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.

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.

Effect of frontal hypothalamic deafferentation on photoperiod-induced changes in the secretion of prolactin in the ewe.

The purpose of these experiments was to determine the effect of frontal hypothalamic deafferentation (FHD) between the suprachiasmatic-preoptic region and the mediobasal hypothalamus on diurnal, seasonal, and photoperiod-induced changes in PRL secretion in ewes. In Exp 1, intact cycling ewes were subjected to either FHD or sham FHD. They were kept outdoors, and sequential hourly blood samples were taken for either 24 or 36 h during two breeding seasons and one nonbreeding season. Mean plasma PRL concentrations were higher (P less than 0.01) during the nonbreeding season than during the breeding season in both FHD and sham FHD ewes. Although FHD did not block the seasonal changes, it reduced (P less than 0.05) the 24-h mean concentration and diurnal peak concentration during the nonbreeding season. During the breeding seasons, there was no consistent diurnal pattern of PRL concentrations. In Exp 2, ovariectomized ewes were placed in a photochamber. After adaptation to an 8-h light, 16-h dark (8L:16D) photoperiod, they were subjected to either FHD or sham FHD. Sequential hourly blood samples were collected on the last day of exposure to each of three sequential lighting periods (120 days 8L:16D, 90 days 16L:8D, and 90 days 8L:16D). The 24-h mean plasma PRL concentrations were higher (P less than 0.01) after exposure to 16L:8D than after exposure to 8L:16D in both FHD and sham FHD ewes. FHD did not affect the 24-h mean concentrations. In both groups, there was a distinct diurnal pattern of plasma PRL after exposure to 16L:8D, but not after exposure to 8L:16D. FHD did not alter the diurnal pattern, but significantly (P less than 0.05) reduced the magnitude of the nocturnal elevation under 16L:8D. In summary, FHD reduced, but did not block, seasonal changes in PRL secretion in intact ewes and did not alter photoperiod-induced changes in 24-h patterns and 24-h mean concentrations of plasma PRL in ovariectomized ewes. These results suggest that direct neural pathways between the supra-chiasmatic-preoptic region and the mediobasal hypothalamus play a relatively minor role in mediating photoperiod-induced changes in PRL secretion in sheep.

A biphasic developmental pattern of circulating leptin in the male rhesus macaque (Macaca mulatta).

To help elucidate the physiological role of leptin during somatic and sexual maturation, circulating concentrations of leptin were measured in 36 male rhesus monkeys of ages ranging from 0-20 yr. The body weight of these animals showed a steady increase of approximately 1 kg/yr during the first decade of life and reached a plateau at approximately 13 yr. In contrast, serum leptin concentrations showed a biphasic developmental pattern, which was highlighted by a strong negative correlation with body weight (r = -0.74, P < 0.001) before the onset of puberty (at approximately 3.5 yr) and by a strong positive correlation afterward (r = 0.77, P < 0.001). Overall, the developmental changes in serum leptin concentrations closely mimicked the expected developmental changes in serum testosterone concentrations (r = 0.62, P < 0.001), which were highly elevated at birth, fell to basal levels during the juvenile phase of development, and gradually rose again after the initiation of puberty. However, mean serum leptin concentrations during the peripubertal period itself (3-5 yr) were significantly lower (P < 0.01) than those observed during the first year of life or those observed in fully mature adults (i.e. > 7 yr) (3.5 +/- 0.3, 1.4 +/- 0.2, and 3.3 +/- 0.6 ng/ml, respectively). These data demonstrate that the role of leptin in energy homeostasis of primates is more than a simple linear relationship, being highly dependent upon the developmental age. Furthermore, the data do not support the hypothesis that leptin plays a major role in triggering the onset of puberty in primates, although the strong correlation between serum concentrations of leptin and testosterone suggests that the secretion of these two hormones may be causally linked.

Preovulatory gonadotropin-releasing hormone surge in ovarian-intact rhesus macaques.

