An increase in in vivo release of LHRH and precocious puberty by posterior hypothalamic lesions in female rhesus monkeys (Macaca mulatta)

We have previously shown that a decrease in γ-aminobutyric acid (GABA) tone and a subsequent increase in glutamatergic tone occur in association with the pubertal increase in luteinizing hormone releasing hormone (LHRH) release in primates. To further determine the causal relationship between developmental changes in GABA and glutamate levels and the pubertal increase in LHRH release, we examined monkeys with precocious puberty induced by lesions in the posterior hypothalamus (PH). Six prepubertal female rhesus monkeys (17.4 ± 0.1 mo of age) received lesions in the PH, three prepubertal females (17.5 ± 0.1 mo) received sham lesions, and two females received no treatments. LHRH, GABA, and glutamate levels in the stalk-median eminence before and after lesions were assessed over two 6-h periods (0600–1200 and 1800–2400) using push-pull perfusion. Monkeys with PH lesions exhibited external signs of precocious puberty, including significantly earlier menarche in PH lesion animals (18.8 ± 0.2 mo) than in sham/controls (25.5 ± 0.9 mo, P < 0.001). Moreover, PH lesion animals had elevated LHRH levels and higher evening glutamate levels after lesions, whereas LHRH changes did not occur in sham/controls until later. Changes in GABA release were not discernible, since evening GABA levels already deceased at 18–20 mo of age in both groups and morning levels remained at the prepubertal levels. The age of first ovulation in both groups did not differ. Collectively, PH lesions may not be a good tool to investigate the mechanism of puberty, and, taking into account the recent findings on the role of kisspeptins, the mechanism of the puberty onset in primates is more complex than we initially anticipated.

erbB-1 and erbB-4 Receptors Act in Concert to Facilitate Female Sexual Development and Mature Reproductive Function

Glial erbB-1 and erbB-4 receptors are key components of the process by which neuroendocrine glial cells control LHRH secretion and the onset of female puberty. We now provide evidence that these two signaling systems work in a coordinated fashion to control reproductive function. To generate animals carrying functionally impaired erbB-1 and erbB-4 receptors, we crossed Waved 2 (Wa-2+/+) mice harboring a point mutation of the erbB-1 receptor with mice expressing a dominant-negative erbB-4 receptor in astrocytes. In comparison to single-deficient mice, double-mutant animals exhibited a further delay in the onset of puberty and a strikingly diminished adult reproductive capacity. Ligand-dependent erbB receptor phosphorylation and erbB-mediated MAPK (ERK 1/2) phosphorylation were impaired in mutant astrocytes. Wa-2+/+ or double-mutant astrocytes failed to respond to TGFα with production of prostaglandin E2, one of the factors mediating the stimulatory effect of astroglial erbB receptor activation on LHRH release. Medium conditioned by Wa-2+/+ or double-mutant astrocytes treated with TGFα failed to stimulate LHRH release from GT1–7 cells. The LH response to ovariectomy was significantly attenuated in mutant mice in comparison with wild-type controls. Although the Wa-2 mutation affects all cells bearing erbB-1 receptors, these results suggest that a major defect underlying the reproductive defects of animals with impaired erbB signaling is a decreased ability of glial cells to stimulate LHRH release. Thus, a coordinated involvement of erbB-1 and erbB-4 signaling systems is required for the normalcy of sexual development and the maintenance of mature female reproductive function.

Stimulation of Luteinizing Hormone-Releasing Hormone (LHRH) Gene Expression in GT1–7 Cells by Its Metabolite, LHRH-(1–5)

