Effect of environmental temperature on growth- and reproduction-related hormones gene expression in the female blue gourami (Trichogaster trichopterus)

Fish are ectothermic vertebrates, and their gonadal development and spawning are affected by changes in environmental temperature. Recent global temperature changes have increased the importance of studying the effect of temperature on reproduction. The aim of this paper was to study the effect of temperature on oogenesis and hormone gene expression related to reproduction and growth in the blue gourami female maintained under non-reproductive and reproductive conditions. In females under non-reproductive conditions, vitellogenic oocytes, gonadotropin-releasing hormone 3 (GnRH3), β luteinizing hormone (βLH) and growth hormone (GH) mRNA levels were affected by temperature changes. In females maintained under reproductive conditions with non-reproductively active males, a percentage of females in the final oocyte maturation (FOM) stage, pituitary adenylyl cyclase activating polypeptide (PACAP and PRP-PACAP), gonadotropins and GH mRNA levels were affected due to temperature changes. In females maintained under reproductive conditions with reproductively active males, also GnRH3 and insulin-like growth factor 1 (IGF-1) were affected by temperature changes. In conclusion, in blue gourami females, changes in environmental temperature affect oogenesis through changes in brain and pituitary hormone mRNA levels.     

Early maturity in the male striped bass, Morone saxatilis: follicle-stimulating hormone and luteinizing hormone gene expression and their regulation by gonadotropin-releasing hormone analogue and testosterone

Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60-64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary beta FSH and beta LH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of beta FSH were elevated during early spermatogenesis, whereas beta LH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in beta FSH mRNA and a rise in beta LH mRNA abundance. Immature males had lower levels of beta LH mRNA than maturing males, but there were no differences in beta FSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in beta FSH and beta LH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on beta FSH and beta LH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.     

Developmental programming: impact of prenatal testosterone excess on pre- and postnatal gonadotropin regulation in sheep

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.     

Changes in gonadotrope responsivity to gonadotropin releasing hormone during development of the rhesus monkey

To assess the changing responsiveness of pituitary gonadotropes to gonadotropin releasing hormone (GnRH) during development, 5 male and 5 female rhesus monkeys were studied. Three monkeys of each sex were tested periodically with a subcutaneous injection of 500 micrograms of GnRH dissolved in 50% polyvinylpyrrolidone (PVP) beginning at 2 to 4 weeks of age and continuing into young adulthood. The remaining 4 monkeys received injections of the vehicle (PVP) alone and served as controls. Serum concentrations of bioactive luteinizing hormone (LH) were determined by an interstitial cell testosterone bioassay, and follicle-stimulating hormone (FSH) levels were measured by radioimmunoassay. Baseline FSH levels in the 5 female neonatal monkeys were higher than those of the 5 male neonatal monkeys during the first 2 months of life. In both sexes, FSH concentrations decreased with age, and FSH was barely detectable by 6 months. Baseline LH values in the 5 female monkeys declined during the first 6 months of the study and were undetectable (less than 0.5 micrograms/ml) at 6 months of age. Baseline LH levels in 4 of the 5 neonatal males also declined to undetectable concentrations by 6 months of age. During the first 3 months of life, there was a striking increase in the serum concentrations of both LH and FSH following GnRH. Although the LH responses to GnRH (delta LH) were similar in males and females of comparable ages, the FSH responses (delta FSH) were considerably greater in the female monkeys. In the males, the delta LH exceeded the delta FSH, whereas in the females, the delta FSH were greater than the delta FSH. In both sexes, the delta LH and delta FSH generally were greatest in the youngest monkeys and decreased gradually with increasing age. By 6 months, the gonadotropin responses to GnRH either were undetectable (males) or very small (females). After 6 months there was no longer an increase in serum gonadotropins after GnRH in either sex until 1.5-4 years (females) or 3 years (males) of age. The delta LH in response to GnRH in the male monkeys 3-5 years of age were comparable to the responses during the first month after birth. Serum concentrations of FSH in the adult males, however, did not increase after GnRH. In the female monkeys, serum levels of LH and FSH increased after GnRH at 1.5 years (1 monkey) and 4 years (2 monkeys) of age. The delta LH were similar to those of the 1- to 2-month-old female monkeys. The delta FSH, however, were variable and were approximately 20% of the neonatal response. In these young adult female monkeys the delta LH exceeded the delta FSH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants

In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%-65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.     

