Isoflurane exposure for three hours triggers apoptotic cell death in neonatal macaque brain.

BACKGROUND: Retrospective clinical studies suggest there is a risk for neurodevelopmental impairment following early childhood exposure to anaesthesia. In the developing animal brain, including those of non-human primates (NHPs), anaesthetics induce apoptotic cell death. We previously reported that a 5 h isoflurane (ISO) exposure in infant NHPs increases apoptosis 13-fold compared with control animals. However, the majority of paediatric surgeries requiring anaesthesia are of shorter durations. We examined whether 3 h ISO exposure similarly increases neuroapoptosis in the NHP developing brain. METHODS: Six-day-old NHP infants ( Macaca mulatta ) were exposed to 3 h of a surgical plane of ISO ( n =6) or to room air ( n =5). Following exposure, NHP brains were screened for neuronal and oligodendrocyte apoptosis using activated caspase-3 immunolabelling and unbiased stereology. RESULTS: ISO treatment increased apoptosis (neurones + oligodendrocyte) to greater than four times that in the control group [mean density of apoptotic profiles: 57 (SD 22) mm -3 vs 14 (SD 5.2) mm -3 , respectively]. Oligodendrocyte apoptosis was evenly distributed throughout the white matter whereas neuroapoptosis occurred primarily in the cortex (all regions), caudate, putamen and thalamus. CONCLUSIONS: A 3 h exposure to ISO is sufficient to induce widespread neurotoxicity in the developing primate brain. These results are relevant for clinical medicine, as many surgical and diagnostic procedures in children require anaesthesia durations similar to those modelled here. Further research is necessary to identify long-term neurobehavioural consequences of 3 h ISO exposure.

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.

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.

Changes in hypothalamic expression of the Lin28/let-7 system and related microRNAs during postnatal maturation and after experimental manipulations of puberty.

Lin28 and Lin28b are related RNA-binding proteins that inhibit the maturation of miRNAs of the let-7 family and participate in the control of cellular stemness and early embryonic development. Considerable interest has arisen recently concerning other physiological roles of the Lin28/let-7 axis, including its potential involvement in the control of puberty, as suggested by genome-wide association studies and functional genomics. We report herein the expression profiles of Lin28 and let-7 members in the rat hypothalamus during postnatal maturation and in selected models of altered puberty. The expression patterns of c-Myc (upstream positive regulator of Lin28), mir-145 (negative regulator of c-Myc), and mir-132 and mir-9 (putative miRNA repressors of Lin28, predicted by bioinformatic algorithms) were also explored. In male and female rats, Lin28, Lin28b, and c-Myc mRNAs displayed very high hypothalamic expression during the neonatal period, markedly decreased during the infantile-to-juvenile transition and reached minimal levels before/around puberty. A similar puberty-related decline was observed for Lin28b in monkey hypothalamus but not in the rat cortex, suggesting species conservation and tissue specificity. Conversely, let-7a, let-7b, mir-132, and mir-145, but not mir-9, showed opposite expression profiles. Perturbation of brain sex differentiation and puberty, by neonatal treatment with estrogen or androgen, altered the expression ratios of Lin28/let-7 at the time of puberty. Changes in the c-Myc/Lin28b/let-7 pathway were also detected in models of delayed puberty linked to early photoperiod manipulation and, to a lesser extent, postnatal underfeeding or chronic subnutrition. Altogether, our data are the first to document dramatic changes in the expression of the Lin28/let-7 axis in the rat hypothalamus during the postnatal maturation and after different manipulations that disturb puberty, thus suggesting the potential involvement of developmental changes in hypothalamic Lin28/let-7 expression in the mechanisms permitting/leading to puberty onset.

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).

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.

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.

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.

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 is required for early follicular development in the mammalian ovary.

