International Journal of Impotence Research (2001) 13, 205±211 ß 2001 Nature Publishing Group All rights reserved 0955-9930/01 $15.00 www.nature.com/ijir

Effects of estrogen on nitric oxide synthase and histological composition in the rabbit and

HN Yoon1, WS Chung1*, YY Park1, BS Shim1, WS Han2 and SW Kwon1

1Department of , Medical Research Center, Seoul, Korea; and 2Department of Pathology, Ewha Womans University College of Medicine, Seoul, Korea

We investigated the functional and histological changes after oophorectomy in the rabbit clitoris and vagina to determine the mechanism responsible for the development of arousal disorder in postmenopausal women. Twenty mature female New Zealand white rabbits were randomly divided into three groups: control; oophorectomy; and estrogen replacement after oophorectomy. We compared the nitric oxide synthase (NOS) activity and the degree of expression of neuronal (nNOS) and endothelial NOS (eNOS) using biochemical and Western blot analysis in clitoral and vaginal tissues. Histological change of smooth muscle and collagen contents in those tissues were also compared using Masson's trichrome staining. NOS activity and the expression of nNOS and eNOS were signi®cantly increased in the oophorectomized group while there was a decrease to the level of the control group in the estrogen replacement group. Histological examination showed that oophorectomy induced a signi®cant increase in collagen and decrease in muscle content in both clitoris and vagina, while the ratio of smooth muscle content was increased signi®cantly after the estrogen replacement. Our results clearly demonstrate that estrogen de®ciency induces compensatory NOS production which may be related to decreases in muscle to collagen ratio in female rabbit genital organs. International Journal of Impotence Research (2001) 13, 205±211.

Keywords: estrogen; NOS; clitoris; vagina; smooth muscle

Introduction Materials and methods

Compared to male , the patho- Animals physiology and treatment of female sexual dysfunc- tion is not well known. Clitoral and vaginal Twenty mature female New Zealand white rabbits congestion that occur in response to sexual stimuli weighing 2.5 ± 3.0 kg between 2 to 6 months old are based on the relaxation of smooth muscle, and were divided into three groups: control (the rabbits similar to penile erection in males, nitric oxide (NO) underwent only sham operation, group N, n ˆ 6); is considered to play an important role during the oophorectomy (group O, n ˆ 7); and estrogen repla- process of sexual excitement in females.1,2 The cement group after oophorectomy (group E, n ˆ 7). function or effects of on NO in the clitoris The animals were fed a normal diet. have not been elucidated, although it was con®rmed to be present in the female vagina and clitoris.3 Estrogen deprivation and replacement In the present study, to determine the mechanism responsible for the development of arousal disorder Estrogen de®ciency was induced by bilateral oo- in postmenopausal women and to establish the phorectomy, and after 2 weeks of the experimental basis for the justi®cation of hormone de®cient state, intramuscular estrogen injections replacement therapy to treat in were given to those rabbits in the estrogen replace- the postmenopausal women, we investigated the ment group (group E). The estrogen used was in the effects of estrogen on the histology and NOS activity form of estradiol cypionate (0.1 mg=kg=1 week, in the female rabbit clitoris and vagina. Hoechst Co., Ltd., Germany).

Tissue preparation and measurement of serum *Correspondence: WS Chung, Department of Urology, Ewha Womans University Mokdong Hospital, 911-1, Mokdong, hormone level Yangcheon Ku, Seoul, 158-710, Korea. E-mail: [email protected] As for tissue sampling, tissues were obtained 2 Received 1 August 2000; accepted 15 March 2001 weeks after oophorectomy from those rabbits in Effects of estrogen on female rabbit genitalia HN Yoon et al 206 group O, and 2 weeks after estrogen replacement Statistical analysis therapy from those rabbits in group E. The tissues were ®xed in 10% formalin for histological exam- ination, and those tissues for the measurement of The obtained data were separated according to each Western blot and NOS activity were snap-frozen in observation method, and the statistical analysis liquid nitrogen and stored at 770C until the next of the average between groups was performed procedure. Serum estrogen levels before and after using the Mann±Whitney U-test. Differences were treatment were measured. considered statistically signi®cant at P < 0.05.

