Gonadotropin-releasing Hormone mRNA Levels Research Articles

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo–pituitary–adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF1 receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF2 receptor) with little or no binding affinity for the CRF1 receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF1 receptor and CRF2 receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF1 receptor, while Ucn2 increased the levels via the CRF2 receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.

Central Injection of Exogenous IL‐1β in the Control Activities of Hypothalamic–Pituitary–Gonadal Axis in Anestrous Ewes

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1β on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, β subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1β on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1β (10 or 50 μg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 μg (60%; p ≤ 0.01) but not with the 10 μg dose was observed. The centrally administrated IL-1β lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1β injection upon the release of LH and FSH. However, the central injection of IL-1β strongly decreased the LHβ mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHβ mRNA in the case of the 50 μg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1β is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.

Elevated estrogen receptor expression in hypothalamic preoptic area decreased by electroacupuncture in ovariectomized rats

In the present study, effects of electroacupuncture (EA) on estrogen receptor alpha (ERα) and beta (ERβ) mRNA and protein expression in the hypothalamus of ovariectomized (OVX) rats were detected by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot analysis. Gonadotropin-releasing hormone (GnRH) release and GnRH mRNA level in hypothalamic preoptic area (POA) were evaluated by push–pull perfusion and qRT-PCR. Our results showed that elevated mRNA and protein expression of ERα and ERβ in hypothalamus were restrained following EA treatment in OVX rats. EA treatment also inhibited GnRH release and GnRH mRNA levels in OVX rats. These results provide novel evidence that EA treatment down regulates the expression of hypothalamic estrogen receptors (ERs), thus restores the response of GnRH neurons to estrogen depression, and partially resets the negative feedback of estrogen to hypothalamus–pituitary–ovary axis (HPOA) in OVX rats, which may be a critical mechanism for EA on female reproductive disorders.

Jak2 Is Necessary for Neuroendocrine Control of Female Reproduction

Gonadotropin-releasing hormone (GnRH) neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity. In vitro, GnRH neuronal cell lines respond to a variety of ligands that activate the Jak (Janus-activated kinase)/STAT (signal transducers and activators of transcription) intracellular signaling pathway. To determine its biological function in reproduction, we used Cre (cAMP response element)/LoxP technology to generate GnRH neuron-specific Jak2 conditional knock-out (Jak2 G(-/-)) mice. GnRH mRNA levels were reduced in Jak2 G(-/-) mice when compared with controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum luteinizing hormone (LH) levels were significantly lower in female Jak2 G(-/-) mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G(-/-) mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However, the activation of GnRH neurons following release from estrogen-negative feedback is retained. Female Jak2 G(-/-) mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity, and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice.

Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (Huso huso) and the disruptive effect of methylmercury on gene expression

Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg(-1) [control]; 0.76 mg kg(-1) [low]; 7.8 mg kg(-1) [medium]; 16.22 mg kg(-1) [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.

Effects of Shortened Photoperiod on Gonadotropin-Releasing Hormone, Gonadotropin, and Vitellogenin Gene Expression Associated with Ovarian Maturation in Rainbow Trout

Reproductive activities of salmonids are synchronized by changes in photoperiod, which control the endocrine system via the brain-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) in the brain regulates synthesis and release of the pituitary gonadotropins (GTHs; FSH and LH). FSH and LH in turn stimulate the production of sex steroids for oocyte growth and maturation-Inducing steroid hormones for oocyte maturation and ovulation, respectively, in female salmonids. To clarify effects of long-term photoperiod manipulations on the reproductive activity of salmonids from early recrudescence to ovulation, we Investigated the gene expression profiles of GnRH, GTHs, and vitellogenin (VTG), and plasma sex steroids in female rainbow trout (Oncorhynchus mykiss). In addition, the percentages of eyed embryos and hatched alevins were examined together with the number of ovulated eggs to evaluate the effects of photoperiod regimes on egg quality. During late summer, the mRNA levels of GnRHs, GTHalpha, and LHbeta, and the plasma level of a maturational steroid (17alpha,20beta-dihydroxy-4-pregnen-3-one; 17,20beta-P) were significantly elevated by a gradually shortened photoperiod under constant temperature, in accordance with accelerated sexual maturation. The percentages of eyed embryos and hatched alevins from fish ovulated in August were comparable to those of control fish observed in December. These results clearly indicate that syntheses of GnRHs, LH, VTG, and 17,20beta-P are effectively accelerated by a programmed long-short photoperiod regime in early recrudescent female rainbow trout, without a marked deterioration in egg quality.

