Penetration Of Zona Pellucida Research Articles

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

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. 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. 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 2in pig sperm. Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2was present within a ∼150kDa 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 2immunoprecipitated 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 (∼53kDa) and Aacrosin binding proteinunder all three conditions and additionally to acrosin (∼35kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2was 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 2and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. 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.

P-263 Clinical factors influencing the incidence of tripronuclear zygotes in IVF

Abstract Study question Is the incidence of tripronuclear zygotes (3PN) in IVF cycles influenced by clinical, patient and treatment parameters? Summary answer Tripronuclear zygotes incidence in IVF cycles was influenced by Antral Follicle Count, number of oocytes retrieved, sperm progressive motility, and the use of fresh semen. What is known already Tripronuclear zygotes are indicative of abnormal fertilisation and are not routinely considered suitable for transfer. It is therefore important to optimise their detection and minimise their occurrence. Tripronuclear zygotes can originate as a result of the combination of one maternal and two paternal sets of chromosomes (fertilisation of a haploid oocyte by two haploid spermatozoa or by one diploid spermatozoon) or the combination of one paternal and two maternal sets of chromosomes (fertilisation of a diploid oocyte by one haploid spermatozoon) (1). Study design, size, duration This is a retrospective study of 550 IVF cycles performed between April 2018- April 2021, in a single UK clinic. IVF cycles either within (group 1) or exceeding (group 2) the key performance indicator (KPI) for the incidence of 3PN (<6%) (2), were assessed for associations with quantitative cycle variables. Proportion of 3PN per cycle was assessed for associations with qualitative variables. Cycles using donor oocytes (n = 73) were excluded for analyses assessing patient (maternal) parameters. Participants/materials, setting, methods Continuous variables including age, body mass index, Antral Follicle Count (AFC), anti-Mullerian hormone (AMH), oocytes recovered, timing from sperm production to insemination or oocyte recovery to insemination, sperm concentration, progressive motility and morphology were analysed by Mann-Whitney U Test. Qualitative variables including patient ethnicity, own/donor oocyte source, fresh/frozen sperm, stimulation regimen and oocyte recovery needle were analysed by logistic regression assuming binomial errors. Significance was assumed at p < 0.05. Main results and the role of chance There was no difference between groups where 3PN incidence exceeded or fell within the KPI when procedure timings and clinical parameters were assessed. There was also no relationship between patient characteristics (age, body mass index, AMH levels, ethnicity, oocyte source (own/donor)), semen concentration or semen morphology and the proportion of 3PN per cycle. However, number of oocytes retrieved in group 2 was significantly higher than group 1 (group 1, n cycles = 307, median oocytes retrieved = 8; group 2, n cycles = 170, median oocytes retrieved = 12 [95% CI -4, -1]) (p < 0.001). Similarly, AFC was significantly increased in group 2 (group 1, n cycles = 254, median AFC = 11; group 2, cycles n = 150, median AFC = 13 [95% CI -4, -1]) (p = 0.009). Likewise, the mean sperm motility parameters were higher for cycles exceeding the 6% 3PN KPI, with 64.6 ± 0.91% vs 68.8 ± 1.11% total motility (p = 0.004), and 57.1 ± 0.97% vs 61.7 ± 1.2% progressive motility (p = 0.003) for group 1 and 2 respectively. Proportion of 3PN per cycle was also significantly higher with fresh semen compared to frozen (6.6 ± 0.39% vs 4.7 ± 0.51%) (p = 0.006). Limitations, reasons for caution This is a retrospective study of a single UK clinic. Further analysis, expanding sample size, are required to confirm these findings. Wider implications of the findings Our finding that number of oocytes recovered influences 3PN incidence is in agreement with previous studies (3, 4). Fresh and high progressively motile sperm may be more capable of zona pellucida penetration leading to polyspermy and 3PN. Adjustment of sperm concentration at insemination could be considered in higher risk circumstances. Trial registration number na

Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation.

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

The conserved fertility factor SPACA4/Bouncer has divergent modes of action in vertebrate fertilization

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

The cell biology of fertilization: Gamete attachment and fusion.

Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm-egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm-egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

Infertility network and hub genes for nonobstructive azoospermia utilizing integrative analysis.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility owing to the absence of sperm during ejaculation as a result of failed spermatogenesis. The molecular mechanisms of NOA have not been well studied. Here, we revealed the dysregulated differentially expressed genes in NOA and related signaling pathways or biological processes. Cluster features of biological processes include spermatogenesis, fertilization, cilium movement, penetration of zona pellucida, sperm chromatin condensation, and being significantly enriched metabolic pathways in proximal tubule bicarbonate reclamation, aldosterone synthesis and secretion, glycolysis and glycogenesis pathways in NOA using Gene Ontology analysis and pathway enrichment analysis. The NOA gene co-expression network was constructed by weighted gene co-expression network analysis to identify the hub genes (CHD5 and SPTBN2). In addition, we used another Gene Expression Omnibus dataset (GSE45887) to validate these hub genes. Furthermore, we used the Seurat package to classify testicular tissue cells from NOA patients and to characterize the differential expression of hub genes in different cell types from different adult males based on the scRNA-seq dataset (GSE106487). These results provide new insights into the pathogenesis of NOA. Of particular note, CHD5 and SPTBN2 may be potential biomarkers for the diagnosis and treatment of NOA.

Effects of latrunculin A on the relocation of sperm IZUMO1 during gamete interaction in mouse.

The sperm protein IZUMO1 plays a central role in gamete fusion. In mouse sperm, IZUMO1 is enriched at the acrosomal cap before the acrosome reaction, and at the equatorial segment following this reaction; its relocation is dependent on filamentous actin. How actin polymerization affects IZUMO1 relocation during gamete interaction remains unknown. The present study addressed these processes using latrunculin A (LatA), an inhibitor of actin polymerization. We report that 25 µM LatA blocked actin polymerization in the capacitated sperm head, resulting in a marked decrease in sperm with relocated IZUMO1 during the A23187-induced acrosome reaction and cumulus layer penetration. Treated sperm also exhibited reduced zona pellucida penetration and fertilizing capacity. Interestingly, LatA-treated sperm present in the perivitelline space of eggs did not show impaired IZUMO1 relocation. Thus, IZUMO1 relocation represents one method by which eggs may select for or rescue sperm that are competent to undergo gamete adhesion/fusion. These data support the hypothesis that dynamic movement of IZUMO1 is essential for gamete fusion during mouse fertilization.

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

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

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Human sperm degradation of zona pellucida proteins contributes to fertilization

BackgroundThe mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process.MethodsPurified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay.ResultsThe results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP.ConclusionsThis study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa

The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (IVF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa–ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were co-incubated for 15 min. A significant increase in spermatozoa–ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa.

Sperm surface glycan modification by chemical reporter conjugation

The sperm surface glycocalyx has long been known to serve an instrumental role in sperm capacitation and zona pellucida penetration; however, utilization of this aspect of sperm for assisted reproduction or diagnostic purposes has been lacking. We describe novel orthogonal chemistry methodologies to characterize the network of sperm sialic acids; this is with the purpose of developing a quantitative measurement of sperm fertility that can be used as a novel sperm separation tool.

Oxidative stress and acrosomal morphology: A cause of infertility in patients with normal semen parameters

Abstract Introduction Acrosome has a vital role in the process of penetration of zona pellucida (ZP). It is highly susceptible to elevated reactive oxygen species (ROS) and oxidative stress (OS). As generally accepted that OS stress has deteriorating effects on sperm functions, we studied the effect of OS on sperm parameters and acrosomal structure in infertile patients with normal semen parameters. Patients and methods 30 infertile patients with normal semen parameters and 20 normal fertile controls were included in this study. Semen analysis was performed according to WHO parameters. Oxidative stress was evaluated by measurement of Malondialdehyde (MDA). Acrosomal anomalies were detected by transmission electron microscopy (TEM). Results Statistically no significant difference was found between infertile patients and controls regarding semen parameters ( p value >0.05). MDA values were statistically highly significant in infertile patients than normal controls ( p value p value Conclusion Percentages of MDA values and acrosomal anomalies were higher in infertile patients than normal subjects. The positive correlation between acrosomal anomalies and MDA values means association between OS and acrosomal anomalies which may indicate negative effects of OS on the acrosomal structure.

Evaluation of sperm subpopulation structure in relation to in vitro sperm–oocyte interaction of frozen-thawed semen from Holstein bulls

The present study examined the relationship between the relative amount of high motile sperm and sperm–oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.