Results

Measurement of NOS activity Changes in NOS activity

NO production was evaluated indirectly by measur- NOS activities were increased signi®cantly in both 27 ing the concentrations of nitrite (NO ) and nitrate clitoris and vaginal tissues of the group O compared (NO37) with the method presented by Malinski et al.4 using the Griess reaction. The concentration of nitrite was calculated using the absorbance curve of sodium nitrite per concentration as the standard curve.

Measurement of NOS expression by Western blot analysis

For quantitative analysis of changes in NOS expres- sion, Western blot analysis was done using SDS- PAGE. As the primary antibody, monoclonal anti neuronal NOS (nNOS) (Transduction laboratories, Lexington, KY) and monoclonal anti endothelial NOS (eNOS) (Transduction laboratories, Lexington, KY) were used, and as the standard marker of protein molecular weight, the low molecular pre- stained marker (Bio-Rad Lab., Hercules, CA) was used to con®rm the expression of nNOS at 155 kDa, and of eNOS at 140 kDa. The measurement of blot density for each protein was done by calculating a relative density of each protein for tubulin using an imaging analysis program.

Histological evaluation of the smooth muscle and collagen contents

In order to observe the changes in the relative Figure 1 Effects of oophorectomy and hormone replacement on composition ratio of smooth muscle and collagen the NOS activity in the rabbit clitoris and vagina. NOS speci®c activity was expressed per gram of tissue (OD=mg protein) at content in the clitoris and vaginal tissues, the 585 nm. All values are expressed in mean Æ s.e., referring to the paraf®n-embedded tissues were stained with Mas- respective intact control series determination. (A) clitoris and (B) son's trichrome. Under 1006magni®cation of a light vagina (a: P < 0.05, analysis of the signi®cance was compared with microscope, 10 ®elds were selected randomly, the that of control. b: P < 0.05, analysis of the signi®cance was number of smooth muscle and collagen cells were compared with that of oophorectomy group. c: P < 0.01, analysis of the signi®cance was compared with that of control. d: P < 0.01, counted, and the numbers were converted into analysis of the signi®cance was compared with that of oophor- percentile and averaged. ectomy group).

International Journal of Impotence Research Effects of estrogen on female rabbit genitalia HN Yoon et al 207 with those of group N (P < 0.05), and were decreased compared to that in the control group or estrogen in those tissues of group E to the level of group N replacement group (Figure 3). (P < 0.05 compared with those of group O)(Figure 1).

Changes in smooth muscle and collagen contents Changes in NOS expression As for histological ®ndings, clitoral tissue showed a In the Western blot analysis, nNOS and eNOS decreased vascularity with tissue atrophy and loss were separated at 155 kDa and 140 kDa, respec- of vaginal mucosal folding with a relatively ¯at tively. Both expressions of nNOS and eNOS pattern in oophorectomized rabbits compared to the increased signi®cantly in clitoris and vaginal control group. The thickness of vaginal mucosa and tissues of group O compared with those of group muscle layer were also decreased (Figure 4). Estro- N(P < 0.01), and signi®cantly decreased in those gen replacement resulted in restoring those changes. of the group E compared with those of group O Quantitative morphometric analysis of smooth mus- (P < 0.01) (Figure 2). Immunohistochemical stain- cle and collagen content revealed marked decreased ing also showed similar results; the degree of smooth muscle and increased collagen content of staining for nNOS and eNOS in clitoris and the group O rabbits and increased smooth muscle vagina was increased in the oophorectomy group and decreased collagen content of group E rabbits

Figure 2 Effects of oophorectomy and hormone replacement on the expressions of nNOS(155 kDa) and eNOS(140 kDa) and relative densitometric analysis of the nNOS and eNOS in the rabbit clitoris and vagina (A) clitoris and (B) vagina (a: P < 0.01, compared with control group, b: P < 0.01, compared with oophorectomy group).

