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It is proposed that the first follicular phase in the lamb is initiated when responsiveness to oestradiol inhibition of LH secretion decreases sufficiently to permit the expression of an inherent hourly LH pulse rhythm. The hourly LH pulse rhythm is believed to drive oestradiol production to levels that induce the first LH surge. This hypothesis is based upon several considerations. First, pulsatile LH secretion invariably occurs at low frequencies in immature lambs whereas hourly pulses are manifest in postpubertal lambs and mature ewes during the follicular phase of the oestrous cycle. Second, intravenous administration of LH at hourly intervals to immature lambs results in an increase in follicular size, induction of an LH surge, ovulation and corpus luteum formation. Third, hourly LH pulses will occur in the immature female if the ovaries are removed; the hourly frequency in the immature ovariectomized lamb can be reduced by exogenous oestradiol. Fourth, in the chronically oestradiol-treated ovariectomized lamb, responsiveness to oestradiol inhibition of LH secretion becomes markedly decreased during the pubertal period, and the hourly LH pulse rhythm is expressed. It is further proposed that a minimum body weight and short daylengths are necessary for the reduction in oestradiol negative feedback. With regard to photoperiod, the delay in onset of cyclicity in lambs born in the wrong season (autumn) may be due to retarded maturation or post-maturational seasonal anoestrus.

The neuroendocrine regulation of GnRH secretion plays a central role in timing gamete release in both sexes. This regulation is more complex in the female because the discontinuous release of ova is more complex than the continuous release of spermatozoa. This review provides an evolving understanding of the sex differences in reproductive neuroendocrine controls and how these differences arise. The rules for sexual differentiation of steroid feedback control of GnRH secretion conceptually parallel the well-established principles that underlie the sexual differentiation of the internal and external genitalia. In the context of the neuroendocrine regulation of the ovarian cycle, and using the sheep as a model, four steroid feedback controls for GnRH secretion are inherent (default). They require no ovarian developmental input to function appropriately during adulthood. Two steroid feedback controls regulate the preovulatory surge mode of GnRH secretion, and two regulate the pulsatile mode. If the individual is a male, three steroid feedback controls of GnRH secretion become unnecessary or irrelevant, and these are abolished or become functionally inoperative through programmed reductions in hypothalamic sensitivity. This central programming occurs through exposure of presynaptic GnRH neurons in the developing male brain to the androgenic and estrogenic actions of testicular steroids. In precocial species such as ruminants, this programming begins well before birth. Understanding how GnRH secretion normally becomes sexually differentiated is of practical importance to determining how inappropriate hormonal environments during development can variously malprogram the neuroendocrine system to produce a variety of reproductive dysfunctions relating to patterning of gonadotropin secretion.

The GnRH system represents a useful model of long-term neural plasticity. An unexplored facet of this plasticity relates to the ontogeny of GnRH neural afferents during critical periods when the hypothalamic-pituitary-gonadal axis is highly susceptible to perturbation by sex steroids. Sheep treated with testosterone (T) in utero exhibit profound reproductive neuroendocrine dysfunctions during their lifespan. The current study tested the hypothesis that these changes are associated with alterations in the normal ontogeny of GnRH afferents and glial associations. Adult pregnant sheep (n = 50) were treated with vehicle [control (CONT)] or T daily from gestational day (GD)30 to GD90. CONT and T fetuses (n = 4–6/treatment per age group) were removed by cesarean section on GD90 and GD140 and the brains frozen at −80°C. Brains were also collected from CONT and T females at 20–23 wk (prepubertal), 10 months (normal onset of puberty and oligo-anovulation), and 21 months (oligo-anovulation in T females). Tissue was analyzed for GnRH immunoreactivity (ir), total GnRH afferents (Synapsin-I ir), glutamate [vesicular glutamate transporter-2 (VGLUT2)-ir], and γ-aminobutyric acid [GABA, vesicular GABA transporter (VGAT)-ir] afferents and glial associations (glial fibrillary acidic protein-ir) with GnRH neurons using optical sectioning techniques. The results revealed that: 1) GnRH soma size was slightly reduced by T, 2) the total (Synapsin-I) GnRH afferents onto both somas and dendrites increased significantly with age and was reduced by T, 3) numbers of both VGAT and VGLUT inputs increased significantly with age and were also reduced by T, and 4) glial associations with GnRH neurons were reduced (

Prenatal testosterone (T) exposure defeminizes reproductive neuroendocrine function in female sheep, although the LH surge dysfunctions are initially less severe in gonadally intact females than in females subject to neonatal ovariectomy and estradiol (E) replacement. Because prepubertal ovarian production of E differs quantitatively and qualitatively from chronic E replacement, we tested the hypothesis that postnatal E exacerbates the consequences of prenatal T on the positive, but not the negative, steroid feedback controls of GnRH secretion. Our approach was to characterize prepubertal sensitivity to E negative feedback, the onset and maintenance of progestagenic cycles, and the LH surge response in ovary intact, prenatally untreated (control), and T-treated (T) sheep that were exposed postnatally to only endogenous E, or exposed to excess E by sc implant. Sensitivity to E negative feedback was reduced in T females, but excess postnatal E did not further increase LH pulse frequency. Excess E prevented ovarian cycles in several control females, and increased cycle irregularity in T females. However, the LH surge mechanism was functional in all control females (regardless of postnatal E exposure) and in some T females without excess E, but nonfunctional in T females with excess E. These findings suggest that postnatal E does not program increased resistance to E negative feedback, but excess postnatal E does disrupt other mechanisms required for ovarian cyclicity. We conclude that in this precocial species, prenatal steroids are sufficient to program controls of tonic LH secretion, but the LH surge mechanism is susceptible to further programming by postnatal E.

Testicular steroids during midgestation sexually differentiate the steroid feedback mechanisms controlling GnRH secretion in sheep. To date, the actions of the estrogenic metabolites in programming neuroendocrine function have been difficult to study because exogenous estrogens disrupt maternal uterine function. We developed an approach to study the prenatal actions of estrogens by coadministering testosterone (T) and the androgen receptor antagonist flutamide, and tested the hypothesis that prenatal androgens program estradiol inhibitory feedback control of GnRH secretion to defeminize (advance) the timing of the pubertal increase in LH. Pregnant sheep were either untreated or treated with T, dihydrotestosterone (DHT) (a nonaromatizable androgen), or T plus flutamide from d 30–90 of gestation. To study the postnatal response to steroid negative feedback, lambs were gonadectomized and estradiol-replaced, and concentrations of LH were monitored in twice-weekly blood samples. Although T and DHT produced penile and scrotal development in females, the external genitalia of T plus flutamide offspring remained phenotypically female, regardless of genetic sex. Untreated females and females and males treated with T plus flutamide exhibited a pubertal increase in circulating LH at 26.4 ± 0.5, 26.0 ± 0.7, and 22.4 ± 1.6 wk of age, respectively. In females exposed to prenatal androgens, the LH increase was advanced (T: 12.0 ± 2.6 wk; DHT: 15.0 ± 2.6 wk). These results demonstrate the usefulness of combining T and antiandrogen treatments as an approach to increasing prenatal exposure to estradiol. Importantly, the findings support our hypothesis that prenatal androgens program sensitivity to the negative feedback actions of estradiol and the timing of neuroendocrine puberty.

