Nitric oxide reverses prostaglandin-induced inhibition in ovarian progesterone secretion in rats

Yuan-Lin Dong, Pandu R.R. Gangula, Li Fang and Chandrasekhar Yallampalli1

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present studies were undertaken to determine whether nitric oxide (NO) is involved in the regulation of ovarian progesterone and oestradiol secretion in rats. Immature female Sprague–Dawley rats at 27 days of age were injected s.c. with 4 IU pregnant mare's serum gonadotrophin (PMSG) and were killed 72 h after the injection. The ovaries were collected, weighed and cultured in Dulbecco's modified Eagle's medium containing saline, NO donor, NO synthesis inhibitor or prostaglandin F2{alpha} (PGF2{alpha}). After 24 h culture, the medium concentrations of progesterone and oestradiol were measured by radioimmunoassay. Results showed that: (i) diethylenetriamine (DETA)/NO (1x10–6, 1x10–5, 1x10–4 M), an NO donor, caused a dose-dependent increase in progesterone synthesis (355 ± 43, 443 ± 46, 647 ± 55 ng/g ovary respectively, P < 0.01) with a concomitant decrease in ovarian oestradiol secretion (408.1 ± 50.7, 272.9 ± 28.2, 132.6 ± 34.6 pg/g ovary respectively, P < 0.01); (ii) neither progesterone nor oestradiol concentrations in the culture medium were altered by DETA without NO; (iii) NG-nitro-l-arginine methyl ester (1x10–4 M), an inhibitor of NO synthesis, did not significantly affect progesterone and oestradiol secretion by rat ovaries; (iv) PGF2{alpha} (1x10–6 M) caused a fall in progesterone and oestradiol synthesis; (v) co-incubation with DETA/NO, significantly reversed the PGF2{alpha}-induced decrease in progesterone concentrations from 184 ± 29 to 388 ± 60 ng/g (P < 0.01), but not that of oestradiol. It can be concluded that NO up-regulates progesterone secretion and down-regulates oestradiol secretion in rat ovaries, and NO can reverse PGF2{alpha}-induced inhibition in ovarian progesterone secretion.

Key words: nitric oxide/oestradiol/ovary/prostaglandin/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is now recognized as an important intracellular and intercellular messenger molecule known to have diverse physiological and pathological roles in multiple systems (Moncada et al., 1991Go). Recent studies of the pregnant rat and rabbit demonstrated that an L-arginine-NO-guanosine cyclic monophosphate (cGMP) system exists in the uterus that inhibits contractility (Sladek et al., 1993Go; Yallampalli et al., 1993Go). The substrate and donors of NO were shown to relax uterine tissues, and these effects were reversed by inhibitors of the NO-cGMP pathway (Yallampalli et al., 1994Go). Reports show that NO production by the rat uterus increases during pregnancy and nitric oxide synthase activity decreases during labour (Yallampalli et al., 1994Go, 1998Go) or before the onset of labour at term (Sladek and Roberts, 1996Go). These data suggested that the NO system inhibits uterine contractility and may play a role in maintaining uterine quiescence during pregnancy. In addition, it has been found that provision of NO prevented prostaglandin F2{alpha} (PGF2{alpha})-induced preterm labour in rats, probably by blocking PGF2{alpha}-induced uterine contractions and attenuating the fall in the serum progesterone concentration (Dong et al., 1997Go). However, the effects of NO on ovarian steroidogenesis remain unclear.

