Sperm-zona Binding Research Articles

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca 2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1 , the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca 2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure.

P-058 Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure

Abstract Study question How does sperm phospholipase C zeta (PLCζ) affect fertilization in conventional in vitro fertilization (cIVF)? Summary answer Absence of PLCζ causes polyspermy, and a decline in the proportion of sperm expressing PLCζ is correlated with a low fertilization rate (FR) after cIVF. What is known already PLCζ is a key sperm-borne factor that triggers Ca2+ oscillations and the subsequent oocyte activation following gamete fusion. Mutations in PLCZ1, the gene encoding PLCζ, cause male infertility and intra-cytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI FR. However, few studies have been published on the relationship between PLCζ and cIVF, an insemination procedure involving several key events that are bypassed in ICSI, e.g., sperm-zona binding and penetration, as well as polyspermy blocking. It is possible that sperm PLCζ may affect fertilization in a different manner from that in ICSI. Study design, size, duration Sixty couples who underwent cIVF treatments were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya between February 2019 and January 2022. Participants/materials, setting, methods We performed whole-exome sequencing in two unrelated males who exhibited infertility and polyspermy in cIVF. Immunofluorescence staining and oocyte-activation testing were employed to evaluate the effects of the candidate variants on PLCζ protein. Subsequent PLCζ analysis was performed in additional 58 males who underwent cIVF. The Pearson correlation coefficient was performed to evaluate the relationships between PLCζ and FRs. The receiver operating characteristic curve and Youden’s index analysis were used to indicate the cutoff value. Main results and the role of chance We identified one previously reported and two novel PLCZ1 variants in two males that were associated with male infertility and polyspermy characterized by excessive sperm–zona binding and a delay in pronuclear (PN) formation. In vitro functional analyses revealed that virtually all sperm from patients lacked functional PLCζ and were thus unable to evoke the physiologic pattern of Ca2+ oscillations. ICSI with an artificial oocyte-activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Subsequent PLCζ analysis were performed in additional 58 males, who were allocated to three groups according to fertilization outcomes: a multiple pronuclear (MPN) group (no. of PN ≥ 3, MPN rate ≥ 50%, n = 9) and low fertilization (LF, total FR ≤ 30%, n = 18) and normal fertilization groups (NF, 2PN ≥ 50%, 1PN + MPN < 30%, n = 31). We found that the proportion of sperm that expressed PLCζ was positively correlated with both 2PN rate (P = 0.0005) and total FR (P = 0.0008) after cIVF, and that the optimal cutoff value below which males were likely to experience low FR (total FR ≤ 30%) after cIVF was 56.7% (area under the ROC curve, 0.705; P = 0.0176). Limitations, reasons for caution Precise mechanisms that underly the roles of PLCζ in cIVF outcomes are still uncovered. Follow-up studies with larger numbers of patients are required to validate the sensitivity, specificity, and accuracy of PLCζ as a diagnostic biomarker in cIVF outcomes. Wider implications of the findings Our study identified PLCZ1 as a novel causative gene of polyspermy in humans,highlighting the potential value of PLCζ as a fertility marker for cIVF outcomes. Trial registration number nor applicable

In vitro adverse effects of amitraz on semen quality: Consequences in bovine embryo development

Veterinary drugs are potential environmental pollutants that interfere with male reproductive function. Infertility has increased, and it is known that environmental toxins contribute to declining sperm parameters. Amitraz {N,N-[(methylamino) dimeth-ylidyne] di-2,4-xylidine} (AMZ) is a formamidine pesticide widely used as an insecticide and an acaricide. The aim of this study was to evaluate the toxicity of AMZ in bovine sperm. Three experiments using frozen-thawed bovine semen incubated with AMZ for 2 h were carried out. Negative and solvent (dimethyl sulfoxide) controls were run simultaneously with treatments. In experiment 1, the AMZ concentrations used were 10, 15 and 25 μg AMZ/ml and the sperm parameters evaluated were viability, mitochondrial activity, acrosomal status, functional membrane integrity and apoptosis. In experiments 2 and 3, 25 μg AMZ/ml was used to evaluate fertilizing capacity, embryo development and blastocyst DNA damage. In experiment 1, 25 μg AMZ/ml decreased sperm viability (P = 0.01), reduced mitochondrial activity (P = 0.03) and induced apoptosis (P < 0.01). Also, 15 and 25 μg AMZ/ml affected functional membrane integrity (P < 0.01). In experiment 2, AMZ did not alter sperm-zona binding (P = 0.40) and pronucleus formation (P = 0.36). In experiment 3, 25 μg AMZ/ml decreased the rate of embryo development (P < 0.01) and increased apoptosis (P = 0.03). These results suggest that AMZ induced alterations in bovine sperm, probably affecting male fertility at concentrations that could be present in the environment.

