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.

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.

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.

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.

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.

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.

Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro.

In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), ß-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14+/PI-), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA+/PI-) and reactive oxygen species (ROS) production (CellRox+/PI-), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.

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.

The Proteome of Equine Oviductal Fluid Varies Before and After Ovulation: A Comparative Study.

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at -80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm-oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

Impaired mammalian sperm function and lower phosphorylation signaling caused by the herbicide Roundup® Ultra Plus are due to its surfactant component.

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3ß phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3ß phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3ß and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

Extracellular Vesicles, the Road toward the Improvement of ART Outcomes.

Nowadays, farm animal industries use assisted reproductive technologies (ART) as a tool to manage herds' reproductive outcomes, for a fast dissemination of genetic improvement as well as to bypass subfertility issues. ART comprise at least one of the following procedures: collection and handling of oocytes, sperm, and embryos in in vitro conditions. Therefore, in these conditions, the interaction with the oviductal environment of gametes and early embryos during fertilization and the first stages of embryo development is lost. As a result, embryos obtained in in vitro fertilization (IVF) have less quality in comparison with those obtained in vivo, and have lower chances to implant and develop into viable offspring. In addition, media currently used for IVF are very similar to those empirically developed more than five decades ago. Recently, the importance of extracellular vesicles (EVs) in the fertility process has flourished. EVs are recognized as effective intercellular vehicles for communication as they deliver their cargo of proteins, lipids, and genetic material. Thus, during their transit through the female reproductive tract both gametes, oocyte and spermatozoa (that previously encountered EVs produced by male reproductive tract) interact with EVs produced by the female reproductive tract, passing them important information that contributes to a successful fertilization and embryo development. This fact highlights that the reproductive tract EVs cargo has an important role in reproductive events, which is missing in current ART media. This review aims to recapitulate recent advances in EVs functions on the fertilization process, highlighting the latest proposals with an applied approach to enhance ART outcome through EV utilization as an additive to the media of current ART procedures.

Boar spermatozoa proteomic profile varies in sperm collected during the summer and winter.

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P <  0.05). Spermatozoa were processed and evaluated using the nano LC-MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater.

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy (1H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved.

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.

Stage-specific metabolomic changes in equine oviductal fluid: New insights into the equine fertilization environment.

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.

Effect of boar semen supplementation with recombinant heat shock proteins during summer.

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 µg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.

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.

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.

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.