As steroids and steroid-like compounds accumulate in the environment, it has become important to understand how low-dose exposure affects reproductive function. Ovary-intact sheep were used in a multigenerational study, to determine whether chronic exposure to low levels of estrogen disrupts reproductive function and behavior. We assessed parameters of reproductive performance in control and postnatally estradiol-treated females (Generation 1, G1), and their offspring (Generation 2, G2). In the G1 animals, 17beta-estradiol (E) was administered continuously from 4 wk of age at two doses via subcutaneous implants (ultralow E [

Findings discussed in this review stress the importance of normal estrogen and androgen signaling at appropriate developmental time points in maintaining normal phenotypic expression, reproductive and metabolic function and document how inappropriate steroid signaling, at inopportune times can have undesirable outcomes. For example, inappropriate testosterone exposure during fetal life alters the developmental trajectory of the female culminating in a suite of disorders, which include intrauterine growth-retardation and postnatal catch up growth, phenotypic masculinization, reproductive neuroendocrine and ovarian disruptions leading to progressive loss of cyclicity and metabolic disruptions manifested as hyperinsulinemia.

Prenatally testosterone (T)-treated female sheep exhibit ovarian and endocrinological features that resemble those of women with polycystic ovarian syndrome (PCOS), which include luteinizing hormone excess, polyfollicular ovaries, functional hyperandrogenism, and anovulation. In this study, we determined the developmental impact of prenatal T treatment on insulin sensitivity indexes (ISI), a variable that is affected in a majority of PCOS women. Pregnant ewes were treated with 60 mg testosterone propionate intramuscularly in cottonseed oil two times a week or vehicle [control (C)] from days 30 to 90 of gestation. T-females weighed less than C-females or males ( P < 0.05) at birth and at 5 wk of age. T-females had an increased anogenital ratio. An intravenous glucose tolerance test followed by an insulin tolerance test conducted after an overnight fast at 5, 20, and 30 wk of age ( n = 7–8/group) revealed that ISI were higher at 5 than 30 wk of age in C-females, reflective of a developing insulin resistance associated with puberty. T-females had higher basal insulin levels, higher fasting insulin-to-glucose ratio, and higher incremental area under the insulin curve to the glucose challenge. The ISI of T-females was similar to that of males. No differences in ISI were evident between groups at 20 and 30 wk of age. Mean basal plasma glucose concentrations and glucose disappearance and uptake did not differ between groups at any age. Our findings suggest that prenatal T treatment leads to offspring with reduced birth weight and impaired insulin sensitivity in early postnatal life.

Exposure of female sheep fetuses to excess testosterone (T) during early to midgestation produces postnatal hypergonadotropism manifest as a selective increase in LH. This hypergonadotropism may result from reduced sensitivity to estradiol (E2) negative feedback and/or increased pituitary sensitivity to GnRH. We tested the hypothesis that excess T before birth reduces responsiveness of LH and FSH to E2 negative feedback after birth. Pregnant ewes were treated with T propionate (100 mg/kg in cotton seed oil) or vehicle twice weekly from d 30–90 gestation. Responsiveness to E2 negative feedback was assessed at 12 and 24 wk of age in the ovary-intact female offspring. Our experimental strategy was first to arrest follicular growth and reduce endogenous E2 by administering the GnRH antagonist (GnRH-A), Nal-Glu (50 μg/kg sc every 12 h for 72 h), and then provide a fixed amount of exogenous E2 via an implant. Blood samples were obtained every 20 min at 12 wk and every 10 min at 24 wk before treatment, during and after GnRH-A treatment both before and after E2 implant. GnRH-A ablated LH pulsatility, reduced FSH by approximately 25%, and E2 production diminished to near detection limit of assay at both ages in both groups. Prenatal T treatment produced a precocious and selective reduction in responsiveness of LH but not FSH to E2 negative feedback, which was manifest mainly at the level of LH/GnRH pulse frequency. Collectively, these findings support the hypothesis that prenatal exposure to excess T decreases postnatal responsiveness to E2 inhibitory feedback of LH/GnRH secretion to contribute to the development of hypergonadotropism.

Prenatal exposure of the female sheep to excess testosterone (T) leads to hypergonadotropism, multifollicular ovaries, and progressive loss of reproductive cycles. We have determined that prenatal T treatment delays the latency of the estradiol (E2)-induced LH surge. To extend this finding into a natural physiological context, the present study was conducted to determine if the malprogrammed surge mechanism alters the reproductive cycle. Specifically, we wished to determine if prenatal T treatment 1) delays the onset of the preovulatory gonadotropin surge during the natural follicular phase rise in E2, 2) alters pulsatile LH secretion and the dynamics of the secondary FSH surge, and 3) compromises the ensuing luteal function. Females prenatally T-treated from Day 60 to Day 90 of gestation (147 days is term) and control females were studied when they were approximately 2.5 yr of age. Reproductive cycles of control and prenatally T-treated females were synchronized with PGF2alpha, and peripheral blood samples were collected every 2 h for 120 h to characterize cyclic changes in E2, LH, and FSH and then daily for 14 days to monitor changes in luteal progesterone. To assess LH pulse patterns, blood samples were also collected frequently (each 5 min for 6 h) during the follicular and luteal phases of the cycle. The results revealed that, in prenatally T-treated females, 1) the preovulatory increase in E2 was normal; 2) the latencies between the preovulatory increase in E2 and the peaks of the primary LH and FSH surges were longer, but the magnitudes similar; 3) follicular-phase LH pulse frequency was increased; 4) the interval between the primary and secondary FSH surges was reduced but there was a tendency for an increase in duration of the secondary FSH surge; but 5) luteal progesterone patterns were in general unaltered. Thus, exposure of the female to excess T before birth produces perturbances and maltiming in periovulatory gonadotropin secretory dynamics, but these do not produce apparent defects in cycle regularity or luteal function. To reveal the pathologies that lead to the eventual subfertility arising from excess T exposure during midgestation, studies at older ages must be conducted to assess if there is progressive disruption of neuroendocrine and ovarian function.

The pubertal transition to adulthood involves both gonadal and behavioral maturation. A developmental clock, along with permissive signals that provide information on somatic growth, energy balance and season, time the awakening of gonadotropin releasing hormone (GnRH) neurons at the onset of puberty. High-frequency GnRH release results from disinhibition and activation of GnRH neurons at puberty onset, leading to gametogenesis and an increase in gonadal steroid hormone secretion. Steroid hormones, in turn, both remodel and activate neural circuits during adolescent brain development, leading to the development of sexual salience of sensory stimuli, sexual motivation, and expression of copulatory behaviors in specific social contexts. These influences of hormones on reproductive behavior depend in part on changes in the adolescent brain that occur independently of gonadal maturation. Reproductive maturity is therefore the product of developmentally timed, brain-driven and recurrent interactions between steroid hormones and the adolescent nervous system.

In sheep, internal and external cues are integrated to time the decrease in sensitivity to steroid negative feedback that increases the frequency of gonadotropin-releasing hormone (GnRH) pulses to drive the transition into adulthood. The neural substrate that processes the information used to time the pubertal increase in GnRH secretion is programmed prenatally by the organizational actions of testosterone produced in the male, but not the female (the default sex), in response to an early increase in GnRH and gonadotropin secretion. In both sexes, metabolic cues provide information that sufficient growth has occurred to begin reproduction. However, the young female, being inherently photoperiodic, cannot express her sexual maturity until day length is appropriate. Once she has experienced the long days of summer followed by the short days of autumn, neuroendocrine sensitivity to steroid negative feedback decreases, and high-frequency GnRH pulses are expressed to initiate ovarian cycles. By contrast, prenatal testicular steroids masculinize the photoneuroendocrine system to reduce the reliance on day length as a cue for puberty, and the male lamb does not use photoperiods to time the expression of high-frequency GnRH pulses. Once metabolic signals indicate that energy balance is sufficient in the male, high-frequency GnRH pulses are expressed. Thus, whereas metabolic information provides permissive signals that time sexual maturity in both sexes, the presence or absence of prenatal programming by testosterone determines whether photoperiod also serves as a permissive cue to begin reproductive activity. Viewed in a broad sense, the neuroendocrine response to internal and external signals timing the transition to adulthood has fetal origins in the sheep.

