Neuromodulatory effect of oestradiol in the metabolism of ovarian progesterone and oestradiol during dioestrus II: participation of the superior mesenteric ganglion.

The aims of the present study were to determine: (1) whether oestradiol (E2) in the superior mesenteric ganglion (SMG) modifies the release of ovarian progesterone (P4), androstenedione (A2) and E2, the activity and gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-HSD and the expression of P450 aromatase (Cyp19a1) and (2) whether any such modifications are related to changes in ovarian nitric oxide (NO) and noradrenaline (NA) levels during dioestrus II. Using an ex vivo SMG-ovarian nervous plexus-ovary system, ovarian P4 release was measured following the addition E2 plus tamoxifen (Txf) (10-6M) to the ganglion, whereas A2, E2, NA and NO were measured following the addition of E2 alone. Steroids were measured by radioimmunoassay, NA concentrations were determined by HPLC and gene expression was evaluated using reverse transcription-polymerase chain reaction. Oestradiol in the ganglion decreased ovarian P4, E2 and NA release, as well as 3ß-HSD activity, but increased the release of A2 and nitrites, as well as the 20α-HSD expression and its activity. No changes were observed in Cyp19a1 gene expression. The addition of E2 plus Txf to the ganglion reversed the effects of E2 alone. The action of oestradiol in SMG favours the beginning of functional luteolysis, due to an increase in NO release and a decrease in NA in the ovary. These results may help elucidate the role of E2 in hormone-dependent pathologies in women.

Noradrenaline modulates the presence of gonadotropin-releasing hormone in ovary. The importance of its interrelation on the ovarian steroidogenesis and apoptosis on dioestrus II in rat.

The aim of this work was to investigate if noradrenaline (NA), added in the coeliac ganglion -superior ovarian nerve- ovary system (CG-SON-O) and in ovary incubation, modifies the release of ovarian progesterone (P4), gonadotropin-releasing hormone (GnRH) and oestradiol (E2), and the expression of 3ß-HSD and 20α-HSD and proapoptotic bax and antiapoptotic bcl-2 on dioestrus II in the rat. The CG-SON-O system and the ovary were removed and placed in one cuvette containing Krebs-Ringer solution (control groups), and NA was added to the ganglion compartment in the ex vivo system and in the ovary compartment in the ovary incubation (experimental groups). P4, GnRH and E2 were measured by RIA, and gene expression was measured by RT-PCR. In the ex-vivo system, the release of ovarian P4 and GnRH and the expression of 3ß-HSD and bax decreased; E2 and bcl-2 increased, and the bax/bcl-2 ratio decreased. However, in the ovary incubation, P4, GnRH, the expression of 3ß-HSD and bax increased; E2, the expression of 20α-HSD and bcl-2 decreased while the bax/bcl-2 ratio increased, thus favoring apoptosis. The peripheral nervous system protected the ovary from the apoptotic mechanisms while in the ovary incubation the effect was reverted. Our results indicate that NA regulates ovarian steroidogenesis and apoptosis by modulating GnRH release from the coeliac ganglion and ovary, being NA a possible generator of a GnRH-gonadotropins axis in the ovary. This work is expected to contribute with new evidence of the clinical importance of catecholamines and GnRH in therapy and prevention of ovarian pathologies.

Involvement of the ganglion cholinergic receptors in gonadotropin-releasing hormone, catecholamines, and progesterone release in the rat ovary.

OBJECTIVE: To investigate whether cholinergic ganglionic stimulus modifies the release of gonadotropin-releasing hormone (GnRH), catecholamines, and progesterone at the ovarian level. DESIGN: Animal study. SETTING: University animal laboratory. ANIMAL(S): Six to eight virgin adult Holtzman rats. INTERVENTION(S): Superior mesenteric ganglion-ovarian nerve plexus-ovary system removed and placed in one cuvette with two compartments, with acetylcholine added to the ganglion in the experimental group. MAIN OUTCOME MEASURE(S): Measurement of ovarian liquid obtained from catecholamines by high-performance liquid chromatography; measurement of progesterone (P(4)), GnRH, and luteinizing hormone (LH) by radioimmunoassay; and measurement of gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULT(S): The study focused on the estrus and diestrus II (DII) stages. On the estrus days, the release of GnRH, NA, and 20α-HSD increased, while P(4) and 3ß-HSD decreased. On the DII days, GnRH, P(4), and 3ß-HSD increased, while 20α-HSD and NA decreased. The ovarian liquid with GnRH showed biologic activity, namely, an increase in LH release during the DII stage and a decrease during the estrus stage. CONCLUSION(S): Neural stimulus from the superior mesenteric ganglion influences the release of NA, adrenaline, and GnRH. We also have demonstrated that these neurotransmitters participate in the atretogenic processes of the ovary, thus providing evidence of the necessity of the sympathetic neural pathway.

