High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function.

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signalling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through pre-pubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of tumour necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signalling of the testis in health and disease.

Amyloidosis increase is not attenuated by long-term calorie restriction or related to neuron density in the prefrontal cortex of extremely aged rhesus macaques.

As human lifespan increases and the population ages, diseases of aging such as Alzheimer's disease (AD) are a major cause for concern. Although calorie restriction (CR) as an intervention has been shown to increase healthspan in many species, few studies have examined the effects of CR on brain aging in primates. Using postmortem tissue from a cohort of extremely aged rhesus monkeys (22-44 years old, average age 31.8 years) from a longitudinal CR study, we measured immunohistochemically labeled amyloid beta plaques in Brodmann areas 32 and 46 of the prefrontal cortex, areas that play key roles in cognitive processing, are sensitive to aging and, in humans, are also susceptible to AD pathogenesis. We also evaluated these areas for cortical neuron loss, which has not been observed in younger cohorts of aged monkeys. We found a significant increase in plaque density with age, but this was unaffected by diet. Moreover, there was no change in neuron density with age or treatment. These data suggest that even in the oldest-old rhesus macaques, amyloid beta plaques do not lead to overt neuron loss. Hence, the rhesus macaque serves as a pragmatic animal model for normative human aging but is not a complete model of the neurodegeneration of AD. This model of aging may instead prove most useful for determining how even the oldest monkeys are protected from AD, and this information may therefore yield valuable information for clinical AD treatments.

Cognition in aged rhesus monkeys: effect of DHEA and correlation with steroidogenic gene expression.

Estradiol supplementation has been shown to enhance cognitive performance in old ovariectomized rhesus macaques (Macaca mulatta). To determine if similar benefits could be achieved in perimenopausal animals using alternative hormonal supplements, we administered dehydroepiandrosterone (DHEA) to old ovary-intact female rhesus macaques for ∼2.5 months. Using computerized touch screen memory tasks, including delayed response (DR) and delayed matching-to-sample (DMS), we observed improved performance with time in all of the animals but failed to detect a significant effect of DHEA. On the other hand, gene expression profiling disclosed a significant correlation between cognitive performance and the expression of several steroidogenic and steroid-responsive genes. The DR performance was positively correlated with hippocampal expression of AKR1C3 and STAR and negatively correlated with the expression of SDRD5A1. A positive correlation was also found between DMS performance and prefrontal cortical expression of AKR1C3 and a negative correlation with STAR, as well as a negative correlation with the hippocampal expression of HSD11B1 and NR3C1. Taken together, the results suggest that steroidogenic gene regulation within the brain may help to maintain cognitive function during the perimenopausal transition period, despite a decline in sex-steroid levels in the circulation.

Leptin and puberty.

Leptin is thought to relay metabolic information to the hypothalamic-pituitary- gonadal axis and to participate in the neuroendocrine control of puberty. To help elucidate the underlying mechanism, Cheung et al. recently performed a diverse series of experiments, the results of which undermine the prevailing hypothesis that leptin acts as a metabolic trigger for the initiation of puberty. Instead, their results suggest that leptin is one of many permissive metabolic factors that allow pubertal development to proceed.

Photoperiodic modulation of luteinizing hormone secretion in orchidectomized Syrian hamsters and the influence of excitatory amino acids.

The effect of N-methyl-D,L-aspartate (NMA) on LH secretion was investigated in Syrian hamsters of the LSH/Ss Lak strain, maintained under either long days (14 h of light, 10 h of darkness) or short days (6 h of light, 18 h of darkness). After 6 weeks of photoperiodic treatment, the animals were orchidectomized. Ten days later, 10-min blood samples were remotely collected from each animal, using surgically implanted intraatrial catheters, and individual pulsatile LH release profiles were subsequently determined by RIA. At the end of the 4-h sampling period, NMA was administered iv (30 mg/kg BW), and the LH response was determined in the next three plasma samples. Hamsters maintained under long days had very high mean plasma LH levels (453 +/- 46 ng/ml) and displayed episodic release patterns characterized by high amplitude pulses. In marked contrast, hamsters that were maintained under short days had significantly (P less than 0.001) lower mean plasma LH levels (90 +/- 12 ng/ml) and showed a significantly (P less than 0.001) lower mean pulse amplitude. The interpulse interval was similar in both of the groups, with LH peaks occurring, on the average, once every 65 min. When challenged with NMA, the long-day hamsters showed only a 37% increase in mean plasma LH levels, which was not statistically significant. In contrast, the short-day hamsters showed a highly significant (P less than 0.01) increase of 294%. Interestingly, the mean plasma LH levels after NMA administration were the same in the two photoperiodic groups despite a marked difference in the plasma levels that preceded the administration. These findings demonstrate that short days can inhibit the neuroendocrine activity of the hamster's reproductive axis independently of gonadal influences. They also suggest that the LHRH neurons have an intrinsic capacity to secrete very high levels of the neuropeptide, regardless of the photoperiod. Taken together, the results support the hypothesis that the breeding season in the hamster is regulated by a gonad-independent mechanism involving a photoperiodic modulation of neuroexcitatory inputs to the LHRH neurons.

