Conditions to optimise the developmental competence of immature equine oocytes.

Optimising the developmental potential of immature equine oocytes and invitro-produced (IVP) embryos was explored through modifications of established media and holding temperature. In Experiment 1, delaying spontaneous resumption of meiosis through the process of simulated physiological oocyte maturation with the addition of the adenylate cyclase activator forskolin (50µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100µM) to overnight holding medium before maturation improved blastocyst production (P<0.05). In Experiment 2, the blastocyst production rate was increased significantly when cumulin (100ng mL-1) was added to the overnight holding or culture media (P<0.05). In Experiment 3, immature oocytes held overnight at 16°C before maturation had improved developmental competence than those held at 20°C and 5°C (P<0.05). There was no difference between maturation rates, but blastocyst formation per cleaved oocyte was significantly greater in oocytes held overnight at 16°C than at 20°C or 5°C. Furthermore, blastocyst formation per recovered oocyte and per fertilised oocyte was greater when oocytes were held before maturation at 16°C than at 5°C (P<0.05). In Experiment 4, the addition of sodium ascorbate (AC; 50µg mL-1) to the maturation and/or culture media of oocytes and IVP embryos did not improve blastocyst production, but did appear to lower cleavage rates compared with oocytes and embryos cultured without AC.

Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes.

Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxide (DMSO) or 3.5% glycerol. Immature equine oocytes were obtained from ovarian follicles of mares during diestrus by transvaginal aspiration (n=128). After invitro maturation, MII oocytes (n=90) were fertilised by ICSI with thawed stallion spermatozoa (n=45 in both the DMSO and glycerol groups). The embryo cleavage rate was greater in the DMSO than glycerol group (73.3% vs 46.7% respectively; P=0.0098), but the blastocyst development rate per fertilised oocyte was similar between the two groups (28.9% vs 15.6% respectively; P=0.128), as was the blastocyst production rate per cleaved embryo (39.4% vs 33.3% respectively; P=0.653). In this study, cryopreservation of equine spermatozoa in 1M DMSO was correlated with significantly higher cleavage rates in IVM oocytes fertilised by ICSI compared with spermatozoa cryopreserved using 3.5% glycerol. Although not statistically significant in this small number of stallions, increased blastocyst production and individual stallion variability was observed among CPA treatments. This warrants further critical examination of cryoprotectants used in equine sperm subpopulations used for ICSI in a larger number of stallions.

X-chromosome inactivation in monkey embryos and pluripotent stem cells.

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.