Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con-and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of Ϸ1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OFexposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoproteinheparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.sperm-oocyte interaction ͉ oviductal fluid ͉ ZP hardening P olyspermy (the penetration of the egg cytoplasm by more than one spermatozoa) is a pathologic condition in placental mammals, usually causing early death of the embryo (1). Although the prevalence of polyspermy under natural conditions is moderate, in in vitro fertilization (IVF) systems polyspermy remains a major obstacle to successful development of viable embryos in different species, including humans (2). Mechanisms underlying the block to polyspermy in mammals have been partially uncovered and characterized, mainly with use of rodents as animal models and usually related to events occurring after sperm entry into the oocyte.The entrance of the spermatozoon into the oocyte's cytoplasm induces the release of cortical granule contents, which modify the vitelline membrane, the zona pellucida (ZP), or both, rendering the oocyte refractory to additional sperm binding and penetration (3) and ending in changes in the mechanical properties and resistance to protease throughout the ZP (4). Yet assuming strong similarities in fertilization mechanisms among rodents and ungulates, observations in ovulated unfertilized porcine and bovine oocytes, showing that ZP resistance to pronase lasts from hours to days (5-8), contras...

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.DOI: http://dx.doi.org/10.7554/eLife.23670.001

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P!0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZPor blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.

Preimplantation embryos experience profound resetting of epigenetic information inherited from the gametes. Genome-wide analysis at single-base resolution has shown similarities but also species differences between human and mouse preimplantation embryos in DNA methylation patterns and reprogramming. Here, we have extended such analysis to two key livestock species, the pig and the cow. We generated genome-wide DNA methylation and whole-transcriptome datasets from gametes to blastocysts in both species. In oocytes from both species, a distinctive bimodal methylation landscape is present, with hypermethylated domains prevalent over hypomethylated domains, similar to human, while in the mouse the proportions are reversed. An oocyte-like pattern of methylation persists in the cleavage stages, albeit with some reduction in methylation level, persisting to blastocysts in cow, while pig blastocysts have a highly hypomethylated landscape. In the pig, there was evidence of transient de novo methylation at the 8-16 cell stages of domains unmethylated in oocytes, revealing a complex dynamic of methylation reprogramming. The methylation datasets were used to identify germline differentially methylated regions (gDMRs) of known imprinted genes and for the basis of detection of novel imprinted loci. Strikingly in the pig, we detected a consistent reduction in gDMR methylation at the 8-16 cell stages, followed by recovery to the blastocyst stage, suggesting an active period of imprint stabilization in preimplantation embryos. Transcriptome analysis revealed absence of expression in oocytes of both species of ZFP57, a key factor in the mouse for gDMR methylation maintenance, but presence of the alternative imprint regulator ZNF445. In conclusion, our study reveals species differences in DNA methylation reprogramming and suggests that porcine or bovine models may be closer to human in key aspects than in the mouse model.

ABSTRACT:In this study, we evaluated the effects of glutathione (L-␥-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5ЊC) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.

Understanding the mechanisms of epigenetic remodeling that follow fertilization is a fundamental step toward understanding the bases of early embryonic development and pluripotency. Extensive and dynamic chromatin remodeling is observed after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate embryonic genome activation. In particular, trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcription repression. Global levels of this epigenetic mark are high in oocyte chromatin and decrease to minimal levels at the time of embryonic genome activation. We provide evidence that the decrease in H3K27me3 observed during early development is cell-cycle independent, suggesting an active mechanism for removal of this epigenetic mark. Among H3K27me3-specific demethylases, Jumonji domain-containing protein 3 (JMJD3), but not ubiquitously transcribed tetratricopeptide repeat X (UTX), present high transcript levels in oocytes. Soon after fertilization JMJD3 protein levels increase, concurrent with a decrease in mRNA levels. This pattern of expression suggests maternal inheritance of JMJD3. Knockdown of JMJD3 by siRNA injection in parthenogenetically activated metaphase II oocytes resulted in inhibition of the H3K27me3 decrease normally observed in preimplantation embryos. Moreover, knockdown of JMJD3 in oocytes reduced the rate of blastocyst development. Overall, these results indicate that JMJD3 is involved in active demethylation of H3K27me3 during early embryo development and that this mark plays an important role during the progression of embryos to blastocysts.G amete nuclei undergo extensive chromatin remodeling soon after fertilization, including alterations to DNA methylation levels and posttranslational histone tail modification. These changes are known to influence chromatin structure and transcriptional readout (1) and also to promote embryonic genome activation (EGA) (2), which occurs in humans and cows after three to four cell divisions (3-5). Because the first few cell divisions occur without embryonic gene transcription, maternally inherited mRNA and proteins present in the oocyte are likely responsible for inducing these epigenetic changes (6).Histone lysine methylation is involved in transcriptional repression and/or activation and plays a key role during lineage specification and cell differentiation (7). Di-and trimethylation of histone 3 lysine 27 (H3K27) are catalyzed by the polycomb repressive complex 2 (PRC2), which has three essential components: enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12) (8-10). These epigenetic marks are specifically associated with gene repression (11) and are typical of many key developmental genes in embryonic stem cells, stem cell maintenance (12-14), and regulation of pluripotency (15). In mouse embryos, asymmetric distribution of trimethylated H3K27 (H3K27me3) i...

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.