Sheep Hypothalamus Research Articles

Early origins of obesity: programming the appetite regulatory system

There is evidence that changes in perinatal nutrition programme the development of relative fat mass and the regulation of appetite in adult life. These studies have been primarily in the rodent utilizing maternal overnutrition or undernutrition imposed at different stages of pregnancy and beyond, mapping of neuropeptide localization and activity and appropriate null mutant models. Whilst the rodent offers significant advantages in terms of a short gestation and the availability of useful transgenic and null mutant models, there are also advantages to using an animal model more akin to the human, in which all components of the 'fat-brain axis' are present before birth, such as the sheep. This review summarizes recent work on the expression and localization of the 'appetite regulatory' peptides in the fetal rodent and sheep hypothalamus and their potential role in the early programming of postnatal appetite and obesity.

Distribution of preprodynorphin mRNA and dynorphin-a immunoreactivity in the sheep preoptic area and hypothalamus

Endogenous opioid peptides (EOP) are important modulators in a variety of neuroendocrine systems, including those mediating reproduction, energy balance, lactation, and stress. Recent work in the ewe has implicated the EOP, dynorphin (DYN), in the inhibitory effects of progesterone on pulsatile gonadotropin releasing hormone secretion. Although DYN is involved in a number of hypothalamic functions in the sheep, little is known regarding the localization of preprodynorphin (PPD) expression and its major product DYN A (1–17). In this study, we determined the distribution of PPD mRNA and DYN A-containing cell bodies in the brains of ovary-intact, luteal ewes. To detect PPD mRNA, an ovine PPD mRNA was subcloned by reverse transcription–polymerase chain reaction from sheep hypothalamus and used to create a 35S-labeled riboprobe for in situ hybridization. Neurons that expressed PPD mRNA and DYN A immunoreactivity were widely distributed in the ovine preoptic area and hypothalamus. PPD mRNA-expressing cells were seen in the supraoptic nucleus, paraventricular nucleus, preoptic area, anterior hypothalamus area, bed nucleus of the stria terminalis, ventromedial nucleus (VMN), dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. All of these regions also contained DYN A-positive cell bodies except for the VMN, raising the possibility that PPD is preferentially processed into other peptide products in the VMN. In summary, based on the expression of both mRNA and peptide, DYN cells are located in a number of key hypothalamic regions involved in the neuroendocrine control of homeostasis in sheep.

Appetite regulatory neuropeptides are expressed in the sheep hypothalamus before birth.

In the adult, a hypothalamic neural network acts to maintain energy balance in response to nutritional feedback from the periphery. Although there is an immediate requirement for this system to be functional at birth, it is unknown whether the components of this central neural network are expressed in the developing brain before birth. We therefore examined in the fetal sheep hypothalamus during late gestation gene expression for leptin receptor (OB-Rb) and neuropeptides that regulate energy balance in the adult. Brains were collected from fetal sheep at 110 days (n = 12) and 140 days of gestation (n = 5) (term = 150 days) and gene expression was detected in all hypothalami using in situ hybridization with radiolabelled riboprobes for OB-Rb, neuropeptide Y (NPY), agouti-related peptide, pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript (CART). All mRNAs were expressed in the arcuate nucleus of fetuses at both time points. Additional sites of mRNA expression were the dorsomedial hypothalamus (DMH) for NPY, the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and lateral hypothalamic area for CART, and the DMH, PVN and VMH for OB-Rb. We have therefore demonstrated that adult-like localization of gene expression for OB-Rb and key appetite regulatory neuropeptides is established in the ovine hypothalamus before birth. Thus, the fetus possesses a central appetite regulatory neural network with the potential to respond to changes in nutrient supply, which could impact on energy balance regulation both before and after birth.

Effects of applying gamma-aminobutyric acid(B) drugs into the medial basal hypothalamus on basal luteinizing hormone concentrations and on luteinizing hormone surges in the female sheep.

Prior investigations have shown that localized infusion by microdialysis of gamma-aminobutyric acid(B) (GABA(B)) agonists into the medial basal hypothalamus of male sheep rapidly increases GnRH and LH pulse amplitude. The objectives of these studies were to determine if infusion of GABA(B) agonists SKF 97541 or baclofen into the medial basal hypothalamus of female sheep would affect basal LH secretion and if infusion of a potent antagonist would alter expression of LH surges induced by injection of estrogen. Infusion of either SKF 97541 (10 or 40 microM) or baclofen (1 mM) into estrogen-treated ovariectomized ewes did not alter basal LH secretory patterns, whereas both drugs significantly elevated mean LH and LH pulse amplitude in ovariectomized ewes during the nonbreeding season. Infusion of the antagonist CGP 52432 (250 or 500 microM) did not affect expression of estrogen-induced LH surges in ovariectomized ewes. These observations support the concept that GABA(B) receptors in the medial basal hypothalamus regulate basal LH secretion but do not regulate the surge mode of LH secretion in the female sheep.

