Precision of progesterone measurements with the use of automated immunoassay analyzers and the impact on clinical decisions for in vitro fertilization.

OBJECTIVE: To compare the precision of progesterone measurements obtained with the use of immunoassays and of liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN: Comparative study. SETTING: Academic, private practice, and in vitro fertilization (IVF) research centers. PATIENT(S): A total of 189 human serum samples were collected during controlled ovarian hyperstimulation and early pregnancy in women undergoing IVF. INTERVENTION(S): Serum progesterone pools (n = 10; 0.2-4 ng/mL) were sent to four laboratory centers that used four different automated immunoassay analyzers. Progesterone was measured by immunoassay in triplicate at three separate time points (n = 9 per pool) and by LC-MS/MS in triplicate once (n = 3 per pool). MAIN OUTCOME MEASURE(S): Inter- and intraassay coefficients of variation (CVs) of progesterone measurements were compared for each analyzer and LC-MS/MS. RESULT(S): Progesterone measurements by immunoassay were highly correlated with those by LC-MS/MS. Only two analyzers had intraassay CVs <10% at all three experimental time points, and only two analyzers had an interassay CV <10%. Mean progesterone levels by the analyzers were different across multiple progesterone pools. CONCLUSION(S): Our results indicate that progesterone threshold measurements used for IVF clinical decisions should be interpreted cautiously and based on laboratory- and method-specific data. A validated progesterone standard incorporated into daily immunoassays could improve medical decision accuracy.

Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin.

BACKGROUND: An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O2) to improve primate follicle growth and oocyte maturation in vitro. METHODS: Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O2 (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30-40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-müllerian hormone (AMH). RESULTS: Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O2, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O2. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O2 than under 20% O2. CONCLUSIONS: This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

In vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female nonhuman primates.

OBJECTIVE: To determine whether sphingosine-1-phosphate (S1P), or the S1P mimetic FTY720 shields ovaries of adult female rhesus monkeys from damage caused by 15 Gy of targeted radiotherapy, allowing for the retention of long-term fertility, and to evaluate whether S1P protects human ovarian tissue (xenografted into mice) from radiation-induced damage. DESIGN: Research animal study. SETTING: Research laboratory and teaching hospital. PATIENT(S): Adult female rhesus macaques (8-14 years of age; n = 21) and two women (24 and 27 years of age) undergoing gynecologic surgery for benign reasons, after informed consent and approval. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian histologic analysis, ovarian reserve measurements, and fertility in mating trials. RESULT(S): Rapid ovarian failure was induced in female macaques by ovarian application of 15 Gy of radiation. Females given S1P or FTY720 by direct intraovarian cannulation for 1 week before ovarian irradiation rapidly resumed menstrual cycles because of maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic before ovarian irradiation also became pregnant in mating trials. Offspring conceived and delivered by radioprotected females developed normally and showed no evidence of genomic instability, as measured by micronucleus frequency in reticulocytes. Adult human ovarian cortical tissue xenografted into mice also exhibited a reduction in radiation-induced primordial oocyte depletion when preexposed to S1P. CONCLUSION(S): S1P and its analogs hold clinical promise as therapeutic agents to preserve ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.

Hypothalamic expression of KISS1 and gonadotropin inhibitory hormone genes during the menstrual cycle of a non-human primate.

