Proteolytic processing and differential distribution of secretogranin-II in goldfish

Secretoneurin (SN) is a 33-34 amino acid neuropeptide derived by endoproteolysis of secretogranin-II (SgII), a chromogranin. A multi-antigenic strategy was used to generate a rabbit polyclonal goldfish SN antiserum that was characterized for Western blot analysis. In the goldfish pituitary two intermediate proteins containing SN and likely processed from the 69.6-kDa SgII precursor were detected. No immunoreactive proteins were observed in the goldfish interrenal, ovary, cerebellum, and telencephalon whereas SgII mRNA was expressed in all these tissues. Immunoreactive levels of the approximately 57 kDa product were higher in the pars distalis (PD) than in the neurointermediate lobe (NIL). The abundance of the approximately 57 kDa protein indicates that this SgII-product containing the SN sequence is a major stored form in secretory granules of the goldfish pituitary. High expression and processing of SN in the hypothalamus and pituitary suggest important roles for SgII-derived peptides in neuroendocrine tissues.     

Seasonal effect of GnIH on gonadotrope functions in the pituitary of goldfish

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release in birds and mammals. To investigate its role in teleosts, we examined the effects of synthetic goldfish (g)GnIH on pituitary LH-β and FSH-β subunit, and gGnIH receptor (gGnIH-R) mRNA levels and LH secretion in goldfish. Intraperitoneal injections of gGnIH increased pituitary LH-β and FSH-β mRNA levels at early to late gonadal recrudescence, but reduced serum LH and pituitary gGnIH-R mRNA levels, respectively, at early to mid-recrudescence and later stages of recrudescence. Static incubation with gGnIH elevated LH secretion from dispersed pituitary cell cultures from prespawning fish, but not at other recrudescent stages; suppressed LH-β mRNA levels at early recrudescence and prespawning but elevated LH-β at mid-recrudescence; and consistently attenuated FSH-β mRNA in a dose-specific manner. Results indicate that in goldfish, regulation of LH secretion and gonadotropin subunit mRNA levels are dissociated in the presence of gGnIH and dependent on maturational status and administration route.     

Estradiol reduces pituitary responsiveness to somatostatin (SRIF-14) and down-regulates the expression of somatostatin sst2 receptors in female goldfish pituitary

Sex steroid hormones have been shown to regulate somatostatin (SRIF) gene expression in goldfish brain, which in turn influences the regulation of GH secretion. In this study, the influences of sex steroids on pituitary responsiveness to SRIF-14 and the pituitary expression of a type two SRIF receptor (sst(2)) were examined. Results from in vitro perifusion of pituitary fragments show that pituitaries from estradiol-primed sexually regressed female fish have significantly lower GH release responsiveness to pulse exposure to SRIF-14 than pituitaries from control or testosterone-treated sexually regressed females. Results from in vitro static culture show that pituitaries from sexually mature female fish have lower GH release responsiveness to SRIF-14 than those from sexually regressed females. In addition, the sst(2) receptor mRNA levels in pituitaries from mature and recrudescent female fish are significantly lower than in sexually regressed female fish. Our results indicate that estradiol acts at the level of the pituitary to regulate GH secretion by influencing the responsiveness to SRIF-14. The underlying mechanism includes, in part, reduction of the expression of sst(2) receptors, presumably leading to the lower number of the receptors available for SRIF binding.     

Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli

Two GnRH receptors (GnRH-R I and GnRH-R II) were obtained in protandrous black porgy (Acanthopagrus schlegeli). We investigated their tissue distribution, developmental/seasonal changes and regulation of expression using in vivo and in vitro (primary cultures of dispersed pituitary cells) approaches. The relative expressions of GnRH-Rs in the pituitary and gonad were as follows: pituitary: GnRH-R I > GnRH-R II; testicular tissue: GnRH-R I > GnRH-R II; ovarian tissue: GnRH-R I = GnRH-R II. GnRH-R I but not GnRH-R II expression was higher in the pituitary during the spawning period as compared to the prespawning. The expression profiles of both forms of GnRH-R were variable in the gonads according to the gonadal stage and season. In vivo, hCG stimualated GnRH-R I and GnRH-R II expression in testis and ovary. The LHRH analog also up-regulated both receptors in testis and but increased only GnRH-R II in the ovary. Sex steroids (estradiol, E2 and testosterone, T) increased the expression of both receptors in the testis and ovary. In the pituitary, sex steroids (E2 and T) increased the expression of GnRH-R I, but not GnRH-II, both in vivo and in vitro. The LHRH analog also specifically up-regulated the expression of GnRH-R I, but not GnRH-R II, by pituitary cells in vitro. All these data suggest that GnRH-R I rather than GnRH-R II may play a major physiological role in the pituitary. In contrast, both GnRH-R I and GnRH-R II may participate in the regulation of gonadal functions, including a possible role during sex change.     