A role for N-methyl-D-aspartate receptors in the control of seasonal breeding.

The neuroendocrine basis for short-day induced testicular regression was studied in Syrian hamsters of the LSH/Ss Lak strain. Adult animals were maintained either under long or short days (14:10D or 6L:18D, respectively) and given single, daily ip injections of N-methyl-D-aspartate (NMDA) (25 mg/kg BW); control animals received injections of saline. As expected, the testes of the short-day controls had completely regressed to a prepubertal condition within 6 weeks, a change that was associated with significantly reduced mean plasma gonadotropin levels. In contrast, the NMDA-treated hamsters from both the long-day and short-day groups, as well as the long-day controls, all maintained large testes and elevated plasma gonadotropin levels, although plasma luteinizing hormone (LH) was partially suppressed in the short-day group. In a second experiment which lasted 2 weeks, short-day hamsters with completely regressed testes were either transferred to long days, maintained further on short days, or maintained on short days and given a daily ip injection of NMDA (25 mg/kg BW). The short-term exposure to long days caused an expected increase in plasma and pituitary concentrations of follicle-stimulating hormone, pituitary LH, and testicular weight. Similar, but even more marked, changes were observed in the short-day hamsters that were treated with NMDA, including significant increases in plasma LH and serum testosterone concentrations. Moreover, histological examination revealed that the recrudescing testes from this latter group already contained mature spermatocytes and in some individuals even spermatozoa. These results demonstrate that NMDA receptors may play a pivotal role in both the termination and onset of the breeding season in photoperiodic species.

The development of afternoon minisurges of luteinizing hormone secretion in prepubertal female rats is ovary dependent.

We have recently disclosed sustained episodes of LH release (minisurges) during the afternoons of the juvenile-peripubertal transition period in the female rat. To determine if these LH minisurges are gonad independent, i.e. develop in the absence of the ovaries, animals were ovariectomized when neonates, and the mode of LH release was examined using a 5-min blood-sampling regimen at one of three ages corresponding to the juvenile, peripubertal, or adult phase of development. In no instance was a minisurge of LH secretion detected. We were concerned, however, that LH minisurges may have been obscured by the exceedingly high level of LH secretion in these long term ovariectomized rats and, therefore, decided to pursue the study employing a short term (48-h) ovariectomy paradigm. Late juvenile rats which had been ovariectomized for 48 h exhibited conspicuous LH pulses, but, again, LH minisurges were not detected, further suggesting that these sustained secretory episodes do not occur in the absence of the ovaries. Next, plasma estradiol (E2) levels were differentially raised in 48-h ovariectomized juvenile rats via sc implantation of Silastic capsules containing the steroid (dissolved in corn oil at various concentrations), and plasma LH was measured at 1-h intervals. The highest E2 concentration elicited a midafternoon LH increase of preovulatory magnitude in all cases, whereas the lowest E2 concentrations consistently suppressed plasma LH. However, intermediate E2 concentrations (producing plasma E2 levels 20-30% greater than those found in intact controls) elicited, in several instances, an increase in LH release of intermediate magnitude. To clarify the nature of these LH responses, we examined, using a 5-min blood-sampling regimen, the afternoon pulsatile pattern of LH release after treatment with appropriate doses of E2. As expected from the results of the infrequent blood-sampling paradigm, the highest E2 dose induced proestrus-like surges of LH secretion, while the lowest dose suppressed pulsatile LH release. Moreover, an intermediate E2 dose which raised plasma E2 levels just above those of intact animals, was again able to cause suppression of LH pulses, a proestrus-like surges of LH secretion, while the lowest dose suppressed pulsatile LH release. Moreover, an intermediate E2 dose which raised plasma E2 levels just above those of intact animals, was again able to cause suppression of LH pulses, a proestrus-like increase in LH output, or, more importantly, a minisurge of LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadal-independent activation of enhanced afternoon luteinizing hormone release during pubertal development in the female rat.

