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Ovarian Hormone Secretory Response to Gonadotropins and Nitric Oxide Following Chronic Nitric Oxide Deficiency in the Rat¹

^a Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201

	ABSTRACT

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Ovarian hormone secretion is regulated by gonadotropins, and it has been demonstrated that this response is modulated by nitric oxide (NO). The focus of this study was to determine the effect of chronic NO deficiency on the secretion of ovarian steroids. Female rats were given N-nitro-L-arginine (L-NNA; 0.6 g/L) in their drinking water, and vaginal smears were obtained daily. By 4 wk of treatment, all the rats were in constant estrus or proestrus. At 6–8 wk the animals were killed; the ovaries were removed and incubated in the presence of eCG (1 IU/ml) and hCG (1 IU/ml) and/or S-nitroso-L-acetyl penicillamine (an NO donor, S-NAP; 0.1 mM) for 4 h. Medium was collected at 30-min intervals, and estradiol, progesterone, and androstenedione were measured. Ovaries from proestrous rats served as controls. Ovaries from L-NNA-treated animals had a greater basal and gonadotropin-stimulated release of estradiol but not of androstenedione or progesterone in comparison to ovaries from untreated controls. S-NAP decreased the gonadotropin-stimulated estradiol, progesterone, and androstenedione in ovaries from NO-deficient rats. Steroid secretion in controls was not responsive to S-NAP. We conclude that chronic NO inhibition produces constant estrus due to increased estradiol production and that NO acts to inhibit estradiol and androstenedione production.

	INTRODUCTION

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Nitric oxide (NO) has emerged as an important regulator of many physiological processes, including vascular tone, platelet function, neurotransmission, host-defense mechanisms [1], the ovary [2, 3], and embryonic development [4]. NO, a highly reactive free radical, is synthesized endogenously via the oxidation of L-arginine by nitric oxide synthase (NOS) to NO and citrulline. NO exerts its biological effects by binding iron-containing enzymes. For example, NO is known to activate the heme-containing guanylate cyclase, causing an increase in intracellular cGMP and vascular smooth muscle relaxation [5]. NO also causes the stimulation of prostaglandin production by activating the heme-containing enzyme cyclooxygenase [6]. The cytochrome P450 steroidogenic enzymes also contain an iron-heme center. It has been demonstrated that the steroidogenic P450 enzyme aromatase can be directly inhibited by NO in vitro [7, 8].

There is supporting evidence of NO's actions in ovarian physiology. NO is synthesized by the rat ovary and is hypothesized to play a role in follicle rupture, ovulation, and steroidogenesis [2, 3, 9, 10]. Administration of NOS inhibitors blocks NO production in the ovary, thereby reducing the rate of ovulation in rats both in vivo and in vitro [9,10]. In addition, ovarian steroid hormone secretion induced by pituitary gonadotropins is modulated by NO. Endogenously derived NO inhibits estradiol secretion by direct inhibition of aromatase in human granulosa-luteal cells [2].

Women with polycystic ovaries have been characterized by hypertension, hyperinsulinemia, anovulation, and an excess ovarian production of androgens and estrogens [11]. The latter observations are consistent with a relative NO deficiency in the ovaries. We were interested in investigating whether long-term administration of N-nitro-L-arginine, an NOS inhibitor, resulted in a polycystic ovary-like function of the hypothalamic-pituitary ovarian axis in rats. Importantly, the effects of chronic NO inhibition in the studies on regulation of ovarian steroidogenesis have not been previously reported, and this was the objective of the present study.

	MATERIALS AND METHODS

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Animals

Sixteen adult female Wistar rats (225–250 g; Harlan, Sprague-Dawley, Indianapolis, IN) were housed in a temperature-controlled room with a 12L:12D cycle and allowed rat chow and water ad libitum. On the third day after arrival, 10 of these rats were placed on drinking water containing the NOS inhibitor N-nitro-L-arginine (L-NNA; 0.6 g/L) for 6 wk. The remaining 6 rats were left untreated during this 6-wk period. Vaginal smears were obtained daily, and systolic blood pressure measurements were made weekly on restrained rats using a tail cuff (NARCO Bio-Systems, Austin, TX). The end-point for NO deficiency was established as the point when the vaginal smears were predominantly cornified cells and, secondly, when systolic blood pressures were 150 mm Hg or greater.

Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Ovary Removal and Incubation

Once rats had entered a period of constant estrus for at least 5 days and showed a significant increase in blood pressure, each animal was anesthetized with sodium pentobarbital, and a terminal blood sample was collected from the abdominal aorta. The ovaries were removed and cut into quarters. Ovarian fragments were randomly and evenly distributed as four explants per well in the culture plates. Ovarian explants were then incubated in medium only or in the presence of eCG and hCG (1 IU/ml each) with or without the NO donor S-nitroso-L-acetyl-penicillamine (S-NAP; 0.1 mM). Ovaries from intact rats killed in the morning of proestrus served as controls. Four ovarian fragments were placed into each well of 24-well tissue culture plates containing 1 ml of alpha modified minimal essential medium and 0.1% BSA. The incubation was carried out for 4 h at 37°C in a humidified atmosphere of 50% O₂:5% CO₂. Ovarian fragments were moved manually at 30-min intervals to wells containing fresh medium. After the 240 min, the ovarian fragments were weighed, and the culture media were frozen (-20°C) until RIA.

