L, P < 0.05), and so did the controls (ï¼»14.44 ± 5.61ï¼½ vs ï¼»26.66 ± 17.69ï¼½ U/L , P < 0.05) and (ï¼»2.09 ± 1.10ï¼½ vs ï¼»2.82±1.08ï¼½ mmol/L, P < 0.05). No statistically significant difference, however, was observed between the two groups after treatment (P > 0.05). CONCLUSION: PRRT can significantly improve semen quality in patients with VC infertility, even more effective than ShengjingCapsules in improving sperm concentration, PMS, sperm survival rate, and sperm acrosin activity, which may be related to its effect of elevating the levels of seminal plasma neutral-α glucosidase and zinc providing sufficient energy for basic sperm metabolism, maturation, energy acquisition and motility.

Immature rat testis sustained long-term development using an integrative model

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

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.

Reduced semen quality in patients with testicular cancer seminoma is associated with alterations in the expression of sperm proteins.

Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.

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.

Assessment of chosen semen characteristics of two colour morphs of the Arctic fox Alopex lagopus L.

The aim of the study was to evaluate semen quality in the two most popular colour morphs of the Arctic fox Alopex lagopus L., blue and white, based on ejaculate parameters, acrosin activity and analysis of sperm morphology. The research material consisted of ejaculates collected once by manual stimulation from 20 one-year-old male Arctic foxes (10 individuals of the blue morph and 10 of the white morph). Ejaculates were evaluated in terms of volume, sperm concentration, total number of spermatozoa and the percentage of spermatozoa with major and minor defects. The study revealed that male blue Arctic foxes produce ejaculates with much higher concentration (148.75 × 106 /ml) and total number of spermatozoa (98.16 × 106 ) compared to white Arctic foxes (42.88 × 106 /ml and 35.2 × 106 respectively). The level of acrosin activity from white foxes seemed to be higher compared to blue foxes but the difference was not statistically confirmed. Semen from Arctic foxes is characterized by high inter-individual variability in sperm morphology. The frequency of morphological changes in sperm from Arctic foxes does not significantly depend on ejaculate volume, sperm concentration or the total number of spermatozoa in the ejaculate, but is associated with acrosin activity.

Expression patterns of male germ cell markers in cryptorchid pig testes.

Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

The association between sperm head elongation and semen quality.

BACKGROUND: Previous studies suggested that sperm head shape may serve as an effective indicator of semen quality. However, there lacks research with large sample and quantitative measurement. OBJECTIVES: The objective of this retrospective study was to explore the association between sperm head elongation (Width/Length ratio) and routine semen parameters. MATERIALS AND METHODS: From January 2012 to December 2017, 63 866 semen samples were collected from male subjects at 18-60 years of age. Sperm head elongation and routine semen parameters (semen volume, sperm concentration, motility, etc.) were examined with computer-assisted semen analysis (CASA) systems in order to evaluate the association between elongation and semen quality. RESULTS: Logistic and linear regression models showed that the value of elongation is negatively correlated with sperm concentration, total sperm count, progressive motility, total motility, percentage of morphologically normal spermatozoa, and acrosin activity (all p < 0.001). DISCUSSION: The results suggested that higher value of elongation is generally associated with higher risks of abnormality in semen quality. The importance of elongation may be explained by abnormal acrosin activity in the round-headed spermatozoa, which has been reported to cause failure of natural pregnancy. CONCLUSIONS: This study provides a new insight into the sperm head shape, which may be used as a complementary parameter in clinical semen examination and academic research.

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.

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.