Kisspeptin, the product of the KISS1 gene, stimulates gonadotropin-releasing hormone (GnRH) secretion; gonadotropin inhibitory hormone (GnIH), encoded by the RF-amide-related peptide (RFRP) or NPVF gene, inhibits the reproductive axis. In sheep, kisspeptin neurons are found in the lateral preoptic area (POA) and the arcuate nucleus (ARC) and may be important for initiating the preovulatory GnRH/luteinizing hormone (LH) surge. GnIH cells are located in the ovine dorsomedial hypothalamic nucleus (DMN) and paraventricular nucleus (PVN), with similar distribution in the primate. KISS1 cells are found in the primate POA and ARC, but the function that kisspeptin and GnIH play in primates has not been elucidated. We examined KISS1 and NPVF mRNA throughout the menstrual cycle of a female primate, rhesus macaque (Macaca mulatta), using in situ hybridization. KISS1-expressing cells were found in the POA and ARC, and NPVF-expressing cells were located in the PVN/DMN. KISS1 expression in the caudal ARC and POA was higher in the late follicular phase of the cycle (just before the GnRH/LH surge) than in the luteal phase. NPVF expression was also higher in the late follicular phase. We ascertained whether kisspeptin and/or GnIH cells project to GnRH neurons in the primate. Close appositions of kisspeptin and GnIH fibers were found on GnRH neurons, with no change across the menstrual cycle. These data suggest a role for kisspeptin in the stimulation of GnRH cells before the preovulatory GnRH/LH surge in non-human primates. The role of GnIH is less clear, with paradoxical up-regulation of gene expression in the late follicular phase of the menstrual cycle.

Sequelae in male rabbits following developmental exposure to p,p'-DDT or a mixture of p,p'-DDT and vinclozolin: cryptorchidism, germ cell atypia, and sexual dysfunction.

Rabbit does (7-9 per group) were treated daily per orum from gestation day 15 through post-natal week 4 to provide per kg body wt 25 micaromol (low) or 250 micromol (high) p,p'-DDT or a mixture of DDT and vinclozolin (12.5 and 125 micromol each). Developmental as well as post-pubertal reproductive sequelae of male progeny were studied. Testicular descent in some pups was impaired by DDT. Serum LH or testosterone was not affected. FSH was lower in mixture- but not in DDT-exposed rabbits. Lack of sexual interest, penile erection and ejaculation were observed in some mixture rabbits. Sperm counts were unaffected, but morphologically normal spermatozoa were fewer; nuclear and acrosomal morphogenesis was disrupted. Atypical germ cells resembling carcinoma in situ were found. Also considering data for vinclozolin [Veeramachaneni DNR, Palmer JS, Amann RP, Kane CM, Higuchi TT, Pau K-YF. Disruption of sexual function, FSH secretion, and spermiogenesis in rabbits following developmental exposure to vinclozolin, a fungicide. Reproduction 2006;131:805-16], we concluded that DDT causes cryptorchidism and germ cell atypia, vinclozolin permanently disrupts FSH secretion and sexual function, and the mixture causes the full spectrum of dysgenesis.

Estradiol increases alpha7 nicotinic receptor in serotonergic dorsal raphe and noradrenergic locus coeruleus neurons of macaques.

Acetylcholine, acting on presynaptic nicotinic receptors (nAChRs), modulates the release of neurotransmitters in the brain. The rat dorsal raphe nucleus (DR) and the locus coeruleus (LC) receive cholinergic input and express the alpha7nAChR. In previous reports, we demonstrated that estradiol (E) administration stimulates DR serotonergic and LC noradrenergic function in the macaque. In addition, it has been reported that E induces the expression of the alpha7nAChR in rats. We questioned whether E increased the expression of the alpha7nAChR in the macaque DR and LC. We utilized double immunostaining to study the effect of a simulated preovulatory surge of E on the expression of the alpha7nAChR in the DR and the LC and to determine whether alpha7nAChR colocalizes with serotonin and tyrosine hydroxylase (TH) in macaques. There was no difference in the number of alpha7nAChR-positive neurons between ovariectomized (OVX) controls and OVX animals treated with a silastic capsule containing E (Ecap). However, supplemental infusion of E for 5-30 hours to Ecap animals (Ecap + inf) significantly increased the number of alpha7nAChR-positive neurons in DR and LC. In addition, supplemental E infusion significantly increased the number of neurons in which alpha7nAChR colocalized with serotonin and TH. These results constitute an important antecedent for study of the effects of nicotine and ovarian steroid hormones in the physiological functions regulated by the DR and the LC in women.