Several lines of evidence support the involvement of NO in ovarian physiology. First, nitrates are secreted from mammalian ovarian dispersates (Ellman et al., 1993Go). Second, NO synthase has been localized to granulosa luteal cells by immunocytochemistry (Van Voorhis et al., 1994Go; Zackrisson et al., 1996Go; Matsumi et al., 1998Go). Third, NO synthase inhibitors block human chorionic gonadotrophin (HCG)-induced ovulation in the rat (Shukovski and Tsafriri, 1994Go). Fourth, endogenously produced NO and NO-releasing agents have been shown to inhibit steroidogenesis in granulosa and lutea cells in the rat and human (Van Voorhis et al., 1994Go; Olson et al., 1996Go). Finally, several studies indicate that there are interactions between NO, prostaglandins and ovarian steroidogenesis (Ahsan et al., 1997Go; Masuda et al., 1997Go; Yamauchi et al., 1997Go; Matsumi et al., 1998Go). These close relations between NO and ovarian functions suggest a positive role for NO in the process of ovulation. In the present study, we examined the actions of NO on ovarian steroidogenesis and on PGF2{alpha}-induced inhibition of progesterone in the in-vitro cultured rat ovaries. These studies were designed to determine if PGF2{alpha}-induced decreases in progesterone synthesis by the ovaries can be rescued by NO and thus maintain the ovarian progesterone secretion.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Immature female Sprague-Dawley rats were purchased from Harlan Sprague–Dawley (Houston, TX, USA) at 21–23 days of age, at which time they weighed 45–50 g. The animals were maintained on a 12 h light and 12 h dark schedule, and allowed free access to food and water. Rats received an s.c. injection of 4 IU of pregnant mare's serum gonadotrophin (PMSG, Sigma, St Louis, MO, USA) in 0.2 ml of saline at 9:00 a.m. on day 27 of age to induce first oestrus and ovulation (Nuti et al., 1975Go). Rats were killed in a CO2 inhalation chamber 72 h after the injection of PMSG, and the ovaries were collected, weighed and cultured as described below. All procedures involving the care and use of animals were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.

Ovarian cultures
Both ovaries from each animal were removed aseptically into sterile Dulbecco's modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) buffered with HEPES (15 mM) and supplemented with L-glutamine and antibiotics in plastic 24 multi-well tissue culture plates (Corning Glass Works, NY, USA). Each well containing 1.0 ml of DMEM with diethylenetriamine–nitric oxide (DETA/NO, 0, 1x10–6, 1x10–5, and 1x10–4 M, was generously provided by Dr L. Keifer, National Cancer Institute, Frederick, MD, USA), NG-nitro-L-arginine methyl ester (L-NAME, 1x10–4 M, Sigma), diethylenetriamine (DETA, 1x10–4M, Aldrich, Milwaukee, WI, USA), prostaglandin F2{alpha} (PGF2{alpha}, 1x10–6 M, Sigma), PGF2{alpha} plus DETA/NO, DETA or indomethacin (1x10–5 M, Sigma). Ovaries from six rats per treatment were incubated in an incubator with a humidified chamber and 5% carbon dioxide at 37°C. After 24 h incubations, the medium was collected to measure the concentration of progesterone and 17ß-oestradiol. Separate aliquots were taken for progesterone and oestradiol determination. Results are expressed as ng progesterone per gram ovary or pg 17ß-oestradiol per gram ovary.

Progesterone and oestradiol measurements
Progesterone and 17ß-oestradiol concentrations were determined in the aliquots of medium incubated with ovaries using commercial radioimmunoassay kits (Diagnostic Systems Laboratories, Inc. Webster, TX, USA). All samples and standards were assayed in duplicate. The inter- and intra-assay coefficients of variation at the concentrations obtained in these experiments were 6.4 and 2.4% respectively for progesterone, and 8.9 and 7.5% respectively for oestradiol.

Statistics
The values (n = 6) were given as mean ± SEM. Differences were tested for significance using one-way analysis of variance (ANOVA) or Student's t-test for paired or unpaired data where appropriate. If the ANOVA test was significant, the individual groups were compared further using Bonferroni's t-test. A P value of < 0.05 was treated as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of nitric oxide on ovarian progesterone synthesis
The progesterone secretion by rat ovaries was increased by DETA/NO in a dose-dependent manner. Progesterone synthesis (ng/g ovary) was 355 ± 43, 443 ± 46 and 647 ± 55 when ovaries were incubated with 1x10–6, 1x10–5 and 1x10–4 M DETA/NO respectively (Figure 1Go). These stimulatory effects of DETA/NO appeared to be specific to NO, since DETA alone at these concentrations was without significant effects on the ovarian progesterone synthesis (Figure 2Go). However, L-NAME, at 100 µM, had no significant inhibitory effects on ovarian progesterone secretion (Figure 2Go).



