Inhibitors of Nitric Oxide Synthase Influence Oocyte Maturation in Rats

Abstract

Objective: To examine the effect of inhibiting nitric oxide synthase (NOS) on the number of ovulated oocytes and on occyte meiotic maturation.

Methods: Female Sprague-Dawley rats (25 days old) were superovulated with a subcutaneous injection of 10 U pregnant mare's serum gonadotropin, followed 52 hours later by a subcutaneous injection of 10 U human chorionic gonadotropin (hCG). Three hours before and 3 hours after hCG injection, the rats were treated orally with the vehicle (0.5% methylcellulose) as the control or with either of two NOS inhibitors, N^ω-nitro-L-arginine methyl ester (L-NAME) and L-N⁶-(1-iminoethyl)-lysine (L-NIL). The rats were killed 20 hours after hCG injection, and oocytes present in the oviduct were flushed, counted, and classified for stages of meiosis. In addition, ovarian occytes (12 hours post-hCG) and ovulated occytes were treated with an immunofluoresect stain for the presence of endothelial NOS (eNOS).

Results: Strong positive staining for eNOS was observed in the cytoplasm of ovarian and ovulated oocytes. Control rats ovulated an average 43.0 ± 4.1 oocytes each, which was lowered with either L-NAME or L-NIL (23.8 ± 4.3 and 23.5 ± 4.0 oocytes per rat, respectively; P < .002). We observed that significantly fewer ovulated oocytes obtained from rats treated with NOS inhibitors were at metaphase II (P < .006), the normal stage of meiosis for unfertilized oocytes, and a significantly greater percentage of oocytes displayed atypical morphology as compared with control oocytes (P < .0001).

Conclusion: Ovarian nitric oxide synthesis is required for maximal ovulation, and a lack of nitric oxide during the periovulatory period results in severe defects in oocyte maturation.