Disruption of sexual function, FSH secretion, and spermiogenesis in rabbits following developmental exposure to vinclozolin, a fungicide.

We studied sequelae of prenatal plus infantile exposure of male rabbits to vinclozolin, because it is ingested by women and children. Female Dutch-Belted rabbits (7-10/group) were treated daily per orum from gestation day 15 through post-natal week 4 to provide 0, 7.2, or 72 mg vinclozolin/kg dam's body weight/day. Vinclozolin had no effect on maintenance of pregnancy, growth of pups, age at testicular descent or weight of organs. Concentrations of serum LH or testosterone at 6, 12, or 24 weeks of age were unaffected. However, FSH was lower (P < 0.05) in both vinclozolin groups at all three ages. Following injection of GnRH at 12 or 24 weeks, the increase in FSH was less (P < 0.05) in both vinclozolin groups, as was testosterone at 12 weeks of age. After full sexual maturity, 2 of 7 low dose rabbits were uninterested in female or male teasers and never achieved erection or ejaculation. Overall, rates of ejaculation failure were: control 0% (0/48), low dose 29% (12/42), and high dose 5% (3/60). Daily sperm production per gram of testis and total number of sperm per ejaculate in both vinclozolin groups were similar (P > 0.1) to controls. However, semen from vinclozolin rabbits contained over two times more (P < 0.05) morphologically abnormal spermatozoa, mostly nuclear and acrosomal defects, than semen from controls. Seminiferous tubules with degenerative changes were more frequent (P < 0.05) in vinclozolin rabbits than in controls. Lesions included syncytia of spherical spermatids and desquamation of germ cells. Hence, developmental exposure to vinclozolin caused presumably permanent changes in copulatory ability, secretion of FSH, and spermiogenesis.

Derivation and characterization of monkey embryonic stem cells.

Embryonic stem (ES) cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes. The promises and limitations of primate ES cell-based therapy are also discussed.

Progress with nonhuman primate embryonic stem cells.

Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

Expression of alpha 4 and alpha 7 nicotinic receptors in the brainstem of female rabbits after coitus.

Coital signaling in the female rabbit involves sequential events in the brainstem and hypothalamus, resulting in a massive release of hypothalamic gonadotropin-releasing hormone (GnRH) that peaks within 1-2 h after mating. The neural connections between coitus and GnRH release involves norepinephrine (NE) and acetylcholine (ACh) since administration of antagonists against NE (dibenamine or phentolamine) or ACh (atropine, alpha-bungarotoxin (alpha-BTX) or scopolamine) blocks or attenuates ovulating events. Moreover, hypothalamic NE release and brainstem tyrosine hydroxylase (TH, the rate-limiting enzyme for NE synthesis) expression in the noradrenergic areas increase prior to, or in concert with, the preovulatory GnRH surge. How ACh is involved in the control of ovulation in the rabbit is lesser known. In the present study, the number of brainstem neurons expressing TH, alpha4 and alpha7 subunits of the nicotinic ACh receptor (nAChR) before and after coitus was determined by immunocytochemistry. Compared to non-mated female rabbits, the number of alpha4, alpha7 and TH single-labeled neurons as well as alpha4/TH and alpha7/TH double-labeled neurons increased in the A1, A2 and A6 brainstem noradrenergic areas at 1 h, but not 2 h, after coitus. The results suggest that the participation of ACh in the control of coitus-induced ovulation may include activation of alpha4beta2 and alpha7 nAChRs in neurons within or adjacent to the brainstem noradrenergic areas in female rabbits.

Chronic exposure to dibromoacetic acid, a water disinfection byproduct, diminishes primordial follicle populations in the rabbit.