The occurrence and profile of the preovulatory hypothalamic GnRH surge in relation to plasma profiles of LH and ovarian steroids, i.e. 17 beta-estradiol (E2) and progesterone (P4), were examined in ovarian intact, freely moving rhesus macaques. Nine monkeys with active ovarian cycles were each fitted with a jugular venous catheter and two push-pull cannulae directed to separate sites within the median eminence (ME). Each female was connected continuously to a tether/swivel device through which daily blood samples or frequent blood samples and ME perfusates (simultaneously at 10- to 20-min intervals for 18-24 h) were obtained without disturbing the animals. An increment in the plasma E2 level (> 150 pg/ml) during the follicular phase (FP) was selected as the preovulatory ovarian signal and served as the index for initiating the ME push-pull perfusion (PPP). Daily increased P4 concentrations of more than 1 ng P4/ml plasma for several consecutive days were consistent with the assumption of ovulation and subsequent formation of a corpus luteum after PPP. A total of 18 PPP trials were completed; each in a fresh ME site. Five of these PPPs were performed during the mid- and late FP (3 were between 6-8 days before and 2 were 4 days before the E2 peak). The remaining 13 PPPs, each of 18- to 24-h duration, were performed between 24 h before and 48 h after the highest daily plasma E2 level, i.e. time zero. Of these 13 PPPs, 2 started within 12 h before (-12 to 0 h), 8 began within 12 h after (0-12 h), and 3 started between 12-24 h after this peak E2 value. During the FP, mean levels of GnRH and LH were less than 2 pg/ml and 20 ng/ml, respectively. During the periovulatory interval (-24 to 48 h around time zero), the release of hypothalamic GnRH (expressed in picograms per ml) increased to 6.63 +/- 2.35 between -12 to 0 h (n = 2), peaked at 20.70 +/- 6.09 between 0-12 h (n = 10), declined to 3.25 +/- 1.39 between 12-24 h (n = 11), and further declined to 0.89 +/- 0.18 between 24-36 h (n = 3). The mean GnRH value from 0-12 h was higher (P < 0.05) than other means (including those during the FP), except for the value between -12 to 0 h. Changes in mean plasma LH values during the same periods paralleled those in GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Third-ventricular infusion of neuropeptide Y suppresses luteinizing hormone secretion in ovariectomized rhesus macaques.

Neuropeptide Y (NPY) has been shown to modulate gonadotropin secretion in an estrogen-dependent manner in the rat and rabbit, and to act centrally in these species to alter GnRH release. The present study examined the ability of centrally administered NPY to affect LH secretion in the primate. Human NPY (hNPY) was administered into the third cerebroventricle of unanesthetized, freely moving, ovariectomized (OVX), or estradiol (E2)-treated OVX rhesus monkeys. LH was measured in blood samples collected remotely at 10-min intervals throughout the experiments. An extensive range of NPY doses was tested in a preliminary study in which OVX monkeys received a 3-h control infusion of Krebs Ringer phosphate buffer (KRP) followed immediately by a 3-h infusion of hNPY (0.1-50 micrograms/h). Only doses greater than or equal to 5 micrograms/h produced a consistent and marked suppression of LH (5 micrograms/h; 35.1 +/- 7.2% reduction, P less than 0.05, n = 4). A longer duration study was performed to better characterize changes in LH pulse frequency and amplitude produced by hNPY treatment. We administered 5 micrograms/h and 15 micrograms/h to OVX (n = 5) and E2-treated OVX (n = 4) monkeys according to the following protocol: 8 h control KRP/8-h hNPY/6-h recovery KRP. In OVX monkeys, LH was suppressed after 2 to 3 h of peptide infusion (P less than 0.01); LH secretion returned to normal after treatment. Both doses of hNPY suppressed mean LH by approximately 55% (P less than 0.05) and LH pulse frequency by approximately 69% (P less than 0.025). LH pulse amplitude was unaffected. In E2-treated OVX monkeys, neither dose of hNPY affected mean LH or LH pulse amplitude. LH pulse frequency was suppressed by approximately 65% (P less than 0.05) during 15-micrograms/h treatment. Because centrally administered hNPY reduced LH pulse frequency and thereby mean LH levels, our results support a central, neural action of NPY to affect the GnRH/LH secretory system. The ability of estrogen feedback to alter the response to NPY treatment supports a physiological role for NPY in controlling reproduction in the primate.

