Preovulatory follicular fluid secretome added to in vitro maturation medium influences the metabolism of equine cumulus-oocyte complexes.

BACKGROUND: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed. RESULTS: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs. CONCLUSIONS: The addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA).

The CASR antagonist NPS2143 induces loss of acrosomal integrity and proteolysis of SPACA1 in boar spermatozoa.

In brief: The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1. Abstract: Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35-45 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35-45 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35-45 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30-32 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35-45 kDa) resulting in a SPACA1 rise at 32 kDa (p32).

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa.

RESEARCH QUESTION: Does sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process? DESIGN: Human spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge. RESULTS: SIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca2+ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237. CONCLUSION: YK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.

Influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction.

We aimed to analyze the influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction. When spermatozoa were incubated at 100 or 200 mill/ml, significant increases in protein tyrosine phosphorylation in the p32 protein were observed, compared to those at 50 mill/ml. In addition, sperm concentration-dependent increases were observed in plasma membrane lipid disorganization (50 mill/ml vs. 200 mill/ml), induction of the acrosome reaction (50 mill/ml vs. 100 mill/ml and 200 mill/ml), and sperm viability (50 mill/ml vs. 100 mill/ml and 200 mill/ml). Our data indicate that an increase in sperm concentration stimulates the induction of capacitation and acrosome reaction in boars.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8-12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

N-acetylcysteine addition after vitrification improves oocyte mitochondrial polarization status and the quality of embryos derived from vitrified murine oocytes.

BACKGROUND: Vitrification is the safest method to cryopreserve oocytes, however the process alters mitochondrial function resulting from increased reactive oxygen species (ROS) production. Our aim was to alleviate ROS stress in vitrified mice oocytes using N-acetylcysteine (NAC at 1 mM), to improve the oocyte's developmental competence. RESULTS: Hence, four experimental groups were compared: fresh oocytes (F-C), vitrified oocytes (V-C), NAC addition prior to oocyte vitrification (V-NAC-Pre) and NAC addition after vitrification (V-NAC-Post). V-NAC-Pre and V-NAC-Post exhibited higher levels of mitochondrial polarization compared to vitrified oocytes (36.5 ± 3.1, 37.7 ± 1.3 and 27.2 ± 2.4 measured as the spatial coefficient of variation/oocyte respectively, mean ± SEM; p < 0.05). However, ROS production increased in vitrified oocytes added with NAC compared to the vitrified control (1124.7 ± 102.1 [V-NAC-Pre] and 1063.2 ± 82.1 [V-NAC-Post] vs. 794.6 ± 164.9 [V-C]; arbitrary fluorescence units/oocyte, mean ± SEM; p < 0.05). ATP significantly decreased in V-NAC-Pre compared to V-NAC-Post oocytes (18.5 ± 6.9 vs. 54.2 ± 4.6 fmol/oocyte respectively, mean ± SEM; p < 0.05), and no differences were observed between V-NAC-Post, F-C and V-C groups. Blastocyst rates derived from F-C oocytes was higher than those derived from V-NAC-Pre (90.7 ± 1.8 vs. 79.1 ± 1.8, respectively, mean % ± SEM,; p < 0.05) but similar to those derived from V-NAC-Post (90.7 ± 1.8, mean % ± SEM, p > 0.05). Total blastomere count of blastocysts derived from V-NAC-Post after in vitro fertilization (IVF) was higher than embryos produced from V-C. CONCLUSIONS: The addition of NAC after vitrification improves the quality of vitrified mature murine oocytes while its addition prior to vitrification is advised against.

Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions.

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 µM CZ, 200 µM W-7, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

Extracellular vesicles derived from endometrial human mesenchymal stem cells improve IVF outcome in an aged murine model.

Advanced age reduces the success of in vitro fertilization (IVF) being this effect partly mediated by an overproduction of reactive oxygen species (ROS) that trigger apoptosis. It has been demonstrated that extracellular vesicles derived from endometrial mesenchymal stem cells (EV-endMSCs) exert an antioxidant effect and can be used as IVF coadjutants. In this work, endMSCs were isolated from human menstrual blood (n = 4) and characterized according to multipotentiality and surface marker expression prior EV-endMSCs isolation. Oocytes were obtained from 21 B6D2 mice (24 weeks) and coincubated with sperm from young males (8-12 weeks). Presumptive zygotes were incubated in the presence of 0, 10, 20, 40 or 80 µg/ml of EV-endMSCs in KSOM medium. Blastocyst yield was evaluated, and 25 blastocysts per group were used for qPCR. Blastocyst rate was 29.4% in control; 45.2% for 10 µg/ml, 62.9% for 20 µg/ml, 55.5% for 40 µg/ml and 53.8% in the 80 µg/ml (n = 124-130 oocytes) being all the increases significantly different when compared against control (p < 0.05). The 20-80 µg/ml treatments decreased the expression of glutathione peroxidase (Gpx1), and the 10-40 µg/ml treatments reduced the expression of superoxide dismutase (Sod1; p < 0.05) compared to control; Bax mRNA expression did not vary. Our results suggest that the increased developmental competence of the embryos could be partly mediated by the EV-endMSCs' ROS scavenger activity.

AMPK Function in Mammalian Spermatozoa.