In vivo and in vitro sex steroids stimulate seabream gonadotropin-releasing hormone content and release in the protandrous black porgy, Acanthopagrus schlegeli

The objective of the present study was to investigate the regulation of seabream gonadotropin-releasing hormone (sbGnRH) release using in vivo and in vitro approaches in the protandrous black porgy, Acanthopagrus schlegeli. Estradiol-17beta (E2), testosterone (T), and 11-ketotestosterone (11-KT) were found to significantly stimulate the increase of sbGnRH levels in pituitary of black porgy after 5-96 h of injection. An in vitro culture system using dispersed brain neurons was also developed to investigate the effects of various steroids on sbGnRH release. Different doses (10(-6) - 10(-12) M) of E2, T, 11-KT, and cortisol were applied during 6 h experiment. KCl stimulated sbGnRH release at a dose- and time-dependent manner. The concentration of sbGnRH increased 2-fold in the highest dose of KCl treatment compared to the control. Treatments with E2, T, 11-KT and cortisol significantly stimulated the release of sbGnRH from the cultured brain neurons. The concentration of sbGnRH in medium was increased by 2-, 1.9-, 2.1-, and 4.9-fold when treated with E2, T, 11-KT, and cortisol, respectively, as compared to the respective control. Cholesterol did not have any stimulatory effects in the release of sbGnRH. The results showed that sex steroids and cortisol had direct effect on brain neuronal cells stimulating the release of sbGnRH.     

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.     

The GnRH test in the assessment of patients with pituitary and parapituitary lesions: results of a 5-year retrospective study

OBJECTIVE: To examine the utility of the GnRH (gonadotrophin-releasing hormone) test in the management of patients with pituitary and parapituitary lesions. PATIENTS AND METHODS: A 5-year retrospective study of LH (luteinizing hormone) and FSH (follicle stimulating hormone) responses to GnRH test in patients with HP (hypothalamic-pituitary) disease in a regional endocrine centre. Serum LH and FSH concentrations were measured at baseline and at 20 and 60 min after an intravenous bolus of 100 mcg (micrograms) of GnRH. The GnRH responses were categorised by tumour size, tumour type, and gonadal status. RESULTS: Of the 104 patients studied, 46 were male and 58 were female. There were 50 normal, 38 subnormal and 16 exaggerated LH responses compared with 34 normal 67 subnormal and three exaggerated responses for FSH. Seventy-four patients (71.2%) were hypogonadal. Normal LH responses were achieved in half of the hypogonadal subjects and normal FSH responses in more than a third. Furthermore, the LH responses were exaggerated in nine hypogonadal patients compared with three for FSH. The GnRH test could not differentiate between pituitary or parapituitary lesions either by size or type of lesion. An exception was the male non-functioning adenoma (NFA) sub-group (10 patients, all were hypopituitary, seven were hypogonadal), which demonstrated significant subnormal LH and FSH responses compared with other male and female tumour type sub-groups. CONCLUSIONS: The data from this study indicate that the GnRH test is unhelpful in the clinical assessment of the HP axis in patients with HP disease.     

