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.

Prenatal hyperandrogenization induces metabolic and endocrine alterations which depend on the levels of testosterone exposure.

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life.

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.

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.

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.