Optimization of dissolved oxygen levels in extender prevents development of cryocapacitation like changes, oxidative stress and augments zona binding capacity of crossbred bull spermatozoa.

The present study was undertaken to determine the efficacy of partial deoxygenation of extender at constant temperature (35°C) in freezability of crossbred bull semen. The dissolved oxygen (DO) levels were reduced by the use of newly developed technique of nitrogen effervescence at a flow rate of 2-3 bubbles per second. Four different levels of oxygen in semen extender, that is 11.7, 2, 4 and 8ppm as control (Group-I), Group-II, Group-III and Group-IV, respectively, were used to assess the effect of partial deoxygenation on semen quality parameters. The 4ppm level of DO resulted in higher (p<0.05) progressive motility in comparison with non-treated group at post-thaw stage, whereas reduction up to 2ppm resulted in drastic fall in motility. Oxidative stress status revealed low superoxide dismutase (SOD) and total antioxidant capacity (TAC) in Group-II, whereas higher (p<0.05) SOD and TAC activities were observed in Group-III in comparison with non-treated group at pre-freeze and post-thaw stages. The sperm-zona binding at 4ppm level of DO was significantly higher than control group, 2 and 8ppm levels of DO. In conclusion, reduction of DO in the extender up to 4ppm reduced oxidative stress and improved in vitro fertility of crossbred bull spermatozoa.

Zinc supplementation within the reference ranges for zinc status in cattle improves sperm quality without modifying in vitro fertilization performance

Zinc (Zn) has important functions in mammalian reproductive processes. In cattle, Zn status can be classified as deficient, marginal, and adequate, depending on the plasma Zn concentration. In addition, Zn deficiency can lead to reproductive failure. The aim of this study was to investigate the effect of maternal Zn status at the beginning of a fixed-time artificial insemination (FTAI) treatment regimen on pregnancy rate in cattle, and evaluate the effect of supplementing in vitro fertilization (IVF) medium with Zn concentrations within the reference range for Zn status on sperm quality and IVF performance. Pregnancy rates of animals with marginal and adequate Zn status did not differ, and there were no Zn-deficient animals detected. Supplementation of 0.8 μg/mL Zn to IVF medium enhanced progressive motility, sperm viability, functional sperm membrane integrity (HOST), acrosomal integrity and sperm-zona binding, without modifying pronuclear formation, or development of embryos to the cleavage or blastocyst stage after IVF. In conclusion, the present results indicate pregnancy rates are not associated with maternal Zn status at the beginning of the FTAI treatment regimen if Zn status is marginal or adequate. Furthermore, supplementation of IVF medium with Zn at concentrations which is considered adequate for Zn status in cattle led to improved sperm quality, without having effects on embryo development in cattle.

An update on the techniques used to measure oxidative stress in seminal plasma.

Reactive oxygen species (ROS) are produced in significant amounts by spermatozoa and leucocytes. They are necessary to carry out various physiological functions such as sperm capacitation, hyperactivation, acrosome reaction, sperm-zona binding and cellular signalling pathways. Oxidative stress (OS) results when the ROS levels overwhelm the available antioxidant reserve. A number of direct and indirect tests have been developed to assess oxidative stress. In this manuscript, we discuss these common direct and indirect tests as well as their advantages and disadvantages. Tests measuring sperm dysfunction secondary to oxidative stress such as lipid peroxidation, DNA fragmentation and protein alterations are also described.

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.

LYPD4, mouse homolog of a human acrosome protein, is essential for sperm fertilizing ability and male fertility†

Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.

Immune regulatory molecules as modifiers of semen and fertility: A review.

Declining fertility rates in both human and animals is a cause for concern. While many of the infertility cases are due to known causes, idiopathic infertility is reported in 30% of the infertile couples. In such cases, 18% of the infertile males carry antisperm antibodies (ASAs). Such data are lacking in livestock, wherein 20-30% of the animals are being culled due to low fertility. In males, the blood-testis barrier (BTB) and biomolecules in the semen provide an immuno-tolerant microenvironment for spermatozoa as they traverse the immunologic milieu of both the male and female reproductive tracts. For example, insults from environmental contaminants, infections and inflammatory conditions are likely to impact the immune privilege state of the testis and fertility. The female mucosal immune system can recognize allogenic spermatozoa-specific proteins affecting sperm kinematics and sperm-zona binding leading to immune infertility. Elucidating the functions and pathways of the immune regulatory molecules associated with fertilization are prerequisites for understanding their impact on fertility. An insight into biomolecules associated with spermatozoal immune tolerance may generate inputs to develop diagnostic tools and modulate fertility. High-throughput sequencing technologies coupled with bioinformatics analyses provides a path forward to define the array of molecules influencing pregnancy outcome. This review discusses the seminal immune regulatory molecules from their origin in the testis until they traverse the uterine environment enabling fertilization and embryonic development. Well-designed experiments and the identification of biomarkers may provide a pathway to understand the finer details of reproductive immunology that will afford personalized therapies.

