Search

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion. • Incubation with 4-Br-A23187 for 20 min induces acrosome reaction in boar spermatozoa. • Glycosylated protein profile changes after acrosome reaction induction. • 50 proteins containing mannose residues showed regulation after acrosome reaction. • 6 transmembrane proteins containing mannose residues were regulated. [ABSTRACT FROM AUTHOR]

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10–24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17–22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium. • Motility parameters of rabbit sperm after prolonged incubation were better in BO medium compared to TALP and HTF. • Fertilization rates were significantly higher in BO medium compared to TALP or HTF. • Sperm pre-incubation for 6 h in BO did not improved fertilization rates compared to no pre-incubation. • Rabbit sperm purified by a density gradient fertilize efficiently COCs collected at 10–12 h after ovulation induction. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 μM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 μM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 μM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are abundant in body fluid and are critical in cell interaction. Seminal plasma contains numerous EVs which affecting sperm function via transferring regulatory cargoes to the sperm. However, the mechanism of seminal plasma extracellular vesicles (SP-EVs) is still not clear. The present study aimed to isolate the boar SP-EVs and explore its potential function, then identify the key protein involved in SP-EVs and sperms interaction, and elucidate mechanism of SP-EVs protein on sperms. Here, we successfully isolated and concentrated boar SP-EVs, the SP-EVs showed a typical vesicle structure under transmission electron microscopy, most of their diameters range between 50 and 200 nm and express EVs biomarkers CD9 and CD63. We proved that SP-EVs could inhibit sperm acrosome reaction and in vitro fertility. Through a data-independent acquisition analysis of protein profiles of noncapacitated sperms, normal capacitated sperms and SP-EVs treated capacitated sperms, we identified that EZRIN was one of the active proteins that participated in SP-EVs and sperms interaction. Furthermore, we tested that the inhibition of EZRIN could promote boar sperm fertility, which is in consistence with the function of SP-EVs. The results may facilitate future research of SP-EVs on sperm function and male infertility. • The boar seminal plasma extracellular vesicles could reduce sperm acrosome reaction and in vitro fertility. • EZRIN protein plays an important role in the interaction between SP-EVs and sperms. • EZRIN phosphorylation is inhibited, which promote the boar sperm acrosome reaction and in vitro fertility. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 μM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the "Computer-assisted Sperm Analysis" system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 μM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 μM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 μM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes. • Phospholipase C reduces the capacitation of cryopreserved ovine spermatozoa. • The U73122 did not affect the kinetics of spermatozoa. • The U73122 did not affect the number of spermatozoa with intact plasmatic membrane. • U73122 did not affect the number of spermatozoa with intact mitochondrial membrane. [ABSTRACT FROM AUTHOR]

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus -denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus -denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus -denuded oocytes in serum-free conditions. [Display omitted] • Exogenous P4 promoted acrosomal exocytosis in vitro capacitated bovine sperm. • Live cell incubation with a fluorescent lectin evidenced different acrosome status. • Patterns of acrosomal exocytosis revealed a heterogeneous sperm population. • Exogenous P4 improved developmental competence of partially cumulus -denuded oocytes. [ABSTRACT FROM AUTHOR]

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria. • Bacterial contamination in the semen deteriorates spermatozoa functions. • Bovine sperm incubated with the bacteria E. coli , S. aureus or P. aeruginosa , revealed sperm-bacteria interaction. • Only E. coli and P. aeruginosa caused an increase in sperm sAR. • Bacteria affects sperm during capacitation and not at the AR step. • PKA and PTP activities, two essential processes that promote capacitation, are inhibited by the bacteria. [ABSTRACT FROM AUTHOR]

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO 2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO 2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs. • FERT medium with heparin it improves sperm capacitation and promotes hypermotility. • Guinea pig may metabolize different types of energy for sperm capacitation. • Density gradient in guinea pig sperm selection supports sperm fertilizing ability. • Guinea pig sperm capacitated with different media penetrate zona-intact bovine oocytes. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2–4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 μM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 μM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 μM), and 100, 250 and 500 μM PRO, but decreased with 1000 μM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 μM: 77.8 ± 10.6%, 100 μM: 63.7 ± 11.7%, 250 μM: 52.1 ± 12.9%, 500 μM: 37.7 ± 11.6%, 1000 μM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 μM: 34.6%, 1000 μM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 μM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs. • Probucol is an ABCA1 specific antagonist and effective in dog spermatozoa membranes. • Increasing probucol decreased the percentage alive cells with cholesterol efflux. • Increasing probucol decreased head and entire tail phosphorylated cells. • Increasing probucol decreased the percentage capacitated cells. • The ABCA1 transporter is involved in sperm capacitation in dogs. [ABSTRACT FROM AUTHOR]

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls. • Frozen-thawed sperm undergo membrane and acrosome remodelation when incubated under capacitating conditions. • Sperm from low-fertility bulls had compromised ability to increase their membrane fluidity in capacitating conditions. • Hyperactivation is impaired in frozen-thawed sperm of low-fertility bulls. [ABSTRACT FROM AUTHOR]

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo , extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. I n vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies. • Oviduct secretions have significant effect on capacitation in stallion sperm. • Extracellular vesicles (EVs) were obtained by endometrial cells and oviductal spheroids. • Spermatozoa incorporate EVs from endometrial cells in 4 h in the head. • Spermatozoa incorporate EVs from oviductal spheroids in 1 h in head and middle tract. • After EV incorporation, spermatozoa are able to capacitate and produce zygotes. [ABSTRACT FROM AUTHOR]

