Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17beta on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.     

Year-to-year differences in plasma levels of steroid hormones in pre-spawning chum salmon

Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST.     

Effects of insulin-like growth factor I on GnRH-induced gonadotropin subunit gene expressions in masu salmon pituitary cells at different stages of sexual maturation

Effects of insulin-like growth factor I (IGF-I) and salmon gonadotropin-releasing hormone (sGnRH) on expression of gonadotropin (GTH) subunit genes were examined using primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Fishes were assessed at three reproductive stages, i.e., in April (early maturation), in June (maturing), and in September (spawning). Amounts of GTH subunit mRNAs in pituitary cells were determined using real-time PCR after incubation with IGF-I and/or sGnRH. IGF-I alone had almost no effects on three GTH subunit mRNAs in both sexes, except for decrease in follicle-stimulating hormone (FSH) beta mRNA in males in June. sGnRH alone was effective in stimulation of FSHbeta and luteinizing hormone (LH) beta gene expression in males in April. Thereafter it had no significant effects on GTH subunit mRNAs, although in September it tended to increase FSHbeta and LHbeta mRNAs in females. Co-administered IGF-I counteracted the sGnRH-induced expression of FSHbeta and LHbeta genes in males in April, but not in females in September. These results suggest that IGF-I is involved in direct regulation of GTH subunit genes during sexual maturation. In particular, IGF-I differently modulates sGnRH-induced GTH subunit gene expression, depending on reproductive stages.     

Effects of endocrine disrupters on the expression of growth hormone and prolactin mRNA in the rainbow trout pituitary

It is now widely accepted that chemical pollutants in the environment can interfere with the endocrine system of animals, thus affecting development and reproduction. Some of these endocrine disrupters (EDs) can have estrogenic or antiestrogenic effects. Most studies to date have focused on the effects of EDs on the reproductive system and sex hormones and only limited information exists on how EDs may affect pituitary gland function. A rainbow trout (Oncorhynchus mykiss) pituitary gland culture system was used for studying the effects of EDs on growth hormone (GH) and prolactin (PRL) mRNA expression. We determined that the pituitary glands actively synthesized and secreted GH and PRL over the experimental time-course. In addition, we found that treatment with 17beta-estradiol (positive control) increased levels of GH and PRL mRNA, in a concentration-dependent manner. Treatment of pituitary glands with 500 and 1000 nM of a xenoestrogen, o,p'-DDT (o,p'-dichlorodiphenyltrichloroethane), resulted in a significant induction of GH and PRL mRNA, with a 20-fold increase for PRL and 3-fold increase for GH following treatment with 1000 nM o,p'-DDT. Co-incubation of pituitary glands with ICI 182 780 (a selective estrogen receptor antagonist) and o,p'-DDT resulted in inhibition of PRL mRNA levels; however, the stimulatory effect of DDT on GH mRNA was not seen in this experiment, nor was the inhibitory effect of ICI 182 780 observed with GH mRNA. To the contrary, ICI 182 780 (2.5 nM) had a stimulatory effect on GH mRNA levels. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which is known to exert antiestrogenic effects, had an estrogenic-like effect that resulted in a concentration-dependent increase in the levels of GH and PRL mRNA. Co-incubation of pituitaries with TCDD and alpha-napthoflavone (ANF), which is an inhibitor of the aryl hydrocarbon receptor (AhR), caused an inhibition of TCDD-induced PRL mRNA at the higher and lower concentrations, but these effects were less consistent on GH mRNA levels. However, the responses of PRL and GH mRNA to co-incubation with TCDD and ANF, at the various concentrations, were bi-phasic wherein stimulation was seen at the low concentrations and inhibition at the high concentrations. Combined, these results suggest that o,p'-DDT and TCDD are xenoestrogens and that their effects on the expression of GH and PRL genes in the rainbow trout pituitary are modulated, in part, through the ER and AhR, respectively.     

Effects of sex steroids,sex, and sexual maturity on cortisol production: an in vitro comparison of chinook salmon and rainbow trout interrenals

Sex steroids appear to be responsible for hyperactivation of the hypothalamus-pituitary-interrenal (HPI) axis that occurs in mature semelparous Pacific salmon as a prelude to post-spawning (programmed) death. This study was undertaken to examine the direct effects of sex steroids on interrenal activity of semelparous (chinook salmon) and iteroparous (rainbow trout) salmonids using an in vitro incubation system. In addition, phenotypic sex differences in cortisol production by interrenals of sexually mature (spawning) rainbow trout and chinook salmon were investigated. Interrenal tissue from juvenile and sexually mature chinook salmon and rainbow trout was incubated for 48 h in culture medium containing either no steroid (controls), 1 microM estradiol (E2) or 1 microM 11-ketotestosterone (11-KT). This tissue was then challenged for 3h with either pregnenolone, dibutyryladenosine 3('):5(')-cyclic monophosphate (dbcAMP) or forskolin, or synthetic human adrenocorticotropic hormone (ACTH(1-24)). Sex differences in in vitro interrenal cortisol production were assessed using separate tissue pools challenged with the same agents. Cortisol in media was measured by radioimmunoassay. E2 suppressed the ability of juvenile chinook salmon interrenals to utilize pregnenolone as substrate for cortisol synthesis. In mature female chinook salmon the suppressive effect of E2 was less pronounced, but was observed as a reduced response of interrenals to both pregnenolone and dbcAMP. E2 did not affect ACTH(1-24) stimulated cortisol production. Immature and mature rainbow trout interrenals were both relatively insensitive to E2. 11-KT did not affect cortisol production by juvenile chinook salmon and juvenile or mature rainbow trout, and had only minor effects in male and female spawning chinook salmon. In mature chinook salmon and rainbow trout, the interrenals of females were more responsive to ACTH stimulation and showed a greater utilization of pregnenolone as a substrate than interrenals of males. Mature female rainbow trout were also more responsive to dbcAMP stimulation than males. The results of this study suggest that the onset of sexual maturation and gonadal steroid production may contribute to sexually dimorphic cortisol responses in vitro.     