Involvement of the oestrogenic receptors in superior mesenteric ganglion on the ovarian steroidogenesis in rat.

Oestradiol (E(2)) is a key hormone in the regulation of reproductive processes. The aims of this work were a) to examine the distributions of oestrogen receptor α (ERα) and ERß in the neurons of the superior mesenteric ganglion (SMG) in the oestrus stage by immunohistochemistry, b) to demonstrate whether E(2) in the SMG modifies progesterone (P(4)), androstenedione (A(2)) and nitrite release in the ovarian compartment on oestrus day and c) to demonstrate whether E(2) in the ganglion modifies the activity and gene expression in the ovary of the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). The ex vivo SMG-ovarian nervous plexus-ovary system was used. E(2), tamoxifen (Txf) and E(2) plus Txf were added in the ganglion to measure ovarian P(4) release, while E(2) alone was added to measure ovarian A(2) and nitrites release. Immunohistochemistry revealed cytoplasmic ERα immunoreactivity only in the neural somas in the SMG. E(2) increased ovarian P(4) and A(2) release at 15, 30 and 60 min but decreased nitrites. The activity and gene expression of 3ß-HSD increased, while the activity and gene expression of 20α-HSD did not show changes with respect to the control. Txf in the ganglion diminished P(4) release only at 60 min. E(2) plus Txf in the ganglion reverted the effect of E(2) alone and the inhibitory effect of Txf. The results of this study demonstrate that ERα activation in the SMG has an impact on ovarian steroidogenesis in rats, thus providing evidence for the critical role of peripheral system neurons in the control of ovarian functions under normal and pathological conditions.

Estradiol promotes luteal regression through a direct effect on the ovary and an indirect effect from the celiac ganglion via the superior ovarian nerve.

There is evidence suggesting that estradiol (E(2)) regulates the physiology of the ovary and the sympathetic neurons associated with the reproductive function. The objective of this study was to investigate the effect of E(2) on the function of late pregnant rat ovaries, acting either directly on the ovarian tissue or indirectly via the superior ovarian nerve (SON) from the celiac ganglion (CG). We used in vitro ovary (OV) or ex vivo CG-SON-OV incubation systems from day 21 pregnant rats. Various concentrations of E(2 )were added to the incubation media of either the OV alone or the ganglion compartment of the CG-SON-OV system. In both experimental schemes, we measured the concentration of progesterone in the OV incubation media by radioimmunoassay at different times. Luteal messenger RNA (mRNA) expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) enzymes, respectively, involved in progesterone synthesis and catabolism, and of antiapoptotic B-cell lymphoma 2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax), were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) at the end of the incubation period. Estradiol added directly to the OV incubation or to the CG of the CG-SON-OV system caused a decline in the concentration of progesterone accumulated in the incubation media. In addition, E(2), when added to the OV incubation, decreased the expression of 3ß-HSD and the ratio of Bcl-2/Bax. We conclude that through a direct effect on the OV, E(2) favors luteal regression at the end of pregnancy in rats, in association with neural modulation from the CG via the SON.

Androstenedione acts on the coeliac ganglion and modulates luteal function via the superior ovarian nerve in the postpartum rat.

