Testosterone-induced downregulation of anti-Müllerian hormone expression in granulosa cells from small bovine follicles.

Polycystic ovary syndrome (PCOS) is characterized by the presence of hyperandrogenism and an increased follicular mass probably determined by deregulation of locally produced factors. Anti-Müllerian hormone (AMH) is a glycoprotein that inhibits follicular recruitment and determines the size of the follicular pool. To evaluate the role of androgens in the regulation of AMH expression in bovine granulosa cells from small follicles, granulosa cells from 3 to 4 mm follicles were isolated and incubated in basal culture media, or in media containing testosterone (T) at 10(-5)M, T 10(-8)M, or estradiol (E2) at 150 ng/ml for 48 h. AMH mRNA levels of these cells were determined using real-time PCR (RT PCR). AMH protein levels and E2 were determined in cell-conditioned media. A 3.4-fold decrease in AMH mRNA levels was observed in granulosa cells exposed to T 10(-5)M (P = 0.03, n = 5), but not in cells exposed to T 10(-8)M. AMH protein levels showed a 1.8-fold reduction in cell-conditioned media from cells exposed to T 10(-5)M (P = 0.01, n = 5), without significant changes in the group exposed to T 10(-8)M. Cells treated with E2 150 ng/ml showed no change in AMH protein levels. We propose that AMH expression is modulated by androgens in bovine granulosa cells from small follicles. Thus, it is possible to speculate that androgens, by inhibiting AMH expression, may promote follicle recruitment, increasing the early growing follicular pool. This new mechanism may have implications for the understanding of PCOS pathophysiology.

Increases in norepinephrine release and ovarian cyst formation during ageing in the rat.

BACKGROUND: Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models. METHODS: Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia. RESULTS: From 8 months old, the induced release of recently incorporated 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs (the proapoptotic/antiapoptotic signals) increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles. CONCLUSION: These data suggest that the ageing process in the ovary of the Sprague-Dawley rat is accompanied by an increased sympathetic tone of the ovary. Consequently, this sympathetic change could be related to a neuroendocrine-driven formation of a polycystic condition similar to that observed in the sympathetic-activated adult ovary.

Catecholamine uptake, storage, and regulated release by ovarian granulosa cells.

Catecholamines present in the mammalian ovary are involved in many normal aspects of ovarian functions, including initial follicle growth, steroidogenesis, and pathological states such as polycystic ovary syndrome. Sympathetic nerve fibers are the largest source of norepinephrine (NE), but not the only one. Surgical denervation of the rat ovary reduces, but does not eliminate, the ovarian content of NE. The aim of this work was to explore which intraovarian cells may participate in the ovarian NE homeostasis and the mechanisms involved. It was found that denervated rat ovaries can take up NE and cocaine considerably, decreased its uptake, suggesting involvement of catecholamine transporters. Granulosa cells of rat ovarian follicles present dopamine transporter and NE transporter. Their functionality was confirmed in isolated rat granulosa cells while cocaine blocked the uptake of NE. Furthermore, the presence of the vesicular monoamine transporter 2, together with the exocytotic protein (synaptosome-associated protein of 25 kDa) in granulosa cells, implies catecholamine storage and regulated release. Regulated calcium-dependent release of NE was shown after depolarization by potassium, implying all neuron-like cellular machinery in granulosa cells. These results in rats may be of relevance for the human ovary because dopamine transporter, NE transporter, vesicular monoamine transporter 2, and synaptosome-associated protein of 25-kDa protein and mRNA are found in human ovarian follicles and/or isolated granulosa cells. Thus, ovarian nonneuronal granulosa cells, after taking up catecholamines, can serve as an intraovarian catecholamine-storing compartment, releasing them in a regulated way. This suggests a more complex involvement of catecholamines in ovarian functions as is currently being recognized.

Functional development of the ovarian noradrenergic innervation.

