In the pituitary Fig. On diestrus 1 and 2, morning levels were higher than afternoon levels. In the ovary Fig. In the pituitary gland Fig. On proestrus, pituitary GnRH-R expression was low in the morning and rose back to diestrous 1 and 2 morning levels at noon, followed by significant transient drops at and h and a second peak at h. Relatively low levels of GnRH-R expression were measured on estrus in this tissue. The lowest expression levels occurred at h on estrus and noon on proestrus.
Reliable use of this technology requires that errors in quantification that might occur at various stages of the experimental procedure e. RNA extraction, RT, etc. The most common method employed to this end consists of normalizing the expression of the gene of interest by that of one, or preferably more, reference genes, whose expression level is invariant across the relevant experimental conditions. Although several tools to identify the most stably expressed genes in a set of experimental samples have been developed 35 , 36 , 38 , 39 , none is, as yet, widely accepted and used.
Therefore, we chose to apply three different approaches described in Materials and Methods to identify the two most suitable reference genes in each of the three tissues studied. We obtained extremely low intersample Ct variability for our candidate reference genes see sd values in Table 2 , which is within the reproducibility range of RT-PCRs This might be a somewhat rare situation, reflecting extremely small technical and biological errors.
Nonetheless, we believe that under such circumstances, measurements of variation in prenormalized DNA quantities provides the most direct means of assessing the stability of candidate reference genes. Therefore, we chose the two most suitable reference genes to be those with the lowest sd. We believe that these reference genes could be used in future studies employing similar experimental systems. When comparing the amounts of GnRH produced by the pituitary or ovary to those found in the hypothalamus, one should take into consideration the different modes of action that this peptide undertakes in the different tissues.
One might therefore expect lower levels of GnRH production in extrahypothalamic tissues compared with the hypothalamic levels. The present study is the first demonstration of a GnRH mRNA expression pattern in the pituitary and ovary throughout the estrous cycle of any species, whereas the pattern of hypothalamic GnRH expression has been studied previously 40 — Our findings corroborate with previous demonstrations of a peak in hypothalamic GnRH mRNA levels in the later afternoon-early evening hours of proestrus, slightly before the gonadotropin surge 42 — Similarly, peak levels of GnRH peptide have been found in the mediobasal hypothalamus 45 , 46 and in the portal blood 47 on the evening of proestrus.
Expression of the receptor for GnRH in the mediobasal hypothalamus was previously shown to be modulated during the rat estrous cycle 48 , such that peak levels were observed on the morning of proestrus and the evening of estrus. It is noteworthy that very few times points of the estrous cycle were sampled in that study. In our investigation, the entire hypothalamus was analyzed rather than specific nuclei, and only nonsignificant fluctuations in the expression of GnRH-R during the estrous cycle were observed.
It is possible that the GnRH-R are differentially modulated at various hypothalamic sublocations. The hypothalamic GnRH-R are probably involved in autoregulatory feedback mechanisms in this tissue. It is possible that the local pituitary GnRH plays a role in the regulation of LH production, because at this time, GnRH levels in the median eminence 50 and portal system 47 are still low.
As mentioned previously, a potential role for pituitary GnRH in the release of LH has been demonstrated in vitro Interestingly, there appears to be a temporal correlation between the expression pattern of GnRH and that of its receptor in the pituitary throughout the estrous cycle, except on estrus. It is possible that endogenous pituitary GnRH participates in the regulation of its receptor in this gland or that the two are coregulated.
Pituitary GnRH-R expression and content as well as GnRH binding to pituitary receptors during the rat estrous cycle have been investigated in the past, although results are somewhat contradictory 52 — Nevertheless, a heightened GnRH-binding capacity of the pituitary during the day or so before the preovulatory gonadotropin surge appears to be a result of increased receptor synthesis.
This increase, which might be generated by locally produced pituitary GnRH, is postulated to induce heightened pituitary responsiveness to hypothalamic GnRH stimulation In an earlier investigation 58 , it was reported that cyclical changes during the estrous cycle in the expression of the ovarian GnRH-R are specific to the stage of follicular development, such that they were observed only in corpora lutea and atretic follicles.
In these two types of follicles, peak GnRH-R levels were observed in the evening of proestrus, with a second increase in the morning of estrus observed only in atretic follicles. The researchers suggested that ovarian GnRH might be involved in follicular atresia and possibly also in the induction of ovulation. The fact that we detected peak GnRH expression concomitantly with peak GnRH receptor expression in the ovary is intriguing and raises the possibility that here too, GnRH regulates the expression of its own receptor or that the two are coregulated.
An interesting observation was recently published 59 , suggesting that oocytes of the gilthead sea bream produce and release gonadotropins, and that this release can be enhanced by a GnRH analog or reduced by a GnRH antagonist.
As the researchers point out, this discovery raises the interesting possibility of a local GnRH-gonadotropin axis within the fish ovary. One could thus envisage local GnRH-gonadotropin axes within the pituitary and ovaries of mammalian females. Such local regulatory axes could contribute to or finely tune the hypothalamic-pituitary-gonadal axis, for instance by priming the relevant organs in preparation for the preovulatory peak in the case of the pituitary and ovulation in the case of the ovaries.