RIA

Progesterone, androstenedione, and estradiol concentrations were determined in the culture medium at each 30-min interval using commercial RIA kits (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA). All samples and standards were assayed in duplicate. Serum estradiol was measured using the Estradiol-6 Coat-a-Count assay kit (Diagnostic Products Corp.), in which rat serum did not have an effect on recovery of estradiol added exogenously. The levels of steroids in each well from a given sample of four ovarian explants were summed across the 4-h period.

Statistics

Statistical analysis was performed using two-way ANOVA followed by Fisher's pair-wise comparison test; p < 0.05 was considered significant.

	RESULTS

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Beginning at 1 wk of L-NNA treatment, systolic blood pressures were significantly higher in all of the animals treated with L-NNA in their drinking water compared to normal cycling controls (Fig. 1). Systolic blood pressure in these animals reached 50 mm Hg above that in the normal animals; it was maintained at this level.

View larger version (49K): [in this window] [in a new window]   FIG. 1. Systolic blood pressure in normal rats and rats receiving the NOS inhibitor, L-NNA, in their drinking water (0.6 g/L) (NO deficient). Each bar represents the mean systolic blood pressure ± SEM obtained by tail cuff measurement in restrained rats. * p < 0.05 vs. normal rats (n = 10 NO deficient; n = 6 proestrous).

Vaginal cytology demonstrated that almost all of the animals were in constant estrus or proestrus/estrus by 25 days of L-NNA treatment and that they remained in estrus/proestrus for the duration of the study.

Rats receiving L-NNA in their drinking water also had serum estradiol levels that were significantly higher (69%) than those of their normal proestrous counterparts (Fig. 2).

View larger version (38K): [in this window] [in a new window]   FIG. 2. Basal serum estradiol obtained at 1000–1200 h in normal proestrous and NO-deficient rats at time rats were killed. Values are the means ± SEM. * p < 0.001 vs. normal proestrous rats (n = 10 NO deficient; n = 6 proestrous).

Similarly, in vitro estradiol release from ovaries of animals treated with L-NNA in medium only (basal) was increased 2.5-fold over that from ovaries of proestrous controls (Fig. 3). However, there was no significant difference in basal androstenedione and progesterone production in the same samples.

View larger version (67K): [in this window] [in a new window]   FIG. 3. The effects of NO deficiency on basal estradiol, androstenedione, and progesterone production from ovaries in vitro. Values are means ± SEM. * p < 0.05 vs. normal proestrous rats (n = 5 per treatment NO deficient; n = 3 per treatment normal proestrous).

Gonadotropin-stimulated release of estradiol from ovarian fragments from L-NNA-treated rats showed a significant increase (167%) compared to the value in medium only (Fig. 4A). The gonadotropin-stimulated response was completely abolished by addition of the NO donor, S-NAP. Treatment with S-NAP alone decreased release of estradiol; however, this effect was not statistically significant in comparison to values in medium only. In ovaries from control proestrous animals, the addition of gonadotropins, S-NAP alone, or gonadotropins plus S-NAP had no effect on estradiol production when compared to values in the appropriate controls (medium only or eCG+hCG) (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Estradiol release in vitro in response to gonadotropins (hCG and eCG [PMSG on figure]) and NO donor (S-NAP). A) Rats receiving L-NNA for 6 wk; values are means ± SEM; * p < 0.001 vs. medium; #, p < 0.0001 vs. hCG+eCG. NS, S-NAP was not significant compared to medium (n = 5 per treatment). B) Normal proestrous rats; none of the treatments were significantly different (n = 3 per treatment).

Androstenedione production from ovaries of NO-deficient rats was increased by 300% by gonadotropins as compared to that in medium alone (Fig. 5A). Addition of S-NAP had no significant effect on androstenedione production as compared with that in medium alone; however, S-NAP did significantly antagonize the gonadotropin-induced response. In ovarian fragments from proestrous rats, the addition of gonadotropins significantly increased (208%) the release of androstenedione production (Fig. 5B). However, S-NAP had no significant effect alone or when added with gonadotropins.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Androstenedione release in vitro in response to gonadotropins (hCG and eCG [PMSG on figure]) and NO donor (S-NAP). A) Rats receiving L-NNA for 6 wk; * p < 0.0001 vs. medium; #, p < 0.0001 vs. hCG+eCG. NS, S-NAP was not significant compared to medium (n = 5 per treatment). B) Normal proestrous rats; * p < 0.005 vs. medium. NS, S-NAP was not significant compared to medium. NS, hCG+eCG+S-NAP was not significant compared to hCG+eCG (n = 3 per treatment).

