Effects of GnRH and PACAP on gonadotropin secretion and subunit messenger RNAs.
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Animals↗
Biomedical subjects
S J Winters
Publications and source records attributed to S J Winters.
Exercise increases serum testosterone and sex hormone-binding globulin levels in older men.
We examined the effects of moderate physical activity on serum luteinizing hormone (LH), sex hormone-binding globulin (SHBG), and testosterone levels in seven sedentary but otherwise healthy men aged 66 to 76 years (mean +/- SD, 70 +/- 4). Blood samples were obtained at 10-minute intervals for 4 hours before, during, and 4 hours after 60 minutes of cycle ergometry. Blood samples were also obtained every 10 minutes for 9 hours during a separate day to control for normal diurnal variation in serum testosterone levels. Serum testosterone increased 39%, SHBG 19%, total serum protein 13%, and the free testosterone index 23% during exercise (P < .01 for all). Testosterone and SHBG levels during the 4-hour sampling period after exercise were similar to values obtained before exercise and on the morning and afternoon of the control day. LH concentrations were unaltered during or after exercise. The change in SHBG levels during exercise correlated positively with the change in testosterone concentrations (r = .74, P = .09). We conclude that short-term exercise produces a transient elevation in serum testosterone levels in elderly men, which is partly due to an increase in serum SHBG concentrations. The concomitant increase in total protein and the rapid return of total protein and SHBG to baseline values after exercise indicate that hemoconcentration partly contributes to the exercise-associated increase in circulating testosterone levels.
Aged↗
Relationship between gonadotropin subunit messenger ribonucleic acid levels and plasma gonadotropin concentrations in intact and orchidectomized adult rats.
To gain further insight into the factors regulating FSH, LH, and uncombined alpha subunit secretion in the male rat, pituitary levels of the gonadotropin subunit mRNAs and plasma levels of FSH, LH, and alpha subunit were measured in 7-wk-old intact male rats and rats castrated 2 wk previously. A highly positive correlation was found between pituitary FSH beta mRNA levels and plasma FSH concentrations in intact (r = 0.60; p = 0.039) but not in castrated rats (r = -0.13; p = 0.66). On the other hand, neither pituitary alpha subunit nor LH beta mRNA levels were predictive of plasma alpha subunit or LH concentrations in intact or orchidectomized rats. This observation is consistent with the proposal that a pretranslational mechanism is the major determinant of FSH secretion in the intact adult male rat but not in the castrated male.
Animals↗
Effects of continuous inhibin administration on gonadotropin secretion and subunit gene expression in immature and adult male rats.
To begin to examine inhibin as a male contraceptive, recombinant human (rh) inhibin-A was administered for 3 days via osmotic minipumps to male rats. Doses of inhibin of 0-150 mg/kg per day did not produce a concentration-dependent suppression of FSH secretion or pituitary FSH beta mRNA levels in adult rats. Treatment of immature rats at a dose of 145 micrograms/kg per day, which was without effect in adults, reduced plasma FSH levels by 49% (p < 0.01), and FSH beta mRNA levels to 47 +/- 11% of control (p < 0.01). Inhibin also decreased levels of LH beta mRNA (63 +/- 8% of control; p < 0.01), alpha-subunit mRNA (86 +/- 10% of control; p < 0.05), and GnRH-receptor mRNA (77 +/- 17% of control; p <0.01) in immature rats. Rh inhibin-A was more effective in immature than in adult animals; plasma inhibin levels were increased (p = 0.03) by rh inhibin-A treatment only in immature rats. Pharmacokinetic studies revealed that the weight-adjusted clearance was greater (p < 0.01), and the elimination half-life of rh inhibin A was shorter (p < 0.01) in adult than in juvenile rats. These data indicate that partial suppression of FSH beta mRNA inhibin is associated with a decline in GnRH receptor gene expression, suggesting that the notion that inhibin can act as a male contraceptive through selective and complete inhibition of FSH production, without effect on LH and Leydig cell function, may be mistaken. In addition, increased inhibin clearance appears to contribute to the fall in plasma inhibin levels with maturation in the male rat.