Nonetheless, research in this direction has yet to be conducted. The earlier increase in GnRH production in the pituitary and ovary compared with the hypothalamus during the proestrous stage of the sexual cycle might indicate that this peptide participates in the preparation of these organs for the imminent preovulatory surge, possibly via local GnRH-gonadotropin axes.
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It is clear that regulation of both synthesis and the secretion of GnRH are effected by neurotransmitter systems in the brain. Thus, steroid regulation of GnRH synthesis and secretion can be direct, but the predominant effects are transmitted through steroid-responsive neuronal systems in various parts of the brain.
LH is a more accurate indicator of GnRH pulse characteristics i. The majority of our understanding of GnRH and GnRH-receptor GnRHR function is based on studies of a single isoform of each; however, recent studies have identified additional forms as described in subsequent sections.
Note also that human gonadotropin-releasing hormone should be abbreviated with all capitals i. As much of the available data have been obtained in non-human species, we have chosen to use the more common abbreviations. While the majority of neural cells arise from neurons within the developing nervous system itself, GnRH neurons are unusual in that they are derived from progenitor cells in the epithelium of the olfactory placode.
These nascent GnRH neurons migrate along the vomeronasal axons, across the cribiform plate and into the mediobasal hypothalamus where migration ceases and the neurons detach from their axonal guides. The identification of genes which direct normal GnRH migration and function is an active area of research. A long list of soluble factors have been identified which appear to be critical for the ultimate development of a network which contains the appropriate number and location of GnRH neurons.
Estimates of the number of GnRH neurons vary, but are in the range of a few thousand, a remarkably small number in view of their critical function. Most GnRH neurons send axonal projections to the median eminence which abuts the hypothalamic—pituitary portal system.
This system consists of capillaries that arise from the superior hypophyseal arteries, traverse the pituitary stalk, and then form a capillary network within the pituitary gland. This anatomic relationship allows minute quantities of GnRH secreted by these axonal terminals to have direct access to the pituitary gonadotropes. The primary direction of this hypophyseal portal system is from the hypothalamus to the pituitary; however, retrograde flow also exists and provides a short feedback loop from the pituitary back to the hypothalamus.
While these projections are not directly involved in the modulation of gonadotropin secretion, they may help to link hormonal status to reproductive behavior.
In an elegant series of experiments, Ernst Knobil and colleagues demonstrated that pulsatile GnRH is required to achieve sustained gonadotropin secretion.
Loss of the GnRH response with continuous treatment is now known to be due to rapid uncoupling of the GnRH receptor from its intracellular signaling molecules followed by downregulation of receptor number. This characteristic is exploited clinically by administration of long-acting GnRH agonists to treat steroid-dependent conditions such as endometriosis, leiomyomas, breast cancer, and prostate cancer.
GnRH neuronal activity varies across the lifespan as is reflected by changes in gonadotropin levels and, ultimately, gonadal steroid and gamete production.
In the human, GnRH is detectable in the hypothalamus by 10 weeks gestational age with FSH and LH produced by 10—13 weeks when the vascular connection between the hypothalamus and pituitary gland has developed.
Gonadotropin levels peak at mid-gestation and then decline towards term due to negative feedback at both the hypothalamus and pituitary by the high levels of placental steroids.
With the withdrawal of placental steroids at birth, gonadotropins rise and remain elevated for the first 1—2 years in girls and first 6 months in boys with a subsequent decrease for the remainder of childhood. GnRH is secreted at a higher frequency pulse rate during the first part of the menstrual cycle leading to a preferential secretion of LH and the LH surge which is noted on Day During the second half of the menstrual cycle, GnRH is secreted at a lower frequency pulse rate which causes a preferential secretion of FSH.
GnRH pulse characteristics change once again at menopause. There has been longstanding consensus that women are born with the full cohort of follicles that they will have during their lifetime. With depletion of follicular number, estrogen levels decrease with subsequent loss of negative feedback and resultant increases in GnRH secretion.
GnRH pulses occur approximately every 50—55 min in younger postmenopausal women which is comparable to a normally cycling woman in the late follicular phase and midcycle surge.
Estradiol likely acts at both the hypothalamus and pituitary gland to exert negative and positive feedback effects on GnRH secretion and gonadotropin release. For many years, it was believed that GnRH neurons lacked estrogen receptor expression, suggesting that all effects on these neurons were achieved via connecting interneurons. However, more recent studies have demonstrated that the estrogen receptor, ERb, is expressed by at least a subset of GnRH neurons.
There is also substantial evidence to support a direct negative effect by estrogens at the pituitary level. Estradiol was able to blunt GnRH-mediated increases in gonadotropin expression, despite a lack of potential feedback at the hypothalamus. Circulating estradiol levels are a reflection of the degree of ovarian follicular development.