Seasonal and individual variation in response to GnRH challenge in male dark-eyed juncos (Junco hyemalis)

Concentrations of gonadal steroids such as testosterone (T) often vary widely in natural populations, but the causes and particularly the consistency of this variation is relatively unexplored. In breeding males of a wild population of the dark-eyed junco (Junco hyemalis), we investigated seasonal and individual variation in circulating T during two breeding seasons by measuring the responsiveness of the HPG axis to a standardized injection of gonadotropin-releasing hormone (GnRH). Individuals were bled prior to and 30min after injection. Pre- and post-challenge levels of T were measured using EIA. Many subjects were sampled repeatedly across multiple breeding stages. Plasma T concentrations nearly doubled in response to GnRH during early spring, but showed significantly smaller increases in later breeding stages. When controlling for seasonal variation in response to challenge, we also found repeatable differences among individuals, indicating individual consistency in the release of T in response to a standardized stimulus. These seasonal and individual differences may arise from comparable variation in responsiveness of the pituitary or a decline in gonadal sensitivity to downstream gonadotropins. In contrast, pre-challenge T showed almost no seasonal changes and did not differ consistently among individuals. To our knowledge, this is the first demonstration of individual repeatability of short-term hormonal changes in a wild population. Such repeatability suggests that hormonal plasticity might evolve in response to changing selection pressures.     

Changes in brain seabream GnRH mRNA and pituitary seabream GnRH peptide levels during ovarian maturation in female barfin flounder

The pleuronectid barfin flounder Verasper moseri expresses three forms of gonadotropin-releasing hormones (GnRHs), i.e., seabream GnRH (sbGnRH), salmon GnRH, and chicken GnRH-II. Among these, sbGnRH is the dominant form in the pituitary, indicating that sbGnRH regulates gonadal maturation. In order to clarify the physiological roles of sbGnRH during ovarian maturation in reared female barfin flounder, the changes in brain sbGnRH mRNA levels and pituitary sbGnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. The fish hatched in April 2002. The gonadosomatic index remained low until August 2004 and increased thereafter until April 2005 when the fish began to ovulate. The sbGnRH mRNA levels per brain increased significantly from April 2004 to April 2005. Pituitary sbGnRH peptide levels also increased significantly during this period. These results indicate that sbGnRH is involved in ovarian maturation and ovulation in the barfin flounder.     

Regional differences in the pituitary distribution of luteinizing hormone in the gonadectomized and proestrous female rat

Previous data have shown that regional differences in the presence of anterior pituitary luteinizing hormone (LH) generally correlate with the comparable disparities in distribution of gonadotropes throughout the gland. In female rats, the differences are apparent over the estrous cycle, but are more prominent during the hours preceding the proestrus surge of LH. The current experiments examined (1) if such regional disparities are present throughout the surge window, (2) if differences are mirrored by release of LH in vitro and (3) if the appearance of regional differences is altered in ovariectomized females. Results showed that a comparative elevation in the rostral portion of the pituitary during the pre-surge period diminishes and finally disappears concurrent with the rise in circulating LH. This increase in rostral LH concentrations is reflected in this region by a comparable effect in vitro on stimulated LH secretion from pituitary fragments, although the effect is somewhat diminished by referencing release against tissue concentrations of LH present in a contralateral rostral fragment. Ovariectomies conducted at 1500h on proestrus, at a time when a significant regional difference has faded, resulted in a prompt increase in LH across all areas of the pituitary, and the emergence of a marked augmentation in rostral concentrations over the ensuing 72h. The effect was not seen when ovariectomies were performed on estrus. These data show that, while a regional disparity in anterior pituitary LH is present as circulating concentrations of estradiol rise prior to the LH surge, the removal of this steroid feedback at a time when LH synthesis is normally amplified accentuates the difference between the rostral region and other areas of the pituitary.     

Seasonal expression of LHbeta and FSHbeta in the male newt pituitary gonadotrophs

Seasonal changes in LHbeta and FSHbeta mRNA levels were examined in the pituitary gland of the adult male newt, Cynops pyrrhogaster, using in situ hybridization histochemistry and a quantitative real-time RT-PCR method. The annual fluctuation of LHbeta mRNA and FSHbeta mRNA levels in the pituitary gland displayed a close relationship with seasonal changes in testicular function. The values obtained by both methods showed similar fluctuation. The levels of LHbeta mRNA were always exceeded those of FSHbeta. The present immunoelectron microscopic observations support the data on the gene expression levels of the beta-subunits of LH and FSH. Gonadectomy in the summer increased the LHbeta and FSHbeta mRNA levels. Testosterone replacement inhibited the expression of LHbeta mRNA, but not of FSHbeta mRNA, suggesting that the expression of FSHbeta is regulated by some non-steroid factor, probably inhibin. In the case of gonadectomy during any other season, the LHbeta mRNA level increased, but not to the same extent as in summer, and androgen concentrations decreased to the minimum of the year. This finding provides new information about the regulation of annual changes in LHbeta and FSHbeta expression in the pituitary gonadotrophs.     

Seasonal changes in expression of LH-beta and FSH-beta in male and female three-spined stickleback, Gasterosteus aculeatus

In teleost fishes, like in other vertebrates, the gonadal development is stimulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). To achieve a better understanding of the role of gonadotropins in teleost reproduction; expression of LH-beta and FSH-beta mRNA and the status of gonads and secondary sexual characters were analyzed over the annual cycle in male and female three-spined sticklebacks, a species in which the development of male secondary sexual characters and spermatogenesis are separated in time. The kidney in the male stickleback hypertrophies during the breeding season and produces a glue used when building nests. Kidney weights, as well as levels of 11-ketotestosterone (11KT), reached a peak in May. Both testosterone (T) levels and the gonadosomatic index (GSI, gonad weight/body weight x 100) in females started to increase in April, and peaked in May as well. Later in summer, after the breeding season, these features declined. In females, LH-beta expression followed the GSI and T levels closely, levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-beta expression peaked earlier, in January and declined slowly over spring. In males, LH-beta expression peaked in May. During June-September, when spermatogenesis was active, LH-beta levels were very low. FSH-beta expression peaked in January, earlier than LH-beta expression did, and reached the lowest levels in July. Thus, when spermatogenesis started at the end of summer, the expression of both GTH-beta mRNAs, and circulating 11KT, displayed their lowest levels.     

The regulation of galanin gene expression in gonadotropin-releasing hormone neurons

In rats, galanin modulates luteinizing hormone (LH) secretion, and gonadotropin-releasing hormone (GnRH) neurons provide a possible source of this galanin. To understand galanin's physiological role in GnRH neurons, we used double-label in situ hybridization and computerized image analysis to examine the regulation of galanin message in GnRH neurons. We found that galanin gene expression in GnRH neurons is regulated by sex steroids, induced coincident with the LH surge, and persists well after the completion of the LH surge, and that the induction of galanin message in GnRH neurons coincident with the LH surge is sexually differentiated neonatally. We postulate that the rise in galanin gene expression in GnRH neurons at the time of the LH surge serves to replenish galanin released with GnRH that is needed for the production of the LH surge, or that galanin is involved in physiological events that occur subsequent to the LH surge.     

Dopamine D2 receptors and secretion of FSH and LH: role of sexual steroids on the pituitary of the female rainbow trout

The role of sexual steroids in the modulation of a dopaminergic inhibitory tone on FSH and LH release was studied in the rainbow trout. The experiments were performed on previtellogenic trout, implanted or not with estradiol (E(2)), and vitellogenic trout. E(2) implant increased the circulating levels of LH and decreased the circulating levels of FSH in previtellogenic fish. The catecholamine inhibitor alphaMPT increased the circulating levels of LH, implanted or not with E(2). AlphaMPT increased circulating levels of LH in vitellogenic fish. This increase could be prevented by the dopaminergic agonist bromocryptine. The dopaminergic drugs had no effect on the circulating levels of FSH in all groups. E(2) decreased mRNA levels of sGnRH1 and sGnRH2 in the telencephalon of previtellogenic fish. The dopaminergic treatments had no effect on mRNA levels of both forms of sGnRH in previtellogenic and vitellogenic fish. Primary cultures of pituitary cells were primed for three days with steroids (E(2) or 17alpha-hydroxy, 20beta-dihydroprogesterone (17alpha20betaP)) before treatment with increasing doses of bromocryptine, associated or not with sGnRH. E(2), but not 17alpha20betaP, potentiated the sGnRH-induced release of LH. Bromocryptine induced a slight dose-dependent decrease of sGnRH-induced release of LH. This decrease was potentiated by 17alpha20betaP. E(2) and 17alpha20betaP had no effect on the release of FSH, but bromocryptine decreased the 10(-8)M sGnRH-induced release of FSH. In conclusion, the development of the dopaminergic inhibitory tone on gonadotropin release, at the onset of vitellogenesis, requires factors other than estradiol. E(2) should contribute in part to decrease the release of FSH. At the end of the reproductive cycle, 17alpha20betaP should reinforce the dopaminergic inhibitory tone.     