To determine if dibromoacetic acid (DBA) affects ovarian folliculogenesis, four groups of female Dutch-belted rabbits were exposed daily to 0, 1, 5, or 50 mg DBA/kg body weight in drinking water beginning in utero from gestation day 15 throughout life. Functionality of the endocrine axis was assessed by measuring serum concentrations of gonadotropins following an im injection of 10 microg GnRH at 12 (prepubertal; n = 6/dose group) and 24 (postpubertal; n = 10/dose group) weeks of age. A day after GnRH challenge, number of ovulation sites and ovarian weights were determined at necropsy. Left ovaries were processed for histopathology, serially sectioned at 6 microm, and every twelfth section stained with hematoxylin and eosin was evaluated. All healthy follicles were categorized as primordial, primary, small preantral, large preantral, or small antral follicles. The area of each section evaluated was measured and the number of follicles in each category expressed per mm2 unit area. In prepubertal animals, DBA caused a reduction in number of primordial follicles (p < 0.05) and total healthy follicles (p < 0.05) at 50 mg/kg dose level. In adult animals, there were fewer primordial follicles in both the 5 (p < 0.01) and 50 (p = 0.1) mg/kg dose groups. No profound changes in gonadotropin profiles were observed. Although chronic exposure to DBA did not appear to have an effect on late follicular development or ovulation, DBA did reduce the population of primordial follicles. The long-term health consequences of diminished primordial follicles are unknown, but it is very likely that reproductive senescence would occur earlier.

Pharmacological effects of oxytocin on gastric emptying and intestinal transit of a non-nutritive liquid meal in female rats.

The effects of oxytocin (OT) on gastric emptying, gastrointestinal transit, and plasma levels of cholecystokinin (CCK) were studied in female rats. Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and Na(2)(51)CrO(4). Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Blood samples were collected for CCK radioimmunoassay. After administration of OT (0.2-0.8 mg/kg), gastric emptying and gastrointestinal transit were inhibited, whereas the plasma concentration of CCK was increased in a dose-dependent manner. Atosiban, an oxytocin receptor antagonist, effectively attenuated the OT- induced inhibition of gastric emptying and gastrointestinal transit. However, administration of atosiban alone had no effect on gastric emptying and gastrointestinal transit. The selective CCK(1) receptor antagonists, devazepide and lorglumide, effectively attenuated the OT-induced inhibition of gastric emptying and gastrointestinal transit. L-365, 260, a selective CCK(2) receptor antagonist, did not alter the OT-induced inhibition of gastric emptying and gastrointestinal transit. These results suggest that OT inhibits gastric emptying and gastrointestinal transit in female rats via a mechanism involving CCK stimulation and CCK(1) receptor activation.

Differentiation of monkey embryonic stem cells into neural lineages.

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

Effects of evodiamine on gastrointestinal motility in male rats.

The effects of evodiamine on gastric emptying, gastrointestinal transit, and plasma levels of cholecystokinin (CCK) were studied in male rats. Evodiamine, isolated from the dry unripened fruit of Evodia rutaecarpa Bentham (a Chinese medicine named Wu-chu-yu), has been recommended for abdominal pain, acid regurgitation, nausea, diarrhea, and dysmenorrhea. Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and Na(2)51CrO(4). Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Blood samples were collected for CCK radioimmunoassay (RIA). After administration of evodiamine (0.67-6.00 mg/kg), both gastric emptying and gastrointestinal transit were inhibited, whereas the plasma concentration of CCK was increased in a dose-dependent manner. The selective CCK(1) receptor antagonists, devazepide and lorglumide, effectively attenuated the evodiamine-induced inhibition of gastric emptying and gastrointestinal transit. L-365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-N'-(3-methylphenyl)-urea), a selective CCK(2) receptor antagonist, did not alter the evodiamine-induced inhibition of gastric emptying and gastrointestinal transit. These results suggest that evodiamine inhibits both gastric emptying and gastrointestinal transit in male rats via a mechanism involving CCK release and CCK(1) receptor activation.