Androstenedione can affect luteal function via a neural pathway in the late pregnant rat. Here, we investigate whether androstenedione is capable of opposing to regression of pregnancy corpus luteum that occurs after parturition, indirectly, from the coeliac ganglion. Thus, androstenedione was added into the ganglionar compartment of an ex vivo coeliac ganglion-superior ovarian nerve-ovary system isolated from non-lactating rats on day 4 postpartum. At the end of incubation, we measured the abundance of progesterone, androstenedione and oestradiol released into the ovarian compartment. Luteal mRNA expression and activity of progesterone synthesis and degradation enzymes, 3ß-hydroxysteroid-dehydrogenase (3ß-HSD) and 20α-hydroxysteroid-dehydrogenase (20α-HSD), respectively, as well as the aromatase, Bcl-2, Bax, Fas and FasL transcript levels, were also determined. Additionally, we measured the ovarian release of norepinephrine, nitric oxide and luteal inducible nitric oxide synthase (iNOS) mRNA expression. The presence of androstenedione in the ganglion compartment significantly increased the release of ovarian progesterone, androstenedione and oestradiol without modifying 3ß-HSD and 20α-HSD activities or mRNA expression. The ovarian release of oestradiol in response to the presence of androstenedione in the ganglion compartment declined with time of incubation in accord with a reduction in the aromatase mRNA expression. Androstenedione added to the ganglion compartment decreased FasL mRNA expression, without affecting luteal Bcl-2, Bax and Fas transcript levels; also increased the release of norepinephrine, decreased the release of nitric oxide and increased iNOS mRNA. In summary, on day 4 after parturition, androstenedione can mediate a luteotropic effect acting at the coeliac ganglion and transmitting to the ovary a signaling via a neural pathway in association with increased release of norepinephrine, decreased nitric oxide release, and decreased expression of FasL.

Modulation of the noradrenergic activity index by neural stimulus, and its participation in ovarian androstenedione release during the luteal phase.

OBJECTIVE: To investigate the participation of catecholamines in the association between peripheral innervation and luteal steroidogenesis. DESIGN: Animal study. SETTING: University animal laboratory. ANIMAL(S): Six to eight virgin adult Holtzman-strain female rats in control and experimental groups on diestrus days 1 and 2. INTERVENTION(S): Removal of the coeliac ganglion-superior ovarian nerve-ovary system, with catecholaminergic agonist or antagonist added in the ganglion compartment (experimental group only). The control group received no treatment. MAIN OUTCOME MEASURE(S): Ovarian neurotransmitters and their catabolites measured by reverse-phase high-pressure liquid chromatography, and A(2) measured by radioimmunoassay. RESULT(S): On day 1, dopamine and catabolite increased whereas norepinephrine decreased, and the noradrenergic neuronal activity index was higher. On day 2, dopamine levels decreased, norepinephrine increased, and dopaminergic neuronal activity was higher. The release of A(2) was decreased by addition of norepinephrine to the ganglions on day 1, but was increased by the norepinephrine antagonist on day 2. Hence, norepinephrine increased A(2) release, and propranolol diminished it. CONCLUSION(S): Ganglionic activity is modified by noradrenergic stimulus, leading to different ovarian A(2) release profiles. The peripheral nervous system is a modulator in these homeostatic mechanisms.

Ovaric physiology in the first oestral cycle: influence of noradrenergic and cholinergic neural stimuli from coeliac ganglion.

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The aims of the work were (1) to investigate whether ganglionic noradrenergic (NE) and cholinergic (Ach) stimulus modify the ovarian steroids and NO release and (2) to examine the effect of those stimuli on the mRNA expression of 3beta-HSD and P450 aromatase in the ovary. The experiments were carried out using the ex vivo coeliac ganglion-superior ovarian nerve-ovary (CG-SON-O) system of rats in the first oestral cycle. The system was incubated in a buffer solution for 120min, with the ganglion and ovary located in different compartments and linked by the SON. NE and Ach were added into the ganglion compartment. Both NE and Ach predominantly induced ovarian release of androstenedione and oestradiol while inhibited progesterone release. Ovarian NO release increased after ganglionic stimulation during proestrous while its secretion decreased during the diestrous. Noteworthily, 3beta-HSD and P450 aromatase expression were modulated by neural stimulation. In the follicular phase, Ach in CG increased 3beta-HSD and NE increased P450 aromatase. In the luteal phase both neurotransmitters increased 3beta-HSD and Ach increased P450 aromatase transcript levels. All above observations suggest that the preponderancy of an either noradrenergic or cholinergic effect would depend on the stage of the first oestral cycle in the rat. The ovarian response to noradrenergic and cholinergic stimuli on GC, via SON, is strongly linked to oestral-stage-specific ovarian structures and their secretion products.