In the present study we investigated the extent to which expression of the diurnal pattern of LH secretion in prepubertal female rats is driven by an ovarian-independent neuroendocrine mechanism. To remove gonadal influences, rats were ovariectomized (OVX) during early juvenile, late juvenile, or peripubertal phases of development (20-22, 26-27, and 32-33 days of age, respectively), and blood samples were collected throughout the day (every 30-60 min) 2 or 4 days after surgery. Morning plasma LH levels were relatively low 2 days after ovariectomy (30-120 ng/ml), but rose during the afternoon to reach levels ranging from 180-300 ng/ml. This afternoon elevation was sustained both in late juvenile and peripubertal rats, but not in early juvenile rats. The predominant change observed 4 days after ovariectomy was an overall 3- to 6-fold increase in plasma LH levels, which masked the afternoon elevation. Analysis of plasma LH profiles from individual rats, however, revealed that at each of the three ages studied the peak plasma LH levels occurred in the afternoon, and these were most pronounced in the peripubertal animals. To further clarify the existence of such a diurnal pattern of LH release in the 4-day OVX animals pulsatile LH release profiles were obtained from individual animals, using a 5-min bleeding paradigm, at either 24 days (in the morning) or 36 days of age (in either the morning or the afternoon). LH release was episodic in all of the animals studied, with pulses occurring on average of about once every 30 min. Analysis of the plasma LH profiles using the PULSAR algorithm revealed that the overall mean plasma LH levels of the peripubertal animals was enhanced during the afternoon, compared to that in the morning or to the afternoon LH levels in the early juvenile rats. Mean nadir and mean peak LH levels were also greater. No differences in LH pulse amplitude and only marginal differences in LH pulse frequency were detected among the three groups studied. The enhancement of afternoon LH secretion in the OVX rats could not be attributed to age-related or diurnal changes in adenohypophyseal responsiveness to LHRH. The results suggest that the initiation of enhanced afternoon LH secretion, previously shown to occur in intact female rats during the juvenile-peripubertal transition period, results primarily from the activation of a central neuroendocrine mechanism. Although the ovaries might play a role in the development of this diurnal pattern, it is clear that its activation is ovarian independent.

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.

A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty.

The physiological role of N-methyl-D-aspartate (NMDA) receptors in controlling LH secretion and the initiation of puberty was investigated using two specific antagonists, MK-801 and DL-2-amino-5-phosphono valeric acid (AP-5). Single daily sc injections of MK-801 (0.1-0.2 mg/kg BW), a noncompetitive NMDA receptor antagonist, given to prepubertal rats significantly delayed but did not prevent the timing of puberty, as determined by the age at vaginal opening and first ovulation. Infusion of MK-801 (5 micrograms/h) via osmotic minipumps for 4 days inhibited the postovariectomy rise of LH secretion in prepubertal rats. Both MK-801 (0.2 mg/kg BW, sc) and AP-5 (4 x 30 mg, iv), a competitive NMDA receptor antagonist, blocked the estradiol-induced LH surge in prepubertal ovariectomized rats. These results demonstrate that blockade of NMDA receptors can prevent the development of enhanced LH secretion in female rats undergoing sexual maturation. Moreover, they support the view that activation of NMDA receptors significantly contributes to the physiological initiation of the pubertal process.

In vitro simulation of prepubertal changes in pulsatile luteinizing hormone release enhances progesterone and 17 beta-estradiol secretion from immature rat ovaries.