Nerve growth factor (NGF) epitomizes a family of proteins known as the neurotrophins (NTs), which are required for the survival and differentiation of neurons within both the central and peripheral nervous system. Synthesis of NGF in tissues innervated by the peripheral nervous system is consistent with its function as a target-derived trophic factor. However, the presence of low- and high-affinity NGF receptors in the gonads suggests another function for the NTs within the reproductive endocrine system. We now report that NGF is required for the growth of primordial ovarian follicles, a process known to occur independently of pituitary gonadotropins. Both the NT receptor p75(NTR) and the NGF tyrosine kinase receptor trkA were found to be expressed in the ovaries of infantile normal mice and mice carrying a null mutation of the NGF gene. The ovaries from homozygote NGF-null (-/-) mutant animals, analyzed after completion of ovarian histogenesis, exhibited a markedly reduced population of primary and secondary follicles in the presence of normal serum gonadotropin levels, and an increased number of oocytes that failed to be incorporated into a follicular structure. Assessment of mitogenic activity using two complementary proliferation markers revealed a conspicuous reduction in somatic cell proliferation in the ovaries of NGF-deficient mice. These results suggest that the delay in follicular growth observed in NGF(-/-) mice may be related to the loss of a proliferative signal provided by NGF to the nonneural endocrine component of the ovary.

Expression of muscarinic receptor types in the primate ovary and evidence for nonneuronal acetylcholine synthesis.

The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.

Direct effects of nerve growth factor on thecal cells from antral ovarian follicles.

TrkA, the nerve growth factor (NGF) tyrosine kinase receptor, is expressed not only in the nervous system, but also in nonneural cells, including discrete cellular subsets of the endocrine and immune system. In the rat ovary, trkA receptor abundance increases strikingly in thecal-interstitial cells during the hours preceding the first ovulation. Blockade of either trkA transducing capacity or NGF biological activity inhibited ovulation, suggesting a role for NGF in the ovulatory process of this species. To identify some of the processes that may be affected by trkA activation in the thecal compartment, we used purified thecal cells/thecal fibroblasts from bovine ovaries (heretofore referred to as thecal cells). Ribonuclease protection assays employing bovine-specific cRNA probes demonstrated the presence of the messenger RNAs (mRNAs) encoding NGF and its receptors, p75 NTR and trkA, in the thecal compartment of small, medium, and large antral follicles and showed that trkA mRNA is also expressed in granulosa cells. In situ hybridization and immunohistochemical examination of intact ovaries confirmed these cellular sites of NGF and trkA synthesis. TrkA mRNA, but not NGF mRNA, was lost within 48 h of placing thecal cells in culture. Thus, to study trkA-mediated actions of NGF on these cells we transiently expressed the receptor by transfection with a vector containing a full-length rat trkA complementary DNA under transcriptional control of the cytomegalovirus promoter. Because ovulation is preceded by an LH-dependent increase in androgen and progesterone production, the ability of NGF to modify the release of these steroids was determined in freshly plated cells still containing endogenous trkA receptors and in cells undergoing luteinization in culture that were transiently transfected with the trkA-encoding plasmid. NGF stimulated both androgen and progesterone release in freshly plated thecal cells, but not in luteinizing cells provided with trkA receptors. As ovulation in rodents requires an increased formation of PGE2 and has been shown to be antedated by proliferation of thecal fibroblasts, we determined the ability of NGF to affect these parameters in trkA-transfected thecal cells. The neurotrophin rapidly stimulated PGE2 release and amplified the early steroidal response to hCG in trkA-expressing cells, but not in cells lacking the receptor. Likewise, NGF stimulated [3H]thymidine incorporation into trkA-containing cells, but not into cells that had lost the receptor in culture. Induction of ovulation in immature rats by gonadotropin treatment verified that an increased cell proliferation in the thecal compartment, determined by the incorporation of bromodeoxyuridine into cell nuclei, occurs 4-5 h before ovulation in this species. These results suggest that the contribution of NGF to the ovulatory process includes a stimulatory effect of the neurotrophin on steroidogenesis, PGE2 formation, and proliferative activity of thecal compartment cells.

Neurotrophic and cell-cell dependent control of early follicular development.

