David C. Bucholtz

Central inhibition of gonadotropin-releasing hormone secretion in the growth-restricted hypogonadotropic female sheep.

Growth retardation induced by dietary restriction results in hypogonadotropism, and thus, puberty is delayed. The present studies determined 1) whether reduced LH secretion in the growth-retarded condition is due to a reduction in the frequency and/or in the amplitude of GnRH secretion, and 2) whether the mechanism regulating LH secretion is being actively inhibited via central mechanisms. To determine whether GnRH pulse frequency and/or amplitude are reduced during growth restriction, blood samples were simultaneously collected from pituitary portal blood for GnRH and from jugular blood for LH determinations over a 4-h period in ovariectomized lambs (52 wk of age) that were either growth restricted (28 kg; n = 8) or growing normally (60 kg; n = 7). As expected, the growth-restricted females were hypogonadotropic and exhibited a long LH interpulse interval compared with the normally growing females. However, although the GnRH interpulse interval was longer in the growth-restricted lambs compared with that...

Central, but not peripheral, glucose-sensing mechanisms mediate glucoprivic suppression of pulsatile luteinizing hormone secretion in the sheep.

Changes in glucose availability are proposed to modulate pulsatile GnRH secretion, and at least two anatomical sites, the liver and hindbrain, may serve as glucose sensors. The present study determined the relative importance of these putative glucose-sensing areas in regulating pulsatile LH secretion in the sheep. Our approach was to administer the antimetabolic glucose analog, 2-deoxy-D-glucose (2DG) into either the hepatic portal vein or the fourth ventricle in gonadectomized females in which LH pulse frequency was high. In the first study, a catheter was placed in the ileocolic vein to determine the effects of local injection of 2DG into the hepatic portal system on the release of LH. After monitoring the pattern of LH secretion for 4 h, 2DG (250 mg/kg) was infused (500 ml/min) into the liver for 2 h. For comparison, animals were also given the same dose of 2DG into a jugular vein for 2 h. Administration of 2DG into either the hepatic portal or jugular vein reduced LH pulse frequency to the same extent. Infusion of the lower dose (50 mg/kg) locally into the hepatic portal vein did not affect plasma LH profiles. Collectively, these results are interpreted to indicate that the liver does not contain special glucosesensing mechanisms for the glucoprivic suppression of LH pulses. In the second study, 2DG (5 mg/kg) was infused (50 ml/min) for 30 min into the fourth ventricle or lateral ventricle. During the subsequent 4-h sampling period, pulsatile LH secretion was significantly suppressed, but there was no significant difference in LH pulse frequency between sites of infusion. Peripheral 2DG concentrations were not detectable after either fourth or lateral ventricle infusions, indicating that the 2DG had acted centrally to suppress LH pulses. Plasma cortisol concentrations increased more in animals infused with 2DG into the fourth ventricle than in those infused into the lateral ventricle, suggesting that 2DG infused into lateral ventricle is transported caudally into the fourth ventricle and acts within the area surrounding the fourth ventricle. Overall, these findings suggest that an important glucose-sensing mechanism is located circumventricularly in the fourth ventricle. Moreover, the liver does not appear to play an important role in detecting glucoprivic action of 2DG to suppress pulsatile LH secretion. (Endocrinology 141: 4472–4480, 2000) R PROCESSES in mammals are influenced by nutritional conditions at various stages of the life cycle. For example, in nutritionally growth-retarded females, the onset of puberty is delayed compared with that in normally growing females [rats (1), cattle (2), humans (3), and sheep (4)]. In adult females, estrous cyclicity is disrupted by restriction of food intake [human (5), sheep (6), rat (7), mouse (8), and hamster (9)]. The broad hypothesis is that reduced nutrition decreases energy intake to inhibit gonadal function through the suppression of GnRH/LH secretion. Indeed, short-term fasting suppresses pulsatile LH secretion in the rat (10, 11), monkey (12), human (13), and sheep (14). More directly, reduced GnRH release has been noted in the pituitary portal vessels of the immature female sheep rendered hypogonadotropic by reduced nutrition (15). Studies in several species have reported that reproductive activity is influenced by the availability of metabolic fuels (for review, see Ref. 16). Among those metabolic fuels, glucose plays an important role as a metabolic regulator of reproductive function. There are several lines of evidence showing that insulin-induced hypoglycemia decreases pulsatile LH secretion in rats (17, 18) and monkeys (19) as well as decreases hypothalamic multiple unit activity in monkeys (20). Pharmacological glucoprivation with 2-deoxy-d-glucose (2DG), an antimetabolic glucose analog that competitively inhibits intracellular glucose oxidation (21), interrupts estrous cyclicity in Syrian hamsters (22). Moreover, iv injection of 2DG immediately suppresses pulsatile LH secretion in rats (23). These data support the hypothesis that changes in glucose availability control reproductive function by regulating GnRH/LH release. The sheep, our experimental model, is a ruminant species in which little glucose is absorbed from the digestive tract, and plasma glucose concentrations are normally only about half those in nonruminant species. Nevertheless, ruminants have an important gluReceived May 17, 2000. Address all correspondence and requests for reprints to: Dr. Douglas L. Foster, Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109-0404. E-mail address: dlfoster@umich.edu. * This work was supported by the U.S.-Japan Cooperative Science Program from NSF (National Science Foundation) and JSPS (Japan Society for the Promotion of Science) (INT-9603310); a grant-in-aid for International Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (Joint Research 09044215); and research grants from the NIH (HD-18258 and HD-18394). A preliminary report of this work was presented at the 29th Annual Meeting of the Society for Neuroscience, Miami Beach, FL, October 1999 (Abstract 166.13). 0013-7227/00/