We have previously demonstrated changes in the episodic pattern of LH secretion in female rats as they approach first ovulation. In the present study, ovaries were taken from peripubertal rats and perifused in vitro with a medium containing FSH and LH. The concentration of FSH in the medium was kept constantly low, whereas the concentration of LH was modulated, simulating as closely as possible the various episodic secretory profiles previously observed in vivo. Medium from the perifusion chambers was collected every 10 min for 5 h and assayed for progesterone (P) and 17 beta-estradiol (E2). Exposure of the ovaries to low amplitude LH pulses (20 ng/ml; comparable to those found in the plasma of peripubertal rats during the morning) did not elicit a significant steroidogenic response. On the other hand, exposure to large amplitude LH pulses (80 ng/ml; as seen during the afternoon) led to a marked increase in the output of both P and E2. Similarly, increased steroid secretion occurred when ovaries were exposed to a 2-h minisurge of LH (160 ng/ml) or to a minisurge preceded by three large amplitude pulses (as seen in some animals during the afternoons of the peripubertal period). Continuous exposure to LH at concentrations similar to either the large amplitude pulses or the LH minisurge increased P and E2 release. However, the increase was not significantly larger than that produced by the discontinuous LH perifusion patterns, even though the total amount of LH reaching the ovaries during the 5-h perifusion period was considerably greater. The results show that episodic LH secretion is functionally much more efficient at eliciting release of P and E2 from immature ovaries than is continuous LH secretion. Furthermore, they strongly suggest that changes in the LH secretory pulse patterns, as seen in vivo close to the time of puberty, are fundamental for the activation of ovarian steroidogenesis that leads to the first preovulatory surge of gonadotropins.

The juvenile-peripubertal transition period in the female rat: establishment of a diurnal pattern of pulsatile luteinizing hormone secretion.

Previous descriptions of pulsatile LH release in immature female rats have been mostly inaccurate because of infrequent blood sampling, discontinuous and unbalanced blood replacement procedures, and possibly because of the use of a heterologous RIA. The present study was undertaken to circumvent these problems and to characterize more precisely the changes in episodic LH secretion previously observed during the juvenile-peripubertal transitional period (27-38 days of age). Changes in LH levels were measured in plasma samples obtained every 5 min from unrestrained conscious animals using a recently developed technique that permitted continuous withdrawal of blood for 4-5 h and simultaneous replacement with an artificial blood mixture. In virtually all of the rats, LH release was pulsatile. In both the juvenile (27-29 days old) and peripubertal (30-38 days old) animals, mean plasma LH levels and LH pulse amplitude were low in the mornings. A similar pattern of LH secretion was found in the afternoon during the juvenile period. In contrast, most of the peripubertal animals exhibited an afternoon increase in LH pulse amplitude. A change in pulse frequency was not detected at this time, but a 1.8-fold increase in mean LH levels suggests that large amplitude pulses may be accompanied by low amplitude, high frequency pulses not revealed by the 5-min sampling paradigm. Some of the peripubertal rats (5 of 12) had plasma LH profiles in which the large amplitude pulses were accompanied by a midafternoon minisurge of secretion which lasted for up to 2 h and reached peak levels of 174 +/- 19 ng/ml; the LH surge at first proestrus was greater than 1000 ng/ml. The results demonstrate the prepubertal development of an afternoon increase in LH pulse amplitude and the appearance of a more prolonged and larger secretory episode of LH secretion (minisurge). Disclosure of a concomitant increase in pulse frequency may necessitate blood sampling at more frequent intervals. It is suggested that the changes in the mode of LH release described here may enhance peripubertal ovarian steroidogenesis and provide an appropriate stimulus for initiating the final stages of ovarian development.

Simultaneous measurement of gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone in the orchidectomized rat.

In the present study two recently developed techniques have been combined to enable the simultaneous in vivo determination of pulsatile release of GnRH, LH, and FSH in the orchidectomized rat. The first of these techniques involves the implantation of two vascular catheters and collecting serial blood samples through one while simultaneously infusing a replacement blood mixture through the other; consequently, blood samples can be collected for an extended period of time, and detailed plasma LH and FSH release profiles can be established for individual animals. The second technique involves push-pull perfusion of the pituitary gland to determine changes in GnRH concentration as might be perceived by the gonadotropes. For each animal (n = 6), blood (150 microliters) and push-pull perfusate (200 microliters) samples were collected at 5- and 10-min intervals, respectively, for approximately 6 h, and the hormone release profiles were determined by RIA. All of the rats showed a clear pulsatile release pattern for GnRH, LH, and FSH. Moreover, the interpulse interval was remarkably similar for each of these hormones (36.9, 41.5, and 43.5 min, respectively, as determined by PULSAR). The percentage of GnRH pulses associated with a gonadotropin pulse was 72% for LH and 76% for FSH; only 14% of the pulses were silent for both gonadotropins. These results demonstrate that in the orchidectomized rat the pulsatile pattern of GnRH release is reflected in the pulsatile pattern of not only LH but also FSH. They may, therefore, be construed to support the concept that the pulsatile secretion of both gonadotropins is primarily orchestrated by a single hypothalamic releasing hormone. Alternatively, if two separate hypothalamic releasing hormones do indeed exist (LHRH and FSH-releasing hormone), it would appear that in the orchidectomized rat their episodic release is tightly coupled to the same hypothalamic pulse generator.