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding

PurposeTo investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)–enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding.MethodsImmunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay.ResultspY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding.ConclusionsThe present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm–zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process.

Insilico characterization of novel PH20 hyaluronidase protein sequences of murrah(Bubalus bubalis)bulls

PH20 hyaluronidase proteins are members of Zona pellucida binding protein family. PH20 hyaluronidase is reportedly bifunctional i.e. have hyaluronidase and sperm-zona binding activities. In this study, we performed insilico characterization of novel PH20 protein using partial nucleotide sequences, obtained from SNP genotyping of Bubalus bubalis bulls, by Sanger's dideoxy sequencingmethod. These gene sequences are novel and first time reported in B. bubalis. Possible glycosylation and mannosylation patterns were also predicted in both the PH20 hyaluronidase sequences which appear responsible for its hyaluronidaseactivity. Further, the potential tertiary structure of PH20 was deduced using Swiss-Model server. The tertiary structure models of PH20 were validated by ProSA server and Ramachandran plots. The tertiary structure models of PH20 were further refined using KoBaMIN server and validated by PROCHECK, ProSA and Ramachandran plots. These structural models were assigned PMDB identifiers PM0080458 and PM0080459. Physico-chemical characterization revealed that B. bubalis PH20 hyaluronidase are more thermostable and more hydrophobic than that of cattle PH20 hyaluro-nidase. The functional annotations were further inferred using Gene Ontolgy based predictions. Present work on elucidated structure of PH20 predicts a reference model for functional genomics studies related to multifunctionality of PH20 hyaluronidase proteins with respect to genetic variants within the species or trans-species. The current work is the first preliminary insilico study on novel bubaline PH20 hyaluronidases.

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P &lt; 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P &gt; 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P &lt; 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P &lt; 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction

Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 105 spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm–zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF.

Spermatozoa protein alterations in infertile men with bilateral varicocele.

Among infertile men, a diagnosis of unilateral varicocele is made in 90% of varicocele cases and bilateral in the remaining varicocele cases. However, there are reports of under-diagnosis of bilateral varicocele among infertile men and that its prevalence is greater than 10%. In this prospective study, we aimed to examine the differentially expressed proteins (DEP) extracted from spermatozoa cells of patients with bilateral varicocele and fertile donors. Subjects consisted of 17 men diagnosed with bilateral varicocele and 10 proven fertile men as healthy controls. Using the LTQ-orbitrap elite hybrid mass spectrometry system, proteomic analysis was done on pooled samples from 3 patients with bilateral varicocele and 5 fertile men. From these samples, 73 DEP were identified of which 58 proteins were differentially expressed, with 7 proteins unique to the bilateral varicocele group and 8 proteins to the fertile control group. Majority of the DEPs were observed to be associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes. Seven DEP were involved in sperm function such as capacitation, motility, and sperm-zona binding. Proteins TEKT3 and TCP11 were validated by Western blot analysis and may serve as potential biomarkers for bilateral varicocele. In this study, we have demonstrated for the first time the presence of DEP and identified proteins with distinct reproductive functions which are altered in infertile men with bilateral varicocele. Functional proteomic profiling provides insight into the mechanistic implications of bilateral varicocele-associated male infertility.

Nuclear DNA fragmentation negatively affects zona binding competence of Y bearing mouse spermatozoa

To investigate the influence of sperm DNA integrity on the zona binding ability of mouse spermatozoa in relation to their sex chromosomal constitution. In this prospective experimental study, the sperm DNA fragmentation was induced by exposing testicular area of Swiss Albino mice (Mus musculus) to different doses of γ-radiation (0, 2.5, 5.0 and 10.0Gy). Sperm DNA fragmentation was quantified by single cell gel electrophoresis (comet assay). In vitro sperm zona binding assay was performed and the numbers of zona bound X and Y bearing spermatozoa were determined using fluorescence in situ hybridization (FISH). The assessment of zona pellucida bound X and Y-bearing spermatozoa using fluorescence in situ hybridization has revealed a unique binding pattern. The number of zona bound Y-spermatozoa declined significantly (P < 0.01 to 0.0001) with increase in the DNA damage. The skewed binding pattern of X and Y-bearing sperm was strongly correlated with the extent of sperm DNA damage. The zona pellucida may have a role in preventing DNA damaged mouse sperm binding especially towards Y-bearing sperm. However, the exact mechanism behind this observation needs to be elucidated further.