Involvement of ganglionic cholinergic receptors on the steroidogenesis in the luteal phase in rat.

The ovarian nervous plexus (ONP) is one of the principal extrinsic innervation pathways reaching the ovary from the superior mesenteric ganglion (SMG). The aims of this work were: (a) to determine if acetylcholine (Ach) in the SMG modifies the release of steroids and ovarian nitrites in an ex vivo SMG-ONP-ovary system on dioestrus (D) I and II, and (b) to demonstrate if the activities and gene expression of the steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) are modified by cholinergic stimulus. The system was incubated in Krebs-Ringer buffer bicarbonate at 37 degrees C in metabolic bath. Ach (10(-6)M) was used as cholinergic agonist. Ach in SMG increased progesterone release at all the incubation times on DI and DII (*p<0.001). Androstenedione increased at 15 and 30min on DI, and at 30min on DII whereas nitric oxide (NO) increased at 30min on DI, and at 15 and 30min on DII. The activity of 3beta-HSD increased whereas the activity of 20alpha-HSD decreased (*p<0.001) on DI and DII. The gene expression of 3beta-HSD showed a significant increase at 120min on DI and DII ((o)p<0.01) and 20alpha-HSD diminished only on DII. The results show the importance of the SMG via the ovarian nervous plexus on the regulation of the steroid secretory activity and on the ovarian release of NO in the luteal phase. The complex synaptic connections in the prevertebral ganglia and the sympathetic ganglionic chain participate in the neuroendocrinological mechanisms that take place during the luteal steroidogenesis.

Administration of noradrenaline in the autonomic ganglia modifies the testosterone release from the testis using an ex vivo system.

The male gonad receives nerve fibres from the autonomic ganglionic system. These fibres converge on the testis along two pathways, the superior and the inferior spermatic nerves. The superior spermatic nerve runs from the superior mesenteric ganglion alongside the testicular artery, whereas the inferior spermatic nerve originates in inferior mesenteric ganglion, accompanies the vas deferens and penetrates the inferior pole of the testis. The aim of this work was to evaluate androgen release after the addition of noradrenaline or adrenoreceptor antagonists (propranolol or phentolamine) to the ganglionic compartment. An ex vivo system used in a previous work was incubated in two separate containers, one for the testis and the other for the ganglion. Both organs remain interconnected (as in vivo) by the respective spermatic nerve. When noradrenaline was added to the inferior mesenteric ganglion, testosterone release in the gonad container underwent a progressive and significant increment. Propranolol diminishes and phentolamine increases the androgen release. When using the superior mesenteric ganglion, no changes were observed. These results indicate that the ganglionic stimulation of the autonomic system clearly participates in testosterone release from the testis. This effect depends on the ganglion involved. These results make it evident that not only the classical and well-known hypothalamus-hypophysial axis, but also the peripheral nervous system, via the autonomic ganglia, are directly involved in the endocrine control of the testis.

Androgen receptors in coeliac ganglion in late pregnant rat.

The ovarian function is controlled by endocrine factors and neural influence. In late pregnant rat, androstenedione, from the coeliac ganglion, has a luteotrophic effect in the ex vivo coeliac ganglion-superior ovarian nerve-ovary system. In this work we investigate the presence of androgen receptors in the coeliac ganglion of late pregnant rats by immunohistochemistry. We also explore, from a physiological point of view, the potential participation of these receptors in the androstenedione ganglionic action on progesterone release and metabolism, as well as on nitrites release in the ovary compartment. The coeliac ganglion was isolated after being fixed in situ and immunohistochemistry was performed. In the system, three experimental groups were used with the addition of (a) androstenedione, (b) flutamide, and (c) androstenedione plus flutamide in the ganglion compartment. Progesterone and nitrite concentrations were determined in the ovary compartment at different incubation times. Corpora lutea samples isolated at the end of incubation were used to determine the expressions and activities of the progesterone synthesis (3beta-hydroxysteroid-dehydrogenase, 3beta-HSD) and degradation (20alpha-hydroxysteroid-dehydrogenase, 20alpha-HSD) enzymes. Immunohistochemistry revealed cytoplasmatic androgen receptor immunoreactivity in neural somas in the coeliac ganglion. In the coeliac ganglion-superior ovarian nerve-ovary system, androstenedione addition increased 3beta-HSD and decreased 20alpha-HSD, showed a tendency to decrease 20alpha-HSD expression, and increased nitrites release in relation to control. Androstenedione plus flutamide decreased progesterone and nitrites release in relation to the androstenedione group. This work demonstrates the presence of androgen receptors in neurons of celiac ganglion and provides evidence for the luteotrophic action of androstenedione via a neural pathway that may be mediated by these receptors.

