Transcriptome Analysis of Neuroendocrine Regulation of Ovine Hypothalamus-Pituitary-Ovary Axis during Ovine Anestrus and the Breeding Season.

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Nutritional status affects the microRNA profile of the hypothalamus of female sheep.

Recent studies on the seasonal regulation of the oestrous cycle in sheep have focussed mainly on the responses to photoperiod. However, the brain systems that control reproductive activity also respond to nutritional inputs, although the molecular mechanisms involved are not completely understood. One possibility is that small, non-coding RNAs, such as micro-RNAs (miRNAs), have significant influence. In the present study, the amounts and characteristics of miRNAs in hypothalamus from oestrous and anestrous ewes, fed low- or high-nutrient diets, were compared using Illumina HiSeq sequencing technology. In total, 398 miRNAs, including 261 novel miRNAs, were identified in ewes with an enhanced nutritional status (HEN), whereas 384 miRNAs, including 247 novel miRNAs, were identified in the ewes with a lesser nutritional status (HAN). There were eight conserved and 140 novel miRNAs expressed differentially between the two libraries. Based on quantitative real-time polymerase chain reaction, six miRNAs were assessed to verify the accuracy of the library database. Moreover, the correlation between the miRNA target and several upstream and downstream genes in the oestrus-related pathways were also verified in hypothalamus nerve cells. According to the results, nutritional status plays an important role in oestrous regulation in sheep, and the hypothalamic processes and pathways induced by nutritional signals (folic acid and tyrosine) are different from those induced by photoperiodic regulation of oestrus. We have expanded the repertoire of sheep miRNAs that could contribute to the molecular mechanisms that regulate the initiation of oestrous cycles in anestrous ewes in response to the influence of nutritional status.

Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase.

Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague-Dawley rats were further analyzed by real-time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor-associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co-localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression.

Prioritization of candidate genes for cattle reproductive traits, based on protein-protein interactions, gene expression, and text-mining.

Reproduction is of significant economic importance in dairy cattle. Improved understanding of mechanisms that control estrous behavior and other reproduction traits could help in developing strategies to improve and/or monitor these traits. The objective of this study was to predict and rank genes and processes in brain areas and pituitary involved in reproductive traits in cattle using information derived from three different data sources: gene expression, protein-protein interactions, and literature. We identified 59, 89, 53, 23, and 71 genes in bovine amygdala, dorsal hypothalamus, hippocampus, pituitary, and ventral hypothalamus, respectively, potentially involved in processes underlying estrus and estrous behavior. Functional annotation of the candidate genes points to a number of tissue-specific processes of which the "neurotransmitter/ion channel/synapse" process in the amygdala, "steroid hormone receptor activity/ion binding" in the pituitary, "extracellular region" in the ventral hypothalamus, and "positive regulation of transcription/metabolic process" in the dorsal hypothalamus are most prominent. The regulation of the functional processes in the various tissues operate at different biological levels, including transcriptional, posttranscriptional, extracellular, and intercellular signaling levels.

Deletion of Otx2 in GnRH neurons results in a mouse model of hypogonadotropic hypogonadism.

GnRH is the central regulator of reproductive function responding to central nervous system cues to control gonadotropin synthesis and secretion. GnRH neurons originate in the olfactory placode and migrate to the forebrain, in which they are found in a scattered distribution. Congenital idiopathic hypogonadotropic hypogonadism (CIHH) has been associated with mutations or deletions in a number of genes that participate in the development of GnRH neurons and expression of GnRH. Despite the critical role of GnRH in mammalian reproduction, a comprehensive understanding of the developmental factors that are responsible for regulating the establishment of mature GnRH neurons and the expression of GnRH is lacking. orthodenticle homeobox 2 (OTX2), a homeodomain protein required for the formation of the forebrain, has been shown to be expressed in GnRH neurons, up-regulated during GnRH neuronal development, and responsible for increased GnRH promoter activity in GnRH neuronal cell lines. Interestingly, mutations in Otx2 have been associated with human hypogonadotropic hypogonadism, but the mechanism by which Otx2 mutations cause CIHH is unknown. Here we show that deletion of Otx2 in GnRH neurons results in a significant decrease in GnRH neurons in the hypothalamus, a delay in pubertal onset, abnormal estrous cyclicity, and infertility. Taken together, these data provide in vivo evidence that Otx2 is critical for GnRH expression and reproductive competence.