Regulation of Pulsatile Luteinizing Hormone Secretion by Insulin in the Diabetic Male Lamb

03.00/0 Vol. 141, No. 12 Endocrinology Printed in U.S.A. Copyright

Glucose Availability Modulates the Timing of the Luteinizing Hormone Surge in the Ewe

Abstract This study tested the hypothesis that LH secretion is modulated by insulin and that the responsiveness to hypoinsulinemia is enhanced by sex steroids. The model was the developing male lamb (12–26 wk of age) rendered diabetic by chemically induced necrosis of insulin-secreting tissue (streptozotocin). Our approach was to monitor LH secretion under diabetic conditions, with or without insulin supplementation, either in the presence or in the absence of gonadal steroids. The first experiment determined if chronic insulin supplementation could sustain LH secretion in diabetic lambs. After documentation of the induced diabetic condition, twice-daily treatment with a long-acting insulin preparation (Lente) minimized diabetes-induced hyperglycemia, sustained growth, and maintained LH pulse frequency at levels comparable to pre-diabetic conditions. A second experiment evaluated the acute regulation of LH secretion by insulin. Twenty-four hours of insulin withdrawal decreased LH pulse frequency, increased circulating glucose levels, increased the concentration of plasma non-esterified fatty acids (NEFAs), and increased urinary output of ketones. LH pulse frequency continued to decline after 96 h of insulin withdrawal. By contrast, 24 h of insulin re-supplementation increased LH pulse frequency, reduced circulating glucose and NEFA concentrations, decreased plasma cortisol, and reduced urinary output of ketones. After 96 h of insulin re-supplementation, LH pulse frequency increased further, to levels comparable with those before insulin withdrawal. A third experiment determined if the effects of insulin withdrawal on LH secretion are influenced by the presence of gonadal steroids. The same individuals were treated with a physiologic dose of estradiol (Silastic capsule, s.c.) and subsequently monitored for changes in LH secretion in the presence and in the absence of exogenous insulin. Prior to insulin withdrawal, estradiol decreased both LH pulse frequency and pulse amplitude. Moreover, after 96 h of insulin withdrawal, estradiol potentiated the decline in LH pulse frequency (47% reduction in LH pulse frequency in the presence of estradiol versus 26% reduction in LH pulse frequency in the absence of estradiol). These findings support the contention that insulin and/or insulin-dependent changes in glucose availability modulate LH(GnRH) pulse frequency, and that such effects are potentiated by, but not dependent upon, gonadal steroids.