Differential expression of gonadotropin-releasing hormone (GnRH) in pancreas during rat pregnancy

Many studies have shown that there is a relationship between gonadotropin-releasing hormone (GnRH) and glucose metabolism, but little is known about the effects of GnRH on the pancreas. Our experiment investigated the effect of GnRH on pancreatic islet cell in Sprague-Dawley (SD) rats fed with high-cholesterol diet before and during pregnancy. We found that although high-cholesterol diet led to no significant difference of GnRH mRNA levels in pancreas in nonpregnant rats, it led to a marked increase of those in pregnant rats. Furthermore, in rats fed with standard laboratory chow, no significant differences were apparent in GnRH mRNA levels before and during gestation; however, when fed with high-cholesterol diet, the GnRH mRNA levels increased significantly in pregnant rats. As results indicated both diets could lead to increase of PG mRNA in pancreas of pregnant rats. It is also demonstrated that the GnRH mRNA levels are positively associated with PG mRNA levels. Moreover, our data showed a significant increase in fasting insulin level in the Gestation group compared with Control. Such changes were contrary to the changes of GnRH level in the pancreas. This may imply that GnRH influences hormones secretion in the pancreas by autocrine and paracrine effects on islet cells.

Naturally-induced endocrine disruption by the parasite Ligula intestinalis (Cestoda) in roach ( Rutilus rutilus)

Fish represent the most frequently used vertebrate class for the investigation of endocrine disruption (ED) in wildlife. However, field studies are complicated by exposure scenarios involving a variety of anthropogenic and natural influences interfering with the endocrine system. One natural aspect rarely considered in ecotoxicological studies is how parasites modulate host physiology. Therefore, investigations were carried out to characterise the impacts of the parasitic tapeworm Ligula intestinalis on plasma sex steroid levels and expression of key genes associated with the reproduction in roach ( Rutilus rutilus), a sentinel species for wildlife ED research. Parasitisation by L. intestinalis suppressed gonadal development in both genders of roach and analysis of plasma sex steroids revealed substantially lower levels of 17β-oestradiol (E2) and 11-ketotestosterone (11-KT) in infected females as well as E2, 11-KT, and testosterone in infected males. Consistently, in both, infected females and males, expression of the oestrogen dependent genes such as vitellogenin and brain-type aromatase in liver and brain was reduced. Furthermore, parasitisation differentially modulated mRNA expression of the oestrogen and androgen receptors in brain and liver. Most prominently, liver expression of oestrogen receptor 1 was reduced in infected females but not in males, whereas expression of oestrogen receptor 2a was up-regulated in both genders. Further, insulin-like growth factor 1 mRNA in the liver was increased in infected females but not in males. Despite severe impacts on plasma sex steroids and pituitary gonadotropin expression, brain mRNA levels of gonadotropin-releasing hormone (GnRH) precursors encoding GnRH2 and GnRH3 were not affected by L. intestinalis-infection. In summary, the present results provide basic knowledge of the endocrine system in L. intestinalis-infected roach and clearly demonstrate that parasites can cause ED in fish.

The Correlation Between Intestinal Gonadotropin-Releasing Hormone (GnRH) and Proglucagon in Hyperlipidemic Rats and Goto-Kakizaki (GK) Rats