Gonadotropin-releasing hormone and norepinephrine release from the rabbit mediobasal and anterior hypothalamus during the mating-induced luteinizing hormone surge.

The release of hypothalamic GnRH in association with the mating-induced LH surge was studied in the rabbit. Push-pull perfusion (PPP) of the mediobasal (MBH) or anterior (AH) regions of the hypothalamus was performed on conscious, unrestrained does for 3 h before and 5 h after exposure to a vasectomized buck. In experiment 1, GnRH concentrations were measured by RIA in 20-min fractions of MBH-PPP. An approximately 100-fold increase in GnRH release was observed within 1 h of coitus (pre, 1.15 +/- 0.29 pg/ml; peak, 106.67 +/- 37.42 pg/ml; n = 6; P less than 0.05). Concomitant surges of LH and PRL in the peripheral circulation were observed. In experiment 2, GnRH and norepinephrine (NE) were measured (the latter by radioenzymatic assay) in 10-min fractions of MBH-PPP. A 218% postcoital rise in NE levels (n = 5; P less than 0.05) in MBH-PPP accompanied an approximately 50-fold peak rise in GnRH in the same samples (pre, 1.57 +/- 0.23 pg/ml; peak, 76.52 +/- 50.14 pg/ml; P less than 0.05). MBH-NE, MBH-GnRH, LH, and PRL release began rising within 10 min of coitus. In experiment 3, GnRH was measured in 20 min fractions of AH-PPP. Coitus induced a marked rise in AH-GnRH release (precoitus, 0.31 +/- 0.03 pg/ml; peak, 2.25 +/- 0.80 pg/ml; n = 4; P less than 0.05) which differed from coitus-induced MBH-GnRH release both quantitatively (i.e. approximately 7-fold increase for AH vs. approximately 50-100-fold increase for MBH; 50-min lag time for AH vs. less than 20 min for MBH) and qualitatively (i.e. AH-GnRH release was discontinuous, while MBH-GnRH release rose sharply, plateaued, and then declined slowly over the 2-5 h following coitus). No changes in MBH-NE, MBH-GnRH, AH-GnRH, LH, FSH, or PRL were observed in sham-mated does (Exp 1, n = 7; Exp 2, n = 3; Exp 3, n = 4). These data support the hypotheses that: 1) hypothalamic GnRH release is a component of reflexive ovulation in the rabbit; 2) increased hypothalamic noradrenergic tone is related to the surge-release of GnRH; and 3) AH-GnRH release is enhanced following coitus.

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep.

The push-pull perfusion technique was used in combination with a sequential bleeding schedule to estimate simultaneously the release patterns of LHRH and LH in unanesthetized ovariectomized sheep and to determine the temporal relationship between the release of these two hormones. Ovariectomized (greater than 30 days) ewes received unilateral push-pull cannula (PPC) implants (od, 0.85 mm) into the median eminence. After at least 6 days of recovery, each ewe was fitted with an indwelling jugular catheter. For push-pull perfusion, a stylette was removed from the outer PPC, and an inner cannula assembly (od, 0.40 mm) was inserted. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 20 microliters/min. Continuous 10-min perfusate fractions were collected, acidified, and stored at -20 C for LHRH RIA. Blood samples were obtained every 10 min via the jugular catheter, each being drawn 5 min after the start of a perfusate collection interval. Plasma LH levels were determined by RIA. The duration of the sampling was 3-7 h. LHRH output was distinctly pulsatile, occurring at a frequency of approximately one pulse every 30-40 min (n = 5 sheep). LHRH pulse amplitude and frequency remained relatively constant throughout each perfusion. Plasma LH values also were pulsatile, and all LH peaks occurred either during the same interval or during the interval after a LHRH pulse. LH pulses always were accompanied or directly preceded by LHRH pulses, but LHRH pulses were not always followed by LH pulses. The amplitudes of LH pulses and corresponding LHRH pulses were highly correlated (r = 0.81; P less than 0.01). Histological examination revealed that detection of LHRH in perfusates depended upon the placement of the PPC tip into either the zona externa of the central median eminence or adjacent areas. These experiments demonstrate that 1) hypothalamic LHRH release in the Ovx ewe occurs in discrete pulses, with a mean interpulse interval of 38.7 +/- 1.5 min, 2) LH pulses invariably are preceded or accompanied by LHRH pulses, and 3) LH pulse amplitude is highly correlated with LHRH pulse amplitude.