Stimulation of cyclic adenosine 3',5'-monophosphate production enhances hypothalamic luteinizing hormone-releasing hormone release without increasing prostaglandin E2 synthesis: studies in prepubertal female rats.

A role for cAMP in the process of LHRH release was suggested several years ago, but only recently has the validity of this notion come under close scrutiny. In the present experiments we have used three probes, which stimulate adenylate cyclase activity via different mechanisms, to determine whether an increase in endogenous cAMP results in LHRH release from the hypothalamus of prepubertal female rats. Median eminences from juvenile, 28-day-old animals were incubated in vitro with either forskolin (F), cholera toxin (CT), or pertussis toxin (PT). All three substances enhanced LHRH release. The estimated ED50 values were 28.7 microM and 20.0 ng/ml, for F and PT, respectively. The effect of CT appeared biphasic and thus no ED50 could be calculated. None of these agents increased the release of prostaglandin E2 (PGE2), an obligatory component in the process of norepinephrine-induced LHRH secretion. Doses of PGE2 and F, which were maximally effective in stimulating LHRH release when administered separately, did not produce any further response when administered concomitantly, thus suggesting that PGE2 and F act along a common pathway. Blockade of phosphodiesterase activity with 1-methyl-3-isobutylxanthine increased LHRH secretion without enhancing PGE2 release, implying that cAMP metabolism was elevated in the median eminence nerve terminals in vitro. Addition of 1-methyl-3-isobutylxanthine augmented the LHRH response to CT and PT, but it did not increase further the already marked LHRH response to PGE2 or F. The results indicate that both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity lead to LHRH release from the median eminence. They also suggest that, upon proper (neurotransmitter?) stimulation, cAMP production increases subsequent to the activation in PGE2 synthesis, which itself causes LHRH release. Furthermore, the capacity of PT to induce LHRH release suggests the involvement of an inhibitory guanine nucleotide-binding regulatory protein in transducing inhibitory inputs impinging on LHRH-secreting neurons.

Mitochondrial uncoupling protein 2 (UCP2) in the nonhuman primate brain and pituitary.

Energy dissipating mechanisms and their regulatory components represent key elements of metabolism and may offer novel targets in the treatment of metabolic disorders, such as obesity and diabetes. Recent studies have shown that a mitochondrial uncoupling protein (UCP2), which uncouples mitochondrial oxidation from phosphorylation, is expressed in the rodent brain by neurons that are known to regulate autonomic, metabolic, and endocrine processes. To help establish the relevance of these rodent data to primate physiology, we now examined UCP2 messenger RNA and peptide expressions in the brain and pituitary gland of nonhuman primates. In situ hybridization histochemistry showed that UCP2 messenger RNA is expressed in the paraventricular, supraoptic, suprachiasmatic, and arcuate nuclei of the primate hypothalamus and also in the anterior lobe of the pituitary gland. Immunocytochemistry revealed abundant UCP2 expression in cell bodies and axonal processes in the aforementioned nuclei as well as in other hypothalamic and brain stem regions and all parts of the pituitary gland. In the hypothalamus, UCP2 was coexpressed with neuropeptide Y, CRH, oxytocin, and vasopressin. In the pituitary, vasopressin and oxytocin-producing axonal processes in the posterior lobe and POMC cells in the intermediate and anterior lobes expressed UCP2. On the other hand, none of the GH-producing cells of the anterior pituitary was found to produce UCP2. The abundance and distribution pattern of UCP2 in the primate brain and pituitary suggest that this protein is evolutionary conserved and may relate to central autonomic, endocrine and metabolic regulation.

A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain.

The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria. Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown.

Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain.

