Evidence for a physiological role for nitric oxide in the regulation of the LH surge: Effect of central administration of antisense oligonucleotides to nitric oxide synthase

Antisense oligonucleotides of brain-nitric oxide synthase (b-NOS) and endothelial-NOS (e-NOS) were used in steroid-primed ovariectomized rats to examine the physiological role of nitric oxide in the regulation of the LH surge. Since macrophage-NOS (m-NOS) is not produced in the hypothalamus under normal conditions, the m-NOS antisense oligonucleotide was used as control for the possible toxicity of the phosphorothioated and propynylated antisense oligonucleotides used. Female rats were ovariectomized on day 70 of age and implanted with a third ventricle cannula on day 77 of age, injected with 5 mu g of estradiol on days 84 and 85 of age at 17.00 h and with 1 mg progesterone or vehicle on day 86 at 09.00 h. Blood samples were collected between 13.00 and 19.00 h on day 86 of age via a jugular cannula inserted on day 85 of age. Antisense oligonucleotides (400 or 800 ng) or vehicle were injected in the third ventricle at 17.00 h on days 84 and 85 just before the estradiol injection and at 06.00 and 12.00 h on day 86. Neither the 400-ng nor the 800-ng dose of m-NOS AS had any effect on the steroid-induced LH surge. In contrast, central administration of the 400-ng dose of e-NOS AS and the 800-ng dose of b-NOS AS significantly attenuated the steroid-induced LH surge. The 40% reduction in LH by e-NOS AS and b-NOS AS was accompanied by a 33 and 28% reduction in their respective protein levels as shown by Western blots. The higher amount of b-NOS AS needed to reduce the LH surge is probably due to the high abundance of b-NOS in the hypothalamus as compared to e-NOS. As a whole, this study provides significant evidence for a physiological role of nitric oxide in mediating the steroid-induced LH surge.