Nitric oxide in prepubertal rat ovary contribution of the ganglionic nitric oxide synthase system via superior ovarian nerve.

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. Considering the existence of the nitric oxide/ nitric oxide synthase system in the peripheral neural system and in the ovary, the aim of this work was to analyze if the liberation of NO in the ovarian compartment of prepubertal rats is of ovarian and/or ganglionic origin. The analysis is carried out from a physiological point of view using the experimental coeliac ganglion--Superior Ovarian Nerve--ovary model with and without ganglionic cholinergic stimulus Acetylcholine (Ach) 10(-6) M. Non selective and selective inhibitors of the synthase nitric oxide enzyme were added to the ovarian and ganglionic compartment, and the liberation of nitrites (soluble metabolite of the nitric oxide) in the ovarian incubation liquid was measured. We found that the non-selective inhibitor L-nitro-arginina methyl ester (L-NAME) in the ovarian compartment decreased the liberation of nitrites, and that Aminoguanidine (AG) in two concentrations in a non-dose dependent form provoked the same effect. The addition of Ach in ganglion magnified the effect of the inhibitors of the NOS enzyme. The most relevant results after the addition of inhibitors in ganglion were obtained with AG 400 and 800 microM. The inhibition was made evident with and without the joint action of Ach in ganglion. These data suggest that the greatest production of NO in the ovarian compartment comes from the ovary, mainly the iNOS isoform, though the coeliac ganglion also contributes through the superior ovarian nerve but with less quantity.

The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system.

The axons that constitute the ovarian nervous plexus originate mostly in the principal neurons of the superior mesenteric ganglion (SMG) that is part of the sympathetic ganglionic chain and exhibits cholinergic receptors. In order to observe the effect of acetylcholine, the main neurotransmitter in the ganglionic transmission, the purpose of the present work was: first, to standardize an integrated ex vivo superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) system in oestrus day rats; secondly, to determine if the ganglionic cholinergic stimulus modifies the release of nitric oxide and steroids in the ovary compartment in the absence of humoral factors; and thirdly, to investigate if there are differences in the responses between the left and right ovaries caused by the neural stimulus. The ex vivo experimental left and right systems were developed and standardized. The systems were incubated in Krebs-Ringer phosphate buffer in a Dubnoff metabolic shaker. The progesterone release was determined to standardize the incubation times, obtaining different responses between the left and right systems, which shows that both systems have their own autonomic tone. Non-specific stimulation with KCl in the ganglion compartment provoked different responses in terms of release of progesterone and oestradiol. Progesterone decreased in the left and right systems. However, oestradiol diminished at short times and increased at 60 and 120 min in the left ovary, whereas it increases at 30 and 60 min in the right ovary. These different responses show the sensitivity and viability of both systems. When acetylcholine was used in the ganglion compartment, the release of nitric oxide, progesterone, androstenedione and oestradiol was evaluated. The liberation of nitrite increased at 15, 30 and 60 min in the left system and decreased in the right system at 120 min. Progesterone showed a decrease in its release at 15, 30 and 120 min and androstenedione at 15 min in the left ovary compartment. In the right ovary, only progesterone decreased in relation to the control at 120 min while androstenedione did not show significant changes. Oestradiol showed an increase in the left ovary compartment at all the studied times, while in the right ovary it did not show any changes. These results indicate that the neural stimulus from the superior mesenteric ganglion through the ovarian nervous plexus is one of the factors modulating the secretory activity of the ovarian steroids and nitric oxide. The system is viable and also shows a different sensitivity of the left ovary in relation to the right one at least in this cycle stage, characterized by marked irrigation and profound structural changes in the ovary.

The celiac ganglion modulates LH-induced inhibition of androstenedione release in late pregnant rat ovaries.

