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Pro-nerve growth factor in the ovary and human granulosa cells.

ABSTRACT: 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.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.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.

SUBMITTER: Meinel S

PROVIDER: S-EPMC4760111 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Pro-nerve growth factor in the ovary and human granulosa cells.

Meinel Sabine S Blohberger Jan J Berg Dieter D Berg Ulrike U Dissen Gregory A GA Ojeda Sergio R SR Mayerhofer Artur A

Hormone molecular biology and clinical investigation 20151101 2

<h4>Background</h4>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.<h4>Materials and methods</h4>Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human gr ...[more]

PMID: 26457789

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Project description:The functioning of the ovary is influenced by the autonomic system (sympathetic and cholinergic intraovarian system) which contributes to the regulation of steroid secretion, follicular development, and ovulation. There is no information on the primary signal that activates both systems. The nerve growth factor (NGF) was the first neurotrophic factor found to regulate ovarian noradrenergic neurons and the cholinergic neurons in the central nervous system. The aim of this study was to determine whether NGF is one of the participating neurotrophic factors in the activation of the sympathetic and cholinergic system of the ovary in vivo and its role in follicular development during normal or pathological states. The administration of estradiol valerate (a polycystic ovary [PCO] phenotype model) increased norepinephrine (NE) (through an NGF-dependent mechanism) and acetylcholine (ACh) levels. Intraovarian exposure of rats for 28 days to NGF (by means of an osmotic minipump) increased the expression of tyrosine hydroxylase and acetylcholinesterase (AChE, the enzyme that degrades ACh) without affecting enzyme activity but reduced ovarian ACh levels. In vitro exposure of the ovary to NGF (100 ng/ml for 3 h) increased both choline acetyl transferase and vesicular ACh transporter expression in the ovary, with no effect in ACh level. In vivo NGF led to an anovulatory condition with the appearance of follicular cysts and decreased number of corpora lutea (corresponding to noradrenergic activation). To determine whether the predominance of a NE-induced polycystic condition after NGF is responsible for the PCO phenotype, rats were exposed to an intraovarian administration of carbachol (100 ?M), a muscarinic cholinergic agonist not degraded by AChE. Decreased the number of follicular cysts and increased the number of corpora lutea, reinforcing that cholinergic activity of the ovary participates in controlling its functions. Although NGF increased the biosynthetic capacity for ACh, it was not available to act in the ovary. Hence, NGF also regulates the ovarian cholinergic system, implying that NGF is the main regulator of the dual autonomic control. These findings highlight the need for research in the treatment of PCO syndrome by modification of locally produced ACh as an in vivo regulator of follicular development.

Project description:BACKGROUND:Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (K(Ca)) of big conductance (BK(Ca)), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of K(Ca) channels (including BK(Ca) beta-subunits), and 2. biophysical properties of BK(Ca) channels. METHODS:GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique. RESULTS:We identified two K(Ca) types in human GCs, the intermediate- (IK) and the small-conductance K(Ca) (SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K(Ca) blockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K(Ca) channels with distinctive features. Both, IK and BK(Ca) channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BK(Ca) channel revealed the presence of mRNAs encoding several BK(Ca) beta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BK(Ca) channels which we observed in electrophysiological recordings. CONCLUSION:Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK(Ca), they express all three K(Ca) types known. We suggest that the plurality and co-expression of different K(Ca) channels and BK(Ca) beta-subunits might allow differentiated responses to Ca2+ signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian K(Ca) channel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.

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Pro-nerve growth factor in the ovary and human granulosa cells.

Publications

Pro-nerve growth factor in the ovary and human granulosa cells.

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