Time- and dose-related effects of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the goldfish pituitary

The goldfish brain contains two molecular forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a preliminary report, we demonstrated the stimulation of gonadotropin hormone (GtH) subunit and growth hormone (GH) mRNA levels by a single dose of GnRH at a single time point in the goldfish pituitary. Here we extend the work and demonstrate time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH gene expression in vivo and in vitro. The present study demonstrates important differences between the time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels. Using primary cultures of dispersed pituitary cells, the minimal effective dose of cGnRH-II required to stimulate GtH subunit mRNA levels was found to be 10-fold lower than that of sGnRH. In addition, the magnitudes of the increases in GtH subunit and GH mRNA levels stimulated by cGnRH-II were found to be higher than the sGnRH-induced responses. However, no significant difference was observed between sGnRH and cGnRH-II-induced responses in vivo. Time-related studies also revealed significant differences between sGnRH- and cGnRH-II-induced production of GtH subunit and GH mRNA in the goldfish pituitary. In general, the present study provides novel information on time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels and provides a framework for further investigation of GnRH mechanisms of action in the goldfish pituitary.     

Differing behavioral and endocrinological effects of two female sex pheromones on male goldfish

Ovulatory female goldfish sequentially release at least two sex pheromones: 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) and a mixture of F prostaglandins (PGFs). This study sought to determine whether these pheromones have different endocrinological and behavioral actions and whether the PGF pheromone, which is released by spawning females, is responsible for increasing the gonadotropin (GtH) and milt (sperm and seminal fluid) levels of spawning males. Grouped and isolated males were exposed to combinations of these pheromones, food odor, and spawning and nonspawning females. 17,20 beta P stimulated GtH increases in both grouped and isolated males but had only minor effects on behavior; because its principal function appears to be physiological it may be considered a "primer" pheromone. In contrast, exposue to the PGFs elicited large increases in sexual behavior but increased GtH only when fish were exposed as groups; this pheromone's principal action appears to be behavioral and it should be considered a "releaser" pheromone. Although males had increased GtH and milt levels after 1 hr of spawning, males allowed to interact with nonspawning females also had elevated GtH; thus, behavioral interactions appear capable of elevating GtH in the absence of either pheromone. The existence of an independent behavioral mechanism which stimulates GtH was supported by the fact that males exposed to 17,20 beta P while spawning had GtH levels much greater than males exposed to only one of these stimuli.     

Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin, and estrogen

Infant (5-day-old) male rats were treated with hormonal regimens to alter their exposure to gonadotropins, prolactin (Prl), and estrogen, and the response of testicular endocrine functions was measured. Human chorionic gonadotropin (hCG) or a potent gonadotropin-releasing hormone agonist analog (GnRH-A) resulted in a short-lived decrease of testicular receptors (R) for luteinizing hormone (LH), but no deleterious effects were found on testicular capacity to produce testosterone (T), which is a typical response of the adult testis. Only GnRH-A, through probable direct testicular action, induced a relative blockade of C21 steroid side-chain cleavage that was observed in vitro upon hCG stimulation. Human chorionic gonadotropin treatment, but not GnRH-A treatment, increased testicular Prl-R. GnRH antagonist analog (GnRH-Ant) treatment did not affect testicular LH-R, but decreased Prl-R and testicular T production. Decrease of serum Prl by bromocriptine had no effect on testicular LH-R or Prl-R, but slightly decreased T production in vitro. Ovine Prl increased binding sites for LH/hCG. The postnatal rats were insensitive to negative effects of diethylstilbestrol when monitored by testis weight, T, and LH-R. In conclusion, the responses to changes in the hormonal environment differed greatly between infant and adult testes. Mainly positive effects of elevated gonadotropin and Prl levels were seen on infant rat Leydig cell functions. Likewise, decreased tropic hormone levels, and exposure to estrogen, were ineffective in bringing about the inhibitory actions seen in the adult.     

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.     

On the regulation of rat testicular steroidogenesis. Short term effect of luteinizing hormone and cycloheximide in vivo and Ca2+ in vitro on steroid production in cell-free systems.

An attempt has been made to correlate the rapid effect of luteinizing hormone on testicular steroid production in vivo with testicular steroid concentrations and in vitro steroid production rates in testis tissue preparations. Within 20 min after intravenous administration of 25 mug luteinizing hormone, increases were observed in testosterone concentrations in testicular venous plasma and in whole testis tissue and in pregnenlone concentrations isolated testis mitochondrial fractions. Testosterone production by whole testis homogenates and pregnenolone production by isolated mitochondrial fractions were significantly increased within 5 min after in vivo administration of luteinizing hormone. Injection of cycloheximide 10 min prior to luteinizing hormone prevented the stimulating effect of luteinizing hormone to steroid levels in testicular venous plasma and testis tissue and on steroid production rates by preparations of rat testis tissue. Cycloheximide treatment of control animals did not significantly alter testosterone concentrations and testosterone production rates vitro, although mitochondrial pregnenolone concentrations and production rates were decreased. Testosterone production by whole testis homogenates as well as the pregnenolone production by isolated mitochondrial fractions obtained from luteinizing hormone treated testes and control glands showed a biphasic time curve A period (5-10 min) of high steroid production was followed by a period lower steroid production. Addition of 25 mug luteinizing hormone or 10(-8)--10(-5) M adenosine 3':5'-monophosphate (cyclic AMP) to the incubation medium had no effect pregnenolone production by isolated mitochondrial fractions. Administration of leuteinizing hormone in vivo markedly enhance the stimulating effect of Ca2+ on testosterone production by whole testis homogenates and on pregnenolone production by isolated mitochondrial fractions.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.     