Suppression of mediobasal hypothalamic gonadotropin-releasing hormone and plasma luteinizing hormone pulsatile patterns by phentolamine in ovariectomized rhesus macaques.

In gonadectomized animals, pulses of LH are secreted concurrently with pulsatile hypothalamic GnRH and it is hypothesized that pulses of GnRH are either driven or modulated by episodes of catecholamine release. The objective of this study was to determine if the alpha-adrenergic antagonist phentolamine (PHEN) can simultaneously block the release of GnRH and LH in ovariectomized (OVX) rhesus macaques. In Exp 1, simultaneous peripheral blood and mediobasal hypothalamic push-pull perfusion (PPP) samples were collected remotely at 10-min intervals for 24 h via a swivel/tether device in eight conscious, freely moving OVX rhesus monkeys. Phentolamine was continuously infused iv for 6 h at the rate of 4 mg/kg BW.h in five animals and 20 mg/kg BW.h in three animals. Infusion started at 6 h after the commencement of PPP. Sampling of PPP and blood continued for 12 h after the cessation of PHEN infusion. Exp 2 was carried out to determine if PHEN affects pituitary responsiveness to exogenous GnRH under conditions similar to those in Exp 1. Exogenous GnRH (5 micrograms, iv) was injected as a single bolus at 10-h intervals before, during, and after either a saline (4 ml/h for 6 h) infusion or, 3 weeks later, a PHEN infusion (4 mg/kgBW.h for 6 h) in three OVX females. The results of Exp 1 show that pulsatile patterns of hypothalamic GnRH and LH were either dampened or abolished by PHEN infusion. During the recovery period after PHEN infusion, pulse amplitudes of LH were enhanced, but pulse amplitudes of endogenous GnRH did not differ, as compared to those of corresponding LH and GnRH before infusion of PHEN. Data from Exp 2 suggested that the alpha-adrenergic blocking agent had no effect on the pituitary LH response to exogenous GnRH administration. These results directly support the hypothesis that adrenergic neuronal activities are critical for the pulsatile release of hypothalamic GnRH which governs the pulsatile release of LH in gonadectomized animals.

Plasma catecholamines in the chronically cannulated sheep fetus: predominance of L-dihydroxyphenylalanine.

Dihydroxyphenylalanine (Dopa) and catecholamines (CA) concentrations were determined in plasma collected from chronically cannulated fetal and maternal sheep from days 125-140 of gestation. Dopa was measured by a radioenzymatic assay, followed by ion exchange chromatography and high performance liquid chromatography. The assay was linear to 2.5 ng Dopa, and its sensitivity was 35-45 pg. Dopamine (DA), norepinephrine, and epinephrine were determined simultaneously by the same radioenzymatic incubation procedure, followed by solvent extraction and two-dimensional thin layer chromatography. The Dopa level in the fetal circulation was 10-25 times higher than that of DA, 5-10 times higher than that of norepinephrine, and 100 times higher than that of epinephrine. Furthermore, Dopa was the only CA that was significantly (P less than 0.05) higher in fetal (3.5-4.5 ng/ml) than in maternal plasma (1-1.5 ng/ml). The data indicate that Dopa is the predominant circulating CA in the sheep fetus. While the physiological importance of this observation is unknown at the present time, fetal Dopa might serve as the source of free DA in fetal urine and/or amniotic fluid.

The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey.

Leptin acts as a metabolic activator of the neuroendocrine reproductive axis in several rodent species, but whether leptin plays a similar role in primates is unknown. To explore this question, we examined the effects of leptin on gonadotropin and testosterone secretion in male rhesus monkeys that were fasted for 2 days. Mean plasma levels of LH and FSH, LH pulse frequency, and LH pulse amplitude were significantly higher in leptin-treated animals compared with saline-treated controls during the second day of the fast. To identify targets for leptin's action, we used in situ hybridization and computerized imaging to map leptin receptor (Ob-R) messenger RNA (mRNA) distribution. Ob-R mRNA was observed in the anterior pituitary and several areas of the brain, including the arcuate and ventromedial nuclei of the hypothalamus. Ob-R mRNA was coexpressed in both POMC and neuropeptide Y neurons in the arcuate nucleus, whereas little or no coexpression of Ob-R mRNA was evident in GnRH neurons. These results suggest that leptin is a metabolic signal to the reproductive axis in primates and imply that both POMC and neuropeptide Y neurons are involved in mediating leptin's effects in the brain.