BACKGROUND: Although the control of ovarian production of steroid hormones is mainly of endocrine nature, there is increasing evidence that the nervous system also influences ovarian steroidogenic output. The purpose of this work was to study whether the celiac ganglion modulates, via the superior ovarian nerve, the anti-steroidogenic effect of LH in the rat ovary. Using mid- and late-pregnant rats, we set up to study: 1) the influence of the noradrenergic stimulation of the celiac ganglion on the ovarian production of the luteotropic hormone androstenedione; 2) the modulatory effect of noradrenaline at the celiac ganglion on the anti-steroidogenic effect of LH in the ovary; and 3) the involvement of catecholaminergic neurotransmitters released in the ovary upon the combination of noradrenergic stimulation of the celiac ganglion and LH treatment of the ovary. METHODS: The ex vivo celiac ganglion-superior ovarian nerve-ovary integrated system was used. This model allows studying in vitro how direct neural connections from the celiac ganglion regulate ovarian steroidogenic output. The system was incubated in buffer solution with the ganglion and the ovary located in different compartments and linked by the superior ovarian nerve. Three experiments were designed with the addition of: 1) noradrenaline in the ganglion compartment; 2) LH in the ovarian compartment; and 3) noradrenaline and LH in the ganglion and ovarian compartments, respectively. Rats of 15, 19, 20 and 21 days of pregnancy were used, and, as an end point, the concentration of the luteotropic hormone androstenedione was measured in the ovarian compartment by RIA at various times of incubation. For some of the experimental paradigms the concentration of various catecholamines (dihydroxyphenylalanine, dopamine, noradrenaline and adrenaline) was also measured in the ovarian compartment by HPLC. RESULTS: The most relevant result concerning the action of noradrenaline in the celiac ganglion was found on day 21 of pregnancy resulting in the inhibition of androstenedione release from the ovarian compartment. In addition on day 15 of pregnancy, LH placed in the ovarian compartment led to an inhibition of the release of androstenedione, and this inhibitory effect was further reinforced by the joint action of noradrenaline in the celiac ganglion and LH in the ovary. The levels of catecholamines in the ovarian compartment showed differences among the experiments; of significance, the joint treatment of noradrenaline in the celiac ganglion and LH in the ovary resulted in a remarkable increase in the ovarian levels of noradrenaline and adrenaline when compared to the effect achieved by either one of the compounds added alone. CONCLUSION: Our results demonstrate that the noradrenergic stimulation of the celiac ganglion reinforces the LH-induced inhibition of androstenedione production by the ovary of late pregnant rats, and that this effect is associated with marked changes in the release of catecholamines in the ovary.

Ganglionic adrenergic action modulates ovarian steroids and nitric oxide in prepubertal rat.

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The purpose of this work was to analyse the ganglionic adrenergic influence on the ovarian release of steroids and NO and the possible steroids/NO relationship. The experiments were carried out in the ex vivo coeliac ganglion-superior ovarian nerve (SON)-ovary system of prepubertal rats. The coeliac ganglion-SON-ovary system was incubated in Krebs Ringer-bicarbonate buffer in presence of adrenergic agents in the ganglionic compartment. The accumulation of progesterone, androstenedione, oestradiol and NO in the ovarian incubation liquid was measured. Norepinephrine in coeliac ganglion inhibited the liberation of progesterone and increased androstenedione, oestradiol and NO in ovary. The addition of alpha and beta adrenergic antagonists also showed different responses in the liberation of the substances mentioned before, which, from a physiological point of view, reveals the presence of adrenergic receptors in coeliac ganglion. In relation to propranolol, it does not revert the effect of noradrenaline on the liberation of progesterone, which leads us to think that it might also have a "per se" effect on the ganglion, responsible for the ovarian response observed for progesterone. Finally, we can conclude that the ganglionic adrenergic action via SON participates on the regulation of the prepubertal ovary in one of two ways: either increasing the NO, a gaseous neurotransmitter with cytostatic characteristics, to favour the immature follicles to remain dormant or increasing the liberation of androstenedione and oestradiol, the steroids necessary for the beginning of the near first estral cycle.

Involvement of the coeliac ganglion in the luteotrophic effect of androstenedione in late pregnant rats.