To penetrate the zona pellucida before sperm-egg binding, sperm must undergo highly time-controlled capacitation and acrosome reaction in the female reproductive tract. Our previous study demonstrated that miR-21-5p is the most abundant miRNA in boar seminal plasma (SP)-derived extracellular vesicles (EVs) and can target Vinculin (VCL) gene, which may participate in boar sperm capacitation. Thus, this study aims to explore the potential role of miR-21-5p from SP-derived EVs in preventing sperm capacitation and its underlying mechanism. We observed that sperm could incorporate miR-21-5p from SP-derived EVs. The roles of SP-derived EVs miR-21-5p in sperm capacitation were then determined using gain- and loss-of-function analyses. In addition, the expression levels of miR-21-5p, VCL, and VCL protein in liquid-preserved boar sperm following transfection were determined using RT-qPCR and Western blotting. Our results revealed that miR-21-5p overexpression inhibited sperm capacitation and acrosome reaction. Similarly, miR-21-5p expression was significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. However, the protein level of VCL was also significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. Furthermore, immunofluorescence analysis showed that VCL protein mainly located in sperm head and sperm capacitation was inhibited after treating with VCL protein inhibitor (Chrysin). In conclusion, our study provides reasonable evidence that miR-21-5p expression in SP-derived EVs could prevent sperm capacitation via VCL inhibition. • Sperm could incorporate miR-21-5p from SP-derived EVs. • MiR-21-5p inhibited sperm capacitation and acrosome reaction. • MiR-21-5p was down regulated in boar sperm during sperm capacitation. • VCL protein was required for sperm capacitation. [ABSTRACT FROM AUTHOR]

In order to fertilize the egg, spermatozoa must undergo a series of biochemical processes in the female reproductive tract collectively called capacitation. Only capacitated sperm can interact with the egg resulting in the acrosome reaction (AR), allowing egg penetration and fertilization. Sperm can undergo spontaneous AR (sAR) before reaching the egg, preventing successful fertilization. Here we investigated the metabolic pathways involved in sperm capacitation and sAR. Inhibition of glycolysis or oxidative phosphorylation did not affect capacitation or sAR levels; however, when both systems were inhibited, no capacitation occurred, and there was a significant increase in sAR. Under such ATP-starvation, the increase in sAR is triggered by Ca2+ influx into the sperm via the CatSper cation channel. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation; there was no change in its activity when a single metabolic system was inhibited, while complete inhibition of was observed when the two systems were inhibited. Protein tyrosine phosphorylation (PTP), also known to occur in sperm capacitation, was partially reduced by inhibition of one metabolic system, and completely blocked when the two metabolic systems were inhibited. We conclude that ATP, PKA and PTP are involved in the mechanisms protecting sperm from sAR. • In order to penetrate the egg, sperm must undergo the acrosome reaction. • Sperm can undergo spontaneous acrosome (sAR) reaction before reaching the egg, which deteriorates successful fertilization. • Inhibition of glycolytic and mitochondrial activities enhanced sAR. • Inhibition of glycolytic or mitochondrial activities does not affect sAR levels. • Protein kinase A and protein tyrosine phosphorylation activities are essential to prevent sAR. [ABSTRACT FROM AUTHOR]

In order to penetrate the egg, spermatozoa must undergo the acrosome reaction in close proximity to the egg. This process can take place only after a series of biochemical changes in the sperm, collectively termed capacitation, occur in the female reproductive tract. Sperm cells can undergo spontaneous-acrosome reaction(sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect sperm from undergoing sAR, and all of them are involved in proper capacitation. Here, we describe the involvement of protein acetylation in the mechanism that protects bovine spermatozoa from sAR. Incubation of bovine sperm under non-capacitation conditions revealed a strong increase in sAR that was significantly reduced in the presence of deacetylase inhibitors. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation, and its inhibition results in high sAR. The reduction in sAR by hyperacetylation was independent of PKA activity. We previously demonstrated that calmodulin-kinase II (CaMKII) activity protects sperm from sAR, and here we show that its activity is essential for reduction in sAR by hyperacetylation. We further show that the 'exchange protein directly activated by Camp' (EPAC) mediates both protein lysine acetylation and the reduced rate of sAR caused by hyperacetylation. In conclusion, these results suggest a PKA-independent and EPAC-CaMKII dependent hyperacetylation mechanism that protects sperm from sAR. • Spontaneous acrosome reaction (sAR) decreases sperm fertilization ability. • Sperm contain several mechanisms to protect against sAR. • Protein hyperacetylation is one of these mechanisms. • PKA-independent and EPAC-CaMKII dependent hyperacetylation occur in bovine sperm. [ABSTRACT FROM AUTHOR]

Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and assess the quality and in vitro capacitation of stallion sperm after thawing. Semen samples from three stallions frozen with membrane stabilizers (cholesterol-loaded cyclodextrin and cholestanol-loaded cyclodextrin) were evaluated in two experiments: i) sperm quality and functional analysis after thawing, and ii) sperm quality and functional analysis after 4 h of post-thaw incubation in capacitating conditions. Plasma membrane integrity, mitochondrial membrane potential, membrane lipid disorder, intracellular Ca2+, tyrosine phosphorylation, acrosome reaction, DNA damage, sperm motility, and binding to the zona pellucida were assessed. The results showed that cholesterol-loaded cyclodextrin was the stabilizer that most efficiently reduced the membrane disruption and post-thaw cell damage. In addition, this stabilizer made it possible to obtain in vitro capacitated sperm showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, binding to the zona pellucida and better response to in vitro capacitating conditions. • Membrane stabilizers were evaluated in stallion semen. • CLC improved post-thaw sperm quality parameters. • Results encourage potential use in reproductive biotechnologies. [ABSTRACT FROM AUTHOR]