Sex differences following gonadectomy in basal gonadotropin secretion rate of rat pituitary fragments in vitro

Sex differences in the acute response of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to withdrawal from gonadal negative feedback in the rat are well established. To investigate postgonadectomy changes at the anterior pituitary level that may underlie dramatic in vivo sex differences, we used a computer-controlled pituitary perifusion system to measure in vitro basal secretion rates (BSRs) of LH and FSH following gonadectomy in the absence of exogenous gonadotropin-releasing hormone (GnRH). We compared BSRs of pituitaries removed from intact rats and from males and females 2 and 6 days post-gonadectomy. Glands were cut into quarters, placed into individual chambers, and perifused in Medium 199 at 10 ml/h for 4 h. In females (n = 12/gp), BSR of LH was not significantly elevated above intact levels by 2 days but had tripled by 6 days post-ovariectomy, while BSR of FSH had already doubled by 2 days and doubled again by 6 days. These changes in BSR in females paralleled changes in serum levels of both hormones. In males (n = 14/gp), although serum LH and FSH had increased 7-fold by 2 days post-orchidectomy, BSRs of LH and FSH had decreased to 75% and 64% of intact levels, respectively, by 6 days. These findings suggest important sex differences at the pituitary level in the responses to withdrawal from gonadal feedback that persist in culture in the absence of direct hypothalamic (GnRH) input.     

Divergent responses of gonadotropin subunit messenger RNAs to continuous versus pulsatile gonadotropin-releasing hormone in vitro

Episodic GnRH input is necessary for the maintenance of LH and FSH secretion. In the current study we have assessed the requirement of a pulsatile GnRH signal for the regulation of gonadotropin alpha- and beta-subunit gene expression. Using a dispersed rat pituitary perifusion system, GnRH (10 nM) was administered as a continuous infusion vs. hourly pulses. Secretion of free alpha-subunit, LH, and FSH were monitored over 5-min intervals for the entire 12-h treatment period before the responses of alpha, LH beta, and FSH beta mRNAs were assessed. Basal release of all three glycoproteins declined slowly over 6-8 h before reaching a plateau. The cells were responsive to each pulse of GnRH, but continuous GnRH elicited only a brief episode of free alpha-subunit, LH, and FSH release, followed by a return to unstimulated levels. Despite the similar patterns of secretion, differences were observed in the responses of gonadotropin mRNAs to the two modes of GnRH. alpha mRNA increased in response to continuous (1.6-fold) or pulsatile (1.7-fold) GnRH. FSH beta mRNA was suppressed to 48% of the control value after continuous GnRH, but was stimulated over 4-fold by the pulses. LH beta mRNA was unresponsive to either treatment paradigm. We conclude that in vitro 1) alpha mRNA levels are increased in response to GnRH independent of the mode of stimulation; 2) under the conditions studied, LH beta mRNA levels are unresponsive to either mode of GnRH input; and 3) the response of FSH beta mRNA to GnRH is highly dependent on the mode of administration, with levels depressed in response to continuous GnRH, but stimulated by pulsatile GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 α-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.     

Courtship interactions stimulate rapid changes in GnRH synthesis in male ring doves

Many birds and mammals show changes in the hypothalamo-pituitary-gonadal (HPG) axis in response to social or sexual interactions between breeding partners. While alterations in GnRH neuronal activity play an important role in stimulating these changes, it remains unclear if acute behaviorally-induced alterations in GnRH release are accompanied by parallel changes in GnRH synthesis. To investigate this relationship, we examined changes in the activity of GnRH neurons in the brains of male ring doves following brief periods of courtship interactions with females. Such interactions have been previously shown to increase plasma LH in courting male doves at 24 h, but not at 1 h, after pairing with females. In the first study, males allowed to court females for 2 h had 60% more cells that showed immunocytochemical labeling for GnRH-I in the preoptic area (POA) of the hypothalamus than did control males that remained isolated from females. To determine whether an increase in GnRH gene expression preceded this increase in GnRH immunoreactivity in the POA, changes in the number of cells with detectable GnRH-I mRNA in the POA were measured by in situ hybridization following a 1 h period of courtship interactions with females. In this second study, courting males exhibited 40% more cells with GnRH-I in this region than did isolated control males. GnRH-immunoreactive neurons in two other diencephalic regions failed to show these courtship-induced changes. Plasma LH was not elevated after 1 or 2 h of courtship. These results demonstrate that the release of GnRH-I in the POA that is presumably responsible for courtship-induced pituitary and gonadal activation is accompanied by a rapid increase in GnRH synthesis that occurs before plasma LH levels increase. We suggest that this increase in GnRH synthesis is necessary to support the extended period of HPG axis activation that is seen in this species during the 5–10 day period of courtship and nest building activity.     

Differences in gonadotrophin concentrations and pituitary responsiveness to GnRH between Booroola ewes which were homozygous (FF), heterozygous (F+) and non-carriers (++) of a major gene influencing their ovulation rate

The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene. The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2-3 times greater in FF compared to ++ ewes with the LH amplitudes for F+ ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined. The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0.1, 0.5 and 25 micrograms i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. less than 2-fold). In the other genotypes a greater than 2-fold response was noted only at the highest GnRH dose (i.e. 25 micrograms). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers. Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.     

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)