Neurotrophins (NTs) and their receptors play an essential role in the differentiation and survival of defined neuronal populations of the central and peripheral nervous systems. Their actions, however, do not appear to be limited to the nervous system, as both NTs and their receptors have been found in non neuronal cells, including cells of the endocrine system. At least four of the five known neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), and their receptors (p75 NTR, trkA, trkB and trkC) are present in the developing ovary. Using mice carrying null mutations of the genes encoding neurotrophins (NGF, NT-4, BDNF) or the receptor that mediates the actions of NT-4 and BDNF (trkB), we have obtained initial results consistent with the notion that neurotrophins are required for the growth of primordial follicles. NGF-deficient mice show a decreased formation of both primary and secondary preantral follicles. Null mutation of the NT-4 gene failed to affect either folliculogenesis or follicular development. However, formation of primary and secondary follicles was compromised in mice carrying a null mutation of both the NT-4 and BDNF genes, suggesting compensation of function by BDNF in NT-4 knockouts. Support for this concept is provided by the similar deficiency in follicular growth observed in animals carrying a null mutation of the gene encoding trkB, the receptors mediating NT-4 and BDNF actions. Initial experiments, using differential display, to isolate genes that may be involved in the process of folliculogenesis and/or early follicular development, resulted in the isolation of a recently identified cell adhesion molecule and a novel transcription factor originally shown to induce cell transformation. It thus appears that formation and development of mammalian follicles requires the concerted action of genes originally thought to be only involved in cell differentiation/survival of neuronal cells, and genes that may control the growth, differentiation, and cell-cell interactions of somatic and germ cells in the ovary.

Alcohol ingestion inhibits the increased secretion of puberty-related hormones in the developing female rhesus monkey.

Alcohol (ALC) use and abuse by adolescents has been rising at an alarming rate. Whether ALC consumption during prepubertal years affects specific hormones and the process of sexual maturation is not known. We used immature female rhesus macaques to assess the effects of ALC on circulating levels of hormones known to be critical for the pubertal process. Ten monkeys averaging 20.3 +/- 0.3 months of age were bled by saphenous vein puncture at 0830 and 2030 h each day for 5 consecutive days to determine baseline levels of GH, insulin-like growth factor I, FSH, LH, estradiol (E2), and leptin. For the next 12 months, each day at 1330 h five monkeys were administered ALC (2 g/kg), and five monkeys were administered an isocaloric sucrose solution via a nasogastric approach. Blood was again collected twice daily on 5 consecutive days at 24, 28, and 32 months for hormone analysis. Food consumption and weight gain were similar for ALC-treated and control animals. The expected night-related increase in serum GH occurred during late juvenile development (28-32 months of age) in control animals, but was suppressed (P < 0.05) in ALC-treated animals. This action was paralleled by a decrease (P < 0.01) in serum insulin-like growth factor I. Serum LH and E2 were also depressed by ALC, with their effects most pronounced at 32 months (LH, P < 0.01; E2, P < 0.001). Serum FSH and leptin were not altered. Although ALC did not affect age at menarche, the interval between subsequent menstruations was lengthened (P < 0.05), thereby showing that ALC affected the development of a regular monthly pattern of menstruation. These results demonstrate the detrimental effects of ALC on the activation of hormone secretion that accompanies puberty in female rhesus monkeys. They also suggest that the subsequent growth spurt and normal timing or progression of puberty may be at risk in human adolescents and teenagers consuming even relatively moderate amounts of ALC on a regular basis.

An increased intraovarian synthesis of nerve growth factor and its low affinity receptor is a principal component of steroid-induced polycystic ovary in the rat.

