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)     

Influence of gonadotropin-releasing hormone pulse amplitude, frequency, and treatment duration on the regulation of luteinizing hormone (LH) subunit messenger ribonucleic acids and LH secretion

The effects of GnRH pulse amplitude, frequency, and treatment duration on pituitary alpha and LH beta subunit mRNA concentrations were examined in castrate-testosterone replaced male rats. Experimental groups received iv GnRH pulses (5, 25, or 125 ng) at 7.5-, 30-, or 120-min intervals for 8, 24, or 48 h. Saline pulses were given to control rats. Acute LH secretion was measured in blood drawn before and 20 min after the last GnRH pulse. In saline controls, alpha and LH beta mRNAs (150 +/- 14, 23 +/- 2 pg cDNA bound/100 micrograms pituitary DNA) fell to 129 +/- 14 and 18 +/- 2, respectively, after 48 h. In animals receiving GnRH pulses (7.5-min intervals), the 125-ng dose stimulated a slight increase (P less than 0.01) in alpha mRNA levels after 8 and 24 h and both LH subunit mRNAs were increased by the 25- and 125-ng doses after 48 h. The 30-min pulse interval injections (25- and 125-ng doses) increased LH beta mRNA levels after 8 h, but alpha mRNAs were not elevated until after 24 h. Maximum (3-fold) increases in alpha and LH beta mRNAs were seen in rats receiving 25-ng pulses every 30 min for 48 h. Using 120-min pulses, LH subunit mRNAs were not increased by any GnRH dose through 48 h. Acute LH release was not seen in rats receiving 5 ng GnRH pulses at any pulse interval.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a gonadotrophin-releasing hormone antagonist on gonadotrophin secretion and gonadal development in neonatal pigs

Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-D2Nal1,D4-Cl-Phe2,D-Trp3,D-Arg6, D-Ala10]- GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P less than 0.01) from 1.7 ng/ml at 0 h to less than 0.5 ng/ml during 4-32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 +/- 8 to 56 +/- 5 ng/ml (P less than 0.05) at 32 h. In males FSH was low (5.7 +/- 0.5 ng/ml) at 0 h and did not change significantly. To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P less than 0.01) to 0.2-0.4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P less than 0.001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P less than 0.05), but not on Day 24. Treatment of females with the GnRH antagonist did not influence (P greater than 0.10) oestradiol-17 beta concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17 beta values (P less than 0.01) and reduced testicular weight (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The frequency of gonadotropin-releasing hormone secretion regulates expression of alpha and luteinizing hormone beta-subunit messenger ribonucleic acids in male rats

The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate, testosterone-replaced male rats. GnRH pulses (25 ng/pulse) or saline to controls, were given via a carotid cannula at intervals of 7.5-240 min for 48 h. alpha and LH beta mRNA concentrations were 109 +/- 23 and 30 +/- 5 pg cDNA bound/100 micrograms pituitary DNA, respectively, in saline controls. GnRH pulse intervals of 15, 30, and 60 min resulted in elevated alpha and LH beta mRNAs (P less than 0.01) and maximum responses (4-fold, alpha; 3-fold, LH beta) were seen after the 30-min pulses. Acute LH release to the last GnRH pulse was seen after the 15-, 30-, and 60-min pulse intervals. In contrast, LH subunit mRNAs were not increased and acute LH release was markedly impaired after the rapid (7.5 min) or slower (120 and 240 min) pulse intervals. Equalization of total GnRH dose/48 h using the 7.5- and 240-min intervals did not increase LH subunit mRNAs to levels produced by the optimal 30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates expression of alpha and LH beta mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit mRNA concentrations are associated with continuing LH responsiveness to GnRH.     

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.     

Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit

The feedback effects of two ovarian steroids, estradiol-17 beta (E2) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha OH), were examined in both intact (INT) and ovariectomized (OVEX) does. We measured steroid-induced alterations in endogenous gonadotropin-releasing hormone (GnRH) from sequential 10-min samples of hypothalamic perfusates, simultaneous changes in peripheral plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the modification of pituitary responsiveness, i.e., increments in plasma LH (delta LH) and plasma FSH (delta FSH), after 50 ng, 250 ng, and 1 microgram of exogenous GnRH in individual does of 6 treatment groups. The groups were: INT does, OVEX does, OVEX does receiving either one (1 E2) or two (2 E2) E2-filled Silastic capsules, OVEX does receiving a 20 alpha OH-filled capsule (20 alpha OH), and OVEX does receiving both capsules of E2 and 20 alpha OH (1 E2 + 20 alpha OH). Ovariectomy enhanced the pulsatile release of hypothalamic GnRH and pituitary LH and FSH, and increased the LH response (delta LH) to exogenous GnRH (OVEX vs. INT, p less than 0.05). Replacement of E2 at the time of ovariectomy prevented the increased GnRH and gonadotropin secretion as well as the enhanced delta LH that were observed in untreated OVEX does. The release of hypothalamic GnRH in the 20 alpha OH group was lower (p less than 0.05) than that in the OVEX group and not different from that in the INT group. The release of pituitary LH and FSH and the delta LH in the 20 alpha OH group was not different from that in the OVEX group, but these parameters were greater (p less than 0.05) than those in the INT group. The hypothalamic GnRH pulse frequency in the 1 E2 + 20 alpha OH group was lower (p less than 0.05) than that in either the 1 E2 or the 20 alpha OH group, but the delta LH in the 1 E2 + 20 alpha OH group was not different from that in either the 1 E2 or the 20 alpha OH group. The highest dose (1 microgram) of exogenous GnRH stimulated a modest increase in FSH in the OVEX, 20 alpha OH, 1 E2 + 20 alpha OH, and 1 E2 groups; but a steroid effect on delta FSH among these 4 groups was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

