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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.

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Growth hormone (GH) secretion regularity and the effects of lighting condition and GH-releasing hormone (GHRH) on GH release were determined in steers. First, steers were kept under 12:12 L : D conditions (light: 06.00-18.00 hours). The animals were then subjected to a 1-h advancement in lighting on/off conditions (05.00 and 17.00 hours, respectively). Blood was sampled for 24 h at 1-h interval on the seventh day of each condition. Second, GHRH was injected intravenously (IV) at 12.00 and 00.00 hours under 12:12 L : D and blood was sampled at 15-min interval for 4-h (1 h before and 3 h after the injection). Plasma GH concentrations were measured by a radioimmunoassay. Periodicity of GH secretory profile was calculated by power spectrum analysis using the maximum entropy method. Plasma GH concentrations showed a characteristic pattern consisting of four distinct peaks. Mean periodicity of GH secretory profile was 5.7 h, and it was not altered by any change in lighting conditions. IV injection of GHRH increased GH secretion during the day and night. The increase in GH secretory volume after GHRH injection during the night was equal to that during the day. The present results suggest that GH secreted from the anterior pituitary have regularity in steers.

ABSTRACT. The effects of melatonin (MEL) injection into the third ventricle (3V) on growth hormone (GH) secretion were investigated in conscious Holstein steers. A stainless steel cannula was stereotaxically implanted in the 3V based on the ventriculogram. In Exp. 1, three doses of MEL (100, 300 or 600 µg) were injected into the 3V through the cannula and the GH concentration after the injection was determined. In Exp. 2, intracerebroventricular (icv) and intravenous (iv) injections of MEL (100 µg) and GH-releasing hormone (GHRH; 0.25 µg/kg body weight), respectively, were performed simultaneously to examine the effect of MEL on GHRH-induced GH release. The icv injection of MEL significantly stimulated GH release at 100 µg. The increase in GH concentrations by 100 µg of MEL was persistent. Intravenous injection of GHRH dramatically increased GH release. The injection of MEL did not alter GHRH-induced GH release. These results suggest that MEL stimulates GH secretion possibly through the hypothalamus in cattle. KEY WORDS: cattle, melatonin, somatotropic axis, stereotaxic, third ventricle.J. Vet. Med. Sci. 68(10): 1075-1080, 2006 Growth hormone (GH) is a very important factor for livestock production [6]. GH-releasing hormone (GHRH) and somatostatin (SS) from the hypothalamus are primary factors controlling GH secretion in mammals. In addition to GHRH and SS, many neuromodulators have been considered to be involved in GH release regulation in many species. Although the central regulatory mechanism of GH secretion has been studied in sheep using hypophyseal portal blood sampling technique [4], such information is not well obtained in cattle.The evidences have shown the possibility that melatonin (MEL) from the pineal gland is one of those factors controlling GH secretion; however, the exact effect of MEL on GH secretion is controversial. For example, in humans it was reported that MEL induced an increase in GH levels [27,29]; however, other groups obtained no effects of MEL on GH secretion [13,31]. The intramuscular injection of MEL decreased GH secretion in prepubertal boys and the same dose of MEL increased GH level in some pubertal subjects [17]. In vitro experiments showed that MEL reduced GH secretion from rat pituitary cells [8]. These variations of the effects of MEL on GH secretion could be caused by the differences in the experimental and physiological conditions, and there might be inter-species differences regarding the role of MEL in the regulation of GH secretion.The purpose of the present study was to determine the role of MEL in the regulatory system of GH secretion in cattle. Since MEL has been reported to stimulate GH release via the hypothalamus [23,29], the technique for the direct injection of MEL into the third ventricle (3V) at three doses (100, 300 and 600 µg) was used. We also assessed whether MEL affects GHRH-induced GH release to reveal the involvement of the hypothalamus in GH secretion modulated by MEL. MATERIALS AND METHODS Animals:Eleven Holstein steers (7 to 8 mo old at the time of su...