Our experiment investigated the mRNA expression of intestinal gonadotropin-releasing hormone (GnRH), proglucagon (PG), and glucagon-like peptide 1 receptor (GLP-1R) in the jejunum, ileum, and colon of rats fed with high-fat diet and Goto-Kakizaki (GK) rats and revealed the physiological role of intestinal GnRH. We found that the GnRH and PG mRNA levels in high-cholesterol (HCh) diet were higher than in the control. However, the GnRH receptor (GnRHR) and GLP-1R mRNA levels did not differ significantly between HCh and control. The GnRH, PG, and GLP-1R mRNA levels in GK rats were lower, respectively, than those in control rats, while the GnRHR levels did not differ significantly between GK rats and control rats. There were no difference in GnRH, PG, GnRHR, and GLP-1R mRNA levels in the ileum and colon tissue between HCh and control rats. The GnRH mRNA levels of GK rats were lower than those in control rats; however, the PG, GLP-1R, and GnRHR levels did not differ significantly between GK and control rats. The GLP-1R mRNA levels of GK rats were lower than those in control rats. The GnRH mRNA expression showed positive correlation with PG mRNA expression in different intestinal sections. The GnRH level in the jejunum showed a significant effect on blood glucose level, while the PG level in the jejunum showed a significant effect on insulin level. This may imply that, compared with the ileum and colon, the jejunum had greater impact on glucose metabolism; furthermore, GnRH might interact with intestinal GLP-1 and GLP-2 through the paracrine and autocrine ways and then regulate glucose metabolism and insulin secretion.

Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines

Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1–7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10 −9 M kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4 h of treatment in both cell lines. Negative regulation by 17β-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17β-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin–GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1–7 and GN11 cell lines.

Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish

In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain.

The Recreational Drug Ecstasy Disrupts the Hypothalamic-Pituitary-Gonadal Reproductive Axis in Adult Male Rats

Reproductive function involves an interaction of three regulatory levels: hypothalamus, pituitary, and gonad. The primary drive upon this system comes from hypothalamic gonadotropin-releasing hormone (GnRH) neurosecretory cells, which receive afferent inputs from other neurotransmitter systems in the central nervous system to result in the proper coordination of reproduction and the environment. Here, we hypothesized that the recreational drug (±)-3,4-methylenedioxymethamphetamine (MDMA; ‘ecstasy’), which acts through several of the neurotransmitter systems that affect GnRH neurons, suppresses the hypothalamic-pituitary-gonadal reproductive axis of male rats. Adult male Sprague-Dawley rats self-administered saline or MDMA either once (acute) or for 20 days (chronic) and were euthanized 7 days following the last administration. We quantified hypothalamic GnRH mRNA, serum luteinizing hormone concentrations, and serum testosterone levels as indices of hypothalamic, pituitary, and gonadal functions, respectively. The results indicate that the hypothalamic and gonadal levels of the hypothalamic-pituitary-gonadal axis are significantly altered by MDMA, with GnRH mRNA and serum testosterone levels suppressed in rats administered MDMA compared to saline. Furthermore, our finding that hypothalamic GnRH mRNA levels are suppressed in the context of low testosterone concentrations suggests that the central GnRH neurosecretory system may be a primary target of inhibitory regulation by MDMA usage.

NMDA Receptor Subunit NR2b: Effects on LH Release and GnRH Gene Expression in Young and Middle-Aged Female Rats, with Modulation by Estradiol

Background/Aims: The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. Methods: Young (3–5 months) and middle-aged (10–13 months) female rats were ovariectomized (OVX), 17β-estradiol (E₂) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 min for 4 h, with ifenprodil (10 mg/kg) or vehicle injected (i.p.) after 1 h of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. Results: Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E₂-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. Conclusion: Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit play a role in GnRH-induced LH release, independent of de novo GnRH gene expression.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

The olfactory organ modulates gonadotropin-releasing hormone types and nest-building behavior in the tilapiaOreochromis niloticus

Direct olfactory inputs to any of the known gonadotropin-releasing hormone (GnRH) containing neurons have not been demonstrated. Therefore, the rationale of this study was to examine whether olfactory inputs might in some way interact with the GnRH system(s) to synchronize reproductive behaviors. In order to establish this, we used anosmic mature male tilapia to investigate changes in reproductive behaviors, gonadal morphology, and GnRH1, GnRH2, and GnRH3 cellular morphology and change in GnRH mRNA levels by real-time polymerase chain reaction. Bilateral removal of the olfactory rosettes followed by occlusion of the nasal cavity (ORX) inhibited nest-building behavior, but had no effect on aggressive and sexual behaviors or gonadal morphology. ORX failed to alter the morphological features of GnRH1, GnRH2, and GnRH3 (cell number, size, GnRH optical density), but significantly decreased copies of GnRH1 and GnRH2 mRNAs. GnRH immunoreactive fibers were not evident in the olfactory nerve and rosettes. DiI application to the olfactory nerve labeled inputs primarily to the glomerular layer of the olfactory bulbs and extrabulbar inputs to the forebrain but not to GnRH neurons. These results provide evidence that the olfactory rosette is crucial for modulating nest-building behavior through second-order olfactory pathways interacting with GnRH1 and GnRH2 neuronal systems.