A substantial fraction of the noradrenergic innervation targeting the mammalian ovary is provided by neurons of the celiac ganglion. Although studies in the rat have shown that noradrenergic nerves reach the ovary near the time of birth, it is unknown how the functional capacity of this innervation unfolds during postnatal ovarian development. To address this issue, we assessed the ability of the developing ovary to incorporate and release (3)H-norepinephrine. Incorporation of (3)H-norepinephrine was low during the first 3 wk of postnatal life, but pharmacological inhibition of norepinephrine (NE) neuronal uptake with cocaine showed that an intact transport mechanism for NE into nerve terminals is already in place by the first week after birth. Consistent with this functional assessment, the mRNA encoding the NE transporter was also expressed in the celiac ganglion at this time. During neonatal-infantile development [postnatal (PN) d 5-20], the spontaneous, vesicle-independent outflow of recently taken up NE was high, but the NE output in response to K(+)-induced depolarization was low. After PN d 20, spontaneous outflow decreased and the response to K(+) increased markedly, reaching maximal values by the time of puberty. Tyramine-mediated displacement of NE stored in vesicles, which displace vesicular NE, showed that vesicle-dependent NE storage becomes functional by PN d 12 and that vesicular release increases during the juvenile-peripubertal phases of sexual development. These results indicate that vesicular release of NE from ovarian noradrenergic nerves begins to operate by the third week of postnatal life, becoming fully functional near the time of puberty.

Participation of vasoactive intestinal polypeptide in ovarian steroids production during the rat estrous cycle and in the development of estradiol valerate-induced polycystic ovary.

Vasoactive intestinal polypeptide (VIP) stimulates estradiol and progesterone release from ovarian granulosa cells in vitro. Very little information is available as to the role VIP plays in the control of steroid secretion during reproductive cyclicity and in ovarian pathologies involving altered steroid secretion. In this study, we determined the involvement of VIP in regulating ovarian androgen and estradiol release during estrous cyclicity and estradiol valerate (EV)-induced polycystic ovarian development in rats. Our findings show that androgen and estradiol release from ovaries obtained during different stages of rat estrous cycle mimic cyclic changes in steroid release observed in vivo with maximal release occurring during late proestrus. VIP increased androgen release from ovaries of all cycle stages except late proestrus and estradiol release from all cycle stages. Increases in VIP-induced androgen and estradiol release were maximal at early proestrus. Inclusion of saturating concentrations of androstenedione increased magnitude of VIP-induced estradiol release at diestrus and estrus but not proestrus. Magnitude of VIP-induced androgen and estradiol release tended to be greater in the ovaries from EV-treated rats with polycystic ovary compared with estrous controls. At the tissue level, ovarian VIP concentration was cycle stage dependent with highest level seen in diestrus. Maximum concentration of VIP was found in EV-treated rats. Changes in VIP were inversely related to changes in ovarian nerve growth factor, a neuropeptide involved in ovarian androgen secretion. These results strongly suggest that intraovarian VIP participates in the control of estradiol secretion during the rat estrous cycle and possibly in the maintenance of increased ovarian estradiol secretory activity of EV-treated rats.

Effects of long-term adrenalectomy on apoptosis and neuroprotection in the rat hippocampus.

Reduction in corticosterone by acute adrenalectomy (5 d) promotes apoptosis in dentate gyrus (DG) granular neurons, an effect concomitant with variations in the expression of the Bcl-2 gene family implicated in apoptotic regulation. However, no studies exist correlating the effect of long-term adrenalectomy (30 d) on the hippocampus in terms of extent of apoptosis and the levels of proteins related to an apoptotic cascade. After 5 d of adrenalectomy, we found an increase in apoptosis of the DG granular region, correlated with an increase in the processing of caspase-9. The magnitude of apoptosis 30 d after adrenalectomy was reduced in the DG granular layer compared with 5 d after adrenalectomy, in close relation to a reduction in the level of processed caspase-9. To understand how the increase in cell survival long after adrenalectomy occurs, we analyzed changes in the expression of genes and proteins related to apoptosis. Long-term adrenalectomy did not change hippocampal pro-apoptotic Bax or antiapoptotic Bcl-2 mRNA levels or protein content with respect to control. However, we found an increase in mRNA levels of the GD's Bcl-x gene, in parallel with the increase in anti-apoptotic BCL-XL protein levels. These results suggest the reduction in apoptosis observed after long-term adrenalectomy occurs through mechanisms that repress proapoptotic genes previously found to be increased at shorter times of adrenalectomy.

Role of stress and sympathetic innervation in the development of polycystic ovary syndrome.

This article presents a review of the role of the sympathetic activity in ovarian pathologies affecting reproductive function. We provide a succinct outline of the findings of our group in this area. The participation of stress as an etiological factor for ovarian pathologies throughout animal models and data in patients with polycystic ovary syndrome give strong support for participation of sympathetic nerves in the ovary function both in normal and pathological status.