Engineering a gene silencing viral construct that targets the cat hypothalamus to induce permanent sterility: An update.

The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference (RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus (AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats.

Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule.

STUDY QUESTION: Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology? SUMMARY ANSWER: DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL. WHAT IS KNOWN ALREADY: DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined. STUDY DESIGN, SIZE, DURATION: Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²âº, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²âº levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments. LIMITATIONS, REASONS FOR CAUTION: Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available. WIDER IMPLICATIONS OF THE FINDINGS: In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).

Applying gene silencing technology to contraception.

Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

Targeted gene silencing to induce permanent sterility.

A non-surgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: it needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article, we discuss this subject and provide a succinct account of our previous experience with: (i) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction and (ii) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

Oxytocin receptors in the primate ovary: molecular identity and link to apoptosis in human granulosa cells.

BACKGROUND: Oxytocin (OT) is produced by granulosa cells (GCs) of pre-ovulatory ovarian follicles and the corpus luteum (CL) in some mammalian species. Actions of OT in the ovary have been linked to luteinization, steroidogenesis and luteolysis. Human IVF-derived (h)GCs possess a functional OT receptor (OTR), linked to elevation of intracellular Ca(2+), but molecular identity of the receptor for OT in human granulosa cells (hGCs) and down-stream consequences are not known. METHODS AND RESULTS: RT-PCR, sequencing and immunocytochemistry identified the genuine OTR in hGCs. OT (10 nM-10 microM) induced elevations of intracellular Ca(2+) levels (Fluo-4 measurements), which were blocked by tocinoic acid (TA; 50 microM, a selective OTR-antagonist). Down-stream effects of OTR-activation include a concentration dependent decrease in cell viability/metabolism, manifested by reduced ATP-levels, increased caspase3/7-activity (P < 0.05) and electron microscopical signs of cellular regression. TA blocked all of these changes. Immunoreactive OTR was found in the CL and GCs of large and, surprisingly, also small pre-antral follicles of the human ovary. Immunoreactive OTR in the rhesus monkey ovary was detected in primordial and growing primary follicles in the infantile ovary and in follicles at all stages of development in the adult ovary, as well as the CL: these results were corroborated by RT-PCR analysis of GCs excised by laser capture microdissection. CONCLUSIONS: Our study identifies genuine OTRs in human and rhesus monkey GCs. Activation by high levels of OT leads to cellular regression in hGCs. As GCs of small follicles also express OTRs, OT may have as yet unknown functions in follicular development.

Prostaglandin involvement in hypothalamic control of gonadotropin and prolactin release.

Prostaglandin E(2) (PGE(2)) injected into the third ventricle of ovariectomized rats increased plasma luteinizing hormone dramatically and follicle stimulating hormone slightly. PGE(1) elevated prolactin; PGF(1alpha) or PGF(2alpha) had no effect. PGE(2) or PGE(1) injected directly into the anterior pituitary were ineffective. These results suggest that specific prostaglandins act at the hypothalamus to control pituitary hormone release.

Transforming growth factor-alpha gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation.

Hypothalamic injury causes female sexual precocity by activating luteinizing hormone-releasing hormone (LHRH) neurons, which control sexual development. Transforming growth factor-alpha (TGF-alpha) has been implicated in this process, but its involvement in normal sexual maturation is unknown. The present study addresses this issue. TGF-alpha mRNA and protein were found mostly in astroglia, in regions of the hypothalamus concerned with LHRH control. Hypothalamic TGF-alpha mRNA levels increased at times when secretion of pituitary gonadotropins--an LHRH-dependent event--was elevated, particularly at the time of puberty. Gonadal steroids involved in the control of LHRH secretion increased TGF-alpha mRNA levels. Blockade of TGF-alpha action in the median eminence, a site of glial-LHRH nerve terminal association, delayed puberty. These results suggest that TGF-alpha of glial origin is a component of the developmental program by which the brain controls mammalian sexual maturation.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

A contactin-receptor-like protein tyrosine phosphatase beta complex mediates adhesive communication between astroglial cells and gonadotrophin-releasing hormone neurones.