Gene expression profiles of intracellular and membrane progesterone receptor isoforms in the mediobasal hypothalamus during pro-oestrus.

Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRalpha, mPRbeta and mPRgamma) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRgamma. The mPRalpha mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRbeta mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRgamma and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRalpha and mPRbeta in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.

Suppression subtractive hybridization and microarray identification of estrogen-regulated hypothalamic genes.

The gonadal steroid estrogen is a pleiotropic hormone that has multiple effects on numerous cellular functions. One of estrogen's major targets is the brain, where the steroid not only affects growth, differentiation, and survival of neurons, but also regulates cell excitability. Because estrogen modulates multiple, overlapping signaling pathways, it has been difficult to scrutinize the transcriptional activity of the steroid. Therefore, we still lack a global picture of how different genes interact and are regulated by estrogen. Herein we report the use of suppression subtractive hybridization followed by custom microarray analysis of thousands of genes that are differentially expressed during the negative feedback phase of the female reproductive cycle. We have found a number of key transcripts that are regulated by estrogen and contribute to the alteration in synaptic transmission and hence excitability of hypothalamic neurons (e.g., GABA neurons). These include gec-1, GABA(B)R2, PI3 kinase subunit p55gamma, and a number of proteins containing pleckstrin homology domains that are critical for plasma membrane targeting. Studies are underway to refine our analysis to individual nuclei and individual cells. However, what has emerged from this highly sensitive microarray analysis is that estrogen affects neuronal plasticity in hypothalamic neurons not only by transcription of new membrane proteins (e.g., receptors and channels), but also by altering expression of downstream signaling molecules and proteins involved in neurosecretory pathways.

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line.

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

Site-specific decrease of progesterone receptor mRNA expression in the hypothalamus of middle-aged persistently estrus rats.

Middle-aged females gradually become acyclic and spontaneously develop a persistently estrus (PE) state. PE rats, acyclic for 30 days (early PE), are unresponsive to the positive feedback action of estrogen, but respond to a progesterone challenge with a luteinizing hormone (LH) surge and ovulation; unlike long-term PE rats, acyclic for 90 days, neither estrogen nor estrogen plus progesterone will elicit an LH surge [10th International Congress of Endocrinology, San Francisco, P3 (1996) 1061]. We hypothesize that the PE state may develop due to a diminished level of estrogen-induced progesterone receptor (PR) expression in the hypothalamus that prevents progesterone from stimulating LH regulating circuits. To test this hypothesis, PR mRNA levels were measured in hypothalamic regions of young, proestrus (2-3 months of age), early PE (10-12 months) and long-term PE (13-15 months) rats. The anteroventral periventricular nucleus (AVPV), an important regulatory site of the LH surge, had decreased PR mRNA levels in early and long-term PE rats compared with proestrus rats. However, PR mRNA levels were reduced only in long-term PE rats in the ventromedial nucleus (VMH) and arcuate nucleus (ARH). In the medial preoptic nucleus (MPN), levels of PR mRNA did not change. A previous report showed that exogenous progesterone stimulates an LH surge in young and early PE animals, indicating that the expression of PR mRNA demonstrated in this study is sufficient to mediate progesterone facilitation of the LH surge in early PE rats. In acyclic, long-term PE rats, diminished estrogen-induced expression of progesterone receptors is correlated with a previously shown inability to respond to exogenous progesterone.

Regulation of hypothalamic neuropeptide Y Y1 receptor gene expression during the estrous cycle: role of progesterone receptors.