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Figure 1. Dose-dependent effects of diethylenetriamine–nitric oxide (DETA)/NO on progesterone secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) with varying doses of DETA/NO (0, 1x10–6, 1x10–5, 1x10–4 M). Progesterone concentration in the medium were determined by radioimmunoassay. Results are means ± SEM (n = 6). *Indicates P < 0.05 versus control (ANOVA).

 


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Figure 2. The effect of nitric oxide synthase (NOS) inhibitor L-NAME (1x10–4 M), NO donor diethylenetriamine–nitric oxide (DETA)/NO (1x10–4 M), or DETA (1x10–4 M) on progesterone secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) alone or with these agents as indicated. Progesterone concentrations in the medium were determined by radioimmunoassay. Results are means ± SEM (n = 6). a,bBars with different superscripts were significantly different (P < 0.05) according to ANOVA and Bonferroni's t-test.

 
The progesterone secretion by rat ovaries was decreased by PGF2{alpha} (1x10–6 M) compared to control (184 ± 29 versus 326 ± 46 ng/g ovary, P < 0.01, Figure 3Go). Co-incubation with DETA/NO (1x10–4 M) reversed the PGF2{alpha}-induced decrease in progesterone concentration (388 ± 60 versus 184 ± 29 ng/g ovary, P < 0.01). Indomethacin (1x10–5 M), an inhibitor of endogenous PGF2{alpha}, significantly increased the ovarian progesterone secretion from 323 ± 47 to 424 ± 43 ng/g ovary (P < 0.01, Figure 3Go), indicating that endogenous prostaglandin inhibits progesterone secretion by rat ovaries.



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Figure 3. The effect of prostaglandin F2{alpha} (PGF2a) (1x10–6 M), PGF2{alpha} (1x10–6 M) plus diethylenetriamine–nitric oxide (DETA)/NO (1x10–4 M), PGF2{alpha} (1x10–6 M) plus DETA (1x10–4 M), and indomethacin (IM, 1x10–5 M) on progesterone secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) alone or with these agents as indicated. Progesterone concentrations in the medium were measured by radioimmunoassay. Results are means ± SEM (n = 6). a,b,cBars with different superscripts were significantly different (P < 0.05) according to ANOVA and Bonferroni's t-test.

 
Effect of nitric oxide on ovarian oestradiol synthesis
The oestradiol secretion by rat ovaries was decreased by DETA/NO in a dose dependent manner. Oestradiol synthesis (pg/g ovary) was 408 ± 51, 273 ± 28 and 133 ± 35 when ovaries were incubated with 1x10–6, 1x10–5 and 1x10–4 M DETA/NO respectively (Figure 4Go). These inhibitory effects of DETA/NO appeared to be specific to NO, since DETA alone at 100 µM had no significant effect on the ovarian oestradiol synthesis (Figure 5Go). However, L-NAME, at 100 µM had no significant stimulatory effects on ovarian oestradiol secretion (Figure 5Go).



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Figure 4. Dose-dependent effects of diethylenetriamine–nitric oxide (DETA)/NO on 17ß-oestradiol secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) with varying doses of DETA/NO (0, 1x10–6, 1x10–5, 1x10–4 M). 17ß-Oestradiol concentrations in the culture medium were determined by radioimmunoassay. Results are means ± SEM (n = 6). *Indicates P < 0.05 versus control (ANOVA).

 


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Figure 5. The effect of NOS inhibitor L-NAME (1x10–4 M), NO donor diethylenetriamine–nitric oxide (DETA)/NO (1x10–4 M), and DETA (1x10–4 M) on 17ß-oestradiol secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) alone or with these agents as indicated. 17ß-Oestradiol concentrations in the medium were determined by radioimmunoassay. Results are means ± SEM (n = 6). a,bBars with different superscripts were significantly different (P < 0.05) according to ANOVA and Bonferroni's t-test.

