Catecholamine Uptake, Storage, and Regulated Release by Ovarian Granulosa Cells

Cystic ovarian disease (COD) is an important cause of abnormal estrous behavior and infertility in dairy cows. COD is mainly observed in high-yielding dairy cows during the first months post-partum, a period of high stress. We have previously reported that, in lower mammals, stress induces a cystic condition similar to the polycystic ovary syndrome in humans and that stress is a definitive component in the human pathology. To know if COD in cows is also associated with high sympathetic activity, we studied isolated small antral (5mm), preovulatory (10mm) and cystic follicles (25mm). Cystic follicles which present an area 600 fold greater compared with preovulatory follicles has only 10 times less concentration of NE as compared with small antral and preovulatory follicles but they had 10 times more NE in follicular fluid, suggesting a high efflux of neurotransmitter from the cyst wall. This suggestion was reinforced by the high basal release of recently taken-up 3H-NE found in cystic follicles. While lower levels of beta-adrenergic receptor were found in cystic follicles, there was a heightened response to the beta-adrenergic agonist isoproterenol and to hCG, as measured by testosterone secretion. There was however an unexpected capacity of the ovary in vitro to produce cortisol and to secrete it in response to hCG but not to isoproterenol. These data suggest that, during COD, the bovine ovary is under high sympathetic nerve activity that in addition to an increased response to hCG in cortisol secretion could participate in COD development.

Section: Discussionmentioning

confidence: 99%

Sympathetic nerve activity in normal and cystic follicles from isolated bovine ovary: local effect of beta-adrenergic stimulation on steroid secretion

Paredes

Salvetti

Diaz

et al. 2011

“…For example, previous evidence showed that human granulosa-luteal cells express M1 and M5 muscarinic receptors [34] as well as P2Y2 purinergic receptors [13,14]; stimulation of either system by acetylcholine or ATP can promote granulosa-luteal cell proliferation. Stimulation of β-adrenergic receptors also modulates steroidogenic activity and ovulation [31] and, given that neurotransmitters released from catecholaminergic terminals might include ATP, it would be of interest to know the effect of activating purinergic receptors in these processes.…”

Section: Discussionmentioning

confidence: 99%

Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells

Vázquez‐Cuevas

Zárate-Díaz

Garay

et al. 2010

BackgroundPurinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC).MethodsTIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA.ResultsRT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 μM, while that for UDP was 3.24 +/- 0.82 μM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 μM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation.ConclusionHere, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.

“…Neurotransmitters, such as catecholamines, have been considered as intraovarian regulators due to the identification of intrinsic catecholamine synthesis and the presence of extensive sympathetic innervation in the mammalian ovary [1-4]. In fact, the mammalian ovary is richly supplied by sympathetic fibers that innervate the interstitium and the perifollicular regions of developing follicles, including the external thecal layer, but do not reach the GC layer [3].…”

Section: Introductionmentioning

confidence: 99%

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Piccinato

Montrezor

Collares

et al. 2012

BackgroundSince noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance.MethodsPrimary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs.ResultsGCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8) M), a non-selective beta-adrenergic antagonist.ConclusionsThe present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.