The volume of a sexually dimorphic nucleus in the ovine medial preoptic area/anterior hypothalamus varies with sexual partner preference.

Sheep are one of the few animal models in which natural variations in male sexual preferences have been studied experimentally. Approximately 8% of rams exhibit sexual preferences for male partners (male-oriented rams) in contrast to most rams, which prefer female partners (female-oriented rams). We identified a cell group within the medial preoptic area/anterior hypothalamus of age-matched adult sheep that was significantly larger in adult rams than in ewes. This cell group was labeled the ovine sexually dimorphic nucleus (oSDN). In addition to a sex difference, we found that the volume of the oSDN was two times greater in female-oriented rams than in male-oriented rams. The dense cluster of neurons that comprise the oSDN express cytochrome P450 aromatase. Aromatase mRNA levels in the oSDN were significantly greater in female-oriented rams than in ewes, whereas male-oriented rams exhibited intermediate levels of expression. Because the medial preoptic area/anterior hypothalamus is known to control the expression of male sexual behaviors, these results suggest that naturally occurring variations in sexual partner preferences may be related to differences in brain anatomy and capacity for estrogen synthesis.

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Several subpopulations of neuropeptide Y– containing neurons exist in the infundibular nucleus of sheep: An immunohistochemical study of animals on different diets

Conversely to rodents, the involvement of hypothalamic neuropeptide Y (NPY) neurons in the control of nutrition is poorly understood in ruminants such as sheep. Therefore, the aim of this work was to describe the NPY neurons of the diencephalon in ewes submitted to different diets. In colchicine-treated animals, large populations of NPY-immunoreactive (-ir) neurons were observed in a ventral and a lateral subpopulation of the infundibular nucleus (IN), in the median eminence, the pituitary stalk, and the dorsomedian and dorsocaudal nuclei. No labeled perikaryon was observed in the magnocellular neurons of the hypothalamus, although numerous labeled fibers were noted in the neural part of the pituitary. The pattern of distribution of NPY-ir neurons in the sheep hypothalamus is similar in many ways to those of rodents, but it presents also many specific characteristics that have not been previously described. In ewes that were fasted for 24 hours, or fed ad libitum, the number of NPY-ir neurons was the same whatever the hypothalamic structures. In underfed ewes (40% of maintenance for 24 weeks), the lateral subpopulation of the IN presented a higher number of NPY-ir neurons than observed in the 100% fed ewes. Conversely, in the ventral subpopulation, the animals refed ad libitum (at least 150% of maintenance for 4 days) presented a lower number of NPY-ir neurons than the other groups. The other NPY neuronal populations of the hypothalamus were not significantly modified by the dietary treatments. For the first time, we demonstrated the presence of two functionally distinct subpopulations of NPY neurons in the sheep IN. The variations of labeled neurons were correlated with plasma nonesterified fatty acid levels but not with leptinemia.

Hypothalamic and zona incerta neurons in sheep, initially only responding to the sight of food, also respond to the sight of water after intracerebroventricular injection of hypertonic saline or angiotensin II

Extracellular single-unit recordings were made from neurons in the lateral hypothalamus (LH) or zona incerta (ZI) of conscious sheep. A small population of neurons (12/83) were found which responded with increased firing rate when the animal looked at food but did not respond when the sheep looked at water. The effects of rapidly inducing intense thirst by the intracerebroventricular (i.c.v.) injection of hypertonic (0.85 M) saline or 200 ng of angiotensin II, or a mixture of the two dipsogenic stimuli, on the response of neurons initially responding only to the sight of food were investigated. Following i.c.v. injection of the dipsogenic stimuli the neurons began to respond strongly to the sight of water. The results demonstrated that changing the animal’s motivational state alters the response of some neurons in the LH and ZI and suggests that the neuronal response is influenced by the animal’s dominant need at the time of testing.

Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus.

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.

Orexin gene expression and regulation by photoperiod in the sheep hypothalamus

Hypothalamic orexin gene expression has not been reported in the ruminant. Here, we describe the localization of preproorexin mRNA in the ovine lateral hypothalamic area (LHA) and zona incerta (ZI) using in situ hybridization. The hypothalamic localization of the orexin gene expression was similar in sheep to rodent models. Since appetite in sheep is seasonally (photoperiodically) regulated, we compared the amounts of preproorexin mRNA between long- (LD) and short-day (SD) photoperiods in both freely feeding (food intake is 20% higher in LD than SD) and food-restricted sheep (50% liveweight maintenance for 11 weeks). Gene expression was higher in SDs than in LDs but was not affected by chronic food restriction. In a second study, hypothalamic orexin gene expression in castrate sheep was not affected by a 4-day fast, irrespective of gonadal steroid (estradiol) replacement, and was not affected by the gonadal steroid per se. The results demonstrate the sensitivity of orexin gene expression to photoperiod, but up-regulation occurs in SDs when the appetite is characteristically low and no sensitivity to imposed changes in food intake. This supports the concept that orexins may not have a primary role in appetite regulation and correction of negative energy balance but since the sheep breed only in SDs, their role in seasonal reproductive activation deserves further study.