In this study, we defined the composition of the culture medium that yield a significant percentage of alive and functional equine spermatozoa during enough time before artificial insemination. The effects of sodium bicarbonate were analyzed in fresh and frozen semen in respect to sperm viability, capacitation, spontaneous acrosome reaction and several kinetic parameters such as total motility, progressive motility, VCL, VSL, ALH, BCF, LIN. Moreover, employing Bayk-6844 and Nifedipine, the involvement of L-type voltage-gated calcium channels in the modulation of intracellular calcium concentrations was investigated. Results evidenced an immediate effect of sodium bicarbonate in reducing fresh sperm viability and in increasing capacitation and spontaneous acrosome reaction of fresh and frozen spermatozoa. Furthermore, it affected total and progressive motility of fresh and frozen semen. Because of the sudden effects of the compound, we think that a buffer lacking sodium bicarbonate is more suitable to preserve the viability and the functional state of equine spermatozoa required to undergo at the right time those modifications necessary for fertilization. We also demonstrated that intracellular calcium modifications induced by Bayk-8644 and Nifedipine are not involved in signals related to vitality, capacitation, spontaneous acrosome reaction and motility. Workflow diagram of the experimental design. A representative scheme describes the protocol used during the breeding season on each of the six stallions enrolled in the study. Twice a month an ejaculated was recovered and divided in two aliquots (f and fz) according to the protocol described in the materials and methods section. Each sample was subdivided in two aliquots to test the effect of sodium bicarbonate (CB) in respect to the control (NCB) and successively analyzed for the effects of Bay k-8644 (Bayk) and Nifedipine (Nif) in respect to the control (C). For each test (to evaluate morphological and kinetic parameters of the semen) the average of the results coming from experiments on the two ejaculates were calculated. • First report analyzing the appropriate buffer composition to maintain equine sperm cell alive and functional until A.I. • Self-acrosome reaction (sAR) decreased reproductive potential of equine fresh or frozen semen. • Sodium bicarbonate added to culture medium decreased viability and functional activity of equine semen. [ABSTRACT FROM AUTHOR]

To understand the effects of urea on sperm functional attributes, fresh bull semen (n = 12) was subjected to four different concentrations (mg/mL) of urea to mimic the physiological (0.04 and 0.13), supraphysiological (0.43) concentrations and control (0 mg/mL). Sperm membrane integrity, kinematics, chromatin integrity, and mitochondrial membrane potential were assessed at different time points (before incubation, 0, 1, 2, and 4 h) of incubation. The concentration of urea in serum and seminal plasma was estimated and correlated with the ejaculate rejection rate and sperm functional attributes. The relative expression of urea transporter gene transcripts (UT-A and UT-B) was assessed in sperm and testis (control) using real-time PCR. The supraphysiological concentration of urea affected sperm kinematics, viability, functional membrane integrity, and acrosome integrity within 1 h of incubation (p < 0.05). Sperm head area decreased (p < 0.05) at 0 h and subsequently increased at 1 h of incubation in all media except supraphysiological (0.43 mg/dL) concentration of urea. Seminal plasma urea concentration showed a significant negative correlation with sperm motility, membrane integrity, and mitochondrial membrane potential (p < 0.05), but had a positive correlation with the ejaculate rejection rate (r = 0.69). Relative expression of the urea transporter genes revealed that UT-A was expressed only in the testis. In contrast, UT-B was expressed in both the testis and sperm, suggesting UT-B 's role in regulating urea transport in sperm. At a supraphysiological level, urea adversely affected sperm functional attributes, osmoadaptation and may affect fertility. • Supraphysiological level of urea affects sperm viability, kinematics, membrane integrity and acrosome reaction in 1 h. • Bovine sperm lose osmoadaptation ability after exposure to supraphysiological level of urea. • Seminal plasma urea showed a positive correlation with ejaculation rejection rate but negative relation with sperm function. • Of all the urea transporter (UT) genes, only UT-B transcript was expressed in the bovine sperm. [ABSTRACT FROM AUTHOR]

In the present study, we standardized an in vitro oviduct explants model for cattle and assessed the oviduct explants binding ability and phenotypic characteristics of spermatozoa obtained from breeding bulls with high- and low-sperm DNA fragmentation index (DFI%). Cryopreserved spermatozoa from Holstein Friesian crossbred breeding bulls (n = 45) with known field fertility were assessed for DFI% and were classified into either high DFI% or low DFI% category. Flow cytometry was used to assess sperm membrane integrity, acrosome reaction status, mitochondrial membrane potential and intracellular calcium concentrations. It was found that spermatozoa from bulls with low DFI% had significantly higher (P < 0.05) membrane integrity, acrosome intactness, and mitochondrial membrane potential. To assess the sperm oviduct binding ability, oviduct explants were prepared by incubating the oviduct cells overnight in TCM-199 medium at 38.5 °C under 5% CO 2. Different sperm concentrations and times of incubation were evaluated and found that 2 million spermatozoa and 1-h incubation yielded high binding index (BI). The BI was also significantly (P < 0.01) higher (>2 times) in the bulls with low-DFI% as compared to high DFI% bulls. The correlation between binding index and DFI% was negative and significant (r = −0.528; P < 0.05). Further, the binding index was positively correlated with conception rate (r = 0.703), intact sperm membrane (r = 0.631) and mitochondrial membrane potential (r = 0.609). It is inferred that sperm phenotypic characteristics and oviduct binding ability are impaired in breeding bulls with high sperm DFI%, which might be associated with low conception rates in these bulls. • Standardized a sperm-oviduct explants binding model for studying binding index. • Sperm-oviduct binding ability was significantly lower for sperm obtained from bulls with high sperm DFI %. • Sperm binding index was positively correlated with conception rates, but negatively corelated with DFI%. • Sperm phenotypic characteristics were significantly altered in bulls with high sperm DFI%. • Conception rates were significantly lower for bulls with high sperm DFI%. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the influence of spermatozoa surface antigenic proteins on the functional competence of bovine neat and frozen-thawed semen. The breeding bulls (n = 38) were screened for seminal antigenic levels in neat semen based on the agglutination titrations with anti-sperm antibody (ASA). Bulls having high (n = 8) and low (n = 7) antigenic levels were selected and spermatozoa functional parameters were analyzed in neat and frozen-thawed semen samples. In neat semen, kinematics such as straightness (73.6 ± 1.0 and 66.9 ± 1.5%), linearity (48.6 ± 1.2 and 40.1 ± 3.9%), curvilinear velocity (103.3 ± 2.6 and 93.4 ± 3.8 μm/s), straight-line velocity (65.7 ± 2.6 and 53.7 ± 2.2 μm/s) and average path velocity (53.8 ± 2.5 and 39.8 ± 2.3 μm/s) were significantly high (p < 0.05) in samples with lower antigenicity. The percentage of spermatozoa that can penetrate mucus (49.9 ± 2.3 and 37.1 ± 3.2) was significantly higher in semen samples with low ASAs. The total motile (84.0 ± 2.5 and 86.0 ± 1.5) and progressive motile (68.4 ± 3.7 and 69.2 ± 1.6) spermatozoa were higher in neat semen samples with higher antigenicity. A significantly (p < 0.05) higher mitochondrial membrane potential was observed in neat (82.5 ± 2.8 and 69.0 ± 2.0%) and post-thaw (28 ± 5. 6 and 16 ± 3.7%) samples of the lower antigenic group. The percentage of acrosome-reacted spermatozoa was significantly (p < 0.05) higher in neat (58.7 ± 2.9 and 52.6 ± 1.8), but reduced significantly (p < 0.05) in post-thaw (32.0 ± 2.0 and 48.0 ± 2.6) semen of higher antigenic groups. The study reveals that higher seminal antigenicity reduces mitochondrial membrane potential and acrosome reaction ability in post-thaw spermatozoa. • Higher levels of seminal antigen affect spermatozoa kinematics and mitochondrial membrane potential in neat semen. • Levels of seminal antigens did not influence spermatozoa kinematics in post-thaw semen samples. • Seminal antigenicity did not influence spermatozoa structural and functional membrane integrities during cryopreservation. • Higher levels of seminal antigens prevent acrosome reaction in post-thaw spermatozoa. • Antigenic levels in semen may affect the fertilization competence of frozen semen samples. [ABSTRACT FROM AUTHOR]