Changes in brain GnRH mRNA and pituitary GnRH peptide during testicular maturation in barfin flounder

The pleuronectid barfin flounder ( Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.

Increased expression of forebrain GnRH mRNA and changes in testosterone negative feedback following pubertal maturation

Pubertal development is associated with increased activity of the gonadotropin releasing hormone (GnRH) neuronal system and rising gonadal steroid levels. The purpose of this study was to determine whether different circulating levels of testosterone affect GnRH mRNA and luteinizing hormone (LH) to the same degree prior to and following pubertal maturation. Pre- and post-pubertal male Syrian hamsters were gonadectomized and treated with timed-release testosterone pellets (0, 0.5, 1.5, or 2.5 mg) for one week. Following treatment, three separate brain tissue dissections containing the majority of GnRH cell bodies, tenia tecta and medial septum (TT/MS), diagonal band of Broca/organum vasculosum of the lamina terminalis (DBB/OVLT), and preoptic area (POA), were analyzed for GnRH mRNA levels by RNase protection assay and terminal plasma luteinizing hormone concentrations were determined by radioimmunoassay. Pre-pubertal animals were more sensitive to testosterone negative feedback on LH. Conversely, the ability of testosterone to reduce GnRH mRNA was much greater after pubertal development. Specifically, GnRH mRNA in the TT/MS was considerably higher in adults, and testosterone reduced GnRH mRNA in a dose-dependent manner only in adults. These data indicate that although testosterone is a powerful suppressor of LH release before puberty, it does not have appreciable control over GnRH mRNA until after puberty. Furthermore, the pubertal increase in GnRH mRNA appears to occur via steroid feedback-independent mechanisms in the male Syrian hamster.

Suppression by ethanol of male reproductive activity

Ethanol has been known to suppress reproductive activity in laboratory animals and humans through the inhibition of luteinizing hormone (LH) release by reduction of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. There are, however, little data is available regarding the effect of ethanol on GnRH gene expression. Thus, the present study was designed to evaluate the effect of ethanol on GnRH gene expression and reproductive activity at all levels of the hypothalamus–pituitary–gonad (HPG) axis simultaneously. To this end, ethanol (3 g/kg i.p., 15% v/v in saline) was administered to adult male rats for 10 days. Serum levels of LH and testosterone were significantly decreased by ethanol. Using Northern blot analysis and in situ hybridization, the present study showed the reduction in GnRH mRNA levels in the hypothalamus by prolonged ethanol administration. The content of LH in the anterior pituitary was also significantly reduced by ethanol. In addition, steroidogenic acute regulatory protein (StAR) mRNA levels were significantly decreased by ethanol, suggesting a cause for the reduced production of testosterone under this condition. These results indicate that ethanol affects the HPG axis at all the levels. Especially, suppressed GnRH mRNA levels in the hypothalamus of ethanol-treated rats strongly demonstrated that hypothalamus is the major action site of ethanol on the HPG axis. Decreased serum LH level may affect the steroidogenesis in the testis, at least in part, through the inhibition of StAR gene expression that induces part of dysfunctions of reproductive activity.

Long-term, low-dose lead exposure alters the gonadotropin-releasing hormone system in the male rat.