Aging↗
Regulation of alpha-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and alpha T3-1 cells.
Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of alpha-subunit protein characteristic of the pituitary. The present study used primary pituitary cell cultures and the alpha T3-1 pituitary cell line to investigate how PACAP affects alpha-subunit mRNA transcripts. Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased alpha-subunit mRNA levels 1.5-fold, whereas GnRH more effectively (P<0.01) stimulated alpha-subunit protein release than did PACAP38 (3.2- vs. 2.0-fold). alpha-Subunit mRNA levels in alphaT3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h (P<0.05), and alpha-subunit protein secretion rose proportionately and in parallel with alpha-subunit mRNA levels. PACAP38 was a 100-fold more potent stimulator of alpha-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors. Type I receptor mRNA transcripts were identified by Northern analysis in alphaT3-1 cells. Depletion of PCK activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing alpha-subunit mRNA levels and alpha-subunit secretion. PACAP38, like 8Br-cAMP and forskolin, stimulated (P<0.05) luciferase (LUC) activity in alphaT3-1 cells transfected with a plasmid containing the first 846 of 180 base pairs of the 5'-flanking region of the human alpha-subunit gene linked upstream to a LUC reporter gene. Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change alpha-subunit mRNA half-life. These findings are consistent with the proposal that PACAP contributes to the high levels of alpha-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating alpha-subunit gene transcription in part by the cAMP/PKA pathway.
8-Bromo Cyclic Adenosine Monophosphate↗
Effects of pulsatile pituitary adenylate cyclase activating polypeptide (PACAP) on gonadotropin secretion and subunit mRNA levels in perifused rat pituitary cells.
The effects of pituitary adenylate cyclase activating polypeptide (PACAP) administered intermittently on gonadotropin secretion and subunit mRNA levels in anterior pituitary cells from 7wk old intact and orchidectomized rats were studied. Cells were perifused for 9 h with hourly pulses of 2.5 nM GnRH or 10 nM PACAP, or with medium alone. Pulsatile PACAP initiated episodes of LH, FSH and alpha-subunit secretion, but was much less effective than GnRH. Responsiveness declined with repeated pulses of PACAP, although interpulse secretion increased gradually throughout the experiment. PACAP was a slightly more effective stimulator of LH release by pituitary cells from castrated than intact rats. At the completion of perifusion, pituitary RNA from castrated rats was extracted for measurement of gonadotropin subunit mRNA levels. Pulsatile PACAP stimulated alpha-subunit and LHbeta mRNA levels but did not affect FSHbeta mRNA. By contrast, continuous PACAP increased alpha-subunit mRNA levels, but suppressed FSHbeta mRNA without affecting LHbeta mRNA. We conclude that pulsatile PACAP is a relatively ineffective stimulator of gonadotropin secretion when administered alone, and regulates gonadotopin subunit gene expression quite differently than continuous PACAP. We propose that the mode of PACAP secretion in vivo may be a determinant of the differential expression of the gonadotropin subunit genes.
Animals↗
Inhibin in normal and abnormal pregnancy: maternal serum concentration and partial characterization.
OBJECTIVE: This study evaluated maternal serum inhibin concentration and molecular distribution in normal and abnormal pregnancies. STUDY DESIGN: Serum inhibin levels were measured by radioimmunoassay in 33 abnormal pregnancies (23 singleton and 10 multiple gestations) and 33 matched controls and were compared with pregnancy variables. RESULTS: In the controls serum inhibin concentrations rose with gestational age (correlation coefficient 0.358, p < 0.01) and were higher in mothers with female fetuses (p < 0.05). Inhibin levels were higher in the abnormal singleton pregnancies (2.69 +/- 1.41 ng/ml) than in controls (1.26 +/- 0.32 ng/ml, p < 0.0001) and increased more rapidly with gestational age in multiple pregnancies (p < 0.05). Gel filtration chromatographic analysis of immunoreactive inhibin revealed three peaks with the major form having an apparent molecular weight of 55 to 60 kd. CONCLUSION: Maternal serum inhibin concentrations are affected by gestational age, fetal gender, and complications of pregnancy. Most of the immunoreactive inhibin in maternal circulation is a large-molecular-weight form.