Although it is well established that gonadotrophin-releasing hormone (GnRH) neurones and astrocytes maintain an intimate contact throughout development and adult life, the cell-surface molecules that may contribute to this adhesiveness remain largely unknown. In the peripheral nervous system, the glycosylphosphatidyl inositol (GPI)-anchored protein contactin is a cell-surface neuronal protein required for axonal-glial adhesiveness. A glial transmembrane protein recognised by neuronal contactin is receptor-like protein tyrosine phosphatase beta (RPTP beta), a phosphatase with structural similarities to cell adhesion molecules. In the present study, we show that contactin, and its preferred in cis partner Caspr1, are expressed in GnRH neurones. We also show that the RPTP beta mRNA predominantly expressed in hypothalamic astrocytes encodes an RPTP beta isoform (short RPTP beta) that uses its carbonic anhydrase (CAH) extracellular subdomain to interact with neuronal contactin. Immunoreactive contactin is most abundant in GnRH nerve terminals projecting to both the organum vasculosum of the lamina terminalis and median eminence, implying GnRH axons as an important site of contactin-dependent cell adhesiveness. GT1-7 immortalised GnRH neurones adhere to the CAH domain of RPTPbeta, and this adhesiveness is blocked when contactin GPI anchoring is disrupted or contactin binding capacity is immunoneutralised, suggesting that astrocytic RPTP beta interacts with neuronal contactin to mediate glial-GnRH neurone adhesiveness. Because the abundance of short RPTP beta mRNA increases in the female mouse hypothalamus (but not in the cerebral cortex) before puberty, it appears that an increased interaction between GnRH axons and astrocytes mediated by RPTP beta-contactin is a dynamic mechanism of neurone-glia communication during female sexual development.

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Origin and ontogeny of mammalian ovarian neurons.

Mammalian ovaries contain sympathetic neurons expressing the low affinity neurotropin receptor (p75NTR). To date neither the role these neurons might play in ovarian physiology nor their embryological origin is known. Immunohistochemistry was used to detect postnatal changes in distribution and number of both p75NTR-positive and tyrosine hydroxylase-positive neurons in rhesus monkey ovaries. Pig fetuses were used to map the pathway of ovarian neuronal migration during embryonic development. Antiserum to p75NTR revealed the presence of isolated neurons and neurons clustered into ganglia in 2-month-old monkey ovaries. After 8 months, the neurons exhibited well-developed processes, and other than being more extensively interlaced, the localization and morphology did not change after 2 yr of age. Total number of p75NTR-positive neurons present decreased gradually between 2 months and 12 yr of age and declined markedly with reproductive aging. Conversely, the subpopulation of neurons immunoreactive to anti-tyrosine hydroxylase increased significantly at puberty and then declined with the loss of reproductive capacity. By d 21 of fetal life in the pig, p75NTR neurons had migrated medially from the neural crest to form the paraaortic autonomic ganglia. Some neurons migrated ventrally from the ganglia and then continued ventrolaterally to enter the genital ridge. By d 27, neurons had entered the developing ovary, and by d 35, the migration was complete with neurons demonstrating immunoreactivity to NeuN, a neuron-specific marker. Results demonstrate that p75NTR-expressing ovarian neurons originate from the neural crest and that a catecholaminergic subset is associated with pubertal maturation of the ovary and subsequent reproductive function.

Nerve growth factor-dependent activation of trkA receptors in the human ovary results in synthesis of follicle-stimulating hormone receptors and estrogen secretion.

CONTEXT: Previous studies showed that nerve growth factor (NGF) induces the expression of functional FSH receptors (FSHR) in preantral follicles of the developing rat ovary. OBJECTIVE: The objective of this study was to determine whether NGF can affect granulosa cell (GC) function in human periovulatory follicles using intact human ovaries and isolated human GCs. PATIENTS AND INTERVENTIONS: Human GCs were obtained from in vitro fertilization patients and normal ovaries from women with elective pelvic surgery for nonovarian indications. RESULTS: In normal ovaries, NGF and trkA (NGF's high-affinity receptor) were detected by immunohistochemistry in GCs of preantral and antral follicles. NGF and trkA are also present in thecal cells of antral follicles. Both freshly collected and cultured GCs contained immunoreactive NGF and trkA in addition to their respective mRNAs. Human GCs respond to NGF with increased estradiol (E(2)) secretion and a reduction in progesterone output. Exposure of human GCs to NGF increased FSHR mRNA content within 18 h of treatment, and this effect was blocked by the trk tyrosine kinase blocker K-252a. Also, cells preexposed to NGF released significantly more E(2) in response to hFSH than cells not pretreated with the neurotropin, showing that the NGF-induced increase in FSHR gene expression results in the formation of functional FSHRs. CONCLUSIONS: These results suggest that one of the functions of NGF in the preovulatory human ovary is to increase the secretion of E(2) while preventing early luteinization via an inhibitory effect on progesterone secretion. NGF stimulates E(2) secretion both directly and by increasing the formation of FSHRs.

Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development.

Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.

Expression of three gene families encoding cell-cell communication molecules in the prepubertal nonhuman primate hypothalamus.