Involvement of cholecystokinin receptor in the inhibition of gastrointestinal motility by estradiol in ovariectomized rats.

BACKGROUND: The effects of estradiol benzoate (EB) on gastric emptying, gastrointestinal transit and plasma levels of cholecystokinin (CCK) were studied in ovariectomized rats. METHODS: Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and Na2 51CrO4. Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Blood samples were collected for E2 and CCK radioimmunoassay. RESULTS: After treatment of EB (4-25 microg/kg), gastric emptying and gastrointestinal transit were inhibited, whereas plasma concentrations of E2 and CCK were increased in a dose-dependent manner. The selective CCK(A) receptor antagonists, devazepide and lorglumide, effectively attenuated the EB-induced inhibition of gastric emptying and gastrointestinal transit. L-365,260, a selective CCK(B) receptor antagonist, did not alter the EB-induced inhibition of gastric emptying and gastrointestinal transit. CONCLUSIONS: The results suggest that EB inhibits gastric emptying and gastrointestinal transit in ovariectomized rats via a mechanism involving CCK stimulation and CCK(A) receptor activation.

Involvement of cholecystokinin receptor in the inhibition of gastric emptying by oxytocin in male rats.

The effects of oxytocin (OT) on gastric emptying and plasma levels of cholecystokinin (CCK) were studied in male rats. Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and Na2(51)CrO4. Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Blood samples were collected for OT and CCK radioimmunoassay. After administration of OT (0.2-0.8 mg x kg(-1)), gastric emptying was inhibited, whereas plasma concentrations of OT and CCK were increased in a dose-dependent manner. Atosiban, an oxytocin receptor antagonist, effectively attenuated the OT-induced inhibition of gastric emptying. However, administration of atosiban alone had no effect on gastric emptying. Devazepide (3 mg x kg(-1)), a selective CCKA receptor antagonist, effectively attenuated the OT-induced inhibition of gastric emptying. L-365, 260, a selective CCKB receptor antagonist, did not alter the OT-induced inhibition of gastric emptying. These results suggest that OT inhibits gastric emptying in male rats via a mechanism involving CCK stimulation and CCKA receptor activation.

Norepinephrine transporter mRNA expression after coitus in the rabbit brainstem.

In the female rabbit, coitus induces a massive release of hypothalamic gonadotropin-releasing hormone (GnRH) within 20 min. The GnRH surge is preceded by an increase in hypothalamic norepinephrine (NE) release. Presumably, coitus stimulates NE, hence GnRH, release by increasing the activity of tyrosine hydroxylase (TH, the rate-limiting enzyme for NE synthesis) and/or decreasing the activity of norepinephrine transporter (NET, the key protein for NE re-uptake). Since NE cell bodies are located primarily in the brainstem, we hypothesize that coital signals are relayed to hypothalamic GnRH-secreting neurons via brainstem NE-containing perikarya. In support of this hypothesis, we found that both c-fos and TH mRNA expressions in brainstem noradrenergic areas, particularly in the A1 and A2 cell groups, increased within 30 min and returned to precoital levels within 60 min after coitus. Here we analyzed coitally induced changes in NET mRNA expression at 0, 15, 30 and 60 min postcoitus in the brainstem by in situ hybridization, using 35S-labeled rabbit NET RNA probes. In comparison with nonmated females (i.e., at 0 min), the expression of NET mRNA significantly increased (P<0.05) within 15 min postcoitus in the A1, but not the A2 area. By 30 min postcoitus, NET gene expression increased in the caudal portion of the A1 and in the caudal and central portion of the A2. By 60 min postcoitus, NET mRNA expression in the caudal and rostral portion of the A1 and the caudal and central portion of the A2 was still higher than NET mRNA expression in nonmated rabbits (P<0.05). No change in NET mRNA expression was observed in the A6. The results suggest that coitus increases NET mRNA expression in A1 and A2 noradrenergic areas within 15-30 min, and this enhanced NET mRNA expression was maintained for at least 60 min, particularly in the A2. These findings, in combination with our previous observation on increased TH gene expression within 30 min, but not 60 min, after coitus, further suggest that the coitus-induced NET transcriptional events within brainstem NE neurons may play an important role in the maintenance, and particularly in the termination, of hypothalamic NE release, hence regulating the size and duration of the coitus-induced GnRH surge.

