Sperm-egg Interaction Research Articles

P–026 Seminal plasma exosomes: a promising source of biomarkers for fertility evaluation

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

P–567 Re-establishment of fertilization competency of the oocyte via CRISPR/dCas9 epigenome edition technology

Abstract Study question Is it possible to increase the decreased levels of the sperm-oocyte binding protein, Juno, to restore the fertilization capacity of the oocyte, via the use of the CRISPR/dCas9 activation system? Summary answer JUNO domain (in the oocyte) suppressed with melamine was opened using the CRISPR/dCas9 system and sperm-oocyte binding and fertilization capability have been restored. What is known already Melamine is a chemical that is widely used in the manufacture of laminates, plastics, etc. Evidence reveals that long-term exposure to melamine could damage reproductive systems in mammals leading to infertility. Izumo1 is the only cell surface protein expressed on sperm that is known to be essential for sperm-egg interaction in vivo. It was in vitro shown that high-dose feeding of melamine to female mice led to a significant decrease of JUNO on the plasma membrane of eggs. CRISPR/dCas9 system can provide the gene activation or repression to activate the target gene via Sam and Tet1 based systems. Study design, size, duration Six different gRNAs were designed for the transfection of oocytes. Six-week-old mice were fed with melamine (50mg/kg/day) for 2 weeks via gavage. Melamine gavage, water gavage, and control groups (n = 15 /each group) were created. CRISPR activation plasmids (SAM) were given by piezo microinjection into the GV oocytes (n = 100 oocytes/each group). Fertilization capacity was evaluated by sperm binding assay, qPCR, Western blotting, and IF staining. Two technical replicates were used in molecular studies. Participants/materials, setting, methods 293T cells were transfected (dCas9 SAM plasmids+gRNA) with Fugene. Mice randomly were assigned to 3 groups (n = 15), as each was given orally a dose of 50mg/kg/d of melamine, only water or no water via gavage. Microinjection of plasmids was performed. Post-injection, oocytes were incubated until MII stage. For binding and fertilization evaluation, motile sperms were incubated with oocytes, and pronuclei were checked. Juno and IZUMO1 levels were evaluated by qPCR, Western blotting, and IF staining. Main results and the role of chance As the SAM system is more efficient compared to the Tet1 system when tested in 293t cells, the SAM system was used for mouse experiments. As a result of qPCR performed in oocytes collected at the end of gavage, it was observed that the JUNO expression levels were decreased by 40 folds in melamine fed mice (p < 0.05). The decrease in the level of JUNO protein was demonstrated by IF stainings, and a decrease in the oocyte count along with abnormal uterine shapes was also observed in this group. IZUMO1 expression in motile sperms was demonstrated by qPCR before sperm binding assay and the position of the IZUMO 1 domain before the acrosome reaction was demonstrated by IF stainings. Sperm binding assay has demonstrated a 70% reduction in fertilization competency of melamine-treated oocytes (p < 0.05). SAM plasmids and JUNO gRNA were given to oocytes by piezo injection. By sperm binding experiments conducted to evaluate fertilization capacities after microinjection, it was shown that the fertilization capacity was restored by 75% (p < 0.05). Re-gaining of Juno expression in the oocytes was supported by a 60 fold increase in qPCR results. Recovery of JUNO protein expression in the oocytes was also demonstrated by IF stainings. Limitations, reasons for caution There is no known promoter region for the JUNO gene in the mouse. Therefore, we designed gRNAs targeting possible promoter regions. However, we have used two activation systems(SAM and Tet1) that are widely used to open a closed gene, but other activation systems (acetylation, etc.) can also be tried. Wider implications of the findings: This study is valuable since: -it presents a possible cure for unsuccessful fertilization in related cases. -it possibly reveals melamine’s unknown way of action. - it presents a new approach as a sperm-binding assay to be used in IVF clinics since Juno can also be expressed in somatic cell lines. Trial registration number non-clinical trials

The Fertilization Enigma: How Sperm and Egg Fuse.

Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm-egg fusion, have remained mysterious for many decades. Here, we summarize our current molecular understanding of sperm-egg interaction, focusing mainly on mammalian fertilization. Given the fundamental importance of sperm-egg fusion yet the lack of knowledge of this process in vertebrates, we discuss hallmarks and emerging themes of cell fusion by drawing from well-studied examples such as viral entry, placenta formation, and muscle development. We conclude by identifying open questions and exciting avenues for future studies in gamete fusion.

Pharmacological Inactivation of CatSper Blocks Sperm Fertilizing Ability Independently of the Capacitation Status of the Cells: Implications for Non-hormonal Contraception.

Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1–20 μM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm’s ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm–egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.

Accumulation of Seminolipid in Sertoli Cells Is Associated with Increased Levels of Reactive Oxygen Species and Male Subfertility: Studies in Aging Arsa Null Male Mice.

Seminolipid (also known as sulfogalactosylglycerolipid-SGG), present selectively in male germ cells, plays important roles in spermatogenesis and sperm–egg interaction. The proper degradation of SGG in apoptotic germ cells is also as important. Sertoli cells first phagocytose apoptotic germ cells, then Sertoli lysosomal arylsulfatase A (ARSA) desulfates SGG, the first step of SGG degradation. We have reported that aging male Arsa−/− mice become subfertile with SGG accumulation in Sertoli cell lysosomes, typical of a lysosomal storage disorder (LSD). Since reactive oxygen species (ROS) levels are increased in other glycolipid-accumulated LSDs, we quantified ROS in Arsa−/− Sertoli cells. Our analyses indicated increases in superoxide and H2O2 in Arsa−/− Sertoli cells with elevated apoptosis rates, relative to WT counterparts. Excess H2O2 from Arsa−/− Sertoli cells could travel into testicular germ cells (TGCs) to induce ROS production. Our results indeed indicated higher superoxide levels in Arsa−/− TGCs, compared with WT TGCs. Increased ROS levels in Arsa−/− Sertoli cells and TGCs likely caused the decrease in spermatogenesis and increased the abnormal sperm population in aging Arsa−/− mice, including the 50% decrease in sperm SGG with egg binding ability. In summary, our study indicated that increased ROS production was the mechanism through which subfertility manifested following SGG accumulation in Sertoli cells.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Mitochondrial Functionality in Male Fertility: From Spermatogenesis to Fertilization.

Mitochondria are structurally and functionally distinct organelles that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), to provide energy to spermatozoa. They can also produce reactive oxidation species (ROS). While a moderate concentration of ROS is critical for tyrosine phosphorylation in cholesterol efflux, sperm–egg interaction, and fertilization, excessive ROS generation is associated with male infertility. Moreover, mitochondria participate in diverse processes ranging from spermatogenesis to fertilization to regulate male fertility. This review aimed to summarize the roles of mitochondria in male fertility depending on the sperm developmental stage (from male reproductive tract to female reproductive tract). Moreover, mitochondria are also involved in testosterone production, regulation of proton secretion into the lumen to maintain an acidic condition in the epididymis, and sperm DNA condensation during epididymal maturation. We also established the new signaling pathway using previous proteomic data associated with male fertility, to understand the overall role of mitochondria in male fertility. The pathway revealed that male infertility is associated with a loss of mitochondrial proteins in spermatozoa, which induces low sperm motility, reduces OXPHOS activity, and results in male infertility.

Anti-sperm antibodies and reproductive failures.