Modulation of goldfish testicular testosterone production in vitro by tumor necrosis factor alpha, interleukin-1beta, and macrophage conditioned media

The relevance of immune-endocrine interactions to the regulation of testicular steroidogenesis in teleosts is virtually unexplored. The objectives of the present study were: 1) to investigate the effects of murine cytokines, tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), and trout (Oncorhynchus mykiss) macrophage conditioned media (MCM) on testosterone (T) production by goldfish (Carassius auratus) testis pieces in vitro; and 2) to identify the site(s) of the inhibitory action of TNFalpha on hCG-stimulated T formation. TNFalpha (0-100 ng/ml) affected basal T production differentially depending on the gonadosomatic index (GSI) value of the fish. TNFalpha stimulated basal T of fish with a relatively low GSI (average 1.99), but inhibited T production by testis of fish with a higher GSI (average 5.14). The remaining studies used fish with only high GSI values. IL-1beta (0-10 ng/ml) inhibited basal T production, while MCM (0-25% v/v) had no effect. The cytokines significantly inhibited hCG-stimulated T production at all doses tested, whereas MCM was inhibitory only at the lower doses of 2.5-5% v/v. TNFalpha did not affect basal or hCG-stimulated cAMP levels, but did inhibit forskolin (0.5 microM; adenylate cyclase activator) and 8-bromo-cAMP (0.15 mM; cAMP analog) stimulated T levels. The inhibitory actions of TNFalpha on T production were greatly reduced by treatment of testis with 25-hydroxycholesterol (1 and 10 microg/ml), pregnenolone (50 and 100 ng/ml), and 17 alpha-hydroxypregesterone (50 ng/ml). TNFalpha caused a moderate decrease in pregnenolone (100 ng/ml)-stimulated T production. Together, these data demonstrate that regulatory actions of TNFalpha may occur at multiple sites within the steroid biosynthetic pathway, but the major effect appears to be related to cholesterol availability in the mitochondria. In conclusion, the results of this study implicate macrophage-derived factors in the regulation of teleost testicular androgen biosynthesis.     

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) β subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshβ mRNA levels in all reproductive stages and lower luteinising hormone β (lhβ) mRNA levels in early recrudescence, whereas gh and fshβ mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish. Mol. Reprod. Dev. 79: 592–602, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

The influence of beta-endorphin on testicular endocrine function in adult rats.

The role of beta-endorphin in testicular steroidogenesis is poorly understood. To address this issue, we treated adult hypophysectomized rats intratesticularly with either saline-50% polyvinylpyrrolidone (SAL-PVP) or human beta-endorphin (0.5 microgram/testis; a total of 1 microgram/rat/day) in SAL-PVP for 3 days. Testicular injections were made under ether anesthesia. On Day 3, rats also received injections (s.c.) of either SAL-PVP or 5 micrograms beta-endorphin in SAL-PVP to minimize the dilution of ether in the testis. One hour later, rats were treated (i.p.) with either saline or ovine LH (25 micrograms/rat). One hour after saline or LH injection, blood was obtained via heart puncture for determination of plasma progesterone (P), androstenedione (A-dione), and testosterone (T) levels. The effects of beta-endorphin (50 ng, equivalent to 13.9 pM; or 250 ng, equivalent to 69.6 pM) on P and androgen secretions in vitro were also examined. Intratesticular injections of beta-endorphin significantly (p less than 0.025) decreased the T response to LH treatment, but failed to affect plasma P and A-dione levels. Response of P to LH treatment was increased (p less than 0.005) in medium containing testicular fragments exposed to 250 ng (69.6 pM) beta-endorphin. However, beta-endorphin attenuated LH effects on A-dione and T production in vitro. These studies demonstrate that beta-endorphin inhibits T secretion, possibly because of its effect on the synthesis of T precursors. Thus, testicular beta-endorphin modulates the endocrine function of the testis in adult rats.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats.

We have examined the role of glucocorticoids in the stress-induced inhibition of testicular steroidogenesis. Immobilization (3 hr) reduced plasma testosterone (T) levels to 24% of control values but did not affect plasma LH levels. This reduction was partially reversed by in vivo injections of the antiglucocorticoid, RU486, prior to the stress session at a dose of 10 mg/kg BW, but not at 1.0 or 50 mg/kg BW. Stressed rats that were treated with 10 mg/kg BW RU486 had twofold higher plasma T levels than vehicle-treated stressed animals. Injections of RU486 did not affect plasma LH levels in control or stressed rats and did not affect T levels of unstressed rats. Stressed rats had eightfold higher plasma corticosterone levels than controls, and RU486 had no effect on control or stress levels of corticosterone. The possible role of glucocorticoids in mediating the effect of stress on testicular T production was investigated also in vitro by incubating testicular interstitial cells from unstressed rats for 3 hr with corticosterone (0, 0.01, 0.1, or 1.0 microM) or dexamethasone (0, 0.001, 0.01, or 0.1 microM), followed by an additional 2 hr with hCG (0, 25, 50, or 100 microIU). Both corticosterone and dexamethasone inhibited hCG-stimulated T production in a dose-dependent manner. Cells incubated with the highest concentration of either of the glucocorticoids showed significantly reduced responses to hCG stimulation. In the absence of hCG, in vitro T production was not affected by dexamethasone or 0.01 and 0.1 microM corticosterone. However, the highest dose of corticosterone (1.0 microM) produced a 63% elevation in basal T production. Coincubation of testicular interstitial cells with corticosterone (1.0 microM) or dexamethasone (0.1 microM) and RU486 (0.01, 0.1, and 1.0 microM) reversed the glucocorticoid-induced suppressions of T production in a dose-dependent manner. Our results suggest that during stress increases in plasma levels of glucocorticoids in male rats act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins, and that the decline of testosterone production during immobilization stress is in part mediated by a direct action of glucocorticoids on the testis.     

Involvement of protein kinase C and arachidonic acid pathways in the gonadotropin-releasing hormone regulation of oocyte meiosis and follicular steroidogenesis in the goldfish ovary

The involvement of protein kinase C (PKC) and arachidonic acid (AA) pathways were investigated in the GnRH regulation of oocyte meiosis and follicular testosterone production in the goldfish ovary. The results clearly demonstrate differences in the postreceptor mechanisms involving the stimulatory and inhibitory actions of GnRH peptides on basal and gonadotropin (GtH)-induced reinitiation of oocyte meiosis and steroidogenesis. In isolated goldfish follicles in vitro, the observed stimulatory effects of both salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) on germinal vesicle breakdown were completely blocked by addition of PKC inhibitors, suggesting the involvement of PKC, presumably through activation of phospholipase C/diacylglycerol pathways in the GnRH-induced reinitiation of oocyte meiosis. Administration of an AA metabolism inhibitor, however, only blocked the stimulatory effect of sGnRH without affecting cGnRH-II-induced meiosis. As observed previously, in the presence of GtH, sGnRH was found to inhibit GtH-induced resumption of meiosis and testosterone production, whereas cGnRH-II was without effect. The inhibitory effect of sGnRH on GtH-induced meiosis and steroidogenesis was completely reversed by addition an AA metabolism inhibitor, whereas PKC inhibitors had no effect. These findings provide functional evidence in support of the novel hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity mediating stimulatory and inhibitory actions of sGnRH and cGnRH in the goldfish ovary.     

高效能的促黄体素释放激素类似物对金鱼促性腺激素分泌的作用

金鱼对LRH-A反应有明显的季节性变化,在早春(2月)生殖季节开始前诱导的血清GtH含量最高;在生殖季节过后(5—6月)GtH的释放反应减弱,而在夏季(8月)性腺处于退化状况时,反应很弱或者没有反应。LRH-A注射剂量的高低对血清GtH含量增高的影响并不都很明显,但高剂量(1.0微克/克体重)能诱导较高的血清GtH含量并能维持24小时。经过多次注射高剂量以后,脑垂体GtH含量下降。温度、注射剂量、注射间隔时间都和诱导的血清GtH含量有密切关系。连续9—10天每日注射LRH-A,能刺激性腺退化的金鱼恢复性腺发育,但对性腺已开始发育的金鱼,却抑制或减慢其性腺进一步发育。       

Characterization of parameters for in vitro culture of isolated ovarian follicles of greenback flounder Rhombosolea tapirina

Isolated ovarian follicles of greenback flounder Rhombosolea tapirina were incubated with a variety of gonadotropins (GtHs) and steroid precursors for periods of up to 42 h, and levels of free and glucuronated testosterone (T) and 17beta-estradiol (E(2)) in the medium, and free T and E(2) from inside follicles were measured by RIA. Short incubations (6 h) generated increases in T and E(2) in response to steroid precursors, but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs stimulated T and, or E(2) production, whereas after 42-h incubation, GtH effects on E(2) production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E(2) glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Instead, this could be explained by accumulation of E(2) in controls as a result of continued basal steroid production. Follicles absorbed substantial amounts of both endogenous and exogenous steroid from the medium, however, this did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed by salmon and carp GtH at approximately 10-fold higher concentrations. Ovarian segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor under certain in vitro conditions. HCG augmented the conversion of 17-hydroxyprogesterone (17P) to T but not T to E(2), consistent with the relative GtH-insensitivity of aromatase in other species. Follicles converted a range of steroid precursors with equal competence, indicating that no step in the cleavage pathway is strongly rate-limited, and that choice of precursor is unlikely to affect the assessment of steroidogenic activity.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.