Tyrosine hydroxylase and norepinephrine transporter mRNA levels increase in locus coeruleus after coitus in rabbits.

Hypothalamic norepinephrine (NE) plays an important role in the control of sexual behavior and in the secretion of gonadotropin. Our previous study showed that coitus induced simultaneous increases in hypothalamic NE and GnRH releases in female but not in male rabbits. To investigate the activities in noradrenergic neurons during the coitus-induced process of an LH surge, we measured tyrosine hydroxylase (TH, the rate-limiting enzyme in NE synthesis) and NE transporter (NET, a key protein for NE cellular reuptake) mRNA levels in locus coeruleus (LC) noradrenergic cells in female New Zealand White rabbits. Changes in LC-TH and LC-NET mRNA levels were also measured in males as controls. Female rabbits were killed before coitus and at 15, 30, 60, 120, and 240 min after coitus (n = 6-7/time point); males were killed before and at 30, 60, and 120 min after coitus (n = 3/time). Individual brainstems were sectioned, the LC neurons punched, and TH and NET mRNAs were quantified by ribonuclease protection assay (RPA). Rabbit-specific TH (330 bp) and NET (503 bp) cDNAs were used as probes in the RPA for gene-specific signals. A rabbit 'house-keeping' cDNA (cyclophilin, 158 bp) was also cloned and used as an internal marker for tissue RNA content. Trunk blood was collected to determine serum LH levels. In female rabbits, serum LH levels rose by 15 min after coitus, reached peak concentrations at 1-2 h, and declined thereafter. The time interval for changes in TH and NET mRNA levels in females was similar to that in serum LH levels. Both TH and NET mRNAs increased significantly by 15 min (73% and 85% respectively) and were elevated for 2 h (87% and 111% respectively). TH mRNA levels returned to basal levels by 4 h after coitus, whereas NET mRNA values were elevated throughout the 4 h of observation. In contrast, LH, TH and NET mRNA levels did not change after coitus in males. The enhanced gene expression of both TH and NET in the LC in females, in accord with our previous demonstration of increased hypothalamic NE release, suggests that regulation of NE synthesis and reuptake is an integral part of the coitus-induced NE/GnRH/LH surge process that includes the initiation, sustenance or recovery of the release and/or storage of these neurochemicals.

Putative estrogen receptor beta and alpha mRNA expression in male and female rhesus macaques.

The profound effects of estrogen on different tissues may involve at least two estrogen receptor (ER) subtypes, ERalpha and the recently discovered ERbeta. Where and how the two ER subtypes differentially or cooperatively mediate estrogen actions, however, are still unknown. In this study, we report the cloning of a specific ERbeta cDNA fragment and the expression of ERalpha and ERbeta mRNAs in various endocrine and non-endocrine tissues of male and female rhesus macaques. Total RNA from monkey tissues was isolated and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using human-specific ERbeta primers. A 126 bp RT-PCR product was identified in ovarian tissue and subsequently transfected into pGEM-T vectors for DNA sequencing. The cloned rhesus monkey ERbeta fragment contained a sequence nearly identical to the corresponding sequence in the human (four mismatched nucleotides out of 126). Because complete monkey ERbeta and ERalpha DNA sequences have not been established, the expression of the ERbeta and ERalpha fragments in monkey tissues by RT-PCR reflects 'putative' ERbeta and ERalpha mRNA expression, respectively. Both ERbeta and ERalpha mRNAs were present in male and female reproductive organs, in several endocrine and non-endocrine tissues, and in various regions of the brain, whereas several tissues, including liver, frontal cortex, caudate nucleus, locus coeruleus and cerebellum, expressed only ERalpha message. In some brain regions, i.e. the putamen, internal capsule, hippocampus and paraventricular hypothalamus, the ERbeta fragment was expressed in the female but not in the male. These data suggest that ERalpha mRNA is widely distributed in both female and male tissues, while ERbeta mRNA is more widely distributed in female than in male brain.