We studied the impact of prenatal androgen exposure on the timing of onset of puberty, maintenance of cyclicity in the first breeding season, and the LH surge mechanism in female sheep. Pregnant sheep were injected with testosterone propionate (100 mg i.m.) twice each week from Day 30 to Day 90 (D30–90) or from Day 60 to Day 90 (D60–90) of gestation (term = 147 days). Concentrations of plasma progesterone and gonadotropins were measured in blood samples collected twice each week from control (n = 10), D60–90 (n = 13), and D30–90 (n = 3) animals. Rate of weight gain and initiation of estrous behavior were also monitored. After the first breeding season, when the animals entered anestrus, competency of the gonadotropin surge system to respond to estradiol positive feedback was tested in the absence or presence of progesterone priming for 12 days. Prenatally androgenized females had similar body weight gain and achieved puberty (start of first progestogenic cycle) at the same time as controls. Durat...

Exposure of the female sheep fetus to exogenous testosterone in early pregnancy permanently masculinizes the reproductive neuroendocrine axis. Specifically, in utero androgens given to female lambs from day 30 to 90 of a 147 day pregnancy dramatically altered the response of the gonadotropin releasing hormone (GnRH) neuronal network in the hypothalamus to both estrogen (E) and progesterone (P) feedback. Elevated concentrations of estrogen stimulated a massive release of GnRH in gonadectomized female sheep; however, male and androgenized female lambs were unable to respond to high E concentrations by producing this preovulatory-like "surge" of GnRH. Further, the inhibitory actions of progesterone (P) were also sexually differentiated and adult males and androgenized females were much less responsive to P-negative feedback than normal ewes. The consequences of these abnormal steroid feedback mechanisms were reflected in the fact that only 72% of ovary-intact androgenized ewes exhibited normal estrous cycles in their first breeding season whereas none had a single estrous cycle during the second breeding season. In contrast, 100% of the control animals exhibited repeated reproductive cycles in both seasons. These data indicate that a relatively short exposure to male hormones during in utero life permanently alters the neural mechanisms that control reproduction and leads progressively to a state of infertility.

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). 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.

Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i.c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 mg/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly lower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 mg of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen. Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.

Pharmacological reduction of glucose availability with 2-deoxyglucose (2DG) suppresses pulsatile luteinizing hormone (LH) secretion in rats and growth-retarded lambs. Gonadal steroids enhance the glucoprivic suppression of LH secretion in rats. The present study determined if corticotropin-releasing hormone (CRH) plays a role in mediating oestrogen-dependent and -independent glucoprivic suppression of LH secretion. The study was conducted in ovariectomized (OVX) rats some of which received Silastic implants containing oestradiol-17beta (OE2) dissolved in peanut oil at 20 microg/ml to produce a physiological plasma level of OE2 (30 pg/ml). Seven days after ovariectomy, the rats were stereotaxically implanted with a guide cannula into the third cerebral ventricle. Seven days later, blood samples were collected through an indwelling atrial cannula every 6 min for 3 h for LH pulse determination. After the first hour of blood sampling, a CRH antagonist, [D-Phe12, Nle21,38]hCRF-(21-41), or vehicle was injected into the third cerebral ventricle through the implanted cannula before 2DG administration through the indwelling atrial cannula. Pulsatile LH secretion was suppressed by 2DG (200 mg/kg b.w.) in the vehicle-treated rats bearing OE2 implants. The CRH antagonist (5.65 nmol) blocked the suppressive effect of 2DG on pulsatile LH secretion in the OE2-treated OVX animals. On the other hand, in the absence of oestrogen, the effect of a twice greater dose of 2DG (400 mg/kg b.w.) was not blocked by five times greater amount of CRH antagonist (28.3 nmol). These results suggest the mechanisms mediating glucoprivic suppression of LH secretion involve two components: one is oestrogen-dependent and the other oestrogen-independent. CRH may be involved in the oestrogen-dependent component of glucoprivic suppression of LH secretion but not the oestrogen-independent one.

Prenatal exposure of sheep to testosterone (T) disrupts ovarian cyclicity and leads to anovulation in adulthood. We propose that the disruption of ovarian function in prenatally-androgenized sheep is mediated via follicular defects stemming from reduced intrafollicular activin availability/action. The intra-follicular activin availability/action that facilitates follicular development is dictated by the relative proportions of activins, inhibins (antagonists of activin action) and follistatins (FS; binding proteins of activin and negator of activin action). Inhibin alpha, beta A, beta B, and FS mRNA expression were determined by in situ hybridization in 5 week-old ovaries from control (C) lambs or those exposed to testosterone (T) or DHT from 30-90 days of gestation. In utero exposure to T, but not DHT, increased total ovarian weight (0.4+/-0.1,1.5+/-0.5 and 0.3+/-0.1 g, C, T and DHT, respectively) and total number of follicles (16.5+/-2.8,37.8+/-7.9, and 18.8+/-3.0). With the exception of two follicles in T animals, all follicles were < or = 2 mm in diameter. All follicles < or = 2 mm in all groups expressed FSH receptor mRNA in the granulosa cells and LH receptor only in the thecal cells. The percentage of follicles expressing FS mRNA was increased (P

We have previously determined that exogenous leptin prevents the inhibition of pulsatile luteinizing hormone (LH) release in the fasting rodent. The present study tested the hypothesis that the mechanism by which leptin facilitates high LH secretion is through an attenuation of the stress response produced by a deficit in energy. Because hypogonadotropism is associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis during both metabolic stress and nonmetabolic stress, our approach included a comparison of whether exogenous leptin could prevent the rise in corticosterone produced by a nonmetabolic stress (immobilization for 2 h), as well as by a widely used metabolic stress (transient glucoprivation by 2-deoxyglucose, 2DG; 400 mg/kg, b.w., i.v.). Each stressor was applied to well-fed ovariectomized rats (n=4‐6 per group), 2 h after leptin (3 mg/g, b.w., i.p.) or vehicle administration. Blood samples were collected through an indwelling atrial cannula every 6 min for 1 h before and for 2 h after the stress treatment to measure LH, leptin and corticosterone. During metabolic stress (acute glucoprivation), circulating leptin decreased, corticosterone increased and LH decreased; leptin administration abolished the increase in corticosterone, but pulsatile LH secretion remained inhibited. In contrast, during nonmetabolic stress (immobilization), leptin secretion was unaffected, but circulating corticosterone increased and LH decreased; leptin treatment did not prevent either the increase in corticosterone or the decrease in LH secretion. An important overall finding is that leptin can differentially alter the HPA axis depending upon the type of stress. In addition, whether the pattern of leptin is altered depends upon the type of stress. Although a glucoprivic-induced decrease in endogenous leptin can be a stressor responsible for the increase in corticosterone secretion, a nonmetabolic stress-induced increase in corticosterone is not mediated by leptin. Moreover, our results reveal that the depression of LH secretion when leptin is low during reduced energy availability is not due to activation of the HPA axis. During an energy deficit, exogenous leptin could not restore high frequency LH secretion when HPA function was restored to normal. Finally, the inability of leptin to increase LH secretion in the face of 2DG supports the notion that the action of leptin is dependent upon the degree of glucose availability.

