Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain.

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.

An increase in glutamate release follows a decrease in gamma aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkeys.

Previously we have shown that release of gamma-aminobutyric acid (GABA) in the stalk-median eminence (S-ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push-pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S-ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid-pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early- and mid-pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid-pubertal monkeys, although mid-pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S-ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S-ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S-ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.

Role of glutamic acid decarboxylase in the prepubertal inhibition of the luteinizing hormone releasing hormone release in female rhesus monkeys.

To investigate further the role of GABA in the onset of puberty, this study examines whether glutamic acid decarboxylase (GAD), the catalytic enzyme for GABA synthesis, is involved in the suppression of luteinizing hormone releasing hormone (LHRH) before puberty in rhesus monkeys. First, both GAD67 and GAD65 mRNAs were detectable by reverse transcription-PCR analysis in the preoptic area, medio-basal hypothalamus, posterior hypothalamic area, and hippocampus of the monkey brain. Second, effects of antisense oligodeoxynucleotides (D-oligos) for GAD67 and GAD65 mRNAs on LHRH release were examined in conscious female rhesus monkeys at the prepubertal stage using a push-pull perfusion method. The GAD67 or GAD65 antisense D-oligos or scrambled D-oligos were infused directly into the stalk-median eminence. Both the GAD67 and the GAD65 antisense D-oligos induced a large and prompt increase in LHRH release, whereas the scrambled D-oligos did not induce any significant effect. The results suggest that the removal of GABA inhibition by interfering with GAD synthesis is effective in increasing LHRH release in prepubertal monkeys. Third, the specificity of the antisense D-oligos on GAD levels was examined by incubating basal hypothalami with D-oligos in vitro and subsequent Western blot analysis. The antisense D-oligos consistently decreased the proteins GAD67 and GAD65 compared with respective control D-oligos. We conclude that the decrease of tonic GABAergic inhibition and maturational changes in GAD synthesis may be critical factors for the onset of puberty in nonhuman primates.

Transplantation of the fetal olfactory placode restores reproductive cycles in female rhesus monkeys (Mucaca mulatta) bearing lesions in the medial basal hypothalamus.

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.

A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey.

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.