In mammals, reproduction is thought to be controlled by a single neuropeptide, gonadotropin-releasing hormone (GnRH-I), which regulates the synthesis and secretion of gonadotropins from the pituitary gland. However, another form of this decapeptide (GnRH-II), of unknown function, also exists in the brain of many vertebrate species, including humans; it is encoded by a different gene and its amino acid sequence is 70% identical to that of GnRH-I. Here we report the cloning of a GnRH-II cDNA from the rhesus macaque (Macaca mulatta), and show for the first time by in situ hybridization that GnRH-II mRNA is expressed in the primate midbrain, hippocampus and discrete nuclei of the hypothalamus, including the supraoptic, paraventricular, suprachiasmatic and arcuate. Because the regional distribution pattern of cells containing GnRH-II mRNA is largely dissimilar to that of cells containing GnRH-I mRNA, it is likely that these two cell populations receive distinct neuroendocrine inputs and thus regulate GnRH synthesis and release differently.

A developmental increase in the expression of messenger ribonucleic acid encoding a second form of gonadotropin-releasing hormone in the rhesus macaque hypothalamus.

GnRH-I is thought to represent the primary neuroendocrine link between the brain and the reproductive axis. Recently, however, a second molecular form of this decapeptide (GnRH-II) was found to be highly expressed in the brains of humans and nonhuman primates. In this study, in situ hybridization was used to examine the regional expression of GnRH-II messenger ribonucleic acid in the hypothalamus of immature (0.6 yr) and adult (10-15 yr) male and female rhesus macaques (Macaca mulatta). Overall, no sex-related differences were observed. In all of the animals (n = 3 animals/group), intense hybridization of a monkey GnRH-II riboprobe was evident in the paraventricular nucleus and supraoptic nucleus and to a lesser extent in the suprachiasmatic nucleus, but no age- or sex-related differences were apparent. Intense hybridization of the riboprobe also occurred in the mediobasal hypothalamus, and this was markedly greater in the adults than in the immature animals. These data show that the expression of GnRH-II messenger ribonucleic acid increases developmentally in a key neuroendocrine center of the brain. Moreover, because GnRH-II can stimulate LH release in vivo, it is plausible that changes in its gene expression represent an important component of the mechanism by which the hypothalamus controls reproductive function.

Effect of 17beta-estradiol on hypothalamic GnRH-II gene expression in the female rhesus macaque.

The hypothalamus of rhesus macaques expresses two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II). However, it is unclear whether these two neuropeptides play similar roles in the control of reproductive neuroendocrine function, especially in the context of positive and negative estrogen feedback. To address this issue, in situ hybridization histochemistry was used to compare the effect of 17beta-estradiol (E) on the expression of GnRH-I and GnRH-II mRNA in the medial basal hypothalamus (MBH) of adult female macaques. GnRH-I mRNA expression was found to be significantly (P<0.01) more abundant in ovariectomized (ovx) animals compared with ovariectomized E-treated (ovx+E) animals. In marked contrast, GnRH-II mRNA expression was found to be significantly (P<0.05) more abundant in ovx+E animals than in the ovx animals. To help elucidate how E exerts this stimulatory action on GnRH-II gene expression, hypothalamic sections were subsequently double labeled using a combination of immunohistochemisty for estrogen receptor (ER) -alpha or -beta and in situ hybridization histochemistry for GnRH-II. Approximately 50% of the GnRH-II positive cells in the MBH were found to express ERbeta, but none expressed ERalpha. Taken together, these data give credence to a novel pathway by which E may control the primate neuroendocrine reproductive axis, one that involves stimulation of GnRH-II release via an ERbeta-mediated mechanism.

Episodic release of LH in gonadectomized male Japanese quail.

Blood samples were taken every 15 min (for 4.75 h) from six castrated quail on three separate occasions. The birds were first bled while under short days (comprising 8 h light : 16 h darkness per 24 h; 8L : 16D) and, subsequently, after 1 and 20 days under long days of 16L : 8D. The photoperiodic alteration produced a marked increase in the mean plasma LH concentration of each bird and in four instances the rise was evident after only 1 long day. Pulsatile release patterns were not detected in the plasma LH profiles obtained during exposure to short days or after 1 long day but were pronounced in all of the birds after 20 long days. The peaks (n = 19) occurred on average every 83 +/- 14 (S.E.M.) min and had an amplitude of 12.3 +/- 1.2 micrograms/l. After 35 days under 16L : 8D three of the quail were bled more frequently (every 8 min for 2.5 h), allowing the LH pulses to be measured more precisely. Each pulse was composed of a sudden increase in secretion followed by a slower decrease which lasted for approximately 30 min. The development and functional significance of episodic LH release in the quail is discussed.