Transsynaptic and glial-neuronal communication are important components of the mechanism underlying the pubertal activation of luteinizing hormone-releasing hormone (LHRH) secretion. The molecules required for the architectural organization of these cell-cell interactions have not been identified. We now show that the hypothalamus of the prepubertal female rhesus monkey expresses a multiplicity of genes encoding three families of adhesion/signalling proteins involved in the structural definition of both neurone-to-neurone and bi-directional neurone-glia communication. These include the neurexin/neuroligin (NRX/NRL) and protocadherin-alpha (PCDHalpha) families of synaptic specifiers/adhesion molecules, and key components of the contactin-dependent neuronal-glial adhesiveness complex, including contactin/F3 itself, the contactin-associated protein-1 (CASPR1), and the glial receptor protein tyrosine phosphatase beta. Prominently expressed among members of the NRX family is the neurexin isoform involved in the specification of glutamatergic synapses. Although NRXs, PCDHalphas and CASPR1 transcripts are mostly detected in neurones, the topography of expression appears different. NRX1 mRNA-containing neurones are scattered throughout the hypothalamus, PCDHalpha mRNA transcripts appear more abundant in neurones of the arcuate nucleus and periventricular region, and neurones positive for CASPR1 mRNA exhibit a particularly striking distribution pattern that delineates the hypothalamus. Examination of LHRH neurones, using the LHRH-secreting cell line GT1-7, showed that these cells contain transcripts encoding NRXs and one of their ligands (NRL1), at least one PCDHalpha (CNR-8/PCDHalpha10), and the CASPR1/contactin complex. The results indicate that the prepubertal female monkey hypothalamus contains a plethora of adhesion/signalling molecules with different but complementary functions, and that an LHRH neuronal cell line expresses key components of this structural complex. The presence of such cell-cell communication machinery in the neuroendocrine brain suggests an integrated participation of their individual components in the central control of female sexual development.

Developmental regulation of the ovary via growth factor tyrosine kinase receptors.

Ovarian development and mature ovarian function are subjected to the regulatory influence of several growth factors exerting their biologic actions via membrane-anchored receptors endowed with intrinsic tyrosine kinase activity. The actions of these growth factors appear to be anatomically, functionally, and temporally coordinated. Whereas transforming growth factor alpha (TGFalpha)-produced in thecal cells- facilitates cell proliferation and slows gonadotropin-dependent biochemical differentiation of both thecal and granulosa cells, IGF-I-produced in granulosa cells-amplifies the effect of gonadotropins on this ovarian compartment. The interactive paracrine-autocrine influence of both growth factors may facilitate the simultaneous occurrence of the two basic processes underlying follicular development: growth and cytochemical differentiation. Upon completion of preovulatory development, fibroblast growth factors (FGFs)-produced by granulosa, and also perhaps by thecal-interstitial cells-may come into play, accelerating cytodifferentiation of thecal cells into luteal cells by reducing the androgenic response of thecal cells to gonadotropins. In addition, FGFs may facilitate proliferative events underlying the organization of pregranulosa cells during early ovarian development. Neurotrophins may exert their greatest impact during these same developmental windows affected by FGF. Neurotrophins may contribute to regulating cell-cell interactive processes related to follicular organization and/or formation during early ovarian development, and to the preovulatory cytodifferentiation of thecal-interstitial cells that precedes ovulatory rupture. Neurotrophins may also regulate the density of follicular innervation. Because each of these growth factors binds to a different receptor tyrosine kinase and expression of each receptor is highly compartmentalized and developmentally regulated, it appears clear that activation of receptor tyrosine kinases is intimately linked to both normal and deranged ovarian development.

Nerve growth factor receptors in the peripubertal rat ovary.

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide regulates cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene expression in granulosa cells from immature rat ovaries.

Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3'-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

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.

Failure to monoaminergic and cholinergic receptor blockers to prevent prostaglandin E2-induced luteinizing hormone release.

Receptor blocking drugs were used to determine whether adrenergic, dopaminergic, serotoninergic, or cholinergic synapses are involved in mediating the LH release induced by intraventricularly injected PGE2. Prostaglandin E2 (5mug) was injected into the 3rd ventricle (3rd V) of ovariectomized rats, and plasma LH concentrations before and after treatment were determined by radioimmunoassay. Phentolamine, 20 or 30 mug, or pronethalol, 20 mug (alpha and beta adrenergic receptor blockers, respectively) injected into the 3rd V failed to alter the elevation of plasma LH evoked by PGE2 injected into the ventricle 10 min later. Likewise, LH release following PGE2 was not changed when a dopaminergic blocker, pimozide (0.63 mg/kg, SC), was injected 2 h prior to PGE2. Two antagonists of serotonin, methysergide maleate (3 mg/kg ip) or cinanserin HC1 (1 mg/kg iv) given 2 h or 45 min before PGE2, respectively, failed to alter the action of PGE2. Atropine (100 or 250 mug) injected into the 3rd V 10 min prior to PGE2 was also ineffective in blocking the increase in plasma LH following PGE2. The results of this study indicate that the effect of PGE2 on LH release is not mediated by adrenergic, dopaminergic, serotoninergic, or cholinergic receptors. They also suggest that PGE2 is not acting trans-synaptically but probably directly on the LHRH neuron to induce the discharge of LHRH into the hypophysial portal vessels which then evokes release of LH from the adenohypophysis.