 
The oestradiol secretion by rat ovaries was decreased by PGF2{alpha} (1x10–6 M) compared to control (166 ± 36 versus 435 ± 55 pg/g ovary, P < 0.01, Figure 6Go). Co-incubations with either DETA/NO or DETA did not significantly change the PGF2{alpha}-induced inhibition in oestradiol secretion (163 ± 34 and 162 ± 50 pg/g versus 166 ± 36 pg/g in control, P = 0.07). Indomethacin at 1x10–5 M concentration had no significant effect on rat ovary oestradiol secretion (Figure 6Go).



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Figure 6. The effect of PGF2{alpha} (1x10–6 M), PGF2{alpha} (1x10–6 M) plus diethylenetriamine–nitric oxide (DETA)/NO (1x10–4 M), PGF2{alpha} (1x10–6 M) plus DETA (1x10–4 M), and indomethacin (IM, 1x10–5 M) on oestrogen secretion by rat ovaries in the cultures. Ovaries were incubated for 24 h in Dulbecco's modified Eagle's medium (DMEM) alone or with these agents as indicated. 17ß-Oestradiol concentrations in the medium were measured by radioimmunoassay. Results are means ± SEM (n = 6). Results are means ± SEM (n = 6). a,bBars with different superscripts were significantly different (P < 0.05) according to ANOVA and Bonferroni's t-test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been demonstrated that NO plays a role in endothelium-dependent vascular relaxation, phagocytic cell cytotoxicity, central nervous system neurotransmission and uterine relaxation (Moncada et al., 1991Go; Moncada, 1992Go; Nathan, 1992Go; Yallampalli et al., 1993Go). In the present studies, it has been demonstrated that NO may also function as a regulator of rat ovarian steroidogenesis. This conclusion is supported by the following observations: (i) DETA/NO, a compound that spontaneously released NO, caused a dose-dependent increase in progesterone synthesis with a concomitant decrease in ovarian oestradiol secretion; (ii) neither progesterone nor oestradiol concentrations in the culture medium were altered when ovaries were incubated with DETA without NO; (iii) co-incubation with DETA/NO significantly reversed the PGF2{alpha}-induced decrease in progesterone secretion, but not that of oestradiol. These findings suggest that NO may modulate ovarian functions and play a significant role in controlling ovarian steroidogenesis by acting as one of the intra-ovarian regulators.

Granulosa cells and luteal cells in the rat ovary synthesize NO (Powers et al., 1995Go; Van Voorhis et al., 1995Go; Olson et al., 1996Go), which has been postulated to play a number of roles in ovarian physiology. It has been reported that endothelial nitric oxide synthase (NOS III) and inducible nitric oxide synthase (NOS II) are expressed in the theca cell layer and stromal cells of pre-ovulatory follicles (Jablonka-Sharrif and Olson, 1997). NO has been suggested to act as a potent and fast-acting signal that regulates the barriers ability to block the entrance of certain blood components into the follicular fluid (Powers et al., 1995Go). The ovarian NO/NOS system also has been suggested to function in ovulation and follicle rupture through its known effects on vascular dilation and ovulatory leukocyte distribution (Shukovski and Tsafriri, 1994Go; Bonello et al., 1996Go). Pharmacological inhibitors of NOS suppress HCG-induced ovulation in rodents (Shukovski and Tsafriri, 1994Go; Powers et al., 1995Go). The physical processes of rupture and cellular reorganization accompanying the transformation of ovulatory follicles into young corpora lutea require extensive tissue remodelling and cell death and NO may participate in these processes. These studies together with the findings of our study support a role for ovarian NO in regulating ovarian steroidogenesis.