Administration of leptin during reduced nutrition improves reproductive activity in several monogastric species and reverses GH suppression in rodents. Whether leptin is a nutritional signal regulating neuroendocrine control of pituitary function in ruminant species is unclear. The present study examined the control of pulsatile LH and GH secretion in sheep. We determined whether exogenous leptin could prevent either the suppression of pulsatile LH secretion or the enhancement of GH secretion that occur during fasting. Recombinant human met-leptin (rhmet-leptin; 50 mg/kg BW; n 5 8) or vehicle (n 5 7) was administered sc every 8 h during a 78-h fast to estrogentreated, castrated yearling males. LH and GH were measured in blood samples collected every 15 min for 6 h before fasting and during the last 6ho ffasting. Leptin was measured both by a universal leptin assay and by an assay specific for ovine leptin. During the fast, endogenous plasma leptin fell from 1.49 6 0.16 to 1.03 6 0.13 ng/ml. The average concentration of rhmet-leptin 8 h after leptin administration was 18.0 ng/ml. During fasting, plasma insulin, glucose, and insulin-like growth factor I levels declined, and nonesterified fatty acid concentrations increased similarly in vehicle-treated and leptintreated animals. In vehicle-treated animals, LH pulse frequency declined markedly during fasting (5.6 6 0.5 vs. 1.1 6 0.5 pulses/6 h; fed vs. fasting; P , 0.0001). Leptin treatment prevented the fall in LH pulse frequency (5.0 6 0.4 vs. 4.9 6 0.4 pulses/6 h; P 5 0.6). Neither fasting nor leptin administration altered GH pulse frequency. Fasting produced a modest increase in mean concentrations of circulating GH in control animals (2.4 6 0.5 vs. 3.4 6 0.6 ng/ml; P 5 0.04), whereas there was a much greater increase in GH during leptin treatment (2.7 6 0.6 vs. 8.6 6 1.6 ng/ml; P 5 0.0001). GH pulse amplitudes were also increased by fasting in control (P 5 0.04) and leptin-treated sheep (P 5 0.007). The finding that exogenous rhmet-leptin regulates LH and GH secretion in sheep indicates that this fat-derived hormone conveys information about nutrition to mechanisms controlling neuroendocrine function in ruminants. (Endocrinology 141: 3965‐3975, 2000)

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, increase...

This study tested the hypothesis that central mechanisms regulating luteinizing hormone (LH) secretion are responsive to insulin. Our approach was to infuse insulin into the lateral ventricle of six streptozotocin-induced diabetic sheep in an amount that is normally present in the CSF when LH secretion is maintained by peripheral insulin administration. In the first experiment, we monitored cerebrospinal fluid (CSF) insulin concentrations every 3–5 h in four diabetic sheep given insulin by peripheral injection (30 IU). The insulin concentration in the CSF was increased after insulin injection, and there was a positive relationship between CSF and plasma concentrations of insulin (r = 0.80, P < 0.01). In the second experiment, peripheral insulin administration was discontinued, and the sheep received either an intracerebroventricular (i.c.v.) infusion of insulin (12 mU/day in 2.4 ml saline) or saline (2.4 ml/day) for 5 days (n = 6) in a crossover design. The dose of insulin (i.c.v.) was calculated...

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...

Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic β-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopos...

In sheep, the control of tonic and surge GnRH secretion is sexually differentiated by testosterone in utero. However, GnRH neurons are not sexually dimorphic with respect to number, distribution, or gross morphology. Therefore, this study tested the hypothesis that prenatal steroids influence synaptic input to GnRH neurons. We compared the number of synapses on GnRH neurons from male, female, and androgenized female lambs (n = 5 each). Androgenized females were exposed to testosterone during mid-gestation. Yearling lambs were perfused, and GnRH neurons were visualized using the LR-1 antibody. Five to seven GnRH neurons from the rostral preoptic area in each animal were viewed at the ultrastructural level. Afferent synapses and glial ensheathment on each neuron were counted in a single section through the plane of the nucleus. GnRH neurons from females received approximately twice as many contacts (3.6 +/- 0.7 synapses/100 microm plasma membrane) as those from male lambs (1.6 +/- 0.3; p < 0.05), similar to previous reports in rats. In addition, the number of synapses on GnRH neurons from androgenized female lambs (1.5 +/- 0.5) was similar to that from male lambs, suggesting that prenatal steroids give rise to sex differences in synaptic input to GnRH neurons.

How nutrition regulates reproductive activity remains a major unsolved question of reproductive biology. Reducing the level of nutrition during adulthood can lead to infertility, primarily through reduction of GnRH secretion. Inquiry about such a mechanism has its roots in the search for cues timing the onset of fertility, because the tempo of sexual maturation is much more closely associated with body growth than with chronological age. Growth depends on the quantity and quality of food intake. When food availability is low, small, short-lived species with high metabolism and reduced growth may not even attain puberty before they die. In longer-lived species, puberty is delayed for months or even years until more food becomes available. To appreciate fully how the pubertal progression is timed will require understanding how peripheral signals relating information about energy metabolism are sensed by the brain and how such information is routed through pathways controlling GnRH secretion. Here, we provide some background and physiologic perspective on the question of whether the fat-derived hormone leptin is the unique peripheral signal, is an important signal, is but one of a constellation of signals, or is not a signal timing puberty.

In many species, the timing of puberty is different in males and females. This does not simply reflect differences in the time course of activation of the testes and ovaries. Rather, sex differences in pubertal onset reside within brain mechanisms controlling GnRH secretion, as exemplified by studies conducted in sheep. Exposure of sheep fetuses to testicular steroids alters the timing of puberty, principally by reducing photoperiod responsiveness. This is manifest as an early increase in LH secretion in males or in females exposed experimentally to testosterone before birth. Steroids also act on non-photoperiodic mechanisms to abolish the preovulatory gonadotrophin surge. In view of these multiple organizational actions of steroids to control postnatal gonadotrophin secretion, it is becoming clear that there are many critical periods of brain development for organizing the GnRH neurosecretory system, and that these may be sensitive to different testosterone metabolites. Although GnRH neurones are not sexually dimorphic with respect to number, distribution or gross morphology, fundamental questions remain as to how steroids exert their effects at the cell through actions on GnRH afferents. Teleologically, these early sex-specific changes in mechanisms timing puberty maximize the chance that reproductive activity will ultimately be successful in each sex.

In sheep, prenatal exposure to androgens during a critical period for sexual differentiation of the brain (30-90 days of gestation; 145 days is term) can advance the timing of puberty in females and prevent the preovulatory LH surge. The present study tests the hypothesis that in sheep, the timing of neuroendocrine sexual maturation is related to the amount of prenatal steroid exposure. In addition, we determined if different steroid requirements exist for sexual differentiation of the tonic and surge modes of gonadotropin secretion. Testosterone was administered weekly to three groups of pregnant ewes from days 30-90 of gestation at doses of 200, 80, or 32 mg/week. The resulting androgenized female lambs together with control males and females (n = 5-7/group) were gonadectomized at 3 weeks of age, and gonadal steroids were replaced with a SILASTIC brand estradiol-filled capsule. LH concentrations were measured from biweekly blood samples. Sustained increases in circulating LH were considered to reflect the initiation of neuroendocrine puberty. In male lambs, LH secretion started to increase at 8.3 +/- 0.9 weeks of age (mean +/- SEM). The two highest doses of prenatal androgen advanced the onset of neuroendocrine sexual maturation in females. In the 200 mg androgenized females, the pubertal LH rise (10.2 +/- 2.0 weeks) began about the same time as in males. In the 80 mg treatment group, LH concentrations increased at 16.2 +/- 1.5 weeks, which was later than in males, but well before that in normal females (27.1 +/- 0.7 weeks). For females treated with the lowest dose of androgen (32 mg), the pubertal LH increase (24.6 +/- 1.9 weeks) began about the same time as in normal females. To test the function of the LH surge system, LH was measured every 2 h for 60 h after an acute increase in circulating estradiol was produced by implanting additional estrogen capsules. All control females produced a surge in response to acute estradiol stimulation. LH surges did not occur in males, 200 mg androgenized females, or 80 mg androgenized females. Of six females from the 32 mg treatment group, two produced LH surges in response to the stimulatory feedback action of estradiol. We conclude that the greater the amount of prenatal testosterone, the earlier the initiation of the pubertal LH rise. Moreover, the finding that low doses of testosterone (32 mg/week) are capable of abolishing the LH surge without significantly advancing the timing of puberty supports our hypothesis that different steroid requirements exist for sexual differentiation of tonic and surge modes of LH secretion.

