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Normal reproductive function requires cyclical follicular and uterine epithelial changes to achieve the release of a viable oocyte and preparation of an adequate environment for the development of the conceptus. These changes are orchestrated by the hypothalamus–pituitary–ovarian (HPO) axis through the integration of various stimulatory and inhibitory signals. Integration occurs through feed-forward, feedback and paracrine loops that control and coordinate the occurrence of several reproductive events in a timely manner. The different components and hormonal factors that regulate the HPO axis along with recent research advances are discussed in this chapter.

Puberty in female mammals is a complex series of developmental events culminating in activation of neuroendocrine centers controlling the secretion of gonadotropin-releasing hormone, onset of high-frequency pulses of luteinizing hormone, and initiation of normal-length ovulatory cycles. These events are controlled by genetics, epigenetics, and nutrition. Nutritional effects are operative prenatally, postnatally, and during juvenile development. Timing of the onset of puberty can be markedly delayed or accelerated by nutrition. The implications of the nutritional modulation of pubertal onset are far-reaching and include not only reproductive efficiency but also health and well-being of adults throughout their lifetime.

The susceptibility of the reproductive system to early exposure to steroid hormones has become a major concern in our modern societies. Human fetuses are at risk of abnormal programming via exposure to endocrine disrupting chemicals, inadvertent use of contraceptive pills during pregnancy, as well as from excess exposure to steroids due to disease states. Animal models provide an unparalleled resource to understand the developmental origin of diseases. In female sheep, prenatal exposure to testosterone excess results in an array of adult reproductive disorders that recapitulate those seen in women with polycystic ovary syndrome (PCOS), including disrupted neuroendocrine feedback mechanisms, increased pituitary sensitivity to gonadotropin-releasing hormone, luteinizing hormone excess, functional hyperandrogenism, and multifollicular ovarian morphology culminating in early reproductive failure. Prenatal testosterone treatment also leads to fetal growth retardation, insulin resistance, and hypertension. Mounting evidence suggests that developmental exposure to an improper steroidal/metabolic environment may mediate the programming of adult disorders in prenatal testosterone-treated females, and these defects are maintained or amplified by the postnatal sex steroid and metabolic milieu. This review addresses the steroidal and metabolic contributions to the development and maintenance of the PCOS phenotype in the prenatal testosterone-treated sheep model, including the effects of prenatal and postnatal treatment with an androgen antagonist or insulin sensitizer as potential strategies to prevent/ameliorate these dysfunctions. Insights obtained from these intervention strategies on the mechanisms underlying these defects are likely to have translational relevance to human PCOS.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone produced by gonadotropes in the anterior pituitary that plays a central role in controlling ovarian folliculogenesis and steroidogenesis in females. Moreover, recent studies strongly suggest that FSH exerts extragonadal actions, particularly regulating bone mass and adiposity. Despite its crucial role, the mechanisms regulating FSH secretion are not completely understood. It is evident that hypothalamic, ovarian, and pituitary factors are involved in the neuroendocrine, paracrine, and autocrine regulation of FSH production. Large animal models, such as the female sheep, represent valuable research models to investigate specific aspects of FSH secretory processes. This review: (i) summarizes the role of FSH controlling reproduction and other biological processes; (ii) discusses the hypothalamic, gonadal, and pituitary regulation of FSH secretion; (iii) considers the biological relevance of the different FSH isoforms; and (iv) summarizes the distinct patterns of FSH secretion under different physiological conditions.

