Biomedical subjects

S J Winters

Publications and source records attributed to S J Winters.

At least 73 records · Page 4Linked to original sources

PubMed1989

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)

Animals↗

PubMed1989

The effect of oxytocin administration upon the pulsatile secretion of luteinizing hormone in humans.

The effect of iv administration of synthetic oxytocin upon the pulsatile pattern of LH secretion was studied in 5 healthy men and 10 healthy women. Five of the women were studied in the follicular phase of a menstrual cycle and the other 5 were studied in the luteal phase of a cycle. Synthetic oxytocin in 0.9% saline or saline alone was administered via continuous iv infusion for 8 h on 2 consecutive days. Infusions were administered using a double-blinded and radomized schedule. The rate of the oxytocin infusion commenced at 1 mU min and was increased 1 mU/min every 40 min to a final rate of 12 mU min. The plasma oxytocin concentration during oxytocin infusion ranged from 2-70 fmol 1. Blood for LH determination was sampled every 20 min in the 5 follicular phase women and every 10 min in the 5 men and 5 luteal phase women. The detect algorithm was used to analyze LH pulsatile secretion. Oxytocin infusion was without significant effect on mean LH, number of LH pulses, or area under the LH curve in men or women studied for the period of observation. Thus it is unlikely that increases in plasma oxytocin regulate the pulsatile secretion of LH in humans.

Adolescent↗

PubMed1988

Effects of inhibin from primate Sertoli cells on follicle-stimulating hormone and luteinizing hormone release by perifused rat pituitary cells.

We used a pituitary cell perifusion system to investigate the time course and selectivity of the inhibin effect on pulsatile GnRH-stimulated LH and FSH release. Dispersed pituitary cells from 7- to 8-week-old male rats were perifused on a Cytodex bead matrix and stimulated with 10 nM GnRH for 2 min every hour for 8-11 h. The addition of a preparation of inhibin partially purified from primate Sertoli cells reduced pulsatile FSH release within 2 h. After removal of inhibin from the perifusion medium, the effect was reversed within 3 h. GnRH-stimulated LH release was also influenced by inhibin, although the decline in LH was less than that in FSH (80 +/- 3% vs. 68 +/- 4% of control; P less than 0.025). Smaller doses of inhibin suppressed GnRH-induced FSH secretion, but had no effect on LH release. Further, prolonged incubation of pituitary cells with inhibin at the higher dose reduced its FSH inhibitory effect and eliminated the effect on LH. These results indicate that inhibin can reduce both LH and FSH secretion in vitro, although the specificity and magnitude of the effect are a function of both the dose and duration of inhibin treatment. Further, the actions of inhibin and GnRH on the pituitary may be interrelated.

Animals↗

PubMed1988

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells.

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.

Animals↗

PubMed1988

Alpha-subunit secretion in men with idiopathic hypogonadotropic hypogonadism.

We studied the regulation of glycoprotein hormone alpha-subunit secretion in four men with idiopathic hypogonadotropic hypogonadism due to presumed GnRH deficiency. Immunoreactive alpha-subunit was present at low but usually detectable levels in blood samples drawn at 10- to 20-min intervals for 12-24 h; however, no characteristic pattern of pulsatile alpha-subunit secretion was found. Serum from each man was examined by gel filtration chromatography. Each sample tested contained an immunoreactive alpha-subunit peak with a slightly lower elution volume than [125I]hCG alpha, as we had previously found in serum from normal men. To determine if this peak represented TSH alpha, two men were treated with 0.3 mg L-T4 daily for 7-14 days. Serum TSH levels decreased to less than 1.5 mU/L, and neither TSH nor alpha-subunit levels increased after the iv administration of 500 micrograms TRH. An alpha-subunit peak that eluted before [125I]hCG alpha was again found in the serum of T4-treated men. We conclude that glycoprotein hormone alpha-subunit is present in the serum of men with idiopathic hypogonadotropic hypogonadism in whom TSH secretion is completely suppressed by L-T4. The gonadotrophs represent the most likely source of this alpha-subunit. The finding of more normal alpha-subunit than LH secretion in these men indicates that the production of the gonadotropin subunits is differentially regulated in man and supports the hypothesis that factors in addition to GnRH regulate alpha-subunit gene expression.