To determine if prenatal androgens prevent activation of GnRH neurons in response to estradiol stimulation, Fos colocalization with GnRH was compared in the brains of normal female lambs, normal males, and androgenized females in response to a surge-inducing dose of estradiol. Blood samples were collected every 1‐2 h for 6 h before estradiol treatment up to the time of sacrifice at 17‐19 h posttreatment. Following perfusion, 60 mm coronal brain sections were immunostained for Fos (151000, Santa Cruz Biochemicals) and GnRH (1540,000, LR-1) using NiCl-enhanced and unenhanced DAB, respectively. Although LH secretion increased in females before sacrifice, no increase was observed in males or androgenized females. Despite differences in LH secretion, the number and distribution of GnRH neurons was not sexually dimorphic. Moreover, Fos immunostaining was visible throughout steroid-responsive limbic regions in all three groups of lambs. However, the colocalization of Fos with GnRH was highly sexually dimorphic. In females perfused after the peak of the LH surge, 65.7% of GnRH neurons in the preoptic area, anterior hypothalamus, and mediobasal hypothalamus expressed Fos, whereas only 1.7% of GnRH neurons were Fos-positive in males and androgenized females. These findings indicate that sex differences in the activation of GnRH neurons in response to estradiol are determined prenatally through the actions of testosterone.

In sheep, the surge mode of gonadotropin secretion is sexually differentiated, i.e. the LH surge is present in the female, but not in the male. The present study tested the hypothesis that sexual differentiation of the LH surge mechanism reflects a sex difference in the pattern of GnRH, and that prenatal androgens abolish the surge mode of GnRH secretion. We monitored the pattern of GnRH secretion in pituitary portal blood after acute treatment with estradiol in gonadectomized postpubertal males (n = 6), females (n = 6), and androgenized females (exposed prenatally to testosterone from day 30-90 in gestation, n = 7). Four capsules, each containing a 30-mm column of estradiol were implanted s.c. into each lamb to produce high physiologic concentrations of the hormone. Beginning 7 h later, portal and peripheral blood samples were collected hourly for 48 h for measurement of GnRH and LH, respectively. All females exhibited a GnRH surge beginning 13.0 +/- 0.4 h after estradiol treatment; this was accompanied by an LH surge. By contrast, only one male produced a small surge in GnRH (1.7 pg/min) with a latency of 32 h; a corresponding increase in LH occurred in this male. Likewise, among the androgenized females, only one exhibited GnRH and LH surges which began at about 22 h after estradiol treatment. Some of the androgenized females had sporadic increases in GnRH which were of lower amplitude than in the control females, and were unaccompanied by rises in LH. These findings provide the first direct evidence that the sex difference in the surge mode of LH secretion results from the sexual differentiation of the pattern of GnRH release. The study also suggests that androgens during prenatal development abolish the GnRH surge and subsequently, the generation of the LH surge.

Photoperiod times the transition to sexual maturity in many seasonal breeders. In male and female sheep, photoperiod influences the timing of puberty differentially. Whereas in females, age at sexual maturity is highly dependent on photoperiod, puberty in males begins at the same age regardless of day length. We have determined that this sex difference is due to the organizing action of androgens during prenatal development. In the present investigation, we studied when during gestation (term: approximately 150 days) androgens defeminize the reproductive response to photoperiod. We compared the age at sexual maturity in female lambs treated with testosterone prenatally from Days 30 to 76 (Early, n = 7) or 89 to 135 (Late, n = 8) to that of normal males (n = 8) and normal females (n = 7). To reveal differential responsiveness to photoperiod, all lambs were maintained from birth under constant long days (16L:8D), a treatment that inhibits puberty in normal females. The age at the pubertal LH rise was determined in a standardized experimental model (lambs gonadectomized and treated with estradiol). As expected in the long day photoperiod, only 1 of 7 normal females had a pubertal rise in LH. In contrast, all males increased LH secretion by 6.7 +/- 0.6 wk. Similarly, in the Early group, a sustained increase in LH occurred in all females, but this was delayed relative to the increase in the males (16.8 +/- 1.7 wk; p < 0.001). The Late group had LH patterns similar to those of the normal females, with only 3 of 8 females having sustained elevations in LH. These data suggest that a "critical period" for the defeminization of the reproductive response to photoperiod occurs early in prenatal development. In addition, it appears that this critical period and the period for defeminization of the surge mode of gonadotropin secretion occur at similar stages in development. When challenged with an acute increase in estradiol, all normal and Late androgenized females responded with an LH surge. In contrast, none of the males and only 1 of 7 Early females produced a robust response to estradiol.

In female sheep, estradiol (E2) stimulates the preovulatory GnRH/LH surge and receptive behavior, whereas progesterone blocks these effects. Prenatal exposure to testosterone disrupts both the positive feedback action of E2 and sexual behavior although the mechanisms remain unknown. The current study tested the hypothesis that both prenatal and postnatal steroids are required to organize the surge and sex differences in reproductive behavior. Our approach was to characterize the LH surge and mating behavior in prenatally untreated (Control) and testosterone-treated (T) female sheep subsequently exposed to one of three postnatal steroid manipulations: endogenous E2, excess E2 from a chronic implant, or no E2 due to neonatal ovariectomy (OVX). All females were then perfused at the time of the expected surge and brains processed for estrogen receptor and Fos immunoreactivity. None of the T females exposed postnatally to E2 exhibited an E2-induced LH surge, but a surge was produced in five of six T/OVX and all Control females. No surges were produced when progesterone was administered concomitantly with E2. All Control females were mounted by males, but significantly fewer T females were mounted by a male, including the T/OVX females that exhibited LH surges. The percentage of estrogen receptor neurons containing Fos was significantly influenced in a brain region-, developmental stage-, and steroid-specific fashion by testosterone and E2 treatments. These findings support the hypothesis that the feedback controls of the GnRH surge are sensitive to programming by prenatal and postnatal steroids in a precocial species.

In most seasonal breeders, photoperiod influences the timing of the transition to sexual maturity. In sheep, males and females express reproductive maturity under different photoperiods. Spring-born males begin the maturational process during lengthening days (spring), whereas females do so during shortening days (autumn). We hypothesized that photoperiod differentially influences the transition to sexual maturity in each sex, and that this difference results from the presence or absence of androgens prenatally. The monitored the timing of sexual maturity in male and female lambs (n=8 each) and in prenatally masculinized female lambs (n=7) maintained under controlled photoperiods (natural-simulate or reverse natural-simulate). Circulating LH was analyzed to reveal the timing of the pubertal gonadotropin rise; lambs were gonadectomized and implanted with estradiol to provide a constant feedback on LH secretion. Under natural-simulate photoperiod, LH increased in males at 7.0±0.3 wk when days were lengthening. In females treated similarly, sexual maturity occurred at 27.0±0.8 wk when day lengths were decreasing. The reverse photoperiod had no effect on males, but it delayed the expression of sexual maturity in females. Thus, LH also increased in males at 7.1±0.9 wk, while none of the females maintained under the same photoperiod had elevated LH secretion when the experiment was terminated (32 wk of age). Prenatal treatment with androgens masculinized the reproductive response to photoperiod; as in the males, LH increased in the androgenized females maintained under a reverse natural-simulate photoperiod at 7.1±0.4 wk. We conclude that a sex difference in the reproductive response to photoperiod exists in developing sheep and that this is due to the organizing action of testicular androgens during fetal development

In female sheep, photoperiod regulates the timing of the transition to adulthood. We tested the hypothesis that photoperiod very early in development influences the timing of the pubertal LH rise that initiates sexual maturation. The first experiment was designed to determine the influence of day length information perceived before birth by varying prenatal photoperiod experience. Two groups that experienced either increasing or constant long days prenatally, and then a gradually decreasing photoperiod postnatally, reached puberty at the same age (Prenatal Increase, 20.4 +/- 0.5 wk vs. Prenatal Long Days [LD], 19.4 +/- 0.8 wk). Puberty in these groups was much earlier than in two control groups exposed to the same photoperiods, but beginning at birth, for 13 wk (Postnatal Increase, 29.6 +/- 1.0 wk; Postnatal LD, 26.2 +/- 1.3 wk). In the second experiment, the role of prenatal photoperiod in timing sexual maturity was also examined through the use of treatments with greater contrast. Lambs were exposed prenatally to either decreasing or increasing day lengths. Beginning at birth, both groups were exposed to a decreasing photoperiod. Although only half of the lambs in each group exhibited the pubertal LH rise, those that attained puberty did so at the same age (Prenatal Decrease, 14.8 +/- 1.0 wk vs. Prenatal Increase, 14.8 +/- 0.3 wk). We therefore conclude that day length cues experienced postnatally predominantly time sexual maturation in the female lamb.

Growth retardation induced by dietary restriction in the lamb results in a decrease in LH pulse frequency and therefore in delayed puberty. Increased circulating cortisol levels have been associated with nutritional restriction in a variety of species. The current study tested the hypothesis that hyperactivity of the adrenal axis sustains hypogonadotropism in the growth-restricted lamb. Our approach was to compare the patterns and levels of circulating cortisol and LH in ovariectomized, growth-restricted (n = 8) and ad libitum-fed (n = 6) lambs. At 37 wk of age, after the growth-restricted lambs had been on the reduced diet for 31 wk, basal cortisol levels were determined hourly for 31 h. In addition, during this period, pulsatile LH and cortisol release was determined during a 4-h period (samples every 12 min). Finally, the cortisol response to a physiologic ACTH stimulus and an audiovisual stimulus (barking dog for 2 min) was determined in frequent samples collected during the last 5 h of the 31-h period. As expected, growth-restricted lambs exhibited a low LH pulse frequency (0.25 +/- 0.10 pulses/h) compared with ad libitum-fed lambs (1.37 +/- 0.07 pulses/h). No diurnal cortisol rhythm was observed in either group, and a similar cortisol pulse frequency occurred in the two groups (1.00 +/- 0.07 pulses/h in growth-restricted lambs and 1.05 +/- 0.10 pulses/h in ad libitum-fed lambs). There was no significant difference between the groups in cortisol pulse amplitude. ACTH administration (i.v.) induced a similar cortisol pulse in 4 of 8 growth-restricted lambs and in 5 of 6 ad libitum-fed lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Two experiments were performed to test the hypothesis that there is a seasonal change in the negative feedback effect of estradiol on episodic secretion of GnRH in the ewe. The first experiment identified a specific estradiol treatment (delivered by s.c. Silastic implant) that produced a 50% decrease in the frequency of pulsatile secretion of LH in ovariectomized ewes during the anestrous season. In the second experiment, this estradiol treatment was administered to ovariectomized ewes during the mid-breeding and anestrous seasons. Separate groups of ovariectomized ewes not treated with estradiol were included during each season to test for a seasonal difference in the effect of estradiol on episodic GnRH and LH secretion. Samples of hypophyseal portal blood (for GnRH) and jugular blood (for LH) were obtained at 5-min intervals approximately one month after placement of the estradiol implants. During the breeding season, no effect of estradiol was observed on either the frequency or size of GnRH and LH pulses. During anestrus, however, estradiol produced a profound suppression of the frequency of GnRH and LH pulses, and an increase in GnRH pulse size. No significant seasonal change was observed in the characteristics of GnRH and LH pulses in ovariectomized ewes in the absence of estradiol treatment. These findings lead to the conclusion that there is a marked seasonal change in the negative feedback effect of estradiol on episodic GnRH secretion in the ewe, with the steroid being maximally effective during anestrus.

This study determined whether chronic exposure of female lambs to the electric and magnetic fields (EMF) of a high voltage transmission line can alter pineal secretion of melatonin and the normal occurrence of puberty. Twenty female Suffolk lambs were assigned randomly in equal numbers to a control and a treatment group. Treatment from 2 to 10 mo of age consisted of continuous exposure within the electrical environment of a 500-kV transmission line (mean electric field 6 kV/m, mean magnetic field 40 mG). Treated lambs were penned directly beneath the transmission line; control lambs were maintained in a pen of similar construction 229 m from the line where EMF were at ambient levels (mean electric field < 10 V/m, mean magnetic field < 0.3 mG). Melatonin was analyzed by RIA in serum of blood samples collected at 0.5-3-h intervals over eight 48-h periods. To assess attainment of puberty, serum concentrations of progesterone were determined by RIA from blood samples collected twice weekly beginning at 19 wk of age. Concentrations of circulating melatonin in control and treated lambs were low during daylight hours and increased during nighttime hours. The characteristic pattern of melatonin secretion during nighttime (amplitude, phase, and duration) did notmore » differ between control and treatment groups. Age at puberty and number of subsequent estrous cycles also did not differ between groups. These data suggest that chronic exposure of developing female sheep to 60-Hz environmental EMF does not affect the mechanisms underlying the generation of the circadian pattern of melatonin secretion or the mechanisms involved in the onset of reproductive activity.« less