It is interesting to note that NO stimulates rat ovarian progesterone secretion in a dose-dependent manner, suggesting a possible new physiological site of action that may contribute to the regulation of ovulation and of luteal support. A neural pathway for the NO effects on ovulation may exist as evidenced by reports identifying hypothalamic NOS-containing neurons that regulate luteinizing hormone releasing hormone secretion via guanylate cyclase (Moretto et al., 1993Go) and NOScontaining nerve fibres present in the rat ovary (Jarrett et al., 1994Go). Further, it is also reasonable to consider NO as a local modulator of steroidogenesis, since many of its actions are mediated by iron-containing enzymes such as guanylate cyclase and cyclooxygenase (Savlemini et al., 1993Go). The cytochrome P-450 steroidogenic enzymes also contain an iron–haem centre (Guengerich, 1989Go), and therefore the activities of these enzymes could be altered by NO. Ovarian cells at all stages of follicular development synthesized NO, and this basal NO production in these cells increases by 6–14 fold when luteinized (Olson et al., 1996Go). Furthermore, modulation of NO synthesis in these cells has also been shown to alter ovarian steroidogenesis (Olson et al., 1996Go). Thus, NO may play a role in ovarian physiology.

Addition of the NO generator, DETA/NO, significantly reduced the oestradiol production by rat ovary in a dose-dependent manner, suggesting that NO inhibits the ovarian synthesis of oestradiol. The observations in this study are consistent with previous studies in which NO has been shown to reduce oestradiol synthesis in human granulosa–luteal cells (Van Voorhis et al., 1994Go), rat luteinized ovarian cells (Olson et al., 1996Go) and rat Leydig cells (Punta et al., 1996Go). The reports from Van Voorhis et al. (1994) revealed that the NO donors S-nitroso-L-acetyl penicillamine and S-nitroso glutathione inhibited both oestradiol secretion and aromatase activity. Further, aromatase activity in microsomal preparations of granulosa cells was inhibited by native NO. It is certainly possible that NO acts in an autocrine manner to regulate the amount of oestradiol produced through modulating aromatase activity.

In the present study, it was found that NO upregulated progesterone and downregulated oestradiol production by rat ovaries, suggesting that NO may contribute in part to the regulatory influence on steroid hormone synthesis. In terms of the mechanisms involved, it can be speculated that an increase in ovarian progesterone production may result from enhanced bioactivity of enzymes involved in the pathway of the conversion of cholesterol to progesterone (Gore-Langton and Armstrong, 1988Go). On the other hand, the decreased oestradiol secretion may be due to inhibition of aromatase P450, which is required for synthesis of oestradiol-17ß (Gore-Langton and Armstrong, 1988Go). The mechanisms by which NO alters the enzymes involved in the steroidogenic pathway are unclear. It is possible that NO could bind to the iron–sulphur moiety of some of these enzymes (Salvemini et al., 1993) and thus alter their activity, or alternatively NO could also affect the gene expression of these enzymes. NO inhibited 11ß-hydroxysteroid dehydrogenase (HSD) type 2 oxidase activity in the human placental trophoblast and reduced the concentrations of type 2 mRNA in the placental syncytiotrophoblast (Sun et al., 1997Go), indicating that NO may affect the activity and gene transcription of the 11ß-HSD type 2. Further, it has been recently reported that NO influences glyceraldehyde-3-phosphate dehydrogenase (GADPH) activity by covalent linkage of NAD+ to the enzyme through ADP-ribosylation (Marin et al., 1995Go), which in turn decreases the NAD+ available for steroid dehydrogenation. Further studies are required to the understanding of mechanisms of NO effects on ovarian steroidogenesis.

The effects of the exogenously added NO on ovarian steroidogenesis are apparent from various studies. However, the effects of endogenous NO in this organ of the rat are not clear. It has been reported that intrabursal administration of L-NAME (0.1 and 1 mg/kg) in equine chorionic gonadotrophin(eCG)–HCG treated immature rats results in 34 and 32% inhibition of ovulation (Shukovski et al., 1994). In in-vitro perfused rabbit ovaries, administration of L-NAME (1x10–8 to 1x10–5 M) to the perfusate inhibited the ovulation induced by HCG in a dose-dependent manner (Yamauchi et al., 1997Go). However, in the current studies, L-NAME at a dose of 1x 10–4 M did not significantly alter the steroidogenesis by the rat ovary. Therefore, it is possible the threshold concentration of L-NAME required to alter ovulation compared with steroidogenesis may be different. Further studies are required to ascertain the effects of endogenous NO synthesis on ovulation and ovarian steroidogenesis using various NO inhibitors, doses and time periods.