Adult↗

PubMed1988

Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size.

This study was designed to determine whether exogenous hCG alone can complete spermiogenesis in men with isolated hypogonadotropic hypogonadism (IHH). hCG was administered to 22 men with IHH until maximal testicular growth was achieved. Their mean testicular volume increased from 5.5 +/- 1.1 (+/- SE) mL (pretreatment) to 10.8 +/- 1.6 mL (maximum) during treatment (P less than 10(-6)). The maximum mean testicular volume was highly positively correlated with initial volume (r = 0.84; P less than 10(-6)). All men attained normal serum testosterone levels, and 7 of 22 men achieved supraphysiological serum estradiol levels. During hCG treatment, 14 of the 22 men had sperm appear in their semen. Six of 11 men with complete gonadotropin deficiency, defined as an initial mean testicular volume less than 4 mL, became sperm positive during hCG treatment. In contrast, 9 of 11 men with partial gonadotropin deficiency (initial mean testicular volume of 4 mL or more) produced sperm during treatment (P less than 0.001). Sperm concentration was highly positively correlated with both pretreatment (r = 0.65; P less than 0.01) and final testicular volume (r = 0.73; P less than 0.0001). Of 13 men attempting to impregnate their partners, 7 were successful in initiating conception; a total of 8 pregnancies ensued. The sperm concentration at the time of conception was less than 10 million/mL in all but 1 man. Our study demonstrates that hCG, in the absence of exogenous FSH, can complete spermiogenesis in men with partial gonadotropin deficiency. The response to hCG in men with IHH is predicted by the initial testicular volume.

Adult↗

PubMed1987

Identification of inhibin secreted by cynomolgus monkey Sertoli cell cultures.

An inhibin was identified in the media of primary Sertoli cell-enriched cultures from the cynomolgus monkey, Macaca fascicularis, and some of its biochemical properties were studied. Conditioned monkey Sertoli cell culture medium (m-SCCM), when added to pituitary cells from 6-week-old male rats, inhibited the basal secretion of FSH but not that of LH. This specificity was lost after the addition of GnRH; mSCCM inhibited not only FSH but also LH release, determined by both RIA and mouse interstitial cell bioassay, from pituitary cells exposed for 6 h to 10 nM GnRH. FSH-inhibiting activity persisted when m-SCCM was boiled for 30 min, but activity was lost after incubation for 1 h at 37 C with 0.1% trypsin. m-SCCM inhibin activity was completely retained by Concanavalin A-Sepharose and could be eluted with 0.2 M alpha-methyl-D-glucoside. Gel filtration high pressure liquid chromatography with a Superose-12 column revealed inhibin activity between 20-60K, with the greatest activity at 40K. Our results indicate that primate Sertoli cells produce an inhibin-like factor which could play a role in controlling gonadotropin secretion in males.

Animals↗

PubMed1986

Assay of primate seminiferous tubule androgen receptors using [3H]mibolerone.

The synthetic radiolabelled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) was used to characterize androgen receptor binding in the seminiferous tubules from Cynomolgus monkey testis. Mibolerone binding was of high affinity (Kd = 0.6-5.4 nM) and limited capacity (37-50 fmol/mg protein), and was androgen specific. Sucrose density gradient centrifugation using a vertical tube rotor permitted the identification of a 9S molybdate-stabilized receptor under low salt conditions. The receptor bound to DEAE-cellulose. Methyltrienolone, but not mibolerone, also bound to a low affinity high capacity binding site in tubule cytosol, which probably represents glucocorticoid receptor binding, since it could be displaced by excess dexamethasone. However, occupancy of this low-affinity binding site by dexamethasone in an androgen receptor assay with [3H]methyltrienolone lead to a 33% underestimation of receptor binding, which appeared to relate to radioactive decomposition. Mibolerone, as well as methyltrienolone, bound to a progestin-binding protein in seminiferous tubule cytosol. These studies provide methods for the study of seminiferous tubule androgen receptors in subhuman primates and indicate that, due to its greater stability and lack of binding to glucocorticoid receptor, mibolerone is a useful new ligand in the study of androgen receptors in testis and its constituent cells.

Animals↗

PubMed1986

Testosterone and estradiol are co-secreted episodically by the human testis.

In spite of a striking pulsatile pattern of luteinizing hormone (LH) secretion, testosterone (T) fluctuations in peripheral blood in normal adult men are irregular and of low amplitude. To determine whether T secretion by the human testis is episodic, T was measured in blood samples drawn at 15-min intervals for 4 h through a catheter placed in the testicular vein of six men with varicocele-associated infertility. Estradiol (E2) concentrations were also determined in each sample. Each subject released testosterone in well-defined pulses. Gonadal vein T levels ranged from 1 to 1,540 ng/ml. Mean (+/- SE) pulse amplitude was 176 +/- 42 ng/ml, with a frequency of 4.0 +/- 0.3 pulses per 4 h. Testicular vein E2 levels ranged from 0.01 to 6.8 ng/ml. E2 secretory episodes were generally coincident with T pulses, and their amplitudes were highly positively correlated (r = 0.90, P less than 0.01). These results indicate that T secretion by the adult human testis is pulsatile, and suggest a functional relationship between intermittent LH secretion and normal testicular steroidogenesis in men. The failure to appreciate these fluctuations as hormone pulses in peripheral blood may relate to their absolute amplitude and frequency. The concordance between E2 and T pulses suggests that the Leydig cell, under LH control, is the source of most of the E2 secreted by the adult human testis.

Adult↗

PubMed1986

Testicular control of prostaglandin E2 production in rat vas deferens.

Orchidectomy decreased and testosterone (T) replacement restored prostaglandin E2 (PGE2) concentrations in adult rat vas deferens. To explain this finding, phospholipase A (PLase-A) and PG synthetic activity were studied in vas tissue from 9-week-old rats orchidectomized with or without T replacement as well as in rats in which the cauda epididymidis was ligated. PG synthetic activity fell to 3% of intact levels in 14-day castrate rats and was restored to normal by T replacement. Although vas PLase-A activity was also significantly (P less than 0.01) reduced to 38% of the control level in 14-day castrate rats, this change appears in part to reflect a castration-related increase in endogenous phospholipid concentrations. Further, T replacement only partially restored PLase-A activity to 59% of intact levels. Ligation of the cauda epididymidis in intact rats reduced vas PLase-A activity to castrate levels without altering vas T concentration. These results demonstrate both a direct effect of T on the biosynthesis of PGs in rat vas deferens as well as a paracrine effect, which appears to be mediated by a factor(s) other than T. These data suggest the existence of a new mechanism through which testicular products contribute to the function of the vas deferens.

Animals↗

PubMed1986

Seminiferous tubule androgen receptors in experimental cryptorchidism.

As an initial approach to the study of seminiferous tubule androgen receptors in disordered spermatogenesis, cytosol androgen receptors were studied in rats with experimental cryptorchidism. Two weeks after the testis had been repositioned in the abdomen of 6-week-old rats, the animals were hypophysectomized to deplete the testis of androgen, and 1 week later they were killed. Androgen receptor binding was studied in seminiferous tubule cytosol using [3H]methyltrienolone as the radiolabelled probe. The androgen-binding capacity of cryptorchid testis, when expressed as fmol bound/testis, was reduced to 50% of control, in parallel with the decline in testis weight. No change in binding affinity was found. Sucrose density gradient centrifugation using a vertical tube rotor revealed a 9S molybdate-stabilized receptor under low-salt conditions in both cryptorchid and scrotal seminiferous tubule cytosol. Receptor-complex stability studies, analysis by gel filtration and DEAE-cellulose chromatography produced similar results in cryptorchid and scrotal tubules. The mechanism for the reduction in testicular receptor content of an abdominal testis remains to be clarified. The demonstration that testicular androgen receptors can be reduced by cryptorchidism suggests that further studies may indicate the role of receptor binding in testicular function.

Animals↗

PubMed1985

An analysis of the age-related decline in testicular steroidogenesis in the rat.

The effect of aging in rats on serum and intratesticular testosterone levels, microsomal steroidogenic enzyme activities and microsomal cytochrome P-450 was studied. Serum testosterone levels were highest in 11-wk-old rats, declined at age 16 wk and further declined between ages 7 and 21 mo. Intratesticular testosterone levels in 21-mo-old rats were significantly lower than those of the other groups. The activity of 17 alpha-hydroxylase and C17-20 lyase, as well as cytochrome P-450, decreased significantly in 21-mo-old rats. The activity of 17 beta-hydroxysteroid oxidoreductase increased from 11 wk to 16 wk of age and then declined by 21 mo of age to the levels of 11-wk-old animals. Similar changes in delta 5-3,3-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase activities were observed, but were not statistically significant. These results suggest that the decline in testosterone production in old rats is predominantly a result of decreased oxygenase activity. Inasmuch as oxygenases are gonadotropin dependent, our results support the hypothesis that gonadotropin deficiency is the major factor responsible for Leydig cell dysfunction in old rats. Further, the decline in the ratio of 17 alpha-hydroxylase to C17-20 lyase with aging suggests that other factors affect these enzymes as well as the reduction in cytochrome P-450.

Aging↗

PubMed1985

Identification of androgen-binding protein from testis cytosol and sertoli cell culture medium of the cynomolgus monkey, Macaca fascicularis.

The biochemical and immunological properties of monkey androgen-binding protein (mABP) from the cynomolgus monkey, Macaca fascicularis, have been examined in testis cytosol and medium of primary Sertoli cell-enriched cultures. mABP in testis tissue was separated from the serum protein testosterone-estradiol-binding globulin (mTeBG) by affinity chromatography on Concanavalin A-Sepharose (Con A). mTeBG from serum extracts was completely retained by the lectin and could be displaced with buffer containing alpha-methyl-D-glucoside. In contrast, mABP from testis extracts either did not interact with the Con A and appeared in the void volume or was partially retained by the column and could be eluted with buffer alone. A third component retained by the Con A may represent mTeBG contamination, a form of mABP which binds to Con A, or both. The specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the void volume of the Con A column was designated mABP and was further studied. [3H]DHT binding to mABP was saturable and of limited capacity (0.163 +/- 19 pmol/mg protein). Scatchard analysis of the data was consistent with a single class of binding sites with an apparent dissociation constant (Kd) at 4 C of 2.6 +/- 0.2 X 10(-9) M. DHT was the most effective competitor of [3H]DHT binding to mABP, followed by 2-methoxyestradiol, testosterone, estradiol, and cyproterone acetate. Concentrated Sertoli cell culture medium subjected to steady state polyacrylamide gel electrophoresis produced a single peak of specifically bound [3H]DHT with a mobility similar to that of other androgen-binding proteins. [35S]Methionine-labeled medium proteins were immunoprecipitated with a rabbit anti-human TeBG antiserum. Two bands, corresponding to mol wt of approximately 46,000 and 48,000, were observed by fluorography, with the lighter component being more intense. After androgen affinity chromatography of radiolabeled medium proteins, these two bands were again observed on sodium dodecyl sulfate-urea-containing polyacrylamide gels. These results demonstrate that 1) mABP may be separated from mTeBG by lectin affinity chromatography, as in humans; 2) hTeBG and mABP are antigenically related; and 3) mABP consists of subunits of different mol wt in unequal ratios.

Androgen-Binding Protein↗

PubMed1985

Pulsatile secretion of immunoreactive alpha-subunit in man.

We compared the secretion of uncombined alpha-subunit with LH secretion, as measured by RIA in six normal men, an agonadal adult man, and an adult woman with testicular ferminization. Blood samples were drawn every 10 min for 12 h to evaluate pulsatile LH and free alpha secretion. Spontaneous alpha pulses occurred every 120 +/- 60 min (mean +/- SD; range, 40-260) in eugonadal men. The mean alpha pulse amplitude was 0.49 +/- 0.11 ng/ml, representing an increase of 60 +/- 18% above the preceding nadir value. alpha pulses were generally associated with LH pulses, and the amplitudes of concordant alpha and LH pulses were highly positively correlated (r = 0.58; P less than 0.01). The absolute amplitude of concordant alpha peaks was 1.5 +/- 0.7% that of corresponding LH peaks. Additional alpha pulses were noted in each subject for which simultaneous LH fluctuations failed to meet the criteria for pulses. Studies in the agonadal man and in the patient with testicular feminization revealed that the frequency of both spontaneous alpha and LH pulses was increased to approximately twice that in normal men. Gel filtration analysis indicated that all alpha radioimmunoactivity in normal male sera coeluted with [125I]alpha. By contrast, in hypogonadal sera, only 50-60% of alpha immunoreactivity coeluted with [125I]alpha, the remaining activity appeared in the region of LH and FSH and resulted from RIA cross-reactivity. We conclude that uncombined alpha-subunit, as well as LH is normally released into the peripheral blood in discrete pulses. In the absence of exposure to androgen, the frequency of these pulses increases, presumably as a consequence of the accelerated release of LRH. However, alpha RIAs overestimate the concentration of uncombined alpha in hypogonadal sera. We propose that the coanalysis of LH and alpha secretory episodes may prove useful in resolving some of the complexities associated with gonadotropin secretory patterns.

Adult↗

PubMed1985

Evidence for a role of endogenous estrogen in the hypothalamic control of gonadotropin secretion in men.

To examine the mechanism by which endogenous estrogens inhibit gonadotropin secretion in men, blood samples were drawn every 10 min for 12 h in five men before and at the completion of 3 weeks of treatment with the estrogen antagonist clomiphene citrate (50 mg twice daily). Samples were analyzed for LH and alpha-subunit by RIA. Clomiphene produced a 3-fold rise in circulating LH levels, which was associated with a 80% increase in pulse frequency and a 70% increase in pulse amplitude. Immunoreactive alpha-subunit secretion was also pulsatile before and after clomiphene treatment. Mean alpha-levels rose 70%, together with a 39% increase in pulse frequency and a 41% increase in pulse amplitude. Circulating testosterone and estradiol levels increased 2-fold and FSH levels increased 3-fold after clomiphene treatment. Insofar as each LH and uncombined alpha-subunit pulse reflects a LHRH secretory episode, our data indicate that endogenous estrogens tonically restrain the hypothalamic release of LHRH. From these results and those of previous studies, we conclude that estrogens as well as androgens are important in the testicular feedback inhibition of the hypothalamic oscillator that governs pulsatile gonadotropin secretion.

Adult↗

PubMed1985

Studies of the human testis. XIX. Preparation of an antibody to human testosterone-oestradiol-binding globulin and its application to the study of testicular androgen-binding protein.

Human testosterone-oestradiol-binding globulin (hTeBG) was purified from pregnancy serum by ammonium sulphate precipitation, preparative flatbed electrofocusing, Concanavalin A-Sepharose affinity chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex chromatography and preparative polyacrylamide gel electrophoresis. The yield was 0.3 mg of hTeBG with a specific acitivity of 1.1 nmoles DHT bound per mg. An antiserum to TeBG was raised in rabbits. Anti-hTeBG IgG was separated from rabbit TeBG by DEAE-Affi-Gel-Blue chromatography. Anti-hTeBG was titrated using protein A-Sepharose which quantitatively binds IgG and therefore bound [3H]DHT-hTeBG-anti-TeBG complexes. The androgen binding components from human testis were separated on Concanavalin A-Sepharose columns into excluded and retained fractions. The antibody bound both testis fractions with titration curves which paralleled that of TeBG, indicating that these androphilic proteins share common immunodeterminants with hTe-BG. The possibility that these testicular proteins are identical in amino acid sequence to TeBG and differ only in carbohydrate content will require further verification. Finally, these results indicate that antibodies to TeBG can be used to study human testicular androgen-binding protein.

Androgen-Binding Protein↗

PubMed1984

Steroidogenesis in the monkey testis: relationship of enzyme organization to endogenous steroids, steroidogenesis and gonadotropin treatment.

To explore testicular factors which determine steroidogenesis, circulating and intratesticular steroid levels were measured and in vitro steroidogenesis in testicular mines and microsomes was investigated using adult rhesus monkeys before and after treatment with pharmacological doses of hCG or hCG together with hMG. After gonadotropin treatment serum and intratesticular levels of 17 alpha-hydroxyprogesterone as well as end product testosterone were markedly elevated. Dynamic studies revealed that 17 alpha-hydroxyprogesterone produced from pregnenolone was partly metabolized to C19-steroids but almost no 17 alpha-hydroxyprogesterone produced from progesterone was utilized by C17-20 lyase. Androstenedione levels increased in serum after both treatments and were elevated in testis tissue after hCG treatment. Androstenedione accumulated in vitro during incubation of untreated and hCG-treated testes with [3H]pregnenolone and [14C]progesterone. The major portion of the accumulated androstenedione was produced from pregnenolone through dehydroepiandrosterone rather than through 17 alpha-hydroxyprogesterone, indicating differential availability of androstenedione to 17 beta-hydroxysteroid oxidoreductase according to its origin. The 3H/14C ratio in product androstenedione was much greater than the ration in product testosterone in both untreated and hCG-treated testis; in testes of animals treated with hCG together with hMG, the ratios in both products were closely linked. Among the enzymes examined, 17 alpha-hydroxylase and C17-20 lyase activities for 5-ene-C21-steroids were increased and 17 beta-hydroxysteroid oxidoreductase activity were decreased by gonadotropin treatment. Our findings confirm the phenomenon of unequal substrate availability to enzymes depending upon the provenance of the substrate and demonstrate that gonadotropin(s) change substrate availability as do direct changes in enzyme activities. The unequal substrate availability validates the assumption, previously proposed by others, that microsome enzymes have an organized arrangement as enzyme complexes.

Animals↗

PubMed1984

Pituitary androgen receptors and the resistance of long-term castrated rams to the androgenic control of luteinizing hormone (LH) secretion.

Luteinizing hormone (LH) responses of short-term (24 h) and long-term (6 mo) castrated rams to testosterone replacement therapy were investigated. Testosterone filled Silastic capsules which maintained physiological concentrations of testosterone in blood (approximately 3.2 ng/ml) prevented the post-castration rise in serum LH of short-term castrated rams but failed to effectively reduce the established elevated serum LH levels in long-term castrated rams. The LH response to exogenous LHRH was also suppressed in the short-term castrated rams, whereas the response was increased in direct proportion to basal LH levels in long-term castrates. The possibility that a change in pituitary androgen receptors may explain the resistance of long-term castrates to testosterone feedback was examined. For this purpose, a cytosolic androgen receptor binding assay was developed using tritiated methyltrienolone (R 1881) as the labeled ligand and dextran-charcoal to separate free and protein-bound steroid. Stable, high affinity (Kd = 0.3-1.5 nM), saturable binding of R 1881 was demonstrated in pituitary cytosol from both intact and castrate rams. Relative binding specificities in intact rams, however, suggested partial binding to a progestin-binding component, whereas receptor binding in long-term castrates was androgen specific. For this reason, androgen receptor binding was studied after addition of triamcinolone acetonide to occupy apparent progestin binding sites. We observed no differences in either androgen receptor concentration (4.10 +/- 0.70 versus 3.23 +/- 0.45 fmol/mg protein; Mean +/- SD) nor ligand affinity at 0 degrees C (Kd = 0.66 +/- 0.34 versus 0.40 +/- 0.08 nM) in 6 mo compared to 24-h castrate rams. These data indicate that the inability of androgens to suppress gonadotropin secretion in long-term castrate rams is unlikely to be related to specific changes in pituitary cytosol androgen receptors.

Animals↗