Seasonal changes of responses to gonadotropin-releasing hormone analog in expression of growth hormone/prolactin/somatolactin genes in the pituitary of masu salmon

Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/SL genes, in the pituitaries of growing and maturing masu salmon (Oncorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones.     

Changes in expression of genes encoding gonadotropin subunits and growth hormone/prolactin/somatolactin family hormones during final maturation and freshwater adaptation in prespawning chum salmon

The pituitary levels of mRNAs encoding gonadotropin (GTH) subunits (GTH alpha2 and IIbeta), prolactin (PRL), and somatolactin (SL) increased in chum salmon during the last stages of spawning migration. In the present study, changes in pituitary levels of mRNAs encoding GTH alpha2, Ibeta, and IIbeta; growth hormone (GH); PRL; and SL were examined in homing chum salmon of Sanriku stock to clarify whether the changes are associated with final maturation or freshwater (FW) adaptation. In 1993, fish were caught at four areas: off the coast of Sanriku (off-coast), the mouth of Otsuchi Bay (ocean), inside of Otsuchi Bay (bay), and the Otsuchi River (river). In addition, effects of hypoosmotic stimulation by transition from seawater (SW) to FW were examined in 1994 and 1995. The amounts of mRNAs were determined by dot-blot analyses or real-time polymerase chain reactions. The levels of GTH alpha2 and IIbeta mRNAs in the ocean, bay, and river fish were two to five times those in the off-coast fish, and the levels of SL mRNAs in the bay fish were two to four times those in the off-coast fish. The levels of GH and PRL mRNAs in the ocean and bay fish were significantly lower than those in the off-coast fish, and those in the river fish were three to five times those in the ocean and bay fish. In the SW-to-FW transition experiment in 1994, the levels of GTH alpha2, Ibeta, and IIbeta mRNAs transiently increased, whereas changes were insignificant in 1995. The levels of GH, PRL, and SL mRNAs increased in both SW and FW environments, and no apparent effects of SW-to-FW transition were observed. The present study suggests that in prespawning chum salmon, expression of genes encoding GTH alpha2, IIbeta, and SL elevates with final maturation regardless of osmotic environment. Hypoosmotic stimulation by transition from the SW-to-FW environment is not critical to modulate expression of genes for PRL. PRL gene expression can be elevated in SW fish that were sexually almost matured.     

Seasonal changes in expression of genes encoding five types of gonadotropin-releasing hormone receptors and responses to GnRH analog in the pituitary of masu salmon

Five types of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, R2, R3, R4, and R5, are expressed in the brain and pituitary of masu salmon (Oncorhynchus masou). In the present study, seasonal changes in the expression of these five genes were examined in the pituitary to elucidate their roles in GnRH action during growth and sexual maturation. In addition, the seasonal variation of these genes in response to GnRH was examined in a GnRH analog (GnRHa) implantation experiment. Pituitary samples were collected 1 week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single pituitaries was determined by real-time PCR assays. Among the five genes, R4 was predominantly expressed in the pituitaries. In the immature fish, the amount of GnRH-R mRNA varied with seasons and subtypes. In the pre-spawning period, R1 and R4 mRNAs in both sexes and R2 and R3 mRNAs in the females increased 4- to 20-fold and then decreased in the spawning season. The effects of GnRHa treatment were significantly different in both sexes. In the females, GnRHa tended to elevate the expression of all the subtypes of GnRH-R genes in various stages during the experimental period, whereas it had almost no apparent effects in the males. These results indicate that the expression of the five GnRH-R genes is seasonally variable and may be related to the responses of the pituitary hormone genes to GnRH, and the regulation of GnRH-R genes by GnRH is different in both sexes.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.     

Effects of AMPK activation and combined treatment with AMPK activators and somatostatin on hormone secretion and cell growth in cultured GH-secreting pituitary tumor cells

We investigated the effects of the AMPK activator AICAR as compared to somatostatin-14 on cell viability and GH secretion in human GH-secreting pituitary adenomas in vitro and in rat GH3 cells.     

Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia,Oreochromis mossambicus

We investigated in vitro effects of insulin-like growth factors (IGF-I and -II) on growth hormone (GH) and prolactin (PRL) release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Pituitaries were removed from freshwater-acclimated adult males and incubated for 2-24h in the presence of human IGF-I or -II at doses ranging from 1-1000 ng/ml (0.13-130 nM). IGF-I at concentrations higher than 10 ng/ml and IGF-II higher than 100 ng/ml significantly inhibited GH release after 8, 16, and 24h. No effect of IGFs was seen during the first 4h of incubation. IGFs at the same concentrations also significantly attenuated GH gene expression after 24h, although no effect was seen at 2h. By contrast, PRL(188) release was stimulated significantly and in a dose-related manner by IGF-I at concentrations higher than 10 ng/ml and by IGF-II at concentrations higher than 100 ng/ml within 2h. No stimulation was observed after 4h. Similarly, both IGFs at concentrations higher than 10 ng/ml increased PRL(177) release within 2h. However, no significant effect of IGF-I or -II was observed on mRNA levels of both PRLs after 2 and 24h at all concentrations examined. These results clearly indicate differential regulation of GH and PRL release and synthesis by IGFs in the tilapia pituitary, i.e., rapid-acting, stimulatory effects of IGFs on PRL release and slow-acting, inhibitory effects on GH release and synthesis.     

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.     

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia,Oreochromis mossambicus

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.