Molecular activation of noradrenergic neurons in the rabbit brainstem after coitus.

Our previous studies indicate that coitus in female rabbits induces a gonadotropin-releasing hormone (GnRH) surge that is preceded by an increase in hypothalamic norepinephrine (NE) release. The additional findings of an enhanced tyrosine hydroxylase (TH) mRNA expression in the female brainstem after coitus, in addition to the appropriate topographic distribution of TH and dopamine-beta-hydroxylase (DBH), lead us to hypothesize that coital signals are relayed to hypothalamic GnRH-secreting neurons via brainstem NE-containing perikarya. Here we analyzed coitally activated areas in the brainstem by in situ hybridization of the oncogene c-fos, as well as the expression of TH mRNA at 0, 30 and 60 min postcoitus using specific 35S-labeled probes for c-fos and TH. To establish the identity of activated brainstem neurons, we immunocytochemically double-labeled cells with specific antibodies against Fos protein and DBH at 90 min postcoitus. Both c-fos and TH mRNAs were present at 0 min (control) in the A1, A2 and A6 brainstem-noradrenergic areas. At 30 min after coitus the expression of both genes significantly increased (P<0.01) in the A1 and A2 areas. By 60 min postcoitus the expression of c-fos mRNA decreased to control levels, while that of TH mRNA remained stimulated. Double-labeling of Fos and DBH indicated that the number of dual-labeled neurons increased (P<0.05) over control levels only in the A1 and A2 areas (not in A6) at 90 min postcoitus. These findings support the hypothesis that coitus activates transcriptional/translational events within brainstem NE neurons that culminate in the release of hypothalamic NE and hence a GnRH surge.

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.

Topographic comparison of the expression of norepinephrine transporter, tyrosine hydroxylase and neuropeptide Y mRNA in association with dopamine beta-hydroxylase neurons in the rabbit brainstem.

In mammalian species, ovulation occurs following a massive release of hypothalamic gonadotropin-releasing hormone (GnRH). Several chemicals, including norepinephrine (NE) and neuropeptide Y (NPY), are responsible for the initiation and/or magnitude and duration of this pre-ovulatory GnRH surge. In the central nervous system, NE neural cell bodies are located in the brainstem; some are co-localized with NPY neurons and/or co-express the NE transporter (NET) gene which dictates NET protein production. The activity of NET at NE terminals is critical for synaptic NE function. In the rabbit, coitus induces a hypothalamic NE release which precedes the GnRH surge. We hypothesize that the coital stimulus is transmitted to the brainstem and transformed and integrated into GnRH-stimulating signals via NE, NET and/or NPY. However, very little is known about the distribution of cells expressing NET, NPY and tyrosine hydroxylase (TH, the rate-limiting enzyme of NE synthesis) in this species. Therefore, we utilized the sensitive in situ hybridization technique to identify the presence of these messages in conjunction with the location of NE cells, the latter being marked by dopamine beta-hydroxylase (DBH), the specific enzyme for NE synthesis. Three non-mated New Zealand White does were perfused with 4% paraformaldehyde and their brainstems were sectioned at 20-micron thick between 2 mm caudal to the obex and the rostral pons. Serial sections were immunohistochemically stained for DBH and hybridized with rabbit-specific TH and NET cRNAs and a human NPY probe. The data suggest that several DBH-positive areas in the medulla expressed one or more messages, i.e. the lateral tegmentum (A1) and the nucleus of the solitary tract (A2) expressed all three mRNAs, the area postrema (AP) contained NET and TH mRNAs but not NPY cells. In the pons, the locus coeruleus (LC), subnucleus of coeruleus (LCs) and lateral tegmental nuclei (A5) expressed NET and TH mRNAs but contained little or no NPY message. The distribution patterns of TH and NET appeared to be similar in the LC, LCs, A2 and AP.

Hypothalamic site-dependent effects of neuropeptide Y on gonadotropin-releasing hormone secretion in rhesus macaques.