The experimental induction of puberty by GnRH administration to prepubertal lambs increases serum bioactive FSH (B-FSH) as measured in the rat Sertoli cell aromatase induction bioassay. Serum immunoreactive FSH (I-FSH) levels are unchanged. The increase in serum B-FSH is associated with an increase in the proportion of less acidic and more biopotent FSH serum isoforms. However, it is unknown if this effect of GnRH on serum FSH microheterogeneity is direct or mediated by gonadal factors. We have used the nutritionally growth-restricted ovariectomized lamb as a model of the neuroendocrine regulation of FSH isoform microheterogeneity. With this model, the hypothalamic-pituitary component of the neuroendocrine axis may be isolated from gonadal factors. In the present study, using the nutritionally growth-restricted ovariectomized lamb as a model, we investigated the role of GnRH on the regulation of FSH microheterogeneity. Specifically, we tested the hypothesis that GnRH increases the proportion of the less acidic (more biopotent) serum FSH isoforms. As an in vitro correlate, we investigated the effect of GnRH on gonadotropin secretion and FSH isoform distribution in ovine pituitary explant cultures. Seven ovariectomized nutritionally restricted lambs were administered GnRH (i.v., 2 ng/kg) for 36 h (at 2-h intervals for 24 h, then hourly for the final 12 h). Six others served as controls. Blood samples were withdrawn at 12-min intervals during the last 4 h for the measurement of serum immunoactive LH (I-LH) and I-FSH. Pituitary homogenates and serum from four animals from each group were individually chromatofocused, and the FSH isoform distribution patterns were determined. Pulsatile administration of GnRH to nutritionally growth-restricted lambs increased circulating I-LH concentrations from 0.6 +/- 1.0 to 5.9 +/- 3.1 ng/ml (P < 0.01), but did not significantly change circulating I-FSH (4.9 +/- 1.8 vs. 11.5 +/- 4.2 ng/ml) nor B-FSH concentrations (3.9 +/- 1.2 vs. 5.7 +/- 1.5 ng/ml). The pituitary content of I-FSH, B-FSH, and I-LH were unchanged. Neither serum nor pituitary FSH isoform distribution patterns were altered by pulsatile GnRH administration. However, compared to the pituitary FSH isoforms, a higher percentage of circulating FSH isoforms eluted in the salt peak of both groups of lambs. Similar to the in vivo studies, in vitro, GnRH increased the release of I-LH, as well as I-FSH, from pituitary explants, but did not significantly change the FSH isoform distribution in either the pituitary explant or media.(ABSTRACT TRUNCATED AT 400 WORDS)

Yellon, S.M., Foster, D.L., Longo, L.D. and Suttie, J.M., 1992. Ontogeny of the pineal melatonin rhythm and implications for reproductive development in domestic ruminants. Anita. Reprod. Sci., 30: 91-112. This review focuses on the important contribution that domestic ruminants have made to understanding the role of environmental photoperiod in the process of reproductive maturation. Evidence in the sheep, goat and deer indicate that the influence of photoperiod on growth and the onset of puberty is mediated by the pineal gland and secretion of the hormone melatonin. The ontogeny of the circadian melatonin rhythm is discussed in relation to its role in the timing of puberty for these ruminant species.

Both the onset of puberty in the lamb and the annual resumption of reproductive activity in adult male and female sheep are characterized by increased secretion of LH due to reduced responsiveness to steroid inhibition. However, the timing of puberty is sexually differentiated, for males undergo a reduction in sensitivity to steroid feedback at 10 wk of age, whereas females remain highly responsive to steroid inhibition until 30 wk. This sex difference is determined by androgens in utero. The present study was conducted to determine whether a sex difference exists in the timing of seasonal transitions in adult males and females. We compared serum UI in gonadectomized, estradiol-treated males (n = 7), females (n = 6), and androgenized females (n = 5) from blood samples collected twice weekly for one year. As detertnined by changes in the pattern of LH secretion, the onset and termination of the autumn breeding season were not different between males, females, and androgenized females (termination: 1 February ± 4 days, mean ± SE all groups; onset: males, 22 August ± 4 days; females, 5 September ± 18 days; androgenized females, 16 September ± 10.5 days). However, there was a transient increase In UI (20 May to 23 June) in males, but not in females or androgenized females. Although no effects of prenatal testosterone were evident in the control of UI secretion in adult androgenized females, UI secretion in androgenized males was elevated throughout the nonbreeding season in 3 of 5 animals, indicating that exogenous testosterone may reduce seasonal increases in responsiveness to steroid inhibition. These data suggest that sex differences in reproductive neuroendocrine ftinction may contribute to sex differences in sexual activity in gonad-intact male and female sheep. However, such differences do not appear to be determined by androgens in utero.

The pubertal process with its multifaceted neuroendocrine control provides an excellent model for the study of the regulation of FSH heterogeneity. We tested the hypothesis that during the pubertal transition in the female lamb 1) an increase in both pituitary and circulating bioactive FSH concentrations occur and 2) that the increase in bioactivity is associated with a change in the distribution pattern of both pituitary and circulating FSH isoforms. Pituitary and serum immunoreactive (I), and bioactive (B, Sertoli cell bioassay) FSH concentrations were measured in six prepubertal lambs (18 +/- 1 weeks, 29.9 +/- 2.8 kg body weight; mean +/- SE) and compared to those of six others (24.2 +/- 2.2 weeks of age, 41.4 +/- 2.5 kg body weight) during the pubertal transition period. Puberty was synchronized by pulsatile iv administration of GnRH (2 ng/kg every 2 h for 24 h and then at hourly intervals for the next 12 h) in a manner mimicking the I-LH pulse patterns observed during the natural transition to adulthood. Blood samples were collected at 12-min intervals for 4 h from both groups of lambs; for the pubertal group this included the final 32-36 h of GnRH administration. At the end of the study, a 25 ml volume of peripheral blood was collected from both prepubertal and pubertal females for the determination of serum FSH distribution patterns; the lambs were then euthanised, and pituitaries were removed for determination of pituitary hormone content and FSH isoform distribution patterns. In addition, the distribution pattern of I-FSH isoforms in the pituitary and serum from both groups of lambs were compared. The pubertal stages of all lambs were verified by measuring the size of follicles, the circulating concentrations of estradiol (E2) and inhibin, and the I-LH pulse patterns. Prepubertal lambs had low frequency I-LH pulses, small (2-3 mm) size ovarian follicles and low circulating concentrations of E2 (4.1 +/- 0.4 pg/ml) and inhibin (38.0 +/- 2.9 U/ml WHO). By contrast, all the pubertal lambs had hourly I-LH pulse frequency (induced with exogenous GnRH), a large (5-6 mm) follicle (in one lamb a 4-mm follicle), follicular phase levels of E2 (7.1 +/- 0.8 pg/ml), and higher concentrations of inhibin (53.2 +/- 3.1 U/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Using nutritionally restricted ovariectomized lambs, we tested the hypothesis that nutritionally regulated endogenous increases in GnRH secretion (as assessed by LH pulsatility) not only alter the quantity of FSLI present in the pituitary and serum, but also alter the pituitary and serum FSH isoform distribution. Eleven lambs were nutritionally restricted from weaning and ovariectomized at 12 wk of age. Beginning at 56 wk, 6 were fed ad libitum for 14 days, and the other 5 were continued on the restricted diet. Jugular blood samples were collected frequently (12-mm interval) for 4 h prior to pituitary removal. Immunoreactive ovine LH (l-0LH) and immunoreactive ovine FSH (l-0FSH) concentrations were measured in sees and pituitary extracts. Bioactive (B) 0FSH and I-oFSH isoform distribution patterns were determined in serum pools and pituitary extracts. Ad libitum feeding increased 1-0th pulsatiity and mean concentrations of pituitary and serum I.oFSH and B-oFSH. The I-oFSH isoform distribution patterns in the pituitaries from the nutritionally restricted animals were not different from those of repleted lambs; in both, the predominant FSFI peak eluted in the pH range of 3.5-5.6. A similar predominance of l-0FSH isoforms was also evident in the serum of ad libiwm.fed animals. This predominance was not demonstrable in 3 of the resthcted-fed animals due to low circulating concentrations of FSH (< 2.5 ng/ml). Subsequent studies, utilizing serum from 4 additional restricted-fed lambs with circulating I-oFSH concentrations in the range of 4-14 ng/ml (but no detectable LH pulses) revealed similar predominance of 0FSH isoforms in the pH 3.5-5.6 range. The ESH isoforms eluting in different pH ranges differed in their Immunological and biological activities. In summary, our results demonstrate that nutritionally regulated changes in endogenous GnRH secretion do not alter the distribution patterns of FSH isoforms in the pituitary or circulation of restricted- and ad libitum-fed ovariectomized lambs.