Efficient artificial insemination (AI) with liquid preserved boar semen is essential for the swine industry. Glucose is the most widely utilized energy source in refrigerated boar semen extenders. However, the relationship between glucose concentration and spermatozoa quality is not fully understood. In the present study, boar spermatozoa were used as a model to investigate the impact of different glucose concentrations on spermatozoa motility, mitochondrial activity, and acrosome integrity at physiological temperature (37 °C) and during refrigeration (17 °C). The proportion of progressively motile spermatozoa and mitochondrial activity in the high glucose group were significantly lower than in the low glucose group when incubated at 37 °C for 3 h or 17 °C for 3 d, but not at 17 °C for 7 d. Lysine acetylation is a reversible post-translational modification that plays a crucial role in spermatozoa function. Our results show that spermatozoa protein acetylation levels were higher in the high glucose group than in the low glucose group. The proportions of progressively motile and acrosome-intact spermatozoa were higher in acetyltransferase inhibitor (WM-1119)-treated spermatozoa than in the control. Spermatozoa acetyl-CoA concentration, which is directly linked to acetylation, was significantly higher in the high glucose group than in the low glucose group. Taken together, spermatozoa motility and acrosome integrity can be altered by changing the concentration of glucose in the extender. High glucose concentration-induced lysine acetylation participates in the regulation of boar spermatozoa motility and acrosome integrity during preservation. These results can provide insights into spermatozoa preservation and AI in the swine industry. • Boar sperm motility were lower in high glucose condition than low glucose condition. • High glucose concentration induced increase in protein lysine acetylation in sperm. • Inhibition of lysine acetylation promotes sperm motility and delays acrosome reaction. [ABSTRACT FROM AUTHOR]

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation. • Periovulatory bovine oviductal fluid modifies sperm functionality. • Oviductal fluid enhances sperm viability and ability to undergo capacitation and acrosome reaction. • In capacitating conditions, periovulatory fluid decreased sperm motion parameters. • Late follicular fluid provides higher DNA stability. [ABSTRACT FROM AUTHOR]

Spermatozoa acquire motility and fertilizing ability during their transit through the epididymis. A wide variety of proteins secreted into the epididymal lumen are added on to the sperm surface to allow morphological and molecular changes involved in sperm maturation. Proteins of the Sperm Associated Antigen 11 (SPAG11) family are known to be localized on the sperm surface. The rat SPAG11A protein was implicated in sperm maturation during epididymal transit in vitro. However, systematic analyses on the significance of SPAG11A in fertility and sperm function is not yet reported in vivo. In this study, using testicular electroporation, we generated transgenic rats that express shRNA to ablate endogenous Spag11a mRNA. Genotyping revealed the integration of the plasmid that expresses shRNA against Spag11a mRNA. Significant decrease in the mRNA levels of Spag11a and its encoded protein was observed in the caput epididymis of transgenic rats. We also generated an active immunization rat model to ablate endogenous SPAG11A protein by administering recombinant SPAG11A protein. Immunized rats had a high antibody titer in the serum and the tissue fluids of caput, cauda and testis. In both these model systems, the litter size and sperm count was significantly reduced. However, spermatozoa obtained from the transgenic or immunized rats underwent capacitation and acrosome reaction and the associated calcium release. Results of this study indicate the role of SPAG11A in fecundity and sperm production and not in sperm function, especially capacitation and acrosome reaction. • Role of SPAG11A in fertility not reported. • Transgenic rats that express shRNA targeting to ablate Spag11a mRNA generated. • Active immunization rat model to ablate SPAG11A protein generated. • Fecundity and sperm count decreased upon Spag11a mRNA or its protein ablation. • Capacitation and acrosome reaction not affected by SPAG11A ablation. [ABSTRACT FROM AUTHOR]

This work was designed to study whether HSP70-1A, HSP90α, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode's medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90α or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90α nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system. • Incubation of porcine oocytes in medium with HSP70-1A, HSP90α, ezrin or PDI4 proteins induces a mild hardening of ZP. • These proteins do not have a detrimental effect on sperm viability or acrosome reaction. • Final porcine IVF efficiency does not increase by adding these exogenous oviductal proteins to fertilization medium. [ABSTRACT FROM AUTHOR]

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm. • Dendrimer based-transfection method increased exogenous DNA binding. • Nanotransfection induced detrimental changes in kinetic and sperm quality parameters. • A total of 218 known and 106 novel miRNAs were identified in the bovine genome. • Dendrimer nanostructure alters bovine sperm miRNA profile. [ABSTRACT FROM AUTHOR]

