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.

Growth and differentiation of ovarian follicles are regulated by systemic and local factors, which may include acetylcholine (ACh). Granulosa cells (GCs) of growing follicles and luteal cells produce ACh and in cultured GCs it exerts trophic actions via muscarinic receptors. However, such actions were not studied in vivo. After having established that rat ovarian GCs and luteal cells express the ACh-metabolizing enzyme ACh esterase (AChE), we examined the consequences of local application of an AChE inhibitor, huperzine A (HupA), by osmotic minipump delivery into the ovarian bursa of hemiovariectomized rats. Saline was used in the control group. Local delivery of HupA for 4 weeks increased ovarian ACh content. Estrus cyclicity was not changed indicating a locally restricted range of HupA action. The number of primordial and primary follicles was unaffected, but small secondary follicles significantly increased in the HupA group. Furthermore, a significant increase in the number of corpora lutea suggested increased ovulatory events. In support, as shown upon mating, HupA-treated females had significantly increased implantation sites and more pups. Thus the data are in support of a trophic role of ACh in follicular development and ovulation and point to an important role of ACh in female fertility.

BackgroundA previous study showed that dopamine (DA), which is contained in follicular fluid (FF) from IVF patients, strongly increased the production of reactive oxygen species (ROS) by cultured human granulosa cells (GCs). ROS, including H2O2, are assumed to play roles in ovarian physiology and pathology. Ovarian DA could be derived from the circulation, ovarian innervation and/or unknown ovarian sources. L-DOPA is the direct precursor of DA in its synthetic pathway. It was not yet described in FF. We examined L-DOPA levels in FF from IVF patients. As it may exert anti-oxidative and ROS-scavenging functions, we studied whether it exerts such actions in human GCs and whether DOPA-decarboxylase (DDC), the enzyme converting L-DOPA to DA, is expressed in the human ovary.ResultsELISA measurements revealed that human IVF-derived FF contains L-DOPA. In cultured human GCs automated confluence analyses showed that L-DOPA enhanced their survival. This is in contrast to the actions of DA, which reduced cell survival. A dose-dependent mode of action of L-DOPA was identified using a fluorescent ROS indicator. The results showed that it antagonized intracellular ROS accumulation induced by exogenous H2O2. DDC was absent in follicular GCs, but immunohistochemistry identified it in theca cells (TCs) of large follicles in the human ovary. Laser micro-dissection followed by RT-PCR corroborated the expression. DDC was also identified in the steroidogenic cells of the corpus luteum.ConclusionsL-DOPA in FF is an antioxidant factor and exerts positive influences on GCs. Ovarian DA is derived from L-DOPA and has opposite actions. Exogenous L-DOPA is a standard therapy for Parkinson’s disease, and the results raise the possibility that it may be able to exert positive actions as an antioxidant in ovarian conditions, as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-016-0269-0) contains supplementary material, which is available to authorized users.

Background Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. Material and Methods Immunohistochemistry of human and Rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. Results and Conclusions Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

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Impairment of puerperal fertility may be a severe problem in some dairy herds. Within the last years, several diagnostic possibilities weredevelopedandvalidated.Theaimofthisstudy wastoevalu-atetheapplicabilityofbloodserumparameterssubstanceP(SP)and vasoactiveintestinalpolypeptide(VIP)fortheiruseinearlydiagnostics of uterine involution disturbances. Blood serum samples of 86 dairycowstakenwithinthefirst20dayspost-partumwereexamined using commercially available ELISA test kits. Animals were divided into two groups (healthy or diseased) depending on the results of clinicalandgynaecologicalexamination.Statisticalanalysisconsisted ofthetimelychangesinbloodserumlevelsaswellasthegroupcomparisonofhealthycowsandcowswithuterinedisease.Bloodserum concentrations for SP increased statistically significant within the first 20days after calving (p<0.04). There was no statistically significantdifferencebetweenthegroups.ConcerningVIP,neitherfor thetimelycoursenorforthegroupcomparisonasignificantdifferencecouldbeshown.SPrepresentsanaccreditedbiomarkerforpain in cattle. An increase of substance P within the first 20days postpartum suggests the presence of persisting pain presumably due to uterine involution processes. Nevertheless, both serum parametersdonotseemsuitableasindicatorsforthepresenceofuterine involutiondisorders. Theobjectivesofthisstudyweretoinvestigatethecasesofcamel dystocia in which fetotomy was indicated, the related complicationsandtheassociatedrisksformaternalmortalityandfertility. Fifty female dromedary camels were handled with fetotomy due to uncorrectable dystocia. The complications during and after fetotomyandpost-operativefertilityrateswererecorded.Logistic regressionwasperformedtoidentifyriskfactorsforthedependentvariablesofmaternalmortalityandfertilityafterfetotomy.The independentvariableswereparity,durationofdystocia,presence of foetal emphysema, number of cuts, duration of fetotomy and occurrence of complications during or after fetotomy. Common indications of fetotomy were head and neck deviations alone or with carpal or shoulder flexions (54%) and breech presentation (14%). Complications included tearing of the soft tissue of the birth canal (24%), uterine prolapse (6%) and retained placenta (4%). Maternal mortality occurred in 26% of the cases. A significantassociationwasdetectedbetweenmaternaldeathsandduration of dystocia (odds ratio=4.67, p=0.03), presence of foetal emphysema (odds ratio=3.93, p=0.04) and occurrence of complications during or after fetotomy (odds ratio=8.9, p=0.003). Post-operative fertility was 62.2%. None of the estimated factors showed a significant relationship with post-operative fertility. In conclusion, postural abnormalities constituted the most common indication of fetotomy in dromedary camels. The duration of dystocia, presenceoffoetal emphysema and occurrence of complications during or after fetotomy represented risks for maternal recovery. The fertilityrateafter fetotomy was generally encouraging. 3 | Effect of the ovarian superstimulation and ovum pick-up procedure on t...