Feeding a high-concentrate diet to heifers during the juvenile period, resulting in increased body weight (BW) gain and adiposity, leads to early-onset puberty. In this study, we tested the hypothesis that the increase in GnRH/LH release during nutritional acceleration of puberty is accompanied by reciprocal changes in circulating leptin and central release of neuropeptide Y (NPY). The heifers were weaned at 3.5 months of age and fed to gain either 0.5 (Low-gain; LG) or 1.0 kg/day (High-gain; HG) for 30 weeks. A subgroup of heifers was fitted surgically with third ventricle guide cannulas and was subjected to intensive cerebrospinal fluid (CSF) and blood sampling at 8 and 9 months of age. Mean BW was greater in HG than in LG heifers at week 6 of the experiment and remained greater thereafter. Starting at 9 months of age, the percentage of pubertal HG heifers was greater than that of LG heifers, although a replicate effect was observed. During the 6-h period in which CSF and blood were collected simultaneously, all LH pulses coincided with or shortly followed a GnRH pulse. At 8 months of age, the frequency of LH pulses was greater in the HG than in the LG group. Beginning at 6 months of age, concentrations of leptin were greater in HG than in LG heifers. At 9 months of age, concentrations of NPY in the CSF were lesser in HG heifers. These observations indicate that increased BW gain during juvenile development accelerates puberty in heifers, coincident with reciprocal changes in circulating concentrations of leptin and hypothalamic NPY release.

Nutrition during the juvenile period has a major impact on timing reproductive maturity in heifers. Restricted growth delays puberty, whereas elevated BW gain advances the onset of puberty. The initiation of high-frequency episodic release of GnRH and, consequently, LH during the peripubertal period is crucial for maturation of the reproductive axis and establishment of normal estrous cycles. Nutritional signals are perceived by metabolic-sensing cells in the hypothalamus, which interact with estradiol-receptive neurons to regulate the secretory activity of GnRH neurons. The orexigenic peptide, neuropeptide Y (NPY), and the anorexigenic peptide derived from the proopiomelanocortin (POMC) gene, melanocyte-stimulating hormone α (αMSH), are believed to be major afferent pathways that transmit inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. The neuropeptide kisspeptin is considered a major stimulator of GnRH secretion and has been shown to mediate estradiol's effect on GnRH neuronal activity. Kisspeptin may also integrate the neuronal pathways mediating the metabolic and gonadal steroid hormone control of gonadotropin secretion. Recent studies in our laboratories indicate that functional and structural changes in the pathways involving NPY, POMC, and kisspeptin neurons occur in response to high rates of BW gain during the juvenile period in heifers. Changes include regulation of expression in NPY, POMC, and KISS1 and plasticity in the neuronal projections to GnRH neurons and within the neuronal network comprising these cells. Moreover, an intricate pattern of differential gene expression in the arcuate nucleus of the hypothalamus occurs in response to feeding high concentrate diets that promote elevated BW gain. Genes involved include those controlling feeding intake and cell metabolism, neuronal growth and remodeling, and synaptic transmission. Characterizing the cellular pathways and molecular networks involved in the mechanisms that control the timing of pubertal onset will assist in improving existing strategies and facilitate the development of novel approaches to program puberty in heifers. These include the use of diets that elevate BW gain during strategic periods of prepubertal development.