Although low estradiol levels feedback negatively as just described, rapidly increasing estradiol levels exert a positive feedback effect and are responsible for generating the pre-ovulatory gonadotropin surge. In the normal physiological situation, estradiol likely acts via both the hypothalamus and pituitary to trigger the LH surge. Hypothalamic GnRH secretion is also increased at the time of the surge, as directly measured in sheep and rats; however, this change may not be a required.
It is possible to generate an estradiol-induced surge experimentally despite holding GnRH pulse frequency and amplitude constant in animals lacking endogenous GnRH activity. Thus, even though GnRH release normally increases at the time of the surge, this increase appears to be facilitory rather than essential for production of a surge.
Progesterone also decreases GnRH secretion at the level of the hypothalamus. It is controversial whether GnRH neurons express progesterone receptors and, therefore, interneurons expressing these receptors may be responsible for feedback effects. It has been clearly established that estrogen priming is necessary to observe a progestational effect, undoubtedly due to the marked ability of estrogen to upregulate progesterone receptor expression.
A number of neurotransmitters and neuropeptides are believed to act as intermediaries between circulating gonadal steroid levels and GnRH pulse secretion.
For example, estrogen promotes endorphin secretion with a further increase in the presence of progesterone. Endorphins, along with other opioids, suppress hypothalamic GnRH release. Thus, endorphin levels peak with the high steroid levels found in the mid-luteal phase, suggesting that opioid tone may act with progesterone to decrease GnRH pulse frequency in this phase relative to the follicular phase. NPY, norepinephrine and dopamine-secreting neurons are also likely to be important for modulation of GnRH neuronal activity.
In addition, it has been demonstrated that CRH inhibits hypothalamic GnRH secretion, both directly and by augmenting endogenous opioid secretion. Women with hypothalamic amenorrhea and women under high levels of stress experience hypercortisolism, suggesting that this may be at least one pathway by which these pathophysiologic states interrupt reproductive function.
The discovery of the kisspeptin neuronal system has substantially advanced our understanding of the regulation of the hypothalamic GnRH system at puberty, across the female reproductive cycle, and at menopause.
The majority of the kisspeptin studies have been performed in rodents, but at least fundamental similarities are being documented in primate models. Humans with mutations in the kisspeptin receptor experience hypogonadotropic hypogonadism, strongly suggesting a key role in GnRH neuronal function in humans. Kisspeptin is a amino acid polypeptide which binds to a specific receptor, termed the G protein-coupled receptor 54 GPR Kisspeptin secreting neurons are located in the arcuate and anteroventral periventricular AVPV nuclei of the hypothalamus.
The majority of kisspeptin neuronal mapping has been done in rodents, but the presence of kisspeptin secreting neurons in the arcuate nucleus has been established in humans. Kisspeptin neurons may act directly or transsynaptically by way of neuorotransmitters.
Arcuate kisspeptin neurons are not as abundant as AVPV neurons, but are in much closer proximity to GnRH neurons in the median eminence.
The onset of puberty is marked by an increase in synaptic connections between Kiss1 and GnRH neurons. Evidence also suggests an overall elevation in kisspeptin tone as well as enhanced kisspeptin signaling efficacy. These changes appear to occur primarily via the AVPV neurons and, in the female, may be triggered by subtle increases in ovarian estrogen production.
Expression of the KISS1 gene is under the control of both estrogens and androgens. Sexual dimorphism also exists in that estrogen is unable to generate a surge in the male, possibly due to the greater number of kisspeptin neurons in the AVPV of adult females compared to males. Kisspeptin expression is further modified by the adipose-derived factor, leptin. A amino acid polypeptide, leptin is secreted by white adipose tissue and regulates body weight by decreasing food intake and increasing energy expenditure.
Within the arcuate nucleus, leptin has been shown to increase activity of the kisspeptin neurons. A third form of GnRH, GnRH3, has been reported in several fish species but is not currently thought to be present in humans. In the human, the gene encoding GnRH1 is found on chromosome 8p11, whereas the gene for GnRH2 is found on chromosome 20p The genes for both forms of GnRH are composed of four exons and three introns which encode an identically organized precursor polypeptide.
Expression of the GnRH1 and GnRH2 genes are driven by different promoter sequences, suggesting that their transcriptional regulation likely differs. Detailed analysis of the GnRH1 gene promoter sequence has identified a number of DNA-regulatory regions which provide for tissue-specific expression.
GnRH1 and GnRH2 are expressed in overlapping tissue patterns; however, as a general rule, GnRH2 can be detected in a broader range of tissues and is present at higher levels outside of the brain. The presence of GnRH1 in human thyrotropes and somatotropes, suggests additional roles for GnRH signaling in the pituitary.
GnRH1 and GnRH2 have been found in reproductive tissues including the ovary, prostate, endometrium, breast and placenta as well as in tumors derived from these tissues. In the central nervous system, both isoforms have been localized to the preoptic and mediobasal regions with additional GnRH2 expression in midbrain regions such as the hippocampus, caudate nucleus, and amygdala. Furthermore, in these same cells, treatment with the progesterone antagonist RU increases GnRH2 transcript number but does not alter GnRH1 expression.
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