AMP-activated protein kinase AMPK regulates cellular energy by controlling metabolism through the inhibition of anabolic pathways and the simultaneous stimulation of catabolic pathways. Given its central regulator role in cell metabolism, AMPK activity and its regulation have been the focus of relevant investigations, although only a few studies have focused on the AMPK function in the control of spermatozoa's ability to fertilize. This review summarizes the known cellular roles of AMPK that have been identified in mammalian spermatozoa. The involvement of AMPK activity is described in terms of the main physiological functions of mature spermatozoa, particularly in the regulation of suitable sperm motility adapted to the fluctuating extracellular medium, maintenance of the integrity of sperm membranes, and the mitochondrial membrane potential. In addition, the intracellular signaling pathways leading to AMPK activation in mammalian spermatozoa are reviewed. We also discuss the role of AMPK in assisted reproduction techniques, particularly during semen cryopreservation and preservation (at 17 °C). Finally, we reinforce the idea of AMPK as a key signaling kinase in spermatozoa that acts as an essential linker/bridge between metabolism energy and sperm's ability to fertilize.

Extracellular calcium regulates protein tyrosine phosphorylation through calcium-sensing receptor (CaSR) in stallion sperm.

Protein tyrosine phosphorylation (PY), a hallmark of sperm capacitation, is inhibited by extracellular calcium in stallion sperm. The objective of this study was to determine the presence and influence of the calcium-sensing receptor (CaSR) in this phenomenon. First, the presence of the CaSR was demonstrated in stallion sperm. We then tested its function in these gametes using its inhibitor NPS2143 or its agonist AC34356. Sperm were capacitated for 4 hr in modified Whitten's medium with 25 mM bicarbonate plus NPS2143 and 2.4 mM calcium or AC34356 alone, followed by analysis of PY. Inhibition of CaSR with NPS2143 prevented the calcium-dependent PY inhibition in a dose-dependent manner (5, 10, and 15 µM) whereas AC34356 (100 µM) inhibited PY similarly to calcium. Stallion sperm motility and viability significantly decreased in presence of 15 µM of NPS2143 whereas only sperm motility decreased with 100 µM of AC34356. CaSR function was also studied in the complete absence of calcium by including 2 mM ethylene glycol tetraacetic acid (EGTA); under these conditions, AC34356 again inhibited PY, but this time induced a significant increase in sperm motility. Inhibition of calmodulin by W-7 did not recover the AC34356-mediated PY inhibition. When stallion sperm were incubated under capacitating conditions (calcium, bicarbonate, plus bovine serum albumin) at elevated pH (7.9 or 8.5) AC34356 did not block PY. These results thus elucidate the effect of extracellular conditions on the regulation of CaSR, and point to its modulatory role on stallion sperm PY, motility, and viability. Mol. Reprod. Dev. 83: 236-245, 2016. © 2016 Wiley Periodicals, Inc.

Regulation of axonemal motility in demembranated equine sperm.

Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm.

Processing of boar spermatozoa with phosphate-buffered saline at 4˚C induces an increase in 32 kDa tyrosine-phosphorylated protein (p32).

We aimed to investigate the impact of processing boar spermatozoa with phosphate-buffered saline (PBS) at 4 ˚C on acrosomal integrity and increase in 32 kDa tyrosine-phosphorylated protein (p32). Following cooled PBS washing, we observed a significant increase in p32 levels and in the proportion of dead spermatozoa with compromised acrosomal integrity compared to sperm washing using PBS at room temperature. Interestingly, this increase in p32 was effectively inhibited when cooled PBS was supplemented with 1 mM AEBSF, a serine protease inhibitor. Our findings suggest that the increase of p32 in response to cooled PBS washing in boar spermatozoa is associated with enhanced protease activity in dead spermatozoa.

Induction of acrosome reaction by 4-Br-A23187 alters the glycoproteomic profile of boar spermatozoa.

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.

The compound YK 3-237 promotes pig sperm capacitation-related events.

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal's sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel.

Description of a new quantitative method to assess mitochondrial distribution pattern in mature equine oocytes.

The Mitochondrial distribution pattern or MDP in mammalian oocytes serves as an indicator of their cytoplasmic maturity, with a heterogeneous pattern associated with mature cytoplasm. Currently, MDP assessment involves fluorescent labelling of mitochondria followed by visual evaluation, as no quantitative method exists. Our objective was to develop a quantitative approach to assess MDP in mature equine oocytes. Equine oocytes, obtained by ovum pick up (OPU) were matured in vitro, and only metaphase II oocytes were used in the study (n = 56). Following denudation, oocytes were fixed, stained with MitoTracker™ Red CMXRos (50 nM in TCM-199 with Hank´s salts and 10% FBS) for 15 min at 38 °C, and then incubated with 2.5 µg/ml Hoechst 33342 for 10 min at 38 °C. Confocal microscope images were acquired, and the oocyte's MDP was visually classified as either homogeneous (HoD; n = 17) or heterogeneous (HeD; n = 39). For quantitative analysis, Fiji-ImageJ software was employed. Background subtraction was performed, and a 1-pixel line along the diameter was drawn to calculate the intensity profile. Fluorescence intensities were normalized, and ratios of peripheral to central fluorescence intensity were determined. Student´s t-test was used for comparations; MDP ratio was (mean ± standard error of the mean): 0.8 ± 0.02 for HoD and 0.3 ± 0.02 for HeD (p < 0.001). These results demonstrate concordance between quantitative and qualitative MDP assessment in mature equine oocytes. Our study describes a new approach to quantify mitochondrial distribution pattern and cytoplasmic maturation in mature equine oocytes.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.

CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.

In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx.