Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

The neurotransmitter norepinephrine (NE) is derived from the sympathetic nervous system and may be involved in the regulation of ovarian functions. Ovarian innervation increases in patients with polycystic ovarian syndrome (PCOS), prompting us to readdress a role of NE in the human ovary. In vitro fertilization-derived granulosa cells (GC), follicular fluids (FF), and ovarian sections were studied. NE was found in FF and freshly isolated GC, yet significantly lower levels of NE were detected in samples from PCOS patients. Furthermore, the metabolite normetanephrine was detected in FF. Together this suggests cellular uptake and metabolism of NE in GC. In accordance, the NE transporter and NE-metabolizing enzymes [catechol-o-methyltransferase (COMT) and monoamine oxidase A] were found in GC, COMT in GC and thecal cells of large human antral follicles in vivo and in cultured GC. Cellular uptake and metabolism of NE also occurred in cultured GC, events that could be blocked pharmacologically. NE, in the range present in FF, is unlikely to affect GC via activation of typical α- or β-receptors. In line with this assumption, it did not alter phosphorylation of MAPK. However, NE robustly induced the generation of reactive oxygen species (ROS). This action occurred even when receptors were blocked but was prevented by blockers of NE transporter, COMT, and monoamine oxidase A. Thus, NE contributes to the microenvironment of preovulatory human follicles and is lower in PCOS. By inducing the production of ROS in GC, NE is linked to ROS-regulated events, which are emerging as crucial factors in ovarian physiology, including ovulation.

Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Progesterone production by the corpus luteum is a process vital for reproduction. In humans its secretion is stimulated by the placental hormone human chorionic gonadotropin (hCG), and this stimulatory action can also be observed in cultured human luteinized granulosa cells (GCs). We now provide evidence that opening of a Ca(2+)-activated K(+) channel, the BK(Ca), is crucially involved in this process. Immunohistochemistry and RT-PCR revealed the presence of the pore-forming alpha-subunit in human luteinized GCs and in luteal cells of human, macaque, and rat, implying that BK(Ca) channels are important throughout species. Blocking of BK(Ca) channels by iberiotoxin attenuated hCG-induced progesterone secretion. The inhibitory action of iberiotoxin suggests that BK(Ca) channels are activated in the course of hCG-induced steroidogenesis. In search of physiological activators we used an electrophysiological approach and could preclude a direct regulation of channel activity by hCG or GC-derived steroids (progesterone and 17beta-estradiol). Instead, the peptide hormone oxytocin and an acetylcholine (ACh) agonist, carbachol, evoked transient BK(Ca) currents and membrane hyperpolarization. These two molecules are both secreted by GCs and act via raised intracellular Ca(2+) levels. The release of oxytocin is stimulated by hCG, and a similar mechanism is likely in the case of ACh. We conclude that BK(Ca) channel activity in GCs is mediated by components of the intraovarian signaling system, thereby interlinking a systemic hormonal and a local neuroendocrine system in control of steroidogenesis.

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