Reproductive failures include infertility and recurrent pregnancy loss (RPL). Although the relative importance of immunological factors in human reproduction remains unclear, there may be immune-mediated reproductive failures in a portion of unexplained infertility and RPL. As a cause of immunological factor, anti-sperm antibodies (ASA) are produced both in men and women. There have been reported several antigens in the surface of sperm that are especially foreign to women. The presence of ASA, especially sperm-immobilizing antibodies, in the sera of infertile women has been shown to inhibit sperm migration in the female genital tract. Therefore, the effectiveness of the treatments for infertile women with sperm-immobilizing antibodies by timed intercourse or intra-uterine insemination is limited. Such antibodies can also exert inhibitory effects on various stages of sperm-egg interaction and subsequent embryo development in vitro. It is suggested that ASA testing for infertile women should be performed before proceeding IVF. The manipulation of gametes and embryos from patients having sperm-immobilizing antibodies should be carefully carried out especially to avoid contaminating patient's serum and follicular fluid in the culture medium in order to overcome the immunological causes of female infertility by ASA, and satisfactory results under suitable conditions for gametes and embryos have been obtained. The relationship between ASA and RPL was controversially reported. Increased miscarriage rates in women with ASA were demonstrated by some authors. In contrast, lack of association between ASA and RPL was reported. In this manuscript, we are focusing the roles of ASA in women with reproductive failures.

Sperm plasma membrane characteristics and boar semen fertility

Much effort is being made to establish relationships between the molecular events that take place in spermatozoa under fertilizing conditions and actual sperm function during fertilization. During capacitation, the process that 'primes' spermatozoa for interaction with the egg, components of the sperm's environment, notably bicarbonate, provoke various specific changes in the architecture and functioning of the sperm plasma membrane in a large number of cells. The individual changes have been found to proceed on different time scales, and may therefore represent sequential stages in the capacitation process. However, each change takes place at different rates in individual cells, revealing considerable functional heterogeneity within the sperm population. Recent work on membrane changes provoked by cooling has indicated similarities with capacitational changes. The effect of cooling may therefore be to induce premature capacitation (and destabilization). Such an effect would greatly compromise sperm fertilizing potential. A pig sperm-egg interaction model was used to examine quantitative details of zona binding and zona penetrating abilities within capacitated sperm populations, and sperm behaviour was found not to accord with generally held beliefs. In particular, individual spermatozoa that have bound to the zona pellucida show great variation in the delay before penetrating: no evidence has been found for a specially competent subgroup. Even in sperm samples incubated to undergo maximal capacitational membrane changes, cells with actual penetrating potential represent less than 15% of the total number that attach initially to the zona pellucida. Thus detection of capacitational membrane changes appears greatly to overestimate zona penetrating capability. Future studies linking sperm membrane characteristics with semen fertility in the field will need to consider differences between in vitro and in vivo conditions. The need for survival in the female tract may require much slower sperm responses than are considered optimal for in vitro fertilization.

Egg-sperm interaction in sturgeon: role of ovarian fluid.

Fertilization of freshwater fish occurs in the environment which negatively affects a lifespan of gametes mostly due to the osmotic shock; therefore, male gametes should reach the female gamete, as soon as possible. The existence of mechanisms controlling the encounter of gametes would be highly expedient in this case. By analogy with other species for which guidance was demonstrated, it is likely that this control may be performed by ovarian fluid or substances released by eggs. The aim was to study the effect of ovarian fluid and egg-released substances on spermatozoa behavior in sterlet. It was found that the presence of a particular concentration of ovarian fluid (30% solution in water) had an inhibiting effect on spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response (using a microcapillary injection of fluids into the suspension of motile spermatozoa) showed no effect of ovarian fluid on spermatozoa behavior, while at the same time, the attracting effect of the egg-conditioned medium was evident (i.e., due to some substances released from the eggs during their contact with freshwater). The results of the fertilization test showed that the presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during a longer period of time. Thus, the combined physicochemical action of "female factors" affects sterlet gametes during fertilization and may be involved in the guidance and selection mechanisms.

Cysteine rich venom protein like isoform X2 from turkey (Meleagris gallopavo) seminal plasma: effects on spermatozoa motility, migration and involvement in sperm-egg interaction

Cysteine rich venom protein like isoform X2 from turkey (Meleagris gallopavo) seminal plasma: effects on spermatozoa motility, migration and involvement in sperm-egg interaction

Evolutionary analysis of genes coding for Cysteine-RIch Secretory Proteins (CRISPs) in mammals