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals. These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.

Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe.

Changes in the secretion of GH induced by long-term alterations in nutritional status are thought to result from alterations in somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) at the level of the hypothalamus. To date however, the effect of nutrition on the gene expression of SRIF and GHRH in a species where GH secretion is increased by food restriction, as is the case for the sheep and human, remains unknown. We determined the effect of under-nutrition on the expression of SRIF and GHRH in the hypothalamus of sheep. Ovariectomised ewes were randomly divided into two groups and either fed an ad lib diet (n=6) or a restricted diet of 500 g lucerne chaff per day (food-restricted; n=5) for 7 months. In situ hybridisation was used to study hypothalamic gene expression for GHRH, SRIF and galanin (GAL). The food-restricted animals had elevated plasma concentrations of GH; this was associated with an increase in GHRH mRNA levels in the arcuate nucleus (ARC) and reduced SRIF in the rostral periventricular nucleus and ventromedial hypothalamic nucleus. The level of gene expression of GAL in the ARC and SRIF in the caudal periventricular nucleus was similar in ad lib and food-restricted animals. In conclusion, the effect of chronic food-restriction on the secretion of GH reflects increased GHRH and reduced SRIF synthesis in the hypothalamus.

Fetal and Maternal Sheep Hypothalamus Pituitary Adrenal Axis Responses to Chronic Binge Ethanol Exposure During the Third Trimester Equivalent

Background : We tested the hypothesis that in utero ethanol exposure results in changes in fetal and maternal adrenocorticotropin (ACTH) and cortisol during the third trimester equivalent, by using a chronically instrumented fetal sheep model. Methods: Pregnant ewes received saline or ethanol intravenously 3 consecutive days per week from day 109 to day 132 of gestation. Fetal and maternal blood samples were collected on days 118 and 132. Results: Maternal and fetal ACTH and cortisol values increased on days 118 and 132 of gestation in response to ethanol infusions that created blood ethanol concentrations (BECs) that are easily achievable by human drinkers. Peak ACTH and cortisol values were detected 30 to 60 min after peak BECs were achieved. Conclusions: Chronic ethanol exposure during the third trimester equivalent in sheep resulted in repeated activation of the hypothalamus-pituitary-adrenal axis in both the mother and fetus. Temporally, the patterns of maternal and fetal responses to ethanol infusion were similar. We conclude that ovine maternal ethanol exposure during the third trimester equivalent increases fetal ACTH and cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects.

Fetal and Maternal Sheep Hypothalamus Pituitary Adrenal Axis Responses to Chronic Binge Ethanol Exposure During the Third Trimester Equivalent

We tested the hypothesis that in utero ethanol exposure results in changes in fetal and maternal adrenocorticotropin (ACTH) and cortisol during the third trimester equivalent, by using a chronically instrumented fetal sheep model. Pregnant ewes received saline or ethanol intravenously 3 consecutive days per week from day 109 to day 132 of gestation. Fetal and maternal blood samples were collected on days 118 and 132. Maternal and fetal ACTH and cortisol values increased on days 118 and 132 of gestation in response to ethanol infusions that created blood ethanol concentrations (BECs) that are easily achievable by human drinkers. Peak ACTH and cortisol values were detected 30 to 60 min after peak BECs were achieved. Chronic ethanol exposure during the third trimester equivalent in sheep resulted in repeated activation of the hypothalamus-pituitary-adrenal axis in both the mother and fetus. Temporally, the patterns of maternal and fetal responses to ethanol infusion were similar. We conclude that ovine maternal ethanol exposure during the third trimester equivalent increases fetal ACTH and cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects.

Localization of the melatonin-related receptor in the rodent brain and peripheral tissues.