Lead is a male reproductive toxicant. Data suggest that rats dosed with relatively high levels of lead acetate for short periods of time induced changes in the hypothalamic gonadotropin-releasing hormone (GnRH) at the molecular level, but these changes were attenuated with increased concentration of exposure. The current study evaluated whether exposure to low levels of lead acetate over longer periods of time would produce a similar pattern of adaptation to toxicity at the molecular and biologic levels. Adult 100-day-old Sprague-Dawley male rats were dosed with 0, 0.025, 0.05, 0.1, and 0.3% lead acetate in water. Animals were killed after 1, 4, 8, and 16 weeks of treatment. Luteinzing hormone (LH) and GnRH levels were measured in serum, and lead levels were quantified in whole blood. Hypothalamic GnRH mRNA levels were also quantified. We found no significant differences in serum LH and GnRH among the groups of animals treated within each time period. A significant dose-related increase of GnRH mRNA concentrations with lead dosing occurred in animals treated for 1 week. Animals treated for more than 1 week also exhibited a significant increase in GnRH mRNA, but with an attenuation of the increase at the higher concentrations of lead with increased duration of exposure. We conclude that the signals within and between the hypothalamus and pituitary gland appear to be disrupted by long-term, low-dose lead exposure.

Long-term effects of the mammary carcinogen 7,12-dimethylbenz(a) anthracene on hypothalamic gonadotropin-releasing hormone and its pituitary receptor gene expression, during the promotion stage, in female Sprague-Dawley rats.

A single intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA) has been shown to induce mammary tumors in young cycling female Sprague-Dawley rats. The appearance of these tumors is preceded by a series of neuroendocrine disturbances, including attenuation of the preovulatory luteinizing hormone (LH) surge and amplification of the preovulatory 17beta-estradiol surge, and gonadotropin-releasing hormone (GnRH) released in vitro. In this study, we examined the hypothesis that DMBA administration decreases levels of GnRH mRNA in the preoptic area-anterior hypothalamus (POA-AH) and GnRH receptor (GnRH Rc) mRNA and protein in the anterior pituitary gland. Sprague-Dawley rats, 55-60 days of age with regular estrous cycles, received a single dose of 15 mg DMBA in 1 ml sesame oil delivered by intragastric intubation. A first series of experiments was performed for the measurement of hypothalamic GnRH mRNA and pituitary GnRH Rc mRNA levels. A second series of experiments was performed for the measurement of pituitary GnRH receptor. In both experiments, animals were sacrificed by decapitation at 11.00, 16.00, 18.00 and 20.00 h on each day of the 7th or 8th estrous cycle (28-32 days) after treatment. GnRH and GnRH receptor mRNAs were quantified using solution hybridization-RNase protection assay. The GnRH Rc was quantified using the 125I-D-Ala6-N-Met-Leu6-des-Gly10-ethylamide GnRH. DMBA-treatment produced no significant effect on the overall mean values of GnRH mRNA. GnRH mRNA levels in control rats rose significantly between 16.00 and 20.00 h on proestrus and between 18.00 and 20.00 h on diestrus I. DMBA-treated rats had a surge in GnRH mRNA levels at 18.00 h on proestrus, and showed additional surges at 18.00h on diestrus II and estrus. GnRH receptor mRNA content in the anterior pituitary gland surged at 16.00h on certain days of the cycle in both groups of rats. In control rats, only the surge on diestrus II proved significant, whereas DMBA-treated rats exhibited significant surges on diestrus I, diestrus II and proestrus. GnRH receptor mRNA values were significantly lower on both days of diestrus in DMBA-treated rats compared with controls. GnRH Rc peptide content, like GnRH receptor in RNA surged at 16.00h in both groups with the exception of a marked fall on proestrus day for DMBA treated rats. A reduction in the amplitude of the surge was also seen on the day of estrous and to a lesser extend on the day of diestrus DII in DMBA treated animal. Overall, there was a disruption of the GnRH Rc pattern which culminate on the day of proestrus in DMBA-treated animals. Interestingly, the daily rise between 11.00 and 16.00h which is the more pronounced on the day of proestrus in control animals, was completely blunted in DMBA-treated rats. Overall, the results are consistent with the hypothesis that the carcinogen attenuates, directly or indirectly, preovulatory biosynthesis of the GnRH receptor and LH release. Obviously, the changes in GnRH might occur simultaneously, independently from mammary tumorigenesis, but may play a role, in association with others DMBA-induced neuroendocrine disorders, in the promotion stage of mammary tumors in the Sprague-Dawley female rat.