Embryological, clinical and ultrastructural study of human oocytes presenting indented zona pellucida

Human oocyte dysmorphisms attain a large proportion of retrieved oocytes from assisted reproductive technology (ART) treatment cycles. Extracytoplasmic defects involve abnormal morphology of the zona pellucida (ZP), perivitelline space and first polar body. The aim of the present study was to describe a novel dysmorphism affecting the ZP, indented ZP. We also evaluated the clinical, embryological and ultrastructural features of these cases. We evaluated all ART treatment cycles during 7 consecutive years and found 13 treatment cycles (six patients) with all oocytes presenting an indented ZP. In addition, these oocytes presented total or partial absence of the perivitelline space, absence of resistance to ZP and oolemma penetration during microinjection, and low ooplasm viscosity during aspiration. This novel described dysmorphism was recurrent and attained all oocytes in three cases that had more than one treatment cycle. When compared with controls, data showed significant low oocyte maturity (42% versus 81.6%) and high cycle cancellation (30.8% versus 8.5%) rates, normal degeneration (3.4% versus 6.3%) and fertilization rates (69% versus 69.5%), and low pregnancy (15.4% versus 33.3%) and live-birth delivery (7.7% versus 27.7%) rates per cycle. Ultrastructure analysis revealed a zona pellucida structure with large empty electrolucent regions, an outer ZP layer with an indented surface with protuberances and a thick inner ZP that obliterated the perivitelline space. There was evidence of exocytosis of ZP material by the oocyte. In conclusion, oocytes with this novel described dysmorphism (indented ZP) are associated with low maturity, pregnancy and live-birth delivery rates.

Co‐evolution of gametes of the Greater Bandicoot Rat, Bandicota indica – a murine rodent from South‐East Asia

AbstractMost Old World mice and rats, subfamily Murinae, have a spermatozoon with an apical hook, a long tail and, as seen typically in eutherian mammals, a bilaterally flattened head. Dramatically different from this are the sperm of the Greater Bandicoot Rat, Bandicota indica. Here, we ask the question has the structure of the sperm head co‐evolved with that of the egg coat, the zona pellucida? For this, we first summarise the morphological features of the spermatozoon of B. indica that may relate to zona pellucida penetration at the time of fertilisation, and we confirm that the sperm head is generally round, not bilaterally flattened, in profile and has a huge acrosome. We then show that the zona pellucida around oocytes in tertiary follicles also differs from that of the other murine rodents in being only about 4 μm thick and, as demonstrated by lectin staining, has an unusual abundance of alpha‐L‐fucose. These findings indicate that both the male and female gametes of this South‐East Asian murine rodent are highly divergent in their structural organisation. One of the functional implications of this probably relates to sperm–zona interactions and the release of acrosomal enzymes that probably facilitate penetration by digestion of the zona matrix at the time of fertilisation.

Evaluation of Zona Pellucida Function for Sperm Penetration During &lt;i&gt;In Vitro&lt;/i&gt; Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.

Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies

The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.

Does the addition of butorphanol have an effect on pharmacologically induced ejaculation in severely injured stallions?

suggested that stimulation of sperm hyperactivity requires calcium influx through a specific cation channel (CatSper) localized on the principal piece of the sperm tail. To study the molecular processes underlying hyperactivation of stallion sperm, it is essential to verify that the genes for the CatSper complex are expressed within the stallion's reproductive tract and that the protein is present onmature sperm. In this study testicular and epididymal tissues were harvested from eight 3-year old DutchWarmblood stallions with acceptable semen quality, immediately after elective castration; in addition, prostate tissue was collected from twomature stallions shortly after euthanasia for medical or surgical problems unrelated to their reproductive function. Prostate tissue was included because binding of prostatederived vesicles (prostasomes) to sperm has been reported to improve their ability to undergo sustained calcium influx (Ren, 2011). The mRNA was isolated, and cDNA was produced from testicular, epididymidal and prostatic tissue using commercial kits. RT-PCR primers were designed for the four subunits of CatSper (CatSper 1, 2, 3 and 4) using the appropriate sequences in the equine genome. Gene expression in the various tissues was then compared using quantitative (real-time) RT-PCR, with relative expression calculated with respect to three stable housekeeping genes (GAPDH, PGK1 and RPL32) using GeNorm analysis. In addition, an antibody against CatSper-1 (H-300, sc-33153: Santa Cruz Biotechnology) was used to verify the presence of CatSper protein (and by inference the cation channel) in stallion spermatozoa and testis by means of dot blots and western blots. Results showed that gene expression for all four Catsper sub-units was high in testicular tissue but more than a factor 100 lower in epididymal tissue (P < 0.001). Interestingly, all four CatSper sub-units were also expressed at low levels in the prostate. CatSper 1 was expressed at a lower level than CatSper 2, 3 and 4 in all tissues examined (P< 0.05). Nevertheless, immunoblotting studies demonstrated that CatSper-1 proteinwas present in stallion testicular tissue and spermatozoa. Having demonstrated expression of the genes for CatSper 1, 2, 3 and 4 in the testicles and prostate of stallions, and the presence of CatSper protein on mature stallion sperm, future studies will focus on the role of CatSper in the induction of hyperactivated motility and zona pellucida penetration.