A form of polycystic ovary (PCO) resembling some aspects of the human PCO syndrome can be induced in rats by a single injection of estradiol valerate (EV). An increase in sympathetic outflow to the ovary precedes, by several weeks, the appearance of cysts, suggesting the involvement of a neurogenic component in the pathology of this ovarian dysfunction. The present study was carried out to test the hypotheses that this change in sympathetic tone is related to an augmented production of ovarian nerve growth factor (NGF), and that this abnormally elevated production of NGF contributes to the formation of ovarian cysts induced by EV. Injection of the steroid resulted in increased intraovarian synthesis of NGF and its low affinity receptor, p75 NGFR. The increase was maximal 30 days after EV, coinciding with the elevation in sympathetic tone to the ovary and preceding the appearance of follicular cysts. Intraovarian injections of the retrograde tracer fluorogold combined with in situ hybridization to detect tyrosine hydroxylase (TH) messenger RNA-containing neurons in the celiac ganglion revealed that these changes in NGF/p75 NGFR synthesis are accompanied by selective activation of noradrenergic neurons projecting to the ovary. The levels of RBT2 messenger RNA, which encodes a beta-tubulin presumably involved in slow axonal transport, were markedly elevated, indicating that EV-induced formation of ovarian cysts is preceded by functional activation ofceliac ganglion neurons, including those innervating the ovary. Intraovarian administration of a neutralizing antiserum to NGF in conjunction with an antisense oligodeoxynucleotide to p75 NGFR, via Alzet osmotic minipumps, restored estrous cyclicity and ovulatory capacity in a majority of EV-treated rats. These functional changes were accompanied by restoration of the number of antral follicles per ovary that had been depleted by EV and a significant reduction in the number of both precystic follicles and follicular cysts. The results indicate that the hyperactivation of ovarian sympathetic nerves seen in EV-induced PCO is related to an overproduction of NGF and its low affinity receptor in the gland. They also suggest that activation of this neurotrophic-neurogenic regulatory loop is a component of the pathological process by which EV induces cyst formation and anovulation in rodents. The possibility exists that a similar alteration in neurotrophic input to the ovary contributes to the etiology and/or maintenance of the PCO syndrome in humans.

Intraovarian excess of nerve growth factor increases androgen secretion and disrupts estrous cyclicity in the rat.

A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.

Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys.

Although progesterone plays an essential role in ovulation and the luteiniziation of the primate follicle, the expression of cellular components required for progesterone synthesis and their control is not well defined. This study was designed to determine the time course and gonadotrophin versus steroid regulation of the transcription of genes involved in progesterone synthesis in peri-ovulatory follicles. Granulosa cells or whole ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or up to 36 h following the administration of an ovulatory human chorionic gonadotrophin (HCG) bolus with or without a 3beta-hydroxysteroid dehydrogenase (3beta-HSD) inhibitor, with or without a non-metabolizable progestin. Granulosa cell concentrations of low density lipoprotein receptor (LDL-R) and steroidogenic acute regulatory protein (StAR) mRNA increased transiently 12 h following HCG administration (P < 0.05) at which time steroid depletion tended to reduce StAR mRNA (P = 0.06). At 36 h post-HCG progesterone suppressed the LDL-R mRNA levels (P < 0.05). P450 side-chain cleavage (P450scc) mRNA decreased in a time-dependent fashion up to 24 h, whereas 3beta-HSD mRNA increased within 12 h of HCG administration (P < 0.05) in a steroid-independent manner. Whole ovarian 17alpha-hydroxylase (P450c17) and granulosa cell P450 aromatase (P450arom) mRNA declined in a time-dependent fashion; by 36 h after HCG administration, steroid depletion increased P450arom mRNA, although progestin replacement did not return aromatase to control values (P < 0.05). These data demonstrate diverse patterns of steroidogenic enzyme expression that generally reflect the conversion of the macaque peri-ovulatory follicle from an oestrogen to progesterone producing gland. Although mRNAs associated with progesterone synthesis and metabolism are primarily regulated by gonadotrophins, cholesterol uptake and utilization may be modulated locally by steroids in luteinizing granulosa cells.

Oocytes are a source of catecholamines in the primate ovary: evidence for a cell-cell regulatory loop.

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine beta-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes-but not those from which the oocytes had been removed-responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a beta-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced beta-adrenoreceptor activation.

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary.

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells.

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.