It was hypothesized that metabolic programming of processes underlying puberty can be shifted temporally through the use of a stair-step compensatory growth model such that puberty is optimally timed to occur at 11 to 12 mo of age. Forty crossbred beef heifers were weaned at approximately 3.5 mo of age and, after a 2-wk acclimation period, were assigned randomly to 1 of 4 nutritional groups: 1) low control (LC), restricted feed intake of a forage-based diet to promote BW gain of 0.5 kg/d until 14 mo of age, 2) high control (HC), controlled feed intake of a high-concentrate diet to promote BW gain of 1 kg/d until 14 mo of age, 3) stair-step 1 (SS-1), ad libitum feed intake of a high-concentrate diet until 6.5 mo of age followed by restricted access to a high-forage diet to promote BW gain of 0.35 kg/d until 9 mo of age, ad libitum feed intake of a high-concentrate diet until 11.5 mo of age, and restricted intake of a high-forage diet to promote BW gain of 0.35 kg/d until 14 mo of age, and 4) stair-step 2 (SS-2), reverse sequence of SS-1, beginning with restricted access to a high-forage diet. Body weight (every 2 wk) and circulating concentrations of leptin (monthly) were determined throughout the experiment. Concentrations of progesterone in blood samples collected twice weekly beginning at 8 mo of age were used to determine pubertal status. Body weight gain followed a pattern similar to that proposed in our experimental design. Circulating concentrations of leptin increased following distinct elevations in BW but decreased abruptly after feed intake restriction. Survival analysis indicated that the percentage of pubertal heifers in the LC group was lower (P < 0.05) than all other groups throughout the experiment. Although heifers in SS-1 were nutritionally restricted between 6.5 and 9 mo of age, the proportion pubertal by 12 mo of age did not differ (P = 0.36) from that of the HC group, with 80% and 70% pubertal in SS-1 and HC, respectively. In contrast, the proportion of heifers pubertal by 12 mo of age in the SS-2 group (40%) was lower (P < 0.05) than both HC and SS-1. However, by 14 mo of age, 90% of heifers in the SS-2 group had also attained puberty compared to only 40% of the LC group. In summary, these data provide evidence that changes in the nutritional and metabolic status during the early juvenile period can program the onset of puberty that occurs months later, allowing optimal timing of sexual maturation in replacement beef heifers.

Onset of the winter anovulatory period in mares is associated with a marked diminution in adenohypophyseal synthesis and release of LH. Native GnRH, unlike its synthetic agonists, stimulates the synthesis and secretion of LH in mares without pituitary refractoriness. Herein we tested the hypotheses that (1) the average Julian day of pregnancy can be accelerated by up to 2 months in winter anovulatory mares treated continuously with native GnRH beginning on February 1 and (2) mares will sustain luteal function and pregnancy after treatment withdrawal. Forty-two winter anovulatory mares were stratified by age, body condition score, and size of the largest follicle across two locations in a randomized design and assigned to one of three groups (n = 14 per group): (1) CONTROL: untreated, (2) GnRH-14: GnRH delivered subcutaneously in saline at a rate of 100 μg/h for 8 weeks (February 1-March 29) using four consecutive 14-day pumps (Alzet 2ML2), or (3) GnRH-28: GnRH delivered as in (2), but using two 28-day pumps (Alzet 2ML4). On development of a 35-mm follicle and expression of estrus, mares were bred the following day and treated with hCG. Pregnancies were confirmed using transrectal ultrasonography on Days 14, 24, 33, and 45, with blood samples collected to assess luteal function. Mares treated with GnRH (GnRH-14 and GnRH-28) did not differ reproductively in their responses and data were pooled for statistical comparisons. Mares treated with GnRH exhibited marked increases (P ≤ 0.04) in the frequency of development of a 35-mm follicle, submission rate for live cover and/or artificial insemination, ovulation, and pregnancy compared with control mares on treatment Day 56 (March 29). Interval to the first 35-mm follicle was 51.8 ± 4.9 and 19.3 ± 3.5 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively. Interval to pregnancy was 65.3 ± 6.7 and 28.6 ± 4.8 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively, excluding one GnRH-14 mare that failed to become pregnant over four cycles. By the end of the treatment period (March 29), only 21% of control mares were pregnant compared with 79% of GnRH-treated mares. Furthermore, mean serum concentrations of progesterone were similar to (GnRH-28; P = 0.26) or greater than (GnRH-14; P = 0.01) that of control mares from Day 0 to 46 postbreeding. Data illustrate that continuous administration of native GnRH is a highly efficient option for managing seasonal anovulation in mares and could be effectively used in the breeding industry if a user-friendly delivery option were available.