Using the ex vivo coeliac ganglion-superior ovarian nerve-ovary system at the end of pregnancy when luteal regression starts, we investigated whether, when administered systemically or when added directly to the ganglion compartment, androstenedione (A2) can reverse such regression, and whether the neural (noradrenaline (NA)) and endocrine (A2) joint action modifies the release of ovarian progesterone. The experimental groups were as follows: group 1--A2 injected systemically 48 h before incubation of the system (A2)s; group 2--A2 directly added to the ganglion compartment (A2)g; group 3--A2 injected 48 h before incubation of the system with NA in the ganglion compartment (A2 + NA); group 4--A2 plus NA added to the ganglion compartment (NA + A2)g. The controls were ex vivo systems without treatment (control), and with the addition of NA alone in the ganglion compartment (NA). The results were as follows. For (A2s) versus control, progesterone increased on days 19 and 21 of pregnancy at all the studied times and only at 180 min on day 20. For (A2 + NA) versus (A2)s, progesterone increased on days 19 and 21. For (A2 + NA) versus NA, progesterone increased at all the studied times on days 19 and 21 and at 180 min on day 20. For (A2)g versus control, progesterone significantly increased every pregnancy day. For (NA + A2)g versus (A2)g, progesterone decreased at 120 and 180 min on day 19. For (NA + A2)g versus NA, progesterone increased on days 20 and 21. We can conclude that A2 can reverse the functional regression of the corpus luteum either systemically or, what is more surprising, when directly added to the coeliac ganglion, whose action on the ovary is exerted via superior ovarian nerve.

Effect of the relation between neural cholinergic action and nitric oxide on ovarian steroidogenesis in prepubertal rats.

The coeliac ganglion and the ovary are related by the superior ovarian nerve, which penetrates into the ovary by the hilium and innervates mainly the ovarian stroma. On the other hand, it is known that the gaseous neurotransmitter nitric oxide (NO) and the two isoforms of its synthesis enzyme, the nitric oxide synthetase (NOS), are present in the ovary. Both innervation and NO participate in ovarian steroidogenesis. Therefore, the purposes of this work were (a) to standardize an in vitro coeliac ganglion-superior ovarian nerve-ovary integrated system in prepubertal rats; (b) to determine the presence of NO in the ovary and analyze the ganglionic cholinergic effect on the ovarian release of androstenedione, progesterone and NO; and (c) to assess the steroids/NO relationship. The system was incubated in buffer solution for 120 min, with the ganglion and ovary located in different compartments and linked by the superior ovarian nerve. From the results obtained, it is concluded that the system is viable and functional. The presence of basal NO is stimulated by the cholinergic action, while the release of the steroids is inhibited, which might indicate that the ganglionic cholinergic effect is probably mediated by NO. To our knowledge, this work constitutes the first study of the relationship between the neural cholinergic action and NO on the ovarian steroidogenesis of prepubertal rats.

Release of ovarian progesterone during the rat oestrous cycle by ganglionic cholinergic influence: the role of norepinephrine.

The coeliac ganglion neurons, whose axons constitute the superior ovarian nerve (SON), contain cholinergic receptors. The aim of this work was to study the effect of cholinergic agents added to the coeliac ganglion on the release of ovarian progesterone in the coeliac ganglion-SON-ovary in vitro system. We also analyzed the release of norepinephrine in the ovarian compartment and its possible relationship with the release of progesterone. After the addition of cholinergic agents in the ganglion compartment, progesterone release was determined by radioimmuneassay (RIA) and norepinephrine by catecholamine assay (HPLC). The release of progesterone and norepinephrine in the ovary compartment was studied during period of 180 min in pre-oestrus (PE), oestrus (E), dioestrus day 1 (D1) and dioestrus day 2 (D2) rats. The most relevant results concerning the action of acetylcholine were found on PE and dioestrus. On PE, the pre-ovulatory peak of progesterone, which is known to respond to the endocrine action, was not modified by neural effect of acetylcholine in our scheme. On the other hand, the progesterone peak occurs in the afternoon of D1, which has been described as independent of the gonadotrophic action but was inhibited by neural effect of acetylcholine in our experimental scheme. This action on D1 was accompanied by a decrease of norepinephrine release in the ovary compartment. We conclude that the action of cholinergic agents varies according to the oestrous cycle stage and constitutes one of the factors governing the secretory activity of the ovarian steroids, in this case, progesterone.