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, Nω-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.
3. Ignarro LJ. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol1990;30:35-60.
4.
4. Xie Q-W, Cho HJ, Calaycay J, et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science1992;256:225-228.
5.
5. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci USA1992;89:6348-6352.
7. Ben-Sholomo I, Kokia E, Jackson MJ, Adashi EY, Payne DW. Interleukin-1β stimulates nitrite production in the rat ovary: Evidence for heterologous cell-cell interaction and for insulinmediated regulation of the inducible isoform of nitric oxide synthase. Biol Reprod1994;51:310-318.
8.
8. Ellman C, Corbett JA, Misko TP, McDaniel M, Beckerman KP. Nitric oxide mediates interleukin-1β induced cellular cytotoxicity. A potential role for nitric oxide in the ovulatory process. J Clin Invest1993;92:3053-3056.
9.
9. Shukovski L, Tsafriri T. The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology1995;135:2287-2290.
10.
10. Chun S-Y, Eisenhauer KM, Kubo M, Hsueh AJW. Interleukin-1β suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology1995;136:3120-3127.
11.
11. Powers RW, Chen L, Russell PT, Larsen WJ. Gonadotropinstimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am J Physiol1995;269:E290-E298.
12.
12. Jabolnka-Shariff A, Olson LM. Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology1997;138:460-468.
13.
13. Bonello N, McKie K, Jasper M, et al. Inhibition of nitric oxide: Effects of interleukin-1β-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol Reprod1996;54:436-445.
14.
14. Yamauchi J, Miyazaki T, Iwasaki S, et al. Effects of nitric oxide on ovulation and ovarian steroidogenesis and prostaglandin production in the rabbit. Endocrinology1997;138:3630-3637.
15.
15. Van Voorhis BJ, Moore K, Strijbos PJLM, et al. Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. J Clin Invest1995;96:2719-2726.
16.
16. Zackrisson U, Mikuni M, Wallin A, Delbro D, Hedin L, Brannstrom M. Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum Repord1996;11:2667-2673.
17.
17. Powers RW, Chambers C, Larsen WJ. Diabetes-mediated decreases in ovarian superoxide dismutase activity are related to blood-follicle barrier and ovulation defects. Endocrinology1996;137:3101-3110.
18.
18. Tao M, Kodama H, Kagabu S, et al. Possible contribution of follicular interleukin-1β to nitric oxide generation in human pre-ovulatory follicles. Hum Reprod1997;12:2220-2225.
19.
19. Jablonaka-Shariff A, Olson LM. The role of nitric oxide in oocyte meiotic maturation and ovulation: Meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes. Endocrinology1998;139:2944-2954.
20.
20. Corbett JA, Wang JL, Misko TP, Shao W, Hickey WG, McDaniel ML. Nitric oxide mediates IL-1β-induced islet dysfunction and destruction: Prevention by dexamethasone. Autoimmunity1993;15:145-153.
21.
21. Olson LM, Jones-Burton CM, Jablonka-Shariff A. Nitric oxide decreases estradiol synthesis of rat luteinized ovarian cells: Possible role for nitric oxide in functional luteal regression. Endocrinology1996;137:3531-3539.
22.
22. Steel RGD, Torrie JH. Principles and procedures of statistics: A biometrical approach, 2nd ed.New York: McGraw-Hill, 1980:137-167.
23.
23. Kassab S, Miller MT, Hester RS, Novak J, Granger JP. Systemichemodynamics and regional blood flow during chronic nitric oxide synthesis inhibition in pregnant rats. Hypertension1998;31:315-320.
24.
24. Wassarman PM, Albertini DF, Knobil E, Neill JD, eds. The mammalian ovum. In: The physiology of reproduction. New York: Raven Press, 1994:79-115.
25.
25. Eppig JJ. Intercommunication between mammalian oocytes and companion somatic cells. BioEssays1991;13:569-574.
26.
26. Sato E, Koide SS. Biochemical transmitters regulating the arrest and resumption of meiosis in oocytes. Int Rev Cytol1987;106:1-33.
27.
27. Tornell J, Billing H, Hillensjo T. Regulation of oocyte maturation by changes in ovarian levels of cyclic nucleotides. Hum REprod1991;6:411-422.
28.
28. Dail WG, Sanborn CR, Ratner A. Immunocytochemical localization of cyclic AMP and cyclic GMP in rat ovarian tissue. Biol Reprod1980;22:281-288.
29.
29. Ratner A, Sanborn CR. Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat. Life Sci1980;26:439-445.
30.
30. Hubbard CJ, Price J. The effects of follicle-stimulating hormone and cyclic guanosine 3′-5′-monophosphate-phosphodiesterase and resumption of meiosis in hamster cumulus-oocyte complexes. Biol Reprod1988;39:829-838.
31.
31. Messinger SM, Albertini DF. Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci1991;100:289-298.
32.
32. Albertini DF. Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oo cytes. Dev Biol1987;120:121-131.