High rates of body weight gain during the juvenile period appear to program molecular events within the hypothalamus, leading to advancement of puberty. Methylation of DNA, an epigenetic mechanism that controls gene expression, is associated with metabolic programming events and is proposed to play a role in the pubertal process. In this study, DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus (ARC) of juvenile heifers fed to gain body weight at low (0.5 kg/d; low-gain, LG, n = 4) or high (1 kg/d; high-gain, HG, n = 4) rates from 4.5 to 8.5 mo of age (earliest puberty expected at 9 mo of age in HG heifers). Using a custom-designed oligonucleotide array targeted to imprinted genes and genes associated with nutritional inputs and the control of puberty, a comparative-genomic-hybridization array was used to identify differentially methylated regions between LG and HG heifers. Differential methylation of genomic regions associated with altered mRNA expression was observed for genes whose activity has been reported to be involved in the modulation of growth and metabolism (GHR) and puberty (HMGA2). Hence, increased rates of body weight gain during the juvenile period alter the methylation pattern of genomic DNA obtained from the ARC and these changes may be involved in programming the age at puberty in heifers.

High rates of body weight gain during the juvenile period appear to program molecular events within the hypothalamus, leading to advancement of puberty. Methylation of DNA, an epigenetic mechanism that controls gene expression, is associated with metabolic programming events and is proposed to play a role in the pubertal process. In this study, DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus (ARC) of juvenile heifers fed to gain body weight at low (0.5 kg/d; low-gain, LG, n = 4) or high (1 kg/d; high-gain, HG, n = 4) rates from 4.5 to 8.5 mo of age (earliest puberty expected at 9 mo of age in HG heifers). Using a custom-designed oligonucleotide array targeted to imprinted genes and genes associated with nutritional inputs and the control of puberty, a comparative-genomic-hybridization array was used to identify differentially methylated regions between LG and HG heifers. Differential methylation of genomic regions associated with altered mRNA expression was observed for genes whose activity has been reported to be involved in the modulation of growth and metabolism (GHR) and puberty (HMGA2). Hence, increased rates of body weight gain during the juvenile period alter the methylation pattern of genomic DNA obtained from the ARC and these changes may be involved in programming the age at puberty in heifers.

Women with polycystic ovary syndrome often manifest insulin resistance. Using a sheep model of polycystic ovary syndrome-like phenotype, we explored the contribution of androgen and insulin in programming and maintaining disruptions in insulin signaling in metabolic tissues. Phosphorylation of AKT, ERK, GSK3beta, mTOR, and p70S6K was examined in the liver, muscle, and adipose tissue of control and prenatal testosterone (T)-, prenatal T plus androgen antagonist (flutamide)-, and prenatal T plus insulin sensitizer (rosiglitazone)-treated fetuses as well as 2-yr-old females. Insulin-stimulated phospho (p)-AKT was evaluated in control and prenatal T-, prenatal T plus postnatal flutamide-, and prenatal T plus postnatal rosiglitazone-treated females at 3 yr of age. GLUT4 expression was evaluated in the muscle at all time points. Prenatal T treatment increased mTOR, p-p70S6K, and p-GSK3beta levels in the fetal liver with both androgen antagonist and insulin sensitizer preventing the mTOR increase. Both interventions had partial effect in preventing the increase in p-GSK3beta. In the fetal muscle, prenatal T excess decreased p-GSK3beta and GLUT4. The decrease in muscle p-GSK3beta was partially prevented by insulin sensitizer cotreatment. Both interventions partially prevented the decrease in GLUT4. Prenatal T treatment had no effect on basal expression of any of the markers in 2-yr-old females. At 3 yr of age, prenatal T treatment prevented the insulin-stimulated increase in p-AKT in liver and muscle, but not in adipose tissue, and neither postnatal intervention restored p-AKT response to insulin stimulation. Our findings provide evidence that prenatal T excess changes insulin sensitivity in a tissue- and development-specific manner and that both androgens and insulin may be involved in the programming of these metabolic disruptions.