BackgroundCysteine-RIch Secretory Proteins (CRISP) are expressed in the reproductive tract of mammalian males and are involved in fertilization and related processes. Due to their important role in sperm performance and sperm-egg interaction, these genes are likely to be exposed to strong selective pressures, including postcopulatory sexual selection and/or male-female coevolution. We here perform a comparative evolutionary analysis of Crisp genes in mammals. Currently, the nomenclature of CRISP genes is confusing, as a consequence of discrepancies between assignments of orthologs, particularly due to numbering of CRISP genes. This may generate problems when performing comparative evolutionary analyses of mammalian clades and species. To avoid such problems, we first carried out a study of possible orthologous relationships and putative origins of the known CRISP gene sequences. Furthermore, and with the aim to facilitate analyses, we here propose a different nomenclature for CRISP genes (EVAC1–4, “EVolutionarily-analyzed CRISP”) to be used in an evolutionary context.ResultsWe found differing selective pressures among Crisp genes. CRISP1/4 (EVAC1) and CRISP2 (EVAC2) orthologs are found across eutherian mammals and seem to be conserved in general, but show signs of positive selection in primate CRISP1/4 (EVAC1). Rodent Crisp1 (Evac3a) seems to evolve under a comparatively more relaxed constraint with positive selection on codon sites. Finally, murine Crisp3 (Evac4), which appears to be specific to the genus Mus, shows signs of possible positive selection. We further provide evidence for sexual selection on the sequence of one of these genes (Crisp1/4) that, unlike others, is thought to be exclusively expressed in male reproductive tissues.ConclusionsWe found differing selective pressures among CRISP genes and sexual selection as a contributing factor in CRISP1/4 gene sequence evolution. Our evolutionary analysis of this unique set of genes contributes to a better understanding of Crisp function in particular and the influence of sexual selection on reproductive mechanisms in general.

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.

Role of Binder of SPerm homolog 1 (BSPH1) protein in mouse sperm-egg interaction and fertilization

In mice, the Binder of Sperm Homolog 1 protein is exclusively expressed in the epididymis. BSP proteins play a role in the membrane modification events that occur during sperm capacitation. In the current study, we investigated the role of mouse recombinant BSP homolog 1 (rec-BSPH1) in sperm-egg interaction. Mouse oocytes were co-incubated with different concentrations of rec-BSPH1 or control proteins and then inseminated with sperm. To establish whether rec-BSPH1 interfered with in vitro fertilization of mouse oocytes, rec-BSPH1 binding to egg and sperm was first tested using an immunodetection assay. In separate experiments, sperm were immuno-neutralized by anti-rec-BSPH1 antibodies to indirectly verify the implication of BSPH1 in sperm-egg interaction and fertilization. The study revealed a dose-dependent inhibition of fertilization when oocytes were pre-incubated with rec-BSPH1. Moreover, sperm immuno-neutralization with anti-rec-BSPH1 antibodies led to dramatic motility changes, followed by compromised fertilization. In view of these results, we conclude that BSPH1 could be a marker of sperm fertility and thus an eventual target for male contraceptive development.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens.

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg's salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg's salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.

Proteome of cat semen obtained after urethral catheterization

The binding of seminal plasma (SP) proteins by spermatozoa plays an important role in the regulation of sperm epididymal maturation, motility gaining in female reproductive tracts and sperm-egg interaction. The aim of the study was to analyze the SP and sperm extracts proteome of cat (Felis catus) semen. The seminal plasma and spermatozoa were obtained by urethra catheterization from 10 male cats. Proteins were extracted using RIPA buffer and separated by electrophoresis (SDS-PAGE). The gels were analyzed using MultiAnalyst software. The proteins were subsequently analyzed using NanoUPLC-Q-TOF/MS. UniProt database-supported identification resulted in 106 proteins identified in the cat SP and 98 proteins in the extracts of spermatozoa. Based on a gene ontology analysis, dominant molecular functions of feline SP proteins were binding, catalytic, and antioxidant activity (56%, 33%, and 11% of cases, respectively). The molecular functions of sperm extracts proteins were mainly involved in catalytic activity (41%) and binding (23%). The proteins present in both, the SP and spermatozoa's extracts, were: serum albumin (ALB), semenogelin 2 (SEMG 2), clusterin (CLU), lactoferrin (LTF), prostatic acid phosphatase (ACPP), prolactin inducible protein (PIP), negative elongation factor E (NELF-E) and ectonucleotide pyrophosphatase (ENPP3). Protein-protein interactions analysis showed significant connection for 12 proteins in the cat semen. The seminal plasma proteins which, with high probability score, participate in important metabolic pathways are: glutathione peroxidases (GPx5 and 6), prostatic acid phosphatase (ACPP), β-hexosaminidase (HEXB), polymeric immunoglobulin receptor (pIgR) and serpin family F member 1 (SERPINF1). For sperm protein extracts it were: pyruvate dehydrogenase (PDHB), succinate-CoA-ligase (SUCLA2), malate dehydrogenase (MDH2), ATP synthase F1 subunit alpha (ATP5F1A) and tubulin beta (TUBB).