Glutathione peroxidases (GPxs) are regarded as important protectors against oxidative stress. Some members of this protein family were reported to play key roles in protecting sperm against oxidative stress. Whether GPx6 a member of the GPx family also plays a role in protection against oxidative stress is not known to date. The objective of the present study was to evaluate the localization and function of glutathione peroxidase 6 (GPx6) in boar accessory sex glands, seminal plasma, and sperm, as well as the effect of GPx6 on vitality and capacitation in boar sperm. qPCR and Western blot analysis demonstrated the presence of GPx6 in testis, epididymis, bulbourethral glands, prostate, seminal vesicle, sperm and seminal plasma. Incubation of sperm with an GPx6 antibody had no significant effect on the viability of boar sperm prior to capacitation. Surprisingly, when capacitated sperm was incubated with the GPx6 antibody for 240 min, sperm vitality was significantly improved. Western blotting showed that in capacitated sperm without prior pretreatment, GPx6 protein content was reduced compared to sperm before capacitation. To further confirm a role for GPx6 in sperm capacitation, we tested sperm acrosome reaction by ACR.2 and FITC-PSA. The results showed that treatment of sperm with the GPx6 antibody significantly increased sperm capacitation and acrosome reaction. Furthermore, we examined the concentration of cAMP in sperm after capacitation. ELISA demonstrated that the cAMP concentration in the sperm exposed to the GPx6 antibody was significantly higher than that of the control group. In addition, the exposure of sperm to the GPx6 antibody significantly increased the concentration of H 2 O 2 , while the expression of SOD3 and CAT were decreased. Based on these observations we would like to postulate that in the boar reproductive tract the GPx6 protein becomes attached to the sperm head preventing the sperm to undergo premature capacitation by affecting components of the antioxidant pathway. How GPx6 expression following ejaculation becomes suppressed to allow sperm capacitation to take place needs further investigation. • Inhibition of GPx6 protein can increase sperm motility after capacitation. • Inhibition of GPx6 protein can promote acrosome reaction of sperm. • GPx6 protein may have the effect of preventing premature capacitation of sperm. [ABSTRACT FROM AUTHOR]

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 μg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 μM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5β1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 μM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 μM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5β1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans. • Fibronectin through α5β1 integrin induces sperm capacitation in bull spermatozoa. • TRPV1 receptor is involved in fibronectin signaling pathway. • PKA phosphorylated substrates and nitric oxide levels are increased during fibronectin-induced sperm capacitation. [ABSTRACT FROM AUTHOR]

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR. • Sperm should reside in the female reproductive tract to undergo biochemical processes collectively called capacitation. • Capacitated sperm can undergo the acrosome reaction, an essential process allowed penetration into the egg and fertilization. • Abrogation of actin polymerization lead to spontaneous acrosome reaction resulting in low fertilization rate.. • Sperm contain at least two pathways, PLD and CaMKII, that prevents sAR. • Ezrin mediates PKA/CaMKII-dependent mechanism that is involved in actin polymerization and protects the sperm from sAR. [ABSTRACT FROM AUTHOR]

The nerve growth factor (NGF), a member of the neurotrophins family, plays an important role in the nervous system but also in the reproductive apparatus and in sperm traits. The aim of this review is to summarize the effect of NGF on sperm functions of mammals. NGF was detected in seminal plasma of many animal species and its receptors, TrkA and p75NTR, are present in both epididymal and ejaculated sperm. The NGF, either endogenous or exogenous, may affects the kinetics and other sperm traits (e.g. capacitation, apoptosis, necrosis and acrosome reaction) and these effects appear modulated by the different receptors involved (TrkA or p75NTR). Although the animal species can affect the localization of receptors, TrkA seems mainly to be confined in the head and p75NTR in the midpiece and tail. Considering that some seminal disorders and sperm traits have been correlated with a lower NGF concentration, this review provides recent insights on the role of NGF on sperm cells and the possible mechanism implicated. • This review provides insights on the role of NGF on sperm and the possible mechanism implicated. • NGF and its receptors TrkA and p75NTR, are widely detected in seminal plasma of many animal species. • NGF affects the kinetics and other sperm traits, which appear modulated by the receptors involved. • TrkA seems mainly confined in the head and p75NTR in the midpiece and tail. [ABSTRACT FROM AUTHOR]

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = −0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility. • Global tyrosine phosphorylation status was assessed in cryopreserved spermatozoa from bulls with varying field fertility. • The proportion of tyrosine phosphorylated spermatozoa was significantly high in below-average fertile bulls. • Tyrosine phosphorylation at acrosome and post-acrosomal area was negatively related to bull fertility. • The proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility [ABSTRACT FROM AUTHOR]

We previously reported that the administration of d -aspartate (D-Asp) in drinking water over a 2-4-week period to 7-week-old mice resulted in higher sperm quality and increased in vitro fertilisation (IVF) rates associated with a systemic increase of luteinizing hormone and testosterone levels in the serum. The goal of this study was to investigate the effects of in vitro treatment with D-Asp on the IVF rate, embryo transfer, and sperm parameters of cryopreserved-thawed C57BL/6NTacCnrm (B6N) spermatozoa derived from young and adult mice. In this study, cryopreserved-thawed B6N spermatozoa from males aging 9, 11, 13, and 16 weeks were treated for 1 h with 4 mM D-Asp during capacitation. Thereafter, the in vitro fertilisation ability and the embryo transfer efficiency were analysed. Also, the kinetic activity of the treated spermatozoa and the acrosome reaction were measured after 1 h, 2 h, and 5 h of incubation. The capacitation rate of spermatozoa was determined after 1 h of pre-incubation. Spermatozoa from 9- and 11-week-old mice, which were treated with D-Asp, led to significantly increased IVF rates. However, spermatozoa derived from 13- and 16-week-old mice did not lead to a significant improvement in the fertilisation rate. At all ages examined, no differences were observed in the birth rate and sperm kinetic parameters. After 1 h incubation under the same conditions as the IVF was performed, the capacitation rate and the acrosome reaction were significantly higher with the D-Asp-treated spermatozoa from 9-week-old (67.5% vs. 41% and 14.5% vs. 10.5%, respectively) and 11-week-old mice (78.5% vs. 41.1% and 21.0% vs. 3.8%, respectively), corresponding to the improved IVF results. Therefore, the present results demonstrate, for the first time, a direct role of D-Asp in the capacitation process and acrosome reaction. • Spermatozoa treated with d -Aspartate in vitro increases IVF rates. • The influence of d -Aspartate is specific for young animals. • Spermatozoa treated with d -Aspartate show a higher capacitation. • d -Aspartate treatment increase acrosome reaction. • d -Aspartate treatment does not negatively affect embryo development. [ABSTRACT FROM AUTHOR]