Increased body weight (BW) gain during the juvenile period leads to early maturation of the reproductive neuroendocrine system. We investigated whether a nutritional regimen that advances the onset of puberty leads to alterations in the hypothalamic neuropeptide Y (NPY) circuitry that are permissive for enhanced gonadotropin-releasing hormone (GnRH) secretion. It was hypothesized that NPY mRNA and NPY projections to GnRH and kisspeptin neurons are reduced in heifers that gain BW at an accelerated rate, compared with a lower one, during the juvenile period. Heifers were weaned at approximately 4 mo of age and fed diets to promote relatively low (0.5 kg/day; low gain [LG]) or high (1.0 kg/day; high gain [HG]) rates of BW gain until 8.5 mo of age. Heifers that gained BW at a higher rate exhibited greater circulating concentrations of leptin and reduced overall NPY expression in the arcuate nucleus. The proportion of GnRH neurons in close apposition to NPY fibers and the magnitude of NPY projections to GnRH neurons located in the mediobasal hypothalamus were reduced in HG heifers. However, no differences in NPY projections to kisspeptin neurons in the arcuate nucleus were detected between HG and LG heifers. Results indicate that a reduction in NPY innervation of GnRH neurons, particularly at the level of the mediobasal hypothalamus, occurs in response to elevated BW gain during the juvenile period. This functional plasticity may facilitate early onset of puberty in heifers.

RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

Reproductive seasonality in the mare is characterized by a marked decline in adenohypophyseal synthesis and secretion of LH beginning near the autumnal equinox. Thus, ovarian cycles have ceased in most mares by the time of the winter solstice. Endogenous reproductive rhythms in seasonal species are entrained or synchronized as a result of periodic environmental cues. In the horse, this cue is primarily day length. Hence, supplemental lighting schemes have been used managerially for decades to modify the annual timing of reproduction in the mare. Although a full characterization of the cellular and molecular bases of seasonal rhythms has not been realized in any species, many of their synaptic and humoral signaling pathways have been defined. In the mare, neuroendocrine-related studies have focused primarily on the roles of GnRH and interneuronal signaling pathways that subserve the GnRH system in the regulatory cascade. Recent studies have considered the role of a newly discovered neuropeptide, RF-related peptide 3 that could function to inhibit GnRH secretion or gonadotrope responsiveness. Although results that used native peptide sequences have been negative in the mare and mixed in all mammalian females, new studies that used an RFRP3 antagonist (RF9) in sheep are encouraging. Importantly, despite continuing deficits in some fundamental areas, the knowledge required to control seasonal anovulation pharmacologically has been available for >20 yr. Specifically, the continuous infusion of native GnRH is both reliable and efficient for accelerating reproductive transition and is uniquely applicable to the horse. However, its practical exploitation continues to await the development of a commercially acceptable delivery vehicle.

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The pubertal process is characterized by an activation of physiological events within the hypothalamic-adenohypophyseal-gonadal axis which culminate in reproductive competence. Excessive weight gain and adiposity during the juvenile period is associated with accelerated onset of puberty in females. The mechanisms and pathways by which excess energy balance advances puberty are unclear, but appear to involve an early escape from estradiol negative feedback and early initiation of high frequency episodic gonadotropin-releasing hormone (GnRH) secretion. Hypothalamic neurons, particularly neuropeptide Y and proopiomelanocortin neurons are likely important components of the pathway sensing and transmitting metabolic information to the control of GnRH secretion. Kisspeptin neurons may also have a role as effector neurons integrating metabolic and gonadal steroid feedback effects on GnRH secretion at the time of puberty. Recent studies indicate that leptin-responsive neurons within the ventral premammillary nucleus play a critical role in pubertal progression and challenge the relevance of kisspeptin neurons in this process. Nevertheless, the nutritional control of puberty is likely to involve an integration of major sensor and effector pathways that interact with modulatory circuitries for a fine control of GnRH neuron function. In this review, observations made in ruminant species are emphasized for a comparative perspective.