Proteomic Analysis of Dpy19l2-Deficient Human Globozoospermia Reveals Multiple Molecular Defects.

To investigate the differences in protein expression between Dpy19l2-deficient human globozoospermia and normozoospermia. Human sperm samples from three globozoospermic donors with Dpy19l2 deletion and three normal controls are subjected to TMT quantitative technology. SPESP1, HIST1H4A, and LYZL1 are randomly selected for western blotting analysis. GO annotations are performed using the Database for Annotation, Visualization, and Integrated Discovery. A total of 2567 proteins are identified, of which 2510 proteins are quantified, and 491 are differentially expressed (fold-change >2), with 370 upregulated and 121 downregulated in globozoospermic patients. The levels of several important proteins, including SPACA 1, IZUMO1, ZPBP1, and PLCZ1, are decreased in globozoospermic sperm. Bioinformatics analysis indicates the Dpy19l2-deficient sperm presented molecular defects in acrosome, chromatin, sperm-egg interaction, and fertilization. The present study is the first to analyze total globozoospermia with Dpy19l2 deletion using high-throughput proteomics. This study may provide insights into the mechanism of globozoospermia.

Expression Analysis of IZUMO1 Gene during Testicular Development of Datong Yak (Bos Grunniens).

Simple SummaryIZUMO1 (IZUMO sperm-egg fusion) is a crucial member of the immunoglobulin superfamily, and it plays a vital part in egg sperm interaction, fusion, and in spermatogenesis. Previous studies identified the fundamental role of the IZUMO1 gene in meiosis of bovine spermatogenesis and spermatogenic cell development and fertility. The current study confirmed the significant expression of the IZUMO1 gene with testicular development and its potential role in fertilization of Datong yak. This research explained the biological role of IZUMO1 in yak reproduction; therefore in the future it might provide a novel prospect to apply data on reproduction and to understand the further regulatory mechanisms of the IZUMO1 gene during spermatogenesis of different mammalian species.The IZUMO1 gene has promising benefits for the national development of novel non-hormonal contraceptives and in the treatments of fertility. Understanding the function of IZUMO1, its mRNA, and protein expression is critical to gain insight into spermatogenesis and promote sperm-egg fusion during reproduction of Datong yak. Therefore, we estimated the IZUMO1 gene expression in different ages of Datong yak by using semi quantitative PCR, qPCR, and western blotting. The results of the qPCR, semi-quantitative PCR and western blotting revealed that the expression level of IZUMO1 mRNA was significantly (p < 0.05) higher in the testis of 30 months and 6 years old followed by 18 and 6 months old Datong yak, respectively. We also predicted secondary and tertiary protein structure of IZUMO1 by using bioinformatics software that the revealed presence of a signal peptide, Izumo domain, immunoglobulin (Ig) like domain, and transmembrane region. Moreover, immunostaining analysis also elucidated that IZUMO1 was more prominent in the testis of 30 months and 6 years old yak, which represented that the IZUMO1 gene expression might be higher during the peak breeding ages (6 to 7 years) of the yak, and play a potential role in spermatogenesis, fertility, and testicular development.