214 SUPEROVULATION IN A/J MICE USING A COMBINATION OF GONADOTROPINS AND THE PTEN INHIBITOR bpV(pic)

Abstract

Strain and individual differences in superovulation constitute a serious problem in mice. Gonadotropins stimulate the maturation of developing ovarian follicles, but not primordial follicle activation (PFA), which is negatively controlled by the Phosphatase and Tensin Homologue Deleted from Chromosome 10 (PTEN). PTEN inhibitors may increase the number of developing follicles by promoting PFA. If so, subsequent gonadotropin injections may make more follicles ovulate. This study tested whether PTEN inhibitors promote superovulation by gonadotropins and examined the fertilizability of ovulated oocytes in vitro. Method: Immature females of the low responder A/J mouse strain were used. Based on preliminary results regarding peaks in the daily changes in the ovary weight/body weight ratio and ovarian anti-Müllerian hormone protein content after injections of the PTEN inhibitor, dipotassium bisperoxo (picolinato) oxovanadate (V) [bpV(pic)], the number of ovulated oocytes was examined using four combinations of the bpV(pic) dose and interval between the bpV(pic) and pregnant mare serum gonadotropin (PMSG) injections: 1) 3 μg and PMSG on Day 3; 2) 3 μg and PMSG on Day 4; 3) 30 μg and PMSG on Day 1; and 4) 30 μg and PMSG on Day 2. Ovulation was induced by hCG 48 h after PMSG injection. In vitro fertilization of the obtained oocytes was performed using TYH medium and epididymal sperm from adult ICR male mice (Day 1). Subsequently, the oocytes were cultured in KSOMaa+0.1% bovine serum albumin for 4 days. The numbers of two-cell embryos and blastocysts were recorded on Days 2 and 5, respectively. Results: The average number of oocytes collected in treatments 1 to 4 was 1) 7.2 ± 0.7 v. 9.4 ± 1.6 [control v. bpV(pic), mean ± s.e.m., n = 5]; 2) 7.8 ± 0.7 v. 9.0 ± 1.4; 3) 9.8 ± 1.6 v. 11.2 ± 1.0; and 4) 9.4 ± 1.0 v. 7.4 ± 1.9. More oocytes tended to be collected in the bpV(pic) groups in treatments 1 to 3, but the differences were not significant on ANOVA. The corresponding percentages of two-cell embryos on Day 2 after insemination were 1) 78.7 ± 7.7% v. 83.7 ± 5.6%; 2) 76.0 ± 4.2% v. 65.2 ± 4.2%; 3) 53.5 ± 13.0% v. 68.6 ± 9.1%; and 4) 78.2 ± 9.7% v. 55.2 ± 5.7%. The respective percentages of blastocysts on Day 5 after insemination were 1) 62.7 ± 6.5% v. 69.5 ± 8.1%; 2) 67.9 ± 7.7% v. 41.6 ± 5.6%; 3) 74.9 ± 11.3% v. 79.2 ± 7.8%; and 4) 78.0 ± 3.8% v. 60.5 ± 4.3%. On weighted ANOVA with angular transformations, the two-cell embryo and blastocyst rates were significantly lower in bpV(pic) groups than in control groups for treatments 2 and 4. Discussion: More oocytes tended to be collected in the bpV(pic) groups in 3 of the 4 experimental treatments. Different injection intervals between bpV(pic) and PMSG influenced both the superovulation efficiency and fertilizability of the oocytes. This new method using both PTEN inhibitors and gonadotropins is a promising method for improving superovulation efficiency, although the optimal injection scheme should be determined.

This work was supported by a grant from the Ministry of Health, Labour, and Welfare of Japan.