GnRH-LH secreting cells axis in the pituitary of the teleost Clarias batrachus responds to neuropeptide Y treatment: an immunocytochemical study

An attempt has been made to immunocytochemically visualize the effect of neuropeptide Y (NPY) on the GnRH fibers and luteinizing hormone (LH) secreting cells in the pituitary of the catfish, Clarias batrachus. Two hours following the intracranial administration of NPY at the dose of 20 ng/g body wt, a significant increase in the area occupied by the GnRH-like immunoreactive fibers, and population and size of the LH cells was observed. The treatment also resulted in considerable augmentation of immunoreactivity in the LH cells. Double immunolabeling revealed NPY fibers directly terminating on the LH cells. The results suggest that NPY may (a) stimulate some GnRH containing hypophysiotropic neuronal group in the brain, (b) promote anterograde transport of GnRH to the pituitary gland, and (c) up-regulate the LH cells.     

Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish

Gonadotrophin-releasing hormone (GnRH) was originally believed to be released by a unique set of hypophysiotrophic neurons to stimulate the release of gonadotrophins from the pituitary, therefore acting as a major initiator of the hormonal cascade controlling the reproductive axis. However, it now appears that each vertebrate species expresses two or three GnRH forms in multiple tissues and that GnRHs exert pleiotropic actions via several classes of receptors. This new vision of the GnRH systems arose progressively from numerous comparative studies in all vertebrate classes, but fish in general, and teleosts in particular, have often plaid a leading part in changing established concepts. To date fish still appear as attractive models to decipher the evolutionary mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest variety of GnRH variants, but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptors. This paper intends to summarize the current situation with special emphasis on interspecies comparisons which provide insights into the possible evolutionary mechanisms leading to the diversification of GnRH functions.     

The role of sex ratio on spawning performance and on the free and conjugated sex steroids released into the water by common dentex (Dentex dentex) broodstock

This paper reports the results of an experiment to determine whether sex ratio has any effect on the spawning efficiency of common dentex, Dentex dentex, broodstock. The paper also reports preliminary data on diurnal changes in water concentrations of steroids (measured on two separate occasions) in the same groups of fish. These latter observations were made to determine whether, using a non-invasive procedure, it was possible to relate hormonal changes in the fish to the "success of spawning." Mature fish were placed in broodstock tanks at three different sex ratios (Group A, 4 female:1 male; Group B, 1 female:1 male; and Group C, 1 female:4 male) and regularly monitored for egg production. At 18 days after the start of spawning, water samples were removed from all three tanks at 130 min intervals over a period of 24 h. At the end of spawning, water samples were removed at 4 h intervals. Free and conjugated steroids were extracted from the water and measured by radioimmunoassay. The highest number of eggs released, percentage of viable eggs, number of spawnings, spawning index, and relative fecundity were observed in Group B (1 female:1 male). The sex steroids testosterone (T), 11-ketotestosterone (11-KT), 17,20beta-P, and 17,20beta, 21-trihydroxy-pregn-4-en-3-one could be readily detected in water from all tanks. Concentrations of the conjugated forms (sulphate and glucuronide) were higher than those of the free form--with the exception of sulphated T and 11-KT. The concentrations of glucuronidated 17,20beta-P, sulphated 17,20beta-P, and free T appeared to be associated with the number of females in the tank and glucuronidated 11-KT concentrations with the number of males. In the first set of water samples (taken during the spawning period) there was also evidence for a cycle in steroid release with an acrophase during the afternoon and early evening, a few hours before the actual act of spawning. We were not able to show any association between steroid concentrations and success of spawning. However, since the data were derived from a "snapshot" of just a single day in what was a prolonged spawning period, this was perhaps not surprising. The results from the present study will help us to design experiments that are more able to answer this question.