Given the central role of the decapeptide LHRH in reproduction and reproductive behavior, it is important to focus on delineating the possible effects of this gene and its products in the regulation of hormone-dependent reproductive processes. In the female, ovulation is preceded by a marked increase in LHRH release; the increase in LHRH release culminates in a preovulatory LH surge, which coincides with a period of sexual receptivity. In contrast to the belief that the proteolytic metabolism of LHRH serves only as a degradative process that removes excess LHRH and attenuates signal transduction through the LHRH receptor, we hypothesized that a metabolite of the decapeptide, LHRH-(1–5), can directly regulate LHRH neuronal function. This study demonstrates the ability of LHRH-(1–5) peptide to regulate LHRH gene expression in the LHRH neuronal cell line, the GT1–7 cell. The results show that LHRH-(1–5) stimulated LHRH gene expression at the posttranscriptional level. In contrast to the LHRH suppression of its own gene expression, the coadministration of LHRH with the metalloendopeptidase, EC 3.4.24.15, an endopeptidase known to cleave LHRH to form LHRH1–5, shows a reversal of effect, a stimulation of LHRH gene expression. Finally, the effect of LHRH-(1–5) on LHRH gene expression appears to be mediated by the calcium/calmodulin-dependent protein kinase. The present study supports the hypothesis that the physiological metabolite of LHRH, LHRH-(1–5), is functionally capable of regulating the reproductive neuroendocrine system.

Vitamin E Stimulates Luteinizing Hormone-Releasing Hormone and Ascorbic Acid Release from Medial Basal Hypothalami of Adult Male Rats

Vitamin E, a dietary factor, is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Previously, we showed that ascorbic acid (AA) acted as an inhibitory neurotransmitter in the hypothalamus by scavenging nitric oxide (NO). Earlier studies have shown the antioxidant synergism between vitamin E and ascorbic acid (AA). Therefore, it was of interest to evaluate the effect of vitamin E on luteinizing hormone-releasing hormone (LHRH) and AA release. Medial basal hypothalami from adult male rats of the Sprague Dawley strain were incubated with Krebs-Ringer bicarbonate buffer or graded concentrations of a water soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS, 22–176 μM) for 1 hr. Subsequently, the tissues were incubated with vitamin E or combinations of vitamin. E + N-methyl-D-aspartic acid (NMDA), an excitatory amino acid for 30 min to study the effect of prior and continued exposure to vitamin E on NMDA-induced LHRH release. AA and LHRH released into the incubation media were determined by high-performance liquid chromatography and radioimmunoassay, respectively. Vitamin E stimulated both LHRH and AA release. The minimal effective concentrations were 22 and 88 μM, respectively. NMDA stimulated LHRH release as previously shown and this effect was not altered in the combined presence of vitamin E plus NMDA. However, AA release was significantly reduced in the combined presence of vitamin E plus NMDA. To evaluate the role of NO in vitamin E-induced LHRH and AA release, the tissues were incubated with vitamin E or combinations of vitamin E + NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NO synthase. NMMA significantly suppressed vitamin E-induced LHRH and AA release indicating a role of NO in the release of both LHRH and AA. The data suggest that vitamin E plays a role in the hypothalamic control of LHRH and AA release and that the release is mediated by NO.

Synchronization of Ca2+ Oscillations Among Primate LHRH Neurons and Nonneuronal Cells In Vitro

Periodic release of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus is essential for normal reproductive function. Pulsatile LHRH release appears to result from the synchronous activity of LHRH neurons. However, how the activity of these neurons is synchronized to release LHRH peptide in a pulsatile manner is unclear. Because there is little evidence of physical coupling among LHRH neurons in the hypothalamus, we hypothesized that the activity of LHRH neurons might be coordinated by indirect intercellular communication via intermediary (nonneural) cells rather than direct interneural coupling. In this study, we used an in vitro preparation of LHRH neurons derived from the olfactory placode of monkey embryos to assess whether nonneuronal cells, play a role in coordinating LHRH neuronal activity. We found that cultured LHRH neurons and nonneuronal cells both exhibit spontaneous oscillations in the concentration of intracellular Ca2+([Ca2+]i) at similar frequencies. Moreover, [Ca2+]i oscillations in both types of cell were periodically synchronized. Synchronized [Ca2+]i oscillations spread as intercellular Ca2+ waves across fields of cells that included LHRH neurons and nonneuronal cells, although waves spread at a higher velocity among LHRH neurons. These results suggest that LHRH neurons and nonneuronal cells are functionally integrated and that nonneuronal cells could be involved in synchronizing the activity of the LHRH neurosecretory network.