Neuropeptide Y (NPY) stimulates the release of GnRH in an estrogen (E2)-dependent manner, which is important in generating preovulatory GnRH surges. We tested the hypothesis that E2 up-regulates NPY's actions by stimulating NPY Y1 receptor (Y1r) gene expression through a mechanism mediated by E2's ability to induce progesterone (P) receptors (PRs). In initial experiments, a specific Y1r antagonist BIBP3226 was used to confirm the involvement of Y1r in the stimulatory effects of NPY on in vivo GnRH release. Hypothalamic Y1r messenger RNA (mRNA) levels were then measured using competitive RT-PCR and were found to be significantly increased at 1000, 1200, and 1400 h on proestrus compared with other times of the day or cycle stage. Ovariectomy eliminated these increases, and E2 treatment restored them. Additional P treatment produced even larger increases in Y1r mRNA levels. To assess the role of PRs in stimulating Y1r expression, proestrous rats were treated with PR antagonist or oil vehicle and killed at 1200 h. Treatment with PR antagonist completely blocked the proestrous rise in Y1r gene expression. In parallel experiments, the same in vivo PR antagonist treatments also blocked NPY stimulation of GnRH release in vitro. Together our findings reveal that 1) Y1r mRNA levels are increased during the late morning and afternoon of proestrus; 2) Y1r mRNA levels are similarly increased by E2, and to an even greater extent by additional P; and 3) PR antagonism blocks both increased Y1r mRNA and induction of GnRH responsiveness to NPY. These observations support the idea that E2 up-regulates GnRH neuronal responses to NPY through stimulation of Y1r gene expression, and that E2's actions are mediated by the induction and subsequent activation of PRs.

Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.

Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.

Gonadal steroid hormones and hypothalamic opioid circuitry.

Endogenous opioid peptides derived from several gene families are localized within hypothalamic regions known to be involved in the regulation of reproduction. For example, the proenkephalin gene products, met- and leu-enkephalin, and the proopiomelanocortin (POMC) gene product, beta-endorphin, are found in the rat medial preoptic area (MPOA). Moreover, the expression of these peptides and their receptors varies across the estrous cycle in the female rat. We have examined the gonadal steroid regulation of mu-opiate receptors and opioid peptides in the MPOA, and POMC mRNA expression in neurons that innervate the MPOA. mu-Opiate receptors in the MPOA are sexually dimorphic and gonadal steroid hormone-dependent. Hormonal priming of ovariectomized rats with estrogen and progesterone (P) upregulates MPOA mu-receptors 27, but not 3, hr after P treatment. Inhibition of protein synthesis during the first 6 hr after P prevents receptor upregulation. The density of beta-endorphin fibers in the MPOA also increases following hormone treatment, and POMC mRNA expression in neurons that innervate the MPOA is induced by hormone treatment beginning 13 hr after P treatment. This delayed response might be ubiquitous among POMC neurons, as those innervating the median eminence also exhibit increased POMC mRNA expression along a similar time course. The results suggest that hormonal feedback regulates opioid peptides which act at mu-receptors in the MPOA to influence reproductive behavior and cyclicity. These opioid functions represent an important component in the complex regulatory processes which control reproduction.

Regulation of galanin and gonadotropin-releasing hormone gene expression in the hypothalamus and basal forebrain of the rat.

Galanin is a cotransmitter in GnRH neurons and is thought to play a role in the control of gonadotropin secretion. The aim of our research has been to learn how galanin mRNA is regulated in GnRH neurons with the goal of understanding galanin's physiological significance. We have used double-label in situ hybridization and computerized image analysis to identify GnRH neurons coexpressing galanin mRNA and to estimate cellular levels of galanin message in these cells under different physiological conditions in the rat. In adult females, levels of galanin mRNA in GnRH neurons increase two- to fourfold with the onset of the proestrous and steroid-induced LH surges. Pharmacological blockade of synaptic transmission with either a general anesthetic (pentobarbital) or an alpha-adrenergic receptor antagonist (phenoxybenzamine) inhibits both the steroid-induced LH surge and the associated induction of galanin expression in GnRH neurons. Compared with the day of diestrus of the estrous cycle, during lactation cellular levels of galanin mRNA in GnRH neurons are profoundly reduced. In contrast to galanin mRNA in GnRH neurons, we could adduce no evidence for changes in cellular levels of GnRH mRNA under any physiological conditions or with any pharmacological manipulations. We conclude that alterations in galanin gene expression play a fundamental role in governing the functional activity of GnRH neurons, possibly by acting presynaptically to shape GnRH pulses, thereby determining the biological efficacy of GnRH action at its target cells in the pituitary.