Regulation of testicular LH receptors by homologous hormone: in vitro studies on receptor occupancy and receptor loss.

A method is described which makes use of 4M MgCl₂ to dissociate the testicular luteinizing hormone-receptor complex without altering either the binding capacity or binding affinity of the receptor. Using this method, it was demonstrated that $\text{in vitro}$ incubation at 4° of decapsulated rat testes with various concentrations of luteinizing hormone or with human chorionic gonadotropin resulted in a reduction in binding capacity. This reduction of binding capacity could not be completely accounted for by occupation of receptors by homologous hormone, suggesting that receptors were lost. Thus negative regulation of LH receptors by LH and hCG was observed. The reduction in LH binding capacity was specific for LH and hCG, dose dependent and time related. FSH, prolactin and growth hormone did not exert the same effect.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Androgen secretion and characteristics of testicