Participation of the extrinsic cholinergic innervation in the action of nitric oxide on the ovarian steroidogenesis in the first proestrous in rats.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system and an ovary without peripheral neural influence from virgin rats in the first proestrous were used to test whether ovarian extrinsic innervation and nitric oxide (NO) affects steroidogenesis in the ovary. The CG and the ovary were placed in separate buffered-compartments, connected by the SON. Stimulation of the CG was achieved by 10(-6)M acetylcholine (Ach). The ovary without peripheral neural influence was placed alone in a buffered-compartment. To test a possible role of NO in the ovarian response to peripheral neural influence, 100µM sodium nitroprusside (SNP, an NO donor) and 100µM N(G)-nitro-l-arginine methyl ester (l-NAME, an inhibitor of NO synthase) were added to the ovarian compartment separately. In the CG-SON-O system, SNP into the ovarian compartment increased the concentration of NO, reduced the release of progesterone and increased the release of estradiol (E2), increasing the mRNAs related to their synthesis enzyme. The addition of l-NAME to the ovarian compartment caused an opposite effect. In the ovary alone, NO manifested an antisteroidogenic effect on both hormones. These results show that the ovarian extrinsic innervation maintains a direct relationship between NO and E2, both needed at high levels during the follicular phase, allowing the continuity of the estrous cycle.

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.

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.

Neural markers expression in rat bone marrow mesenchymal stem cell cultures treated with neurosteroids.

The aim of the present investigation is to study the effects of DEX or E(2) treatment during differentiation towards neural cell line of rat BM-MSCs in culture. In order to better characterize biochemically our in vitro model, we evaluate by western blotting and immunocytochemical analysis some neural lineage markers (nestin, neurofilament, ß-tubulin) and MAP-Kinases. An enhanced expression of the neural markers and MAP-Kinase in DEX-treated BM-MSCs cultures is found. In addition, E(2)-treatment increases MAP-Kinase and ß-tubulin expression, but it decreases nestin and neurofilament expression. In conclusion, our findings highlight a significant up and down modulation of nestin, neurofilament, ß-tubulin and MAP-Kinases expression in neurosteroids-treated BM-MSCs. In particular, our results clarify the molecular mechanism involved during eventual differentiation of these stem cells treated with DEX and E(2), addressed towards a neural cell line, that may express neurotrophic receptors and release neurotrophines particularly implicated during neurogenesis processes.

Growth factors and steroid mediated regulation of cytoskeletal protein expression in serum-deprived primary astrocyte cultures.

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them together. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressful conditions.

Neurosteroid-growth factor cross-talk induces up and down regulation of GFAP and vimentin expression in serum free astrocyte cultures.

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under the following experimental condition: 24h pretreatment with bFGF, subsequent 72h switching in serum-free medium (SFM) and final addition of GFs, only one or more in the last 24h, after a prolonged (60h) DEX treatment. Western blot analysis data showed a marked GFAP expression in cultures submitted to our experimental condition comparing results to untreated or treated controls. In particular, the maximum level of GFAP expression is observed when EGF or INS or both together are added in a prolonged 60h DEX treatment. This finding well correlates with differentiative role played by glucocorticoids interacting with the "competence" factor bFGF and demonstrates as increased GFAP expression mostly depends on maturation, rather than proliferating status of astroglial cells in culture. VIM expression was instead significantly reduced after GFs addition in the last 24h of 60h DEX treatment, respect to control DEX-pretreated ones. Comparing the data to untreated controls, VIM expression was significantly enhanced after GFs addition. Collectively, our findings evidence an interactive effect between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressfull conditions.