Previous studies have provided a limited examination of the expression of the orphan melatonin-related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin-related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase-polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin-related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

Localization of long-form leptin receptor in the somatostatin-containing neurons in the sheep hypothalamus

Reduction in the adiposity or dietary restriction increases plasma growth hormone (GH) concentrations, and in sheep this appears to be due, at least in part, to a reduction in the concentrations of somatostatin (SRIF) in hypophyseal portal blood. Leptin is a hormone secreted by the adipocytes and it is possible that the effects of altered adiposity or fasting on GH secretion could be due to regulation of SRIF neurons by leptin. To ascertain the extent to which leptin may act on these neurons, we have used immunohistochemistry to examine co-localization of long-form of the leptin receptor (OB-Rb) and SRIF in the sheep hypothalamus. In the hypothalamic periventricular area (PeriV), 44.5±10% of SRIF cells were found to co-stain for OB-Rb. In the dorsomedial hypothalamic, ventromedial hypothalamic and arcuate nuclei, 100% of SRIF immunoreactive neurons expressed OB-Rb. These findings provide a basis for the direct action of leptin on SRIF neurons. Thus, it is possible that leptin stimulates the secretion of SRIF in relatively obese individuals. The significance of the lower number of SRIF cells in the PeriV co-localizing OB-Rb expression is not clear at present.

Effect of feeding on Fos protein expression in sheep hypothalamus with special reference to the supraoptic and paraventricular nuclei: an immunohistochemical study

Effect of feeding on Fos protein expression in sheep hypothalamus with special reference to the supraoptic and paraventricular nuclei: an immunohistochemical study

Effect of feeding on Fos protein expression in sheep hypothalamus with special reference to the supraoptic and paraventricular nuclei: an immunohistochemical study

The hypothalamus plays an important role in the control of food intake in different species, but there is little relevant information for ruminants like sheep. In order to study the putative role of several hypothalamic nuclei in food intake in sheep, Fos expression, a marker of cellular activity, was compared by immunohistochemistry between fed and unfed ewes. The expression of Fos protein was stimulated in the supraoptic nucleus of fed ewes, whereas it was increased in the paraventricular nucleus of unfed animals. In the latter nucleus, Fos immunoreactivity was mainly localized close to the third ventricle, an area corresponding to the parvocellular system of the nucleus, but never in the magnocellular system. In the paraventricular nucleus, the number of corticotrophin releasing factor-immunoreactive neurons and the number of Fos/corticotrophin releasing factor double-labelled neurons were not affected by feeding or lack of feeding. The number of Fos-immunoreactive neurons was higher in the lateral septum, the infundibular, the ventromedial and in the dorsomedial nuclei of unfed ewes than in those of fed ewes. Our results show for the first time that the dorsomedial and ventromedial nuclei are involved in the control of feeding in sheep as in rodents. The supraoptic nucleus of sheep is activated by the same conditions as in rodents but, conversely, the paraventricular nucleus is activated in unfed sheep, whereas in rodents and primates, this nucleus is activated by satiety as well as by fasting. In sheep, unlike in rodents, corticotrophin releasing factor did not appear to be involved in short-term regulation of food intake.

The Distribution of Cells Containing Estrogen Receptor- (ER ) and ER Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females

The Distribution of Cells Containing Estrogen Receptor- (ER ) and ER Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females

The similarity of FSH-releasing factor to lamprey gonadotropin-releasing hormone III (l-GnRH-III).

To validate further the existence of a specific hypothalamic follicle stimulating hormone releasing factor (FSHRF), stalk-median eminence (SME) fragments from sheep and whole hypothalami from male rats were purified by gel filtration on Sephadex G-25, and the gonadotropin-releasing activity on hemipituitaries of rats incubated in vitro was determined by bioassay and compared with the radioimmunoassayable luteinizing hormone releasing hormone (LHRH) and lamprey gonadotropin releasing hormone (l-GnRH) activities in the fractions. The FSH-releasing fractions eluted in the same sequence of tubes from the Sephadex column found earlier by in vivo bioassay and were clearly separated from the immunoassayable and bioassayable LHRH. The radioimmunoassay (RIA) for l-GnRH recognized equally l-GnRH-I and -III but had negligible cross-reactivity with LHRH. Fractionation of rat hypothalamic extract by gel filtration on Sephadex G-25 revealed three peaks of l-GnRH determined by RIA, all of which eluted prior to the peak of LHRH. Only the second peak had FSH-releasing but not LH-releasing activity. To determine if this FSH-releasing activity was caused by the presence of l-GnRH in the fraction, the pituitaries were incubated with normal rabbit serum or the l-GnRH antiserum (1:1000), and the effect on the FSH- and LH-releasing activity of the FSH-releasing fraction and the LH-releasing activity of LHRH was determined. The antiserum had no effect on basal release of either FSH or LH but eliminated the FSH-releasing activity of the active fraction without altering the LH-releasing activity of LHRH. Since l-GnRH-I has little activity to release FSH or LH, and its activity is nonselective, whereas previous experiments have shown that l-GnRH-III highly selectively releases FSH with a potency equal to that of LHRH to release LH, the results support the hypothesis that the FSH-releasing activity observed in these experiments was caused by l-GnRH-III or a closely related peptide.