Ovarian influence on gonadotropin and prolactin release in mated rabbits.

In 17beta-estradiol (E)-treated ovariectomized (OVX) rabbits, the coitus-induced luteinizing hormone (LH) surge is only one fourth that in ovarian-intact rabbits. In this study, we determined the pattern of the coitus-induced gonadotropin release, i.e., LH and follicle-stimulating hormone (FSH), in OVX + E animals without or with continuous 3-wk treatment of 20-alphahydroxypregn-4-en-3-one (20alphaP). For positive and negative experimental controls, ovarian-intact rabbits were either mated or sham mated, respectively. The pituitary hormones prolactin (PRL) and growth hormone (GH) were measured to serve as collateral controls for gonadotropins. The addition of continuous 20alphaP in OVX + E does fail to stimulate a coitus-induced LH surge equal in magnitude and duration to the LH surge in ovarian-intact rabbits. Postcoital levels of FSH were greater in OVX + E + 20alphaP animals than those in OVX + E rabbits. Coitus induced a PRL surge in ovarian-intact and OVX + steroid-treated females, but not in mated males, thereby suggesting a gender difference in this neuroendocrine circuit. Neither coitus nor steroids altered plasma GH values in female or male animals. We conclude that chronic administration of neither E nor E + 20alphaP can restore full-scale gonadotropin surges in OVX rabbits, whereas replacement of one or both of these steroids is sufficient for a coitus-induced PRL surge. Moreover, the presented observation that activin stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release suggests a possible involvement of ovarian proteins in the production of a full-scale coitus-induced GnRH/LH surge.

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques.

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

Coitus-induced activation of c-fos and gonadotropin-releasing hormone in hypothalamic neurons in female rabbits.

Copulation induces hypothalamic release of neuropeptides and catecholamines, especially gonadotropin-releasing hormone (GnRH) and norepinephrine, in female rabbits. The forebrain distribution of GnRH cells and the cellular events responsible for the coitally induced GnRH surge have not been identified. We characterized the expression of c-fos mRNA before (0 min) and up to 60 min after coitus in forebrain tissues of mated and nonmated females and compared these findings with those in which single- and double-labeled GnRH/Fos protein cells were identified by immunocytochemistry (ICC). Enhanced expression of fos-mRNA occurred 30 min after coitus, especially in the anteroventral periventricular nucleus (AVPV), the encapsulated portion of the bed nucleus of the stria terminalis (BNSTe) and the ventrolateral hypothalamus (VLH); this increased fos-mRNA activity remained elevated at 60 min in the AVPV and VLH, and was reflected by Fos protein expression 90 min postcoitus. Both ICC Fos-labeled and ICC GnRH-labeled cells were widely distributed throughout the forebrain with postcoital increased double-labeling in the preoptic-septal areas, the anterior-medial hypothalamus and the VLH. The increased number of dual-labeled and unchanged number of single-labeled GnRH cells after coitus suggest some GnRH neurons were non-detected before coitus. Many dual-labeled neurons were adjacent to Fos-labeled cells, suggesting enhanced interneuronal input to GnRH cells after coitus. Collectively, the results suggest that coitus activates hypothalamic GnRH neurons via several loci that include the AVPV, BNSTe and VLH. The distinct anatomical location of the AVPV, BNSTe and VLH further suggests that coital signals may reach the hypothalamus via separate neural pathways that are likely developed within the brainstem.