236 CREATION OF PARTHENOGENETIC SHEEP EMBRYOS: EFFECTS OF SERUM AND BREEDING SEASON

Abstract

The monoparental embryo (with only the maternal genome, termed a parthenogenote, or with only the paternal genome, termed an androgenote) is a powerful model to study the epigenetic status of developmentally regulated genes, including imprinted genes (those expressed only when inherited from one parent). Therefore, to use monoparental embryos for future study of placental development in normal and compromised pregnancies, the objective of this study was to test, validate and optimize the methodologies necessary to create parthenogenetic sheep embryos. In Exp. 1, cumulus–oocyte complexes (COC) were collected during the breeding season from nonpregnant and early pregnant ewes (n = 18) and matured overnight in vitro. The oocytes were then activated using ionomycin (a calcium ionophore) and 6-dimethylaminopurine (DMAP; a protein kinase inhibitor) in medium with (n = 47 COC) or without (n = 112 COC) 2% serum. In Exp. 2, COC were collected from nonpregnant, seasonally anestrous (n = 7; 79 COC) and late pregnant (n = 4; 44 COC) ewes, matured in vitro and activated as in Exp. 1 in the presence of serum. In Exp. 1, the rates of activation and blastocyst formation were not affected by reproductive status (nonpregnant v. pregnant). Activation of oocytes in serum-containing v. serum-free medium resulted in 76.3% v. 4.35% (P < 0.0001) cleavage rates and 21.9% v. 8.3% (P < 0.006) blastocyst formation rates, respectively. In Exp. 2, cleavage rates were similar for nonpregnant and pregnant ewes (31.9 v. 37.5%), but blastocyst formation was 13% in nonpregnant and 0% in pregnant ewes. These data demonstrate that (1) during the breeding season, the presence of serum in activation medium enhances cleavage and blastocyst formation rates; (2) in out-of-season ewes blastocyst formation but not cleavage rates are greater for nonpregnant than pregnant ewes, and (3) cleavage and blastocyst formation rates are greater (P < 0.0001 and P < 0.04, respectively) during the breeding season than out-of-season. Thus, oocytes obtained from ewes during the breeding season and activation medium containing serum should be used for creating parthenogenetic embryos in sheep. This study also demonstrated that creation of parthenogenotes in sheep, which can be used in the future to study parentally imprinted genes, is feasible but requires specific season and culture conditions.

Supported by USDA-NRI grant 2007-01215 to LPR and ATGB, and NIH grant P20 RR016741 (INBRE program of the NCRR, NIH).