Case-Control Studies↗
Replacement with recombinant human inhibin immediately after orchidectomy in the hypophysiotropically clamped male rhesus monkey (Macaca mulatta) maintains follicle-stimulating hormone (FSH) secretion and FSH beta messenger ribonucleic acid levels at precastration values.
This study directly tested the inhibin hypothesis by examining the ability of replacement with recombinant human (rh-) inhibin, either alone or in combination with testosterone (T), to maintain FSH secretion and FSH beta messenger RNA (mRNA) at intact levels after orchidectomy in the hypophysiotropically clamped juvenile rhesus monkey. Thirteen male monkeys (11-21 months of age) received an intermittent i.v. infusion of GnRH (0.1 microgram/min for 3 min every 3 h). After 4-6 weeks of GnRH stimulation, 10 animals were orchidectomized, and 3 monkeys were sham castrated. Hormone replacement was initiated at castration and maintained for 4 days. Three monkeys received a combination of inhibin and T replacement, 4 monkeys received replacement with inhibin alone, and 3 monkeys received T replacement alone. A continuous i.v. infusion of rh-inhibin (832 ng/h.kg) was used to replace the testicular protein, whereas SILASTIC capsules were implanted sc for T replacement. The FSH response to castration and hormone replacement was determined by measuring circulating concentrations of this gonadotropin before a GnRH pulse and for 3 h thereafter on the day before surgery and on days 2 and 4 postcastration. Circulating immunoactive inhibin was measured by a RIA that recognizes the free alpha-subunit of inhibin as well as inhibin dimers. At the end of the study, anterior pituitaries were collected for analysis of steady state levels of FSH beta, LH beta, and alpha-subunit mRNAs. Steroid replacement alone, which produced circulating T concentrations in the upper physiological range, failed to prevent the postcastration increases in circulating FSH concentrations and pituitary FSH beta mRNA levels. In contrast, when circulating immunoactive inhibin in T-replaced monkeys was maintained at precastration levels (approximately 2 ng/ml) by infusion of rh-inhibin, FSH secretion and synthesis were held at control values. When T was omitted from combined replacement, the FSH-suppressing action of the recombinant hormone was not compromised. These results demonstrate that rh-inhibin is biologically active in the monkey, and the action of inhibin to suppress FSH synthesis and secretion does not require a concomitant action of T. Moreover, because the hypophysiotropic drive to the pituitary-testicular axis was clamped, the FSH-suppressing action of rh-inhibin must be at the pituitary.
Analysis of Variance↗
Role of testosterone in the induction of hepatic peroxisome proliferation by clofibrate.
Hepatic peroxisome proliferation is induced by a number of agents, including clofibrate. Sustained proliferation of peroxisomes is associated with the development of hepatocellular carcinoma. In the present study, we have investigated the role of testosterone in peroxisome proliferation induced by clofibrate. Three groups of male rats (intact, castrated, and castrated replaced with testosterone) were studied. Proliferation of peroxisomes was induced by feeding clofibrate (0.25%, 0.50%, and 1.0% of diet) for 2 weeks. Peroxisome proliferation was monitored by measuring total peroxisomal beta-oxidation activity. In intact rats, the peroxisomal beta-oxidation activity (nmol/min/mg protein) increased in a dose-dependent manner and was 7.2 +/- 0.4, 52.6 +/- 7.5, 63.2 +/- 3.7, and 92.4 +/- 4.0 at clofibrate doses of 0%, 0.25%, 0.50%, and 1.0%, respectively. In contrast, in castrated rats, the total peroxisomal beta-oxidation activity was significantly (P < .01) lower at clofibrate levels of 0.25% and 0.50% (25.8 +/- 2.7 and 42.5 +/- 2.2, respectively), but not at the clofibrate level of 1.0% (85.0 +/- 6.3). Testosterone replacement of castrated rats restored the peroxisomal beta-oxidation activity. To determine whether the above results were related to the metabolism of clofibrate in the absence or presence of testosterone, we measured serum clofibrate levels. These levels were 50% lower in castrated rats than in intact rats or in testosterone-treated castrated rats. The activity of hepatic uridine diphosphate (UDP)-glucuronyltransferase, the enzyme catalyzing the glucuronidation of clofibrate, was measured using either bilirubin or 4-methylumbelliferone as substrates and was found to be unaffected by castration or testosterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary cells.
Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Pattern of oxytocin concentrations in the peripheral blood of healthy women and men: effect of the menstrual cycle and short-term fasting.
A sensitive and specific radioimmunoassay was used to measure basal oxytocin (OT) concentrations in samples of peripheral blood drawn q 10 min for 8 hrs from women during the follicular and luteal phases of the menstrual cycle and from men before and after 20 hrs of fasting. Few, if any, pulses of OT were found in the blood of men and women in this study. The pattern of OT concentrations in the circulation was the same during both stages of the menstrual cycle and was not affected by short-term fasting. The use of frequent sampling and the present assay system for OT did not detect a pulsatile pattern of OT concentrations in the blood of men and women in the basal state.
Adult↗
Endocrine evaluation of testicular function.
This article reviews currently available endocrine tests that are used to evaluate adult testicular function. In many patients, all that is needed is a thorough medical history and physical examination with measurement of total testosterone, FSH, and LH levels. For others, various secondary tests are appropriate. The usefulness and pitfalls of these endocrine tests are discussed.
Estrogens↗
Endocrine consequences of weight loss in obese, hyperandrogenic, anovulatory women.
OBJECTIVE: To determine whether weight loss in obese, hyperandrogenic, anovulatory women is associated with resumption of ovulation and/or with changes in insulin, androgen, and gonadotropin concentrations. DESIGN: Prospective, randomized, controlled study. SETTING: University research center. PATIENTS: Twelve obese, hyperandrogenic, anovulatory women. INTERVENTIONS: Twelve-week weight loss program in treatment (n = 6); 12-week "waiting list" in control group (n = 6). MAIN OUTCOME MEASURES: [1] Ovulation; [2] fasting insulin and glucose measurements; [3] sex hormone-binding globulin (SHBG), total and non-SHBG T concentrations; [4] LH pulse frequency, amplitude, and concentration; and [5] FSH concentration. RESULTS: In contrast with the control group who showed no change in weight, ovulation status, or hormone levels, women in the treatment group lost an average of 16.2 kg and showed a significant increase in SHBG, a significant decline in non-SHBG T, and a decline (though nonsignificant) in fasting insulin. Four of six subjects resumed ovulation. However, no changes were evident in LH pulse frequency or amplitude or in mean LH and FSH concentrations. CONCLUSIONS: Weight loss in obese, hyperandrogenic, anovulatory women appears to reduce insulin and non-SHBG T concentrations despite the absence of a change in gonadotropin secretion and may lead to resumption of ovulation.
Adult↗
The follicle stimulating hormone-inhibin feedback loop in male primates.
This paper reviews our current knowledge of the role of testicular inhibin in the regulation of follicle stimulating hormone (FSH) secretion in the rhesus monkey. Species differences between monkey and rat are described, and evidence for and against the hypothesis that control of FSH secretion in the human male is similar to that for the monkey is presented.
Animals↗
Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids.
cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.
8-Bromo Cyclic Adenosine Monophosphate↗
Increased luteinizing hormone and alpha-subunit secretion in women with hyperandrogenic anovulation.