Prss37 Is Required for Male Fertility in the Mouse1

In order to understand the mechanisms of mammalian fertilization, studies using genetically manipulated animals have provided us with plenty of interesting and valuable information on the genetic factors affecting male fertility. In the present work, we demonstrate for the first time that Prss37, a previously uncharacterized putative trypsin-like serine protease, is required for male fertility. Prss37 is highly and exclusively expressed in the testis of adult mice, especially in the elongating spermatids during spermiogenesis, and almost vanishes in the mature sperm of mice. Mice deficient for Prss37 show male infertility, but their mating activity, spermatogenesis, sperm morphology, and motility remain unaffected. In vivo fertilization assays revealed that Prss37(-/-) mice exhibited a markedly decreased fertilization rate (2.3% vs. 70% of that in control mice) accompanied by the defect in sperm migration from uterus into oviduct. In vitro study further showed sperm were incapable of sperm-egg recognition/binding when zona-intact eggs were exposed to Prss37(-/-) sperm, in which mature Adam3 was completely undetectable. Interestingly, however, Prss37(-/-) sperm were able to fertilize cumulus-intact oocytes in vitro. These data clearly indicate that Prss37 deficiency causes the absence of mature Adam3 in sperm and a defect in sperm migration from uterus into oviduct, which mainly accounts for male infertility of Prss37-null mice, while the defect in sperm-zona binding seems irrelevant to the fertilizing ability of Prss37(-/-) sperm.

261 SUPPLEMENTATION OF SHEEP OOCYTE MATURATION MEDIA WITH NITRIC OXIDE SYNTHASE INHIBITORS MODIFIES THE ZONA PELLUCIDA AND DECREASES SPERM ZONA BINDING

Nitric oxide plays a critical role in several reproductive functions, including sperm and egg maturation. Nitric oxide has complex and sometimes antagonistic biological effects. At low and controlled levels, NO aids gamete maturation, but at high levels, it has detrimental effects and often causes cell death. Both the female tract and egg vestment contain endothelial NO synthase (eNOS) and inducible NOS (iNOS). The knockout mouse for specific NOS has been shown to impair egg maturation and ovulation. The aim of this work was to study the effect of NOS inhibitors on in vitro sheep oocyte maturation and function. We evaluated the cumulus cell expansion, sperm zona binding, and time required to fully digest the zona pellucida (ZP). The 3 different NOS inhibitors used were 1 mM l-NAME (N-ω-nitro-l-arginine methyl ester, an eNOS inhibitor), 1 mM l-NMMA (NG-monomethyl-l-arginine acetate, a competitive inhibitor of all three isoforms of NOS), and 1 mM aminoguanidine (eNOS and iNOS inhibitors). We also used a NO donor, S-nitroso-glutathione (GSNO; 50 µM). Oocytes were matured in TCM without supplementation or supplemented with GSNO or NOS inhibitors for 24 h at 38°C, 5% CO2. The effect of the inhibitors on the ZP was assessed by determining the time necessary to complete digestion of the ZP with pronase (0.1%). To evaluate the number of sperm attached to the ZP, oocytes matured as described above were denuded and co-incubated for 4 h (38°C, 5% CO2) with epididymal sheep sperm (1 × 106 spermatozoa mL–1 in TALP medium). At the end of the incubation, sperm unbound to the ZP were eliminated by several washings. The oocytes were fixed and stained with Hoechst to stain the sperm nucleus, and the number of sperm bound per zona was measured under an epifluorescence microscope. The times required for complete ZP digestion and spermatozoa attachment were analysed by one-way ANOVA. Mean digestion times (seconds) and numbers of spermatozoa bound to the ZP in the control, GSNO, l-NAME, aminoguanidine, and l-NMMA groups were 234.6a, 297.8a, 243.2a, 323.0ab, 398.4b, and 20.2a, 12.0ab, 11.1b, 7.7b, and 8.1b, respectively (a,bP &lt; 0.001). Our results showed that 1) egg maturation, as measured by cumulus expansion, was not affected by the NOS inhibitors or GSNO, 2) the time necessary to completely digest the ZP increased in the presence of aminoguanidine and l-NMMA, and 3) all three NOS inhibitors decreased the number of spermatozoa that could be attached to the ZP. The GSNO showed no effect because these levels represent physiological concentration observed during normal egg maturation. In conclusion, these results are in accordance with the importance of NO for egg maturation, and this study shows for the first time the possible involvement of NO in maintaining the structure of the ZP, which is important for sperm–egg binding and further fertilization. Such an effect on the ZP proteins could be due to a change in protein S-nitrosylation, which is a post-translational modification attributable to NO.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

Treatment of human sperm with serine protease during density gradient centrifugation

Seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. (i) Pooled semen samples (n = 9) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n = 5) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing. Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.