In rodents and rabbits, neuropeptide Y (NPY) has a bimodal effect on gonadotropin-releasing hormone (GnRH) secretion. Intracerebroventricular (icv) administration or direct infusion of NPY into the median eminence (ime) suppresses GnRH release in ovariectomized (OVX) animals, but stimulates GnRH release in intact or OVX animals treated with ovarian steroids. Specific ovarian steroid-dependent NPY effects are, however, not obvious in non-human primates. In OVX rhesus monkeys, icv administration of NPY has been shown to suppress luteinizing hormone (LH) secretion whereas ime infusion of NPY stimulates GnRH pulses. In such animals, estrogen replacement does not reverse the inhibitory NPY effect on LH release, although estrogen enhances the stimulatory NPY effect on GnRH secretion. These observations led us to speculate that the bimodal NPY effects in non-human primates may depend on either the site of NPY action or the nature of the steroid milieu. This study utilized the push-pull perfusion (PPP) technique to examine the effects of either ime or icv infusion of NPY on GnRH release in OVX monkeys treated with or without both ovarian steroids. Without exception, ime infusion of NPY increased GnRH concentrations in push-pull perfusates regardless of the steroid status of the animals. In contrast, GnRH levels were reduced during icv infusion of NPY in both untreated and estrogen/progesterone-treated, OVX monkeys. These results indicate that, unlike other mammalian species, in the rhesus monkey the stimulatory and inhibitory effects of NPY on GnRH release depend on the site of NPY infusion within the brain rather than the ovarian steroidal environment.

Gonadectomy alters tyrosine hydroxylase and norepinephrine transporter mRNA levels in the locus coeruleus in rabbits.

The central noradrenergic system has a major regulatory role on gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in rabbits. Exogenous administration of norepinephrine (NE) alters GnRH/LH release in a sex steroid-dependent manner, i.e. NE stimulates GnRH/LH release in oestrogen-primed ovariectomized (OVX) animals but not in non-primed individuals. To investigate how gonadal steroids influence noradrenergic neuronal activities in the locus coeruleus (LC), mRNA levels of tyrosine hydroxylase (TH) and NE transporter (NET), two key factors regulating NE synthesis and uptake, were compared 3 weeks after gonadectomy (GDX). Intact male (n = 5) and female (n = 6) New Zealand White rabbits were sacrificed along with castrated males (n = 4) and OVX females (n = 5). The brainstem from each individual was sectioned and the LC was punched for detection of TH and NET mRNA levels using the ribonuclease protection assay (RPA). Trunk blood was collected to determine immunoactive serum LH values. Levels of LH were elevated in both males and females after GDX. Luteinizing hormone concentrations averaged 0.10 +/- 0.05 ng/ml in intact males vs 1.64 +/- 0.31 ng/ml in castrated males (P < 0.01) and 0.30 +/- 0.08 ng/ml in intact vs 9.80 +/- 3.50 ng/ml in OVX females (P < 0.05), respectively. Removal of the gonads also increased TH mRNA levels in the LC in both males and females. In intact males, TH mRNA levels were 0.796 +/- 0.181 pg/microgram total RNA, whereas in castrates mRNA levels averaged 1.667 +/- 0.345 pg/microgram total RNA (P < 0.05). In intact females, TH mRNA levels were 0.617 +/- 0.054 pg/microgram total RNA while the OVX group averaged 1.084 +/- 0.202 pg/microgram total RNA (P < 0.05). Similar increases in NET mRNA were noted after GDX in both sexes. In males, NET mRNA levels were 1.461 +/- 0.401 pg/microgram total RNA in intacts vs 3.666 +/- 0.649 pg/microgram total RNA in castrates (P < 0.05). In females, NET mRNA levels averaged 1.336 +/- 0.212 pg/microgram total RNA and 3.297 +/- 0.835 pg/microgram total RNA in the intact and OVX groups, respectively (P < 0.05). The data indicate that GDX enhances gene expression of both TH and NET. The results support the hypothesis that the feedback regulation of sex steroids on LH secretion in rabbits of both sexes involves transcriptional/translational processes of at least TH and NET in brainstem NE cells.