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (1H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa. • The metabolome of pre- and post-ovulatory equine oviductal fluid was analyzed. • Differences existed in the metabolome of equine pre- and post-ovulatory fluid. • Sperm capacitation was achieved following incubation with oviductal fluid. • Equine OF induced protein tyrosine phosphorylation in stallion spermatozoa. • OF did not induce hyperactivated motility or acrosome reaction. [ABSTRACT FROM AUTHOR]

During freezing and thawing procedures, sperm are exposed to chemical and/or physical stressors that may cause adverse and harmful changes to sperm membranes. Accurate evaluation of the structural and functional integrity of fresh as well as cryopreserved sperm is highly important in predicting sperm fertilization capacity and success of artificial insemination (AI). The herbicide atrazine (ATZ) and its major metabolite, diaminochlorotriazine (DACT) are considered a ubiquitous environmental contaminants and endocrine disruptors, which deleteriously effect sperm function. Taking into consideration possible damage caused by environmental contaminants to sperm membranes, additive effects during cryopreservation cannot be ruled out. The aim of the current study was to evaluate the effect of ATZ (0.1 or 1 μM) and DACT (1 or 10 μM) exposure during or after cryopreservation on bovine sperm cryotolerance. Sperm membrane integrity and functionality were evaluated using fluorimetric probes: (1) double-stranded DNA was examined by 4′,6-diamidino-2-phenylindole; (2) plasma membrane integrity was examined by propidium iodide; (3) acrosome reaction (AR) was examined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; mitochondrial membrane potential (ΔΨm) was examined by 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe. The findings demonstrate, that exposure of sperm to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during cryopreservation increased the proportion of dead sperm relative to the control (P < 0.09); exposure to DACT (1 or 10 μM) increased ΔΨm (P < 0.03). Neither ATZ nor DACT affected spontaneous AR. In contrast, the proportion of sperm with Ca++ ionophore-induced AR was lower after exposure to 1 μM DACT (P < 0.05). Following freezing and thawing procedures, exposing sperm to 1 μM ATZ increased the proportion of dead sperm relative to the control (P < 0.05), but had no significant effect on sperm ΔΨm or AR. In conclusion, exposing sperm to endocrine-disrupting chemicals such as ATZ or DACT during cryopreservation reduces sperm cryotolerance and resistance post-thawing. • ATZ/DACT and cryopreservation have an additive harmful effect on sperm membranes. • Exposure to DACT during cryopreservation reduced Ca++ ionophore-induced AR. • Exposure of frozen-thawed sperm to ATZ/DACT has no effect on AR and ΔΨm. [ABSTRACT FROM AUTHOR]

Energy balance is an important feature for spermatozoa functions. The 5′-AMP-activated protein kinase (AMPK), a sensor of cell energy, has been implicated as a mediator between spermatozoa functions and energy balance. We recently identified and localized the AMPK protein in chicken spermatozoa and showed that its activation with non-specific activators significantly modified spermatozoa quality. The aim of the present study was to determine more directly the role of AMPK activation induced by the specific activator A-769662 and the interaction between AMPK and another pathway, the protein kinase A (PKA) signaling pathway in bird spermatozoa. The results showed that A-769662 induced at the low dose 50 μM an increase in spermatozoa motility, viability, and acrosome reaction through AMPK activation. Furthermore, phospho-Thr172-AMPK levels were greatly decreased by the PKA inhibitor H89 that also decreased spermatozoa quality. The inhibitory action of H89 was also efficient on A-769662 AMPK phosphorylation. We conclude that AMPK activity in bird spermatozoa is stimulated by low dose of A-769662 with consequent increase in spermatozoa quality, and that AMPK is upstream regulated by PKA pathway in this model. • This manuscript is the first demonstration that AMPK is activated by A-769662 and that AMPK and PKA interact in chicken sperm. • A-769662 induced at the low dose 50µM an increase in chicken sperm motility, viability and acrosome reaction. • AMPK phosphorylation is activated rapidly at low dose 50µM of A-769662 and is regulated upstream by PKA in chicken sperm. [ABSTRACT FROM AUTHOR]

Prior to interaction with the oocyte, spermatozoa must undergo capacitation, which involves a series of physio-chemical transformations that occur in the female tract. As capacitation is a pre-requisite for successful fertilisation, it is a topic of great interest for sperm biologists, but the complexity of the numerous biochemical and biophysical processes involved make it difficult to measure. Capacitation is an extremely complex event that encompasses numerous integrated processes that can occur concurrently during this window of time. The identification of techniques to accurately assess and quantify capacitation is therefore crucial to gain a meaningful insight into this fascinating sperm maturation event. Whilst there are extensive reviews in the literature that focus on the functional changes to spermatozoa during capacitation, few have examined the methods required to measure these changes. The aim of this review is to highlight frequently used methods to quantify different stages of capacitation and identify promising novel techniques. Factors that are able to modulate various capacitation processes will also be discussed. The overall outcome is to provide researchers with a toolbox of methods that can be used to gain a deeper understanding of the intricacies of capacitation in spermatozoa. [ABSTRACT FROM AUTHOR]