The sheep exhibits a marked sex difference in the timing of the pubertal increase in luteinizing hormone (LH). Male lambs undergo a reduction in sensitivity to inhibitory steroid feedback, leading to an increase in LH by 10 weeks of age, but females remain hypersensitive until 30 weeks of age. Endogenous opioids suppress LH secretion in the female lamb prepubertally and in adult male and female sheep. It has been suggested that a reduction in opioid inhibition of LH secretion is the signal to time puberty. Therefore, if a decrease in opioid tone occurs during sexual maturation, it should begin earlier in the male lamb than in the female. The objective of this study was to compare opioid inhibition of LH secretion in male and female lambs in relation to the timing of puberty. Our approach was to examine the response to the opioid antagonist naloxone at various ages in both sexes. To determine the timing of the pubertal LH rise in the presence of constant inhibitory steroid feedback, male and female lambs (n = 5 each) were gonadectomized at 3 weeks of age and implanted with a Silastic capsule of estradiol. They were then challenged with naloxone at 5, 11, and 23 weeks of age; blood samples were collected every 12 min for 8 hours, and lambs received naloxone (1 mg/kg i.v.) at hours 4, 5, 6, and 7. Mean LH before and during naloxone treatment was compared at each age. During the 4-hour pretreatment period, no LH pulses were evident in prepubertal females at 5, 11, or 23 weeks of age, and mean LH remained low (0.8 ± 0.05 ng/ml). In prepubertal males, mean LH was 1.5 ± 0.2 ng/ml at 5 weeks of age and rose at puberty to 6.8 ± 0.3 ng/ml by 23 weeks of age. Despite this sex difference in the timing of the pubertal LH rise, neither male nor female lambs increased LH secretion in response to naloxone until 23 wseeks of age. At this time, mean LH in females increased by 3.2 ± 0.7 ng/ml over pretreatment values in response to naloxone and increased by 5.0 ± 0.9 ng/ml in males. Thus, although opioid tone is expressed prepubertally in the female lamb, it is not evident until after puberty in the male. In a second experiment, we determined if increasing the concentration of estradiol in circulation affects the gonadotropin response to naloxone. LH pulse frequency during naloxone treatment did not differ in ovariectomized prepubertal females bearing 3-, 10-, or 30-mm implants of estradiol, although the larger implants of estradiol suppressed LH during the pretreatment period. These data suggest that increasing steroid negative feedback does not increase opioid tone, thereby reducing the gonadotropin response to a single dose of naloxone. Together, these studies provide evidence that in sheep the timing of the pubertal rise in LH secretion is not causally related to changes in opioid inhibition; rather, other factors including age or season may influence when endogenous opioids begin to serve a major role in modulating gonadotropin secretion in this species.

Puberty in the female lamb is accompanied by an increased frequency of LH pulses, and during normal development this is preceded by a decline in GH. Conversely, in the growth-retarded lamb, when LH levels are depressed by low nutrition, GH secretion is elevated. Based upon this inverse relationship, we tested the hypothesis that GH may act as a metabolic signal from the brain to inhibit the secretion of LH, and that the decline in GH times puberty. Our approach was to extend high circulating GH levels far beyond the early postnatal period, in a physiological pattern and level, in an attempt to block the pubertal LH rise. To evaluate the pattern of LH as a continuous variable under conditions of constant estradiol negative feedback, the gonadotropin was measured in blood samples collected by jugular venipuncture twice weekly; the lambs were ovariectomized and treated chronically with estradiol (Silastic capsule) beginning at 3 weeks of age. Nine lambs served as untreated controls, and 7 were infused iv with pituitary-derived bovine GH (bGH) between 5 and 28 weeks of age. A programmable backpack infusion pump delivered bGH as hourly pulses, with a total dose of 18 micrograms/kg.24 h, to maintain a physiological pattern and level of GH. At various ages, blood samples were collected at 12-min intervals for 6 h to monitor patterns and levels of peripheral LH and GH. Circulating GH in untreated and treated lambs averaged 7.7 +/- 1.5 ng/ml over a 6-h period at 4 weeks of age and declined to 1.1 +/- 0.2 ng/ml by 19 weeks in the untreated lambs; in contrast, bGH-infused lambs averaged 10.4 +/- 0.9 ng/ml at 19 weeks. Although body weights did not differ, back fat depth and quantity of perirenal fat were reduced in bGH-treated females compared to that in controls. Moreover, insulin-like growth factor-I levels were higher in bGH-treated compared with control lambs, and the bGH-treated lambs exhibited glucose intolerance, thus confirming that infused bGH was biologically active. Neuroendocrine sexual maturity, however, was not different in bGH-treated and control lambs, and it occurred at 21-22 weeks of age. The results do not support our hypothesis that decreasing GH secretion is a requirement for puberty in the sheep. Moreover, unlike in children with delayed puberty, exogenous bGH did not advance normal puberty in the lamb.(ABSTRACT TRUNCATED AT 400 WORDS)

This report provides evidence that an increment in serum gonadotropin levels occurs at puberty in the sheep and that this reflects the critical hormonal event culminating in first ovulation in this species. Blood samples were collected from 6 female lambs at 4-h intervals for a period of approximately 2 mo around the expected time of puberty (32 wk of age) until behavioral estrus was observed and ovulation was verified by assay of serum progesterone. Patterns of circulating LH, FSH, progesterone, and estradiol concentrations were characterized during the peripubertal period for each lamb. A rise in serum levels of both LH and FSH began approximately 7-10 days before the first preovulatory surge of gonadotropins. Although the increase in gonadotropin levels occurred gradually over several days, serum estradiol levels rose only during the final 40-60 h prior to the preovulatory surge of gonadotropin. Serum progesterone profiles revealed, however, that normal (14-16-day) luteal phases were induced in only 2 of 6 females as a result of the first surge. In four lambs, a short luteal phase of 2.5 days' duration occurred, which was followed by another estradiol rise and a preovulatory surge that then resulted in a full luteal phase of 14 days' duration. These data demonstrate clearly that the precipitating event at puberty in the female sheep is an increase in circulating gonadotropin levels and that the estradiol secreted from the newly stimulated follicle provides the signal for the first preovulatory surge.

In spring-born female lambs, the long days of summer, followed by their gradual decrease, provide the seasonal cue necessary to time puberty to early autumn (approximately 30 wk of age). Male lambs begin spermatogenesis during mid-summer, some 20 wk before puberty occurs in females. Unlike young female lambs, male lambs attain puberty at the same age under a variety of photoperiodic manipulations, raising the possibility that sexual maturation in males is not affected by photoperiod. We have reinvestigated the role of photoperiod on puberty in the male lamb, using a more precise indicator of reproductive activation--the decreased sensitivity of the hypothalamo-pituitary axis to inhibitory steroid feedback leading to increased LH secretion. To test whether photoperiod can influence the onset of neuroendocrine sexual maturation in male lambs, this study compared the timing of the decrease in sensitivity to inhibitory steroid feedback in two groups of males under opposite photoperiodic conditions. Eight males were reared indoors from 2 wk of age under conditions simulating the natural increasing and decreasing day lengths around the summer solstice; an additional 7 males were exposed to a reversed simulated natural photoperiod in which the changes in day length were amplified and accelerated relative to outdoor conditions. Both groups of lambs were castrated and received s.c. implants of Silastic estradiol capsules to provide a constant steroid feedback signal. The timing of reduction in sensitivity to estradiol negative feedback, measured as a sustained increase in circulating of LH above 1.0 ng/ml, was used to define neuroendocrine sexual maturity.(ABSTRACT TRUNCATED AT 250 WORDS)