FSH regulates acetycholine production by ovarian granulosa cells.

BACKGROUND: It has been previously shown that cultured granulosa cells (GCs) derived from human ovarian preovulatory follicles contain choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. They also produce ACh and express functional muscarinic ACh receptors. ACh can act on GCs to increase proliferation, disrupt gap junctional communication, alter intracellular calcium levels, as well as expression of transcription factors, suggesting an unrecognized role of ACh in GC function. To gain further insights into the possible role of ACh in the ovary, we examined ChAT expression in the gland before and after birth, as well as in adults, and studied the regulation of ACh production by FSH. METHODS: ChAT immunohistochemistry was performed using ovarian samples of different species and ages (embryonic, postnatal and adult rats and mice, including embryonic ovaries from mice null for ChAT, neonatal and adult rhesus monkeys and adult humans). ACh was measured by HPLC and/or a fluorescence based method in rat ovaries and in a FSH receptor-expressing cell line (rat GFSHR-17) cultured with or without FSH. RESULTS: In adult rat, as well as in all other species, ovarian ChAT immunoreactivity is associated with GCs of antral follicles, but not with other structures, indicating that GCs are the only ovarian source of ACh. Indeed ACh was clearly detected in adult rat ovaries by two methods. ChAT immunoreactivity is absent from embryonic and/or neonatal ovaries (mouse/rat and monkey) and ovarian development in embryonic mice null for ChAT appears normal, suggesting that ACh is not involved in ovarian or follicular formation. Since ChAT immunoreactivity is present in GCs of large follicles and since the degree of the ChAT immunoreactivity increases as antral follicles grow, we tested whether ACh production is stimulated by FSH. Rat GFSHR-17 cells that stably express the FSH receptor, respond to FSH with an increase in ACh production. CONCLUSION: ACh and ChAT are present in GCs of growing follicles and FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs, we suggest that ACh may act in the growing ovarian follicle as a local mediator of some of the actions ascribed to FSH.

Two types of calcium channels in human ovarian endocrine cells: involvement in steroidogenesis.

Nature, regulation, and functional role of ion channels of human ovarian endocrine cells are not well known. In our present study, we show two types of voltage-activated Ca(2+) currents (I(Ca)) in cultured human luteinized granulosa cells (GCs), as assessed by whole-cell patch-clamp experiments. Electrophysiological properties, namely low threshold of activation, pronounced time-dependent inactivation, slow and voltage-dependent deactivation kinetics, insensitivity to SNX-482, and high sensitivity to Ni(2+), defined the predominant I(Ca) as a T-type Ca(2+) current (I(Ca.T)). In 4% of cells a Ni(2+)-insensitive I(Ca) was measured alone or together with I(Ca.T). This Ca(2+) current was high voltage activated and highly sensitive to dihydropyridine, indicative of an L-type Ca(2+) current. RT-PCR analysis demonstrated the presence of mRNA coding for alpha(1)-subunits of two different Ca(2+) channels (T-type Ca(v)3.2 and L-type Ca(v)1.2) in GCs. In addition, these two types were detected in the human corpus luteum by RT-PCR (Ca(v)3.2) and immunohistochemistry (Ca(v)1.2). Although stimulation of cultured GCs with human chorionic gonadotropin did not change the characteristics of recorded I(Ca.T), it markedly increased the percentage of cells displaying I(Ca) from 29 to 63% and significantly increased (2.2-fold) the density of I(Ca.T). Furthermore, the stimulatory effect of human chorionic gonadotropin on progesterone production was diminished by pharmacological blockage of I(Ca.T) by Ni(2+) or flunarizine. Thus, our study provides evidence that human GCs in vivo and in vitro express T- and L-type Ca(2+) channels and that the Ca(v)3.2 (also called alpha(1H)) isoform is involved in a fundamental endocrine function of these cells.

Stimulation of TM3 Leydig cell proliferation via GABA(A) receptors: a new role for testicular GABA.

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA(A) and GABA(B) receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA(A) receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA(A) receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA(A) receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA(A) agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA(A) antagonist bicuculline, implying a role for GABA(A) receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA(A) receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.

Voltage-dependent K+ channel acts as sex steroid sensor in endocrine cells of the human ovary.

Molecular targets of rapid non-genomic steroid actions are not well known compared to those of the classical transcription pathway, but ion channels have recently been identified to be steroid-sensitive. Especially, in the ovary, the very organ producing high amounts of sex steroids, their rapid actions are not well examined. We now identified a yet unknown target for sex steroids, a voltage-dependent K+ channel (Kv4.2) that contributes to a transient outward K+ current (I(A)) in human granulosa cells (GCs). Sex steroid hormones at concentrations typical for the ovary (1 microM) blocked Kv4.2 thereby attenuating I(A) by about 25% within seconds. We also found both Kv4.2 (KCND2) mRNA and protein in endocrine cells of the human and rhesus macaque ovary, emphasizing the physiological relevance of this channel. Therefore, we propose a role as fast-responding steroid sensor for the Kv4.2 channel. The direct regulation of K+ channel activity by sex steroids might represent a yet unknown mechanism of rapid steroid action in close proximity to the site of steroid production in the primate ovary. Our data might also be important for Kv4 channels in the brain and the cardiovascular system where rapid steroid effects are discussed in the context of prevention of cell death.