319 REDUCED SUPEROVULATION EFFICIENCY BY HIGH-DOSE TREATMENT OF DEHYDROEPIANDROSTERONE IN MICE

Abstract

Strain differences in in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse oocytes, we examined the effect of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, in female mice on oocyte fertilizability. The DHEA pellets (0.25, 1.5, or 5 mg pellet^–1, 21-day release form, Innovative Research of America, Sarasota, FL, USA) or corresponding placebo pellets were implanted subcutaneously in 9-week-old female 129X1/SvJJmsSlc mice. On Day 18 of implantation, superovulation was induced in these females by injections of pregnant mare serum gonadotropin (PMSG) and hCG 48 h apart. On Day 21, IVF was conducted using oocytes collected from the oviducal ampullae of these females and epididymal sperm from Slc : ICR male mice using TYH medium. Then, the embryos were cultured in vitro in Whitten’s medium for 96 h. Plasma steroid levels and expression of 5 ovarian proteins (PTEN and receptors for FSH, androgen, oestrogen, and progesterone) at oocyte collection were measured by enzyme immunoassay and quantitative Western blots, respectively. Embryo development into 2-cell and blastocyst stages at each dose at 24 h and 96 h after insemination, respectively, were compared between DHEA and placebo groups using weighted ANOVA with angular transformations. Other observed values were compared using Student’s t-test. Treatment with DHEA suppressed the numbers of ovulated oocytes in the 1.5 and 5 mg groups (DHEA v. placebo: 21.0 ± 2.6 v. 32.5 ± 2.8 and 19.9 ± 1.4 v. 27.1 ± 1.6, respectively, n = 10; P < 0.05), but not in the 0.25 mg group (26.6 ± 3.2 v. 24.8 ± 2.4). Treatment with DHEA did not affect the percentages of 2-cell embryo formation at any dose, ranging 50 to 60%. In the 0.25 mg group, DHEA treatment tended to increase blastocyst formation rate (1.8 ± 0.8% v. 0.4 ± 0.4%; not significant). However, the treatment at 1.5 mg suppressed the rate (0.0 ± 0.0% v. 3.1 ± 0.7%; P < 0.05) and treatment at 5 mg did not affect the rate (1.3 ± 0.9% v. 0.8 ± 0.6%). Plasma testosterone levels were increased by DHEA at 1.5 and 5 mg (338.2 ± 39.8 v. 197.0 ± 8.9 pg mL^–1 and 534.9 ± 111.4 v. 241.8 ± 34.4 pg mL^–1, respectively, n = 6; P < 0.05), but not at 0.25 mg (247.4 ± 22.0 v. 252.5 ± 35.6 pg mL^–1, n = 6). No significant difference was induced by DHEA in plasma DHEA, progesterone, or oestradiol at any doses. Ovarian PTEN protein was more abundant in DHEA group than in placebo group at 5 mg (P < 0.05), tended to be more abundant at 1.5 mg (P ≈ 0.14), and was not different at 0.25 mg (P ≈ 0.35). The amounts of the 4 receptor proteins were not significantly changed by DHEA at any dose. These results suggest that DHEA at a low dose (e.g. 0.25 mg pellets for 21 days) might have a potential to improve in vitro fertilizability of mouse oocytes. Higher doses of DHEA reduced superovulation efficiency, perhaps because of the high testosterone level induced by the high-dose treatment of DHEA. The high testosterone level might upregulate ovarian PTEN expression, which might suppress ovarian primordial follicle activation. A more detailed study is needed to determine the optimal dose, timing, and duration of DHEA treatment for the improvement of female fertilizability.