S. Yavin

Seasonal effects on gene expression, cleavage timing, and developmental competence of bovine preimplantation embryos

We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec-Apr) and hot (May-Nov) seasons. Matured oocytes were chemically activated and cultured in vitro. The developmental peak to the two- and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P<0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, matured in vitro, fertilized, and cultured for 8 days. In each season, early- and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers for GDF9, POU5F1, and GAPDH using 18S rRNA as the reference gene. In both seasons, the expression of all examined genes was higher (P<0.05) in early- versus late-cleaved embryos. POU5F1 expression was higher (P<0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.

Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush

BACKGROUND Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination. METHODS An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN. RESULTS Ultrarapid cooling of murine embryos at 32 200 degrees C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 +/- 3.5%, 106/196) than cooling at 1700 degrees C/min in 0.25 ml straws (10 +/- 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains approximately 5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 +/- 3.6%, 99/126) than cryopreservation in 100% VS (31 +/- 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 +/- 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4-8-cell stage formed blastocysts at rates of 68 +/- 4.5 and 60 +/- 3.7%, respectively, after vitrification in 87.5% VS. CONCLUSIONS This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

The antioxidant epigallocatechin gallate (EGCG) moderates the deleterious effects of maternal hyperthermia on follicle-enclosed oocytes in mice

Hyperthermia-induced oxidative stress is one of the mechanisms suggested to underlie loss of developmental competence in mouse embryos. In this study, we examined whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) can alleviate the negative effects of hyperthermia on developmental competence of the ovarian pool of oocytes and improve embryonic development. Female mice (CB6F1) were synchronized (eCG+hCG) and injected with 0.4 ml EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated mice were exposed to heat stress (HS; 40 degrees C, 65% RH) or kept under normothermal conditions (Control; 22 degrees C, 45% RH). In vivo-derived zygotes were recovered 20 h after hCG administration and cultured in vitro. Maternal hyperthermia attenuated embryonic cleavage rate in association with further disruption in embryonic early cleavage and subsequently, with embryonic development. While pretreatment with EGCG did not affect the proportion of zygotes that cleaved to the two-cell stage, it appeared to moderate the effect of hyperthermia on both cleavage timing and developmental rate, as reflected by an increased rate of early cleaved embryos and blastocyst formation. Blastocyst developmental competence was also improved, as indicated by the increased total cell number and percentage of embryos that underwent hatching, in association with reduced apoptotic status, as reflected by the percentage of TUNEL-positive cells and intensity of caspase activity for the HS-EGCG embryos vs. HS-saline ones. In summary, while hyperthermia disrupts the competence of the follicle-enclosed oocyte, in vivo administration of the antioxidant EGCG improves developmental competence and the quality of the embryos that develop from these oocytes.