Hypothalamic versus Pituitary Stimulation of Luteinizing Hormone Secretion in the Prepubertal Female Lamb

To determine if glucose availability modulates the timing of the positive feedback action of oestrogen on gonadotropin secretion, we monitored the estradiol‐induced luteinizing hormone (LH) surge in sheep (n=5/group) made transiently hypoglycemic by insulin. Experiment 1 determined an effective insulin treatment, one which would depress tonic LH secretion. Two injections of insulin (5 IU/kg iv) 4 h apart were found to induce extended hypoglycemia (10–13 h) and to decrease the LH pulse frequency for 8 h (5.0±0.32 pulses/4 h before versus 2.5±0.34 pulses/4 h after insulin; P<0.05; mean±SEM). Using this same paradigm, experiment 2 determined the influence of the transient hypoglycemia on the LH surge mechanism. In control sheep, estradiol (subcutaneous implants at hour 0) evoked an LH surge with a latency period of 12.4±0.5 h. When insulin was administered either before (hours −4 and 0) or after the estradiol stimulus (hours 4 and 8, or 12 and 16), the onset of the LH surge was delayed to 29.0±2.4 h (average of all three time groups, P<0.05). Infusion of glucose from hours 12–30, along with insulin, prevented hypoglycemia and restored the normal timing of the oestrogen‐induced LH surgeto that of controls (15.4±0.93 h, P>0.05). These findings suggest that not only is the tonic mode of LH secretion sensitive to metabolic fuel availability, but the surge mode of LH secretion is as well.

Toward an Understanding of Interfaces Between Nutrition and Reproduction: The Growth-Restricted Lamb as a Model

Glutamate and aspartate have been hypothesized to function as neurotransmitters in the regulation of the gonadotropin-releasing hormone (GnRH) neurosecretory system. We, therefore, determined if hypothalamic stimulation of luteinizing hormone (LH) secretion in the intact prepubertal female lamb could be achieved by intravenous injection of N-methyl-D,L-aspartate (NMA), a glutamate agonist. A pilot study determined a dose of NMA that would induce physiologic pulses of LH (GnRH). Subsequently, we compared the ability of NMA with exogenous GnRH to induce ovulation in the prepubertal lamb when administered chronically. Eighteen prepubertal lambs (21 weeks of age, 34.2 +/- 1.5 kg body weight) were treated intravenously with either NMA (2 mg/kg, n = 6) or GnRH (68 ng/injection or approximately 2 ng/kg, n = 6) for 3 days, every 2 h on day 1 and every 1 h on days 2 and 3, or received no treatment (controls, n = 6). Gonadotropin surges were detected only in GnRH-treated lambs (5/6 lambs, onset = 54.0 +/- 4.5 h from the start of study, mean +/- SE). Compared to 83% of GnRH injections inducing LH pulses, only 47% of NMA injections induced LH pulses. Because each injection of NMA did not induce a pulse of LH, a second experiment was performed in an attempt to optimize the LH response to NMA. Ten prepubertal lambs (25 weeks of age) were injected every 2 h for 24 h with higher doses of NMA, either 4 mg/kg (n = 5) or 16 mg/kg (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic Interfaces between Growth and Reproduction. I. Nutritional Modulation of Gonadotropin, Prolactin, and Growth Hormone Secretion in the Growth-Limited Female Lamb*

For many basic and clinical scientists who focus on mechanisms underlying female puberty, the fundamental question has become, “How does the developing female determine when she has grown sufficiently to begin reproductive cycles?” Other investigators, interested in the reason for cessation of reproductive cycles after puberty in response to weight loss or high level exercise, are seeking an understanding about how the reproductive system senses these altered physiologic states.