Effect of Estrogen Withdrawal on Nitric Oxide Synthase Expression and Apoptosis in the Rat Vagina☆

Abstract 

Objectives. The physiology of the female sexual response and its molecular mediators remain poorly understood. Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. In this study, we hypothesize that vaginal atrophy and declining sexual function during menopause may be NO dependent. Using the rat as an experimental model, we examined the expression and topologic localization of vaginal NO synthase (NOS) and the concomitant induction of apoptosis under normal and estrogen-depleted conditions.

Methods. Thirty rats were categorized into six groups on the basis of phase of the estrous cycle or estrogen status after oophorectomy. The expression and cellular localization of NOS was examined in frozen sections using specific antibodies against neuronal (N-NOS) and endothelial NOS (E-NOS). Apoptotic cells were identified in situ using the terminal transferase technique (TUNEL). Trichome staining was performed in all specimens to determine smooth muscle/collagen ratios.

Results. N-NOS immunoreactivity was localized to nerve fibers supplying vaginal smooth muscle, perivascular nerve plexuses, and lamina propria. E-NOS was localized to vascular endothelium and perivascular smooth muscle fibers. Both E-NOS and N-NOS expression in intact cycling animals was highest during proestrous and lowest during metestrous. After oophorectomy, levels of both N-NOS and E-NOS declined substantially compared with those of intact animals, and there was a parallel induction of apoptosis. Estrogen withdrawal also resulted in increased vaginal atrophy, intramural collagen accumulation, and perivascular wall thickening, as identified by trichome staining. Estrogen replacement resulted in a significant increase in E-NOS and N-NOS expression, as well as diminished apoptosis and vaginal atrophy.

Conclusions. This cellular distribution of NOS in the rat vagina suggests that NO may modulate both vaginal blood supply and vaginal smooth musculature. Estrogen appears to play a critical role in concomitantly regulating vaginal NOS expression and apoptosis in nerves, smooth muscle, vascular endothelium, and epithelium of the rat vagina. These findings may have significant clinical implications for the pathophysiology of postmenopausal female sexual dysfunction.