The GnRH precursor is present in the anterior pituitary gland and an important increase of its content precedes serum LH surge induced by estradiol-17 beta in female primates

Estradiol-17 beta (E2 17 beta) is well known to evoke a preovulatory-like LH surge in ovariectomized monkeys even in the absence of the integrity of the hypothalamo-pituitary connections. LH release from the anterior pituitary (AP) is reliant on stimulation by hypothalamic GnRH which is derived from proteolytic cleavage of a precursor (designated Pro-GnRH-GAP) which also results in the production of an associated peptide (GAP). The present study examined the effects of E2 17 beta on the hypothalamic content of Pro-GnRH-GAP, GnRH and GAP while incidental observations revealed the presence of Pro-GnRH-GAP and its products in the AP. Changes in GnRH and GAP were closely related at all times after E2 17 beta treatment. However, the pattern of change in the hypothalamus and AP was inversely related. Pro-GnRH-GAP levels remained unchanged in the hypothalamus whereas in the AP the peptide increased markedly (48 hrs. post E2 17 beta) prior to the LH surge and declined to low levels (72 hrs. post E2 17 beta) at the time of the LH surge. The increase in Pro-GnRH-GAP in the AP that precedes the rise in GnRH and accompanying LH surge by 24 hrs. strongly indicates that AP GnRH is more important than hypothalamic GnRH for the mediation of the E2 17 beta-induced LH surge in female primate.     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Estradiol-17beta triggers luteinizing hormone release in the protandrous black porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotropin-releasing hormone control.

We investigated the mechanism of estradiol-17beta (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17beta also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17beta potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2) induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2in the sex change observed in the protandrous black porgy.     

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.     

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.     

Neuroendocrine consequences of prenatal androgen exposure in the female rat: absence of luteinizing hormone surges, suppression of progesterone receptor gene expression, and acceleration of the gonadotropin-releasing hormone pulse generator

Preovulatory GnRH and LH surges depend on activation of estrogen (E2)-inducible progesterone receptors (PGRs) in the preoptic area (POA). Surges do not occur in males, or in perinatally androgenized females. We sought to determine whether prenatal androgen exposure suppresses basal or E2-induced Pgr mRNA expression or E2-induced LH surges (or both) in adulthood, and whether any such effects may be mediated by androgen receptor activation. We also assessed whether prenatal androgens alter subsequent GnRH pulsatility. Pregnant rats received testosterone or vehicle daily on Embryonic Days 16-19. POA-hypothalamic tissues were obtained in adulthood for PgrA and PgrB (PgrA+B) mRNA analysis. Females that had prenatal exposure to testosterone (pT) displayed reduced PgrA+B mRNA levels (P < 0.01) compared with those that had prenatal exposure to vehicle (pV). Additional pregnant animals were treated with vehicle or testosterone, or with 5alpha-dihydrotestosterone (DHT). In adult ovariectomized offspring, estradiol benzoate produced a 2-fold increase (P < 0.05) in PgrA+B expression in the POA of pV females, but not in pT females or those that had prenatal exposure to DHT (pDHT). Prenatal testosterone and DHT exposure also prevented estradiol benzoate-induced LH surges observed in pV rats. Blood sampling of ovariectomized rats revealed increased LH pulse frequency in pDHT versus pV females (P < 0.05). Our findings support the hypothesis that prenatal androgen receptor activation can contribute to the permanent defeminization of the GnRH neurosecretory system, rendering it incapable of initiating GnRH surges, while accelerating basal GnRH pulse generator activity in adulthood. We propose that the effects of prenatal androgen receptor activation on GnRH neurosecretion are mediated in part via permanent impairment of E2-induced PgrA+B gene expression in the POA.     

Biosynthesis of steroids in ovarian follicles of red seabream,Pagrus major (Sparidae,Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.