Women with hyperandrogenic anovulation (HAA) have increased circulating levels of LH relative to those of FSH. The cause of this disturbance in gonadotropin secretion is uncertain. Previous investigations have sought to determine if increased GnRH drive is responsible for the excessive LH concentrations. Because previous results have conflicted, we addressed this question by comparing the 24-h secretory patterns of alpha-subunit and LH in women with HAA (n = 9) to those in eumenorrheic women in the midfollicular phase (n = 9). The mean (+/- SEM) pulse frequency was increased in women with HAA compared to that in eumenorrheic women of comparable age and percent ideal body weight for both LH (23.0 +/- 0.7 pulses/24 h vs. 3 17.1 +/- 1.7; P = 0.002) and alpha-subunit (23.0 +/- 0.8 vs. 19.1 +/- 1.2; P = 0.02). LH and alpha-subunit, but not FSH, responses to a submaximal dose of exogenous GnRH were increased in HAA, as were basal LH and alpha-subunit levels (P < 0.01). The present observations provide evidence for increased GnRH drive, including pulse frequency, in HAA. Although the results confirm the presence of a disturbance in gonadotropin secretion and suggest that its proximate cause may be of hypothalamic origin, they do not exclude the possibility that other factors, perhaps of ovarian origin, play a role in the establishment and/or maintenance of the altered gonadotropin secretory patterns and the chronic anovulation characteristic of HAA.
Adult↗
Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin.
In primary cultures of rat pituitary cells, inhibin and follistatin reduce steady state levels of FSH beta mRNA to less than 10% of control within 4-6 h, while activin increases this mRNA 2- to 3-fold after 2-4 h of treatment. The effects of these three gonadal polypeptide hormones on the LH beta and common alpha-subunit mRNAs are more gradual and of lesser magnitude. The present study was designed to determine whether inhibin, activin, and/or follistatin act at the posttranscriptional level by altering the stability of the gonadotropin subunit mRNAs. To determine the decay rates of FSH beta, LH beta, and alpha-subunit mRNAs, primary pituitary cell cultures were treated for 1-24 h with either of two transcriptional inhibitors, actinomycin-D or 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB), in the presence or absence of recombinant human inhibin-A, recombinant human activin-A, or purified bovine follistatin. The decay of preexisting gonadotropin subunit mRNAs was followed by Northern blot analysis. Levels of LH beta and alpha-subunit mRNAs remained constant or increased during the 24-h exposure to transcriptional inhibitors; therefore, it was not possible to calculate their half-lives. The stability of these mRNAs was not altered by inhibin, activin, or follistatin. In contrast, FSH beta mRNA turned over rapidly: the estimated half-life was 2.6 +/- 0.19 h (mean +/- SEM of eight determinations) after actinomycin-D treatment and 1.9 +/- 0.14 h (mean +/- SEM of 12 determinations) after DRB treatment. When new RNA synthesis was blocked by either actinomycin-D or DRB, there were no significant effects of inhibin, activin, or follistatin on the stability of FSH beta mRNA (n = 2-4 for each hormone). The decay of FSH beta mRNA in the presence of inhibin or follistatin alone, however, was even more rapid than that determined after the administration of transcriptional inhibitors (P < 0.005). After an initial lag of 1-2 h, the half-life of FSH beta mRNA was 0.88 +/- 0.15 h (n = 4) or 0.62 +/- 0.11 h (n = 3), in the presence of inhibin or follistatin, respectively. The most likely interpretation of these results is that inhibin/follistatin reduces steady state levels of FSH beta mRNA by inducing a labile protein that accelerates the degradation of this mRNA species, and the synthesis of this protein is blocked by actinomycin-D or DRB treatment. It is not clear at present whether inhibin, follistatin, and activin have additional effects on transcription of the gonadotropin subunit genes.
Activins↗
Expanding the differential diagnosis of male hypogonadism.
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Adult↗