International Journal of Impotence Research Effects of estrogen on female rabbit genitalia HN Yoon et al 208

Figure 3 Immunoreactivity pattern for nNOS and eNOS of the rabbit clitoris. Similar patterns of change were observed in the vagina. (A) nNOS expression in nerves or ganglia of the rabbit clitoris (6200, oophorectomy group). (B) eNOS expression in the vascular endothelium of the rabbit clitoris (6200, oophorectomy group). (C) nNOS expression in the estrogen replacement group (6200, clitoris). (D) eNOS expression in the estrogen replacement group (6200, clitoris).

compared to that of group N rabbits (P < 0.01) in both secondary to declining estrogen levels and altera- clitoral and vaginal tissue of the rabbit (Figure 5). tions of NOS levels.16 For the changes of NOS expression after estrogen withdrawal in the female animals, different results Discussion are seen according to the type of species. Berman et al.17 reported that the expression of NOS in the rat vagina was decreased after oophorectomy In male penile erection, it is being accepted that NO and estrogen replacement resulted in a signi®cant plays an important role in corpus cavernosal smooth increase in NOS expression. On the contrary, muscle relaxation, and NOS is testosterone-depen- Batra and Al-Hijji18 reported that NOS expression dent in male corpus cavernosum.5±8 However, there and activity were increased in estrogen de®cient is little knowledge about hormonal effects on the female rabbit vaginal tissues and NOS was female genital organ. Female genital organs are down-regulated by estrogen replacement. In the known to receive a complex spectrum of neuro- present study, we found that NOS expression and transmitters and modulators, including NO.9±11 activity were increased with estrogen de®ciency Recent studies have demonstrated increased NOS in both clitoral and vaginal tissues of the female activity and gene expression in the female rabbit. With hormone replacement, its expression and fallopian tubes in response to estrogen.12 ± 15 and activity were decreased and returned to the With aging and the , the majority of original level of the control group like the similar women experience some change in sexual function. ®ndings in the female rabbit lower urinary tract.19 It is believed that symptoms related to alterations in Therefore, there seems to be different patterns of genital sensation and blood ¯ow are, in part, response on NOS production according to the

International Journal of Impotence Research Effects of estrogen on female rabbit genitalia HN Yoon et al 209 species by the change of estrogen level. We need The role of estrogen in maintaining vaginal to ®nd how estrogen regulates NOS production in mucosa, vascular endothelium, and uterine and humans. vaginal blood ¯ow has been recognized.12,20 ± 22 Estrogens have been shown to be vasoprotective and to enhance genital sensory ®eld enlargement possibly through a NO-dependent mechan- ism.20,21,23 However, there is little published data on NOS activity in the female rabbit genital organ. To our knowledge, this is the ®rst study demonstrat- ing an association between estrogen withdrawal- induced histological change and NOS expression in female rabbit external genital organs including the clitoris. The data of the current study showed that estrogen de®ciency had the effect of increasing ®brosis in the clitoris and vagina of the female rabbit. When we considered the changes of histology and the alterations of NOS expression and activity

Figure 4 Masson's trichrome staining of the clitoral section from each group of rabbit (6200). Similar patterns of change were observed in the vaginal tissue. (A) Normal control, (B) oophor- ectomy, ®broblasts are markedly increased compared to those of Figure 5 Effects of oophorectomy and hormone replacement on control group, (C) estrogen replacement. Collagen components are the collagen and smooth muscle content in the rabbit clitoris and decreased and light red stained smooth muscle components are vagina. (A) clitoris and (B) vagina (a: P < 0.01, compared with markedly increased compared to those of oophorectomy group. control group, b: P < 0.01, compared with oophorectomy group).

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