Prostaglandins are important physiological regulators of granulosa cell proliferation during ovarian follicular development and luteal function. It has been demonstrated that PGF2{alpha} dose-dependently inhibited forskolin-induced cAMP and progesterone synthesis as well as the progesterone synthesis induced by dibutyryl-cAMP in isolated rat luteal cells (Kenny and Robinson, 1986Go). These data suggested that the antigonadotrophic effect of PGF2{alpha} has more than one locus of action, i.e. it both inhibits an adenylate cyclase event associated with cAMP generation and blunts the cellular response to cAMP (Kenny and Robinson, 1986Go). Also, it has been shown that progesterone concentrations in the corpora lutea declined significantly by 12 and 24 h after PGF2{alpha} treatment (Hehnke et al., 1994Go), indicating an inhibitory influence of PGF2{alpha} on ovarian progesterone synthesis. The current experiments support these in-vivo studies, since PGF2{alpha} caused a decrease in progesterone synthesis by the rat ovary in culture. Interestingly, in this study it was found that co-incubation with DETA/NO significantly reversed the PGF2{alpha}-induced decrease in progesterone secretion. Conversely, indomethacin, an inhibitor of endogenous PGF2{alpha}, significantly increased the ovarian progesterone synthesis, indicating that endogenous prostaglandins inhibit progesterone secretion by rat ovaries. Since NO has been reported to inhibit prostaglandin production in some tissues (Stadler et al., 1993Go), it is possible that suppression of endogenous prostaglandin production by the NO may be a cause for the increase in progesterone secretion by DETA/NO. Together with our previous findings that NO can prevent PGF2{alpha}-induced preterm labour in the rat (Dong et al., 1997Go), these results may have important implications in the understanding of the role NO and prostaglandins during pregnancy, parturition, and in the control of luteolysis.

PGF2{alpha} inhibited both progesterone and oestradiol secretion from the PMSG-stimulated ovary, but these inhibitions were differentially modulated by DETA/NO. The NO donor reversed the decreases in progesterone synthesis, but was unable to restore oestradiol synthesis. This inability of the NO donor to reverse the PGF2{alpha}-induced decrease in oestradiol synthesis is not surprising, since the NO donor by itself inhibited oestradiol synthesis in a dose-dependent manner, in similarity to other studies (Van Voorhis et al., 1994Go; Olson et al., 1996Go), as discussed earlier. Although we did not measure aromatase activity directly in this study, results from our studies and those of others suggest that NO inhibits aromatase activity. On the other hand, enhanced progesterone synthesis by NO may involve enhancement of the progesterone synthetic pathway. While the data from indomethacin treated ovaries support a role for prostaglandins in progesterone synthesis but not for oestradiol synthesis. Thus both NO donor and indomethacin have differential effects on progesterone and oestradiol synthesis. From the current studies, the mechanisms responsible for these differential effects are not clear and will require further mechanistic studies.

In conclusion, this study revealed that NO stimulates progesterone and inhibits oestradiol secretion by rat ovaries. These findings may have important significance in the understanding of normal physiological functions of the ovary, including the control of ovarian steroidogenesis. We speculate that elevated NO synthesis during pregnancy could reduce or prevent luteolytic effects of prostaglandins during pregnancy and thus maintain adequate progesterone. This isolated ovarian culture has provided an ideal model to conduct detailed studies of this organ independent of systemic influence, such as pregnancy, gonadotrophins and other endocrine factors. Further investigations are required to define better the functional role of different isoforms of NO synthase in the ovary in controlling steroidogenesis during pregnancy.


    Acknowledgments
 
This work was supported by grants HD 30273, HL 58144, from the National Institutes of Health to C.Yallampalli.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, 301 University Boulevard, Medical Research Building Room 11.138, Galveston, Texas 77555–1062, USA Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 11, 1998; accepted on September 24, 1998.