The addition of gonadotropins significantly increased (45%) the release of progesterone production by the ovaries from NO-deficient rats (Fig. 6A), and the addition of S-NAP resulted in a significant reduction of progesterone production (40%). However, S-NAP actually enhanced the gonadotropin-induced progesterone response by 23%. In ovaries from normal proestrous rats, the addition of gonadotropins significantly increased (70%) the release of progesterone compared to that in medium only (Fig. 6B). Adding S-NAP in the presence or absence of gonadotropins had no effect on progesterone production when compared to that in the appropriate control.

View larger version (38K): [in this window] [in a new window]   FIG. 6. Progesterone release in vitro in response to gonadotropins (hCG and eCG [PMSG on figure]) and NO donor (S-NAP). A) Rats receiving L-NNA for 6 wk; * p < 0.0001 vs. medium; #, p < 0.004 vs. hCG+eCG; ** p < 0.0007 vs. medium (n = 5 per treatment). B) Normal proestrous rats; * p < 0.0001 vs. medium. NS, S-NAP was not significant compared to medium. NS, hCG+eCG+S-NAP was not significant compared to hCG+eCG (n = 3 per treatment).

	DISCUSSION

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Our results support and extend the hypothesis that NO plays a key tonic inhibitory role in ovarian hormone secretion. We found that a chronic 6-wk treatment with the NOS inhibitor L-NNA in the drinking water resulted in the rapid induction of hyperestrogenemia. Previous studies have examined the effects of acute NO deficiency; however, to our knowledge no one has shown the long-term effects of NO deficiency on ovarian steroidogenesis in vivo. The condition of constant estrus/proestrus was supported by higher serum estradiol levels (Fig. 2) and by a significant increase in estradiol production as shown by the in vitro data (Figs. 3 and 4A). As expected, the chronic treatment with L-NNA also rapidly induced hypertension (Fig. 1), while the initial decrease in blood pressure in normals was likely due to adaptation to the procedure.

Estradiol is synthesized in the ovary by aromatase, a cytochrome P450 enzyme, which converts androgens to estrogens [8]. Several lines of evidence support a relationship between NO and aromatase. NO can directly inhibit aromatase activity [8], thereby regulating ovulation [9, 10] and estradiol synthesis [2, 3, 12]. The biological responses observed in the NO-deficient rats in the current study were consistent with what has been previously reported. The reduction of NO by the NOS inhibitor L-NNA induced increased estradiol production by the ovary to the degree that the estrous cycle was terminated in persistent estrus/proestrus. This is consistent with previous studies in which NOS inhibitors reduced the rate of ovulation in rats both in vivo and in vitro [9, 10]. Our in vitro data agree with previous reports showing that NO negatively regulated steroidogenesis in human granulosa cells [2], rat luteinized ovarian cells [12], and rabbit ovarian cells [3].

However, our results extend the concept that NO regulates steroidogenesis with the demonstration that NO also inhibits androstenedione production. Although basal androstenedione production in the NO-deficient ovaries was not different from that in control proestrous rats, the NO donor S-NAP antagonized the gonadotropin-induced androstenedione production in the NO-deficient animals. It appears, therefore, that NO inhibits gonadotropin-induced steroidogenesis at a higher level than aromatase. The exact site of action is not clear. Several enzymes responsible for androstenedione production are cytochrome P450 enzymes. These data suggest that the reduction in estradiol by NO or a NO donor such as S-NAP may also result, in part, from reduced androgen precursor. This observation that NO reduces androstenedione indicates that perhaps NO may also regulate testicular steroidogenesis [13, 14].

NO may have a dual effect at the level of progesterone by the ovary. Basal progesterone production by the NO-deficient ovaries was not different from that in control proestrous rats (Fig. 3), but the NO donor S-NAP when given alone appeared to be inhibitory, compared to medium only, in NO-deficient animals. However, S-NAP did not antagonize the gonadotropin-induced response. Instead, NO enhanced the level of gonadotropin-induced progesterone by ovaries from NO-deficient rats. The action of NO on progesterone levels is perplexing, since 3-hydroxyl-3-methylglutaryl coenzyme A reductase is also a cytochrome P450 enzyme to which NO should bind. Perhaps NO affects enzymes that metabolize progesterone as well as hMG reductase.

In conclusion, chronic NO deficiency produces high blood pressure, constant estrus, and high serum estradiol levels. Our data show that NO inhibits estradiol production directly and by reducing the ovarian synthesis of androstenedione.

	FOOTNOTES

 
¹ This work was supported by grants NIH-MH-47181 and NIH-GM-08167.

² Correspondence: Joseph C. Dunbar, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. FAX: 313 577 5494; jdunbar{at}med.wayne.edu

Accepted: November 20, 1998.

Received: June 30, 1998.

	REFERENCES

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