Acid extrusion and intracellular alkalisation are the key events during sperm capacitation and these are mediated through proton gated channels (Hv1). Role of Hv1 in regulating sperm motility, capacitation and acrosome reaction has been documented in human spermatozoa; but no such data is available in bull spermatozoa; therefore, the present study was undertaken in Hariana bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls to investigate the functional involvement of Hv1 in regulation of sperm motility, capacitation and acrosome reaction in bull spermatozoa. Immunoblotting revealed the presence of a single band of 31.8 kDa corresponding to Hv1 in Hariana bull spermatozoa and immunoflourescence confirmed the positive immune-reactivity at principal piece of spermatozoa for Hv1. Functional study was carried out using 200 μM 2-Guanidinobenzimidazole (2-Guanidinobenzimidazole,selective Hv1 blocker) and 1 mM zinc chloride (potent Hv1 blocker), and 0.3 μM Anandamide (AEA), an activator of Hv1. Blocking of Hv1 resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM). Activation of Hv1 with AEA also resulted in significant (P < 0.05) reduction in PSM till 1 h and thereafter, the PSM was restored and reduction was almost similar to that in control. However, during activation and inactivation of Hv1, per cent spermatozoa showing hyperactive motility were found to be markedly increased (20–30%) compared to 10–20% in control. 2-Guanidinobenzimidazole, zinc and AEA treated spermatozoa revealed significant (P < 0.05) increase in B-pattern of spermatozoa indicating induction of capacitation. Downstream signalling of Hv1 activation or inactivation seemed to be mediated through cAMP and PKA pathways. Catsper channels were found to be intimately associated with Hv1 function in regulating sperm motility. Blocking as well as activation of Hv1 resulted in significant (P < 0.05) reduction in sperm livability, spermatozoa having intact membrane, intact acrosome, and high mitochondrial transmembrane potential (MTP). Our findings evidently suggest that Hv1 channels are present in bull spermatozoa and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through complex interplay between different pathways involving cAMP, PKC, and Catsper. Further studies are required to find out the possible relationship between Hv1 channels and other channels in regulating spermatozoa functions. • First study to report the functional role of Hv1 in regulating sperm functions. • Hv1 mediates sperm capacitation and acrosome reaction through soluble adenyl cyclase (sAC) and PKA pathway. • Modulation of Hv1 results in sperm capacitation and acrosome reaction. • Functions of Hv1 are dependent on Catsper channel. [ABSTRACT FROM AUTHOR]

In order to fertilize the egg, the spermatozoon should undergo a process called acrosomal exocytosis or acrosome reaction (AR), a process which can take place only after a series of biochemical changes collectively called capacitation occur in the female reproductive tract. We present here for the first time the involvement of protein-kinase A (PKA) and phosphatidyl-inositol-3-kinase (PI3K) in protecting sperm from undergoing spontaneous AR (sAR) which decreases fertilization rate. Previously we showed that Calmodulin-kinase II (CaMKII) and phospholipase-D (PLD) prevent the occurrence of sAR in two distinct pathways by enhancing actin polymerization. Here we show that PKA mediates PLD activation, and inhibition of PKA resulting in an increase of sAR and a decrease of F-actin levels, two functions which can be recovered by adding phosphatidic acid (PA), the product of PLD activity. PKA is known to induce CaMKII activation. Inhibition of CaMKII, also enhanced sAR and reduced F-actin levels which can be recovered by adding PA. Inhibition of the PLD pathway resulted in an increase in sAR and a decrease in F-actin levels which can be artificially reversed by inhibition of protein - phosphatase 1 (PP1), and this effect is mediated by PI3K. All together, we showed that PKA via PLD and PI3K activation protects the sperm from undergoing sAR by enhancing actin polymerization. • Prevention of spontaneous acrosome reaction (sAR). • PKA, PLD, CaMKII and PI3K are involved in the mechanism that protect sperm from undergoing sAR. • PLD activation is mediated by PKA activity. [ABSTRACT FROM AUTHOR]

In view of the limited information available on functional significance of TRPV1 in regulating sperm functions, present study was undertaken on bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls and were used for molecular and functional characterisation of TRPV1. Immunoblotting using TRPV1 specific antibody revealed the presence of a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Indirect immuno fluorescence revealed positive immune-reactivity to TRPV1 at acrosomal, pre-acrosomal, post acrosomal and flagellar regions of spermatozoa. Based on the results of pilot study dose-response analysis, doses of anandamide (AEA; 0.3 μM) and capsazepine (Cp; 10 μM) were selected for further studies. Three groups of semen samples (control 100 μL diluted semen having 1 × 106 spermatozoa; anandamide (3 μL AEA+97 μL of diluted semen containing 1 × 106 spermatozoa and Cp (1 μL Cp+99 μL of diluted semen containing 1 × 106 spermatozoa) were used to study the functional involvement of TRPV1 in bull spermatozoa. Blocking of TRPV1 with Cp resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM) as compared to control. With activation of TRPV1 using AEA also, PSM was significantly (P < 0.05) decreased till 1h and thereafter the PSM was sustained to the level as observed in control. However, both during blocking and activation of TRPV1, per cent spermatozoa showing hyperactive motility were significantly (P < 0.05) increased (20–30%) compared to the control. Treatment with both Cp and AEA revealed significant (P < 0.05) increase in B-pattern of spermatozoa in chlortetracycline hydrochloride (CTC) staining indicating induction of capacitation. Inhibition of soluble adenyl cyclase (sAC) with 99 nM KH7and protein kinase A (PKA) with 3 μM P9115 significantly (P < 0.05) decreased PSM both in the presence of Cp and AEA. Blocking as well as activation of TRPV1 showed significant (P < 0.05) reduction in sperm livability, intact membrane, intact acrosome, high mitochondrial transmembrane potential; hence indicating the involvement of TRPV1 in regulation of sperm functions in bulls. From the study-it was concluded that TRPV1 channels are found in bull spermatozoa and mediate number of sperm functions like motility, hypermotility, capacitation and acrosome reaction. Further studies are required to find out the possible relationship between TRPV1 channels and other channels in regulating spermatozoa function and possible mechanisms associated with TRPV1 activation as well as its role in sperm function regulation. • Functional significance of TRPV1 in bull spermatozoa. • TRPV1 mediates sperm capacitation through soluble adenyl cyclase (sAC) and PKA pathway. • Both activation and inactivation of TRPV1 mediates sperm capacitation. [ABSTRACT FROM AUTHOR]

Abstract This study was conducted to investigate the complex interactions between oviducts and cryopreserved spermatozoa. Herein we report the dynamic changes in bull sperm functions during in vitro incubation with bovine estrus and luteal oviductal fluid. Frozen-thawed bull spermatozoa was incubated either in non-capacitating medium, capacitating medium, non-capacitating medium containing 20% v/v estrus oviductal fluid or non-capacitating medium containing 20% v/v luteal oviductal fluid for 6 h at 38 °C under 5% CO 2. At hourly interval spermatozoa were evaluated for kinematics, tyrosine phosphorylation and acrosome reaction. The sperm velocity parameters were higher (P < 0.05) in capacitating medium compared to the other treatments. At 4 and 5 h of incubation, the proportion of live tyrosine phosphorylated spermatozoa was higher (P < 0.05) in estrus oviductal fluid compared to all other treatments. From 4 to 6 h of incubation the proportion of live acrosome reacted spermatozoa was higher (P < 0.05) in estrus oviductal fluid compared to the other treatments. We conclude that estrus oviductal fluid induced tyrosine phosphorylation and acrosome reaction in a higher proportion of frozen-thawed bull spermatozoa compared to luteal oviductal fluid, although sperm kinematics were not significantly influenced by oviductal during incubation. Highlights • Estrus oviductal fluid induced more tyrosine phosphorylation and acrosome reaction in bull spermatozoa compared to luteal oviductal fluid. • Capacitating medium induced an increase in velocity; while sperm velocity were not significantly influenced by oviductal fluid. • Four tyrosine phosphorylated proteins were found in frozen-thawed bull spermatozoa. [ABSTRACT FROM AUTHOR]

It was hypothesized here that sperm-bound antisperm antibodies (ASAs) impair the ability of bovine spermatozoa to undergo capacitation, bind to the zona pellucida, and complete the acrosome reaction. The effect of ASA binding on these functions was evaluated in frozen/thawed spermatozoa from four bulls before and after induction of ASAs. Ejaculates were divided into ASA negative (<10% immunoglobulin [Ig]G- and IgA-bound spermatozoa) or ASA positive (≥20% IgG and/or IgA-bound spermatozoa). The percentage of capacitated (Merocyanine 540 positive) live spermatozoa in response to heparin was lower in ASA-positive than ASA-negative ejaculates (P < 0.0001). Treatment with heparin resulted in a higher percentage of capacitated spermatozoa compared with control treatments in ASA-negative but not ASA-positive ejaculates. The percentage of capacitated spermatozoa after heparin treatment was negatively correlated with IgA (P = 0.02, R 2 = −0.48) but not IgG binding. Sperm binding to the zona pellucida was lower in IgA-positive (six spermatozoa/oocyte; 3–10 spermatozoa/oocyte) than IgA-negative ejaculates (seven spermatozoa/oocyte; 4–13 spermatozoa/oocyte) (P = 0.019). Zona binding was negatively correlated with the percentage of IgA-bound spermatozoa (P = 0.04; R 2 = −0.24) but not IgG-bound spermatozoa. The percentage of acrosome-reacted spermatozoa was higher in calcium ionophore A23187-treated than control aliquots in both ASA-negative and ASA-positive ejaculates (P < 0.0001). However, the percentage of acrosome-reacted spermatozoa did not differ between ASA-positive and ASA-negative samples, and no correlation was identified with IgG or IgA binding. It was concluded that sperm-bound IgA affected the ability of bovine spermatozoa to undergo capacitation. ASAs inhibited the changes in plasma membrane fluidity associated with capacitation and binding of spermatozoa to the zona pellucida. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0, and 9.0 for 4 hours at 38 °C, 5% CO 2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group); viability with mitochondrial activity, motility, and acrosome integrity were measured. In addition, a homologous binding assay was carried out: stallion spermatozoa were capacitated 1 hour at 38 °C, 5% CO 2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group). Oocytes were then added to sperm suspensions and coincubated for 1 hour. Our results indicate that AP at pH 9.0 significantly increases the percentage of living cells with active mitochondria, whereas it significantly reduces the percentage of acrosome-damaged cells at pH 8.0. No significant differences were registered in motility parameters. The homologous binding assay showed a strong effect of AP, that increased the number of sperm bound to the oocyte's zona pellucida at all pHs tested. In conclusion, AP can induce some modifications on sperm membranes thus enhancing their capacity to bind to the zona pellucida of equine oocytes. [ABSTRACT FROM AUTHOR]

Parthenogenesis, embryonic development without fertilization, resembles very early embryonic mortality in fertilized eggs. Also, parthenogenesis alters egg albumen characteristics in virgin Chinese Painted quail hens genetically selected for parthenogenesis (PV). When these PV hens are mated (PM), hatchability is reduced versus control mated (CM) hens that were not genetically selected for parthenogenesis. However, it is unclear if parthenogenesis, which occurs in PM hens, reduces hatchability due to infertility and altered albumen characteristics. Sperm–egg penetration (SEP) holes are indicative of true fertilization and may be useful in identifying if eggs from PM hens exhibit a decrease in fertility versus CM hens. Therefore, the objectives of this study were to determine if parthenogenesis in PM hens (1) decreases SEP, (2) alters albumen characteristics similar to parthenogenesis in eggs from PV hens, and (3) yields albumen characteristics similar to fertilized eggs containing early mortality. Daily, PV and PM eggs were collected, labeled, and incubated for 10 days, then broken out to determine the incidence of parthenogenesis and albumen characteristics. Also daily, fresh PM and CM quail eggs were macroscopically examined to determine if an egg was infertile with no embryonic development, parthenogenetic, or fertile. Each of these eggs was then microscopically examined for SEP. For both PV and PM incubated eggs, parthenogenesis decreased albumen pH, O 2 , and protein concentrations yet increased Ca 2+ and CO 2 concentrations versus eggs with no development. For incubated PM eggs, albumen pH and O 2 were lower, yet CO 2 was higher for eggs containing parthenogens or early dead embryos versus infertile eggs. For SEP, fresh eggs classified as infertile or parthenogenetic from PM and CM hens had similar SEP holes but only one sixth as many SEP holes as eggs classified as fertilized. Eggs from CM hens had 3.5 times as many SEP holes as PM eggs. In conclusion, parthenogenesis that occurs in mated quail hens inhibits fertility and alters albumen characteristics similarly to parthenogenesis in unfertilized eggs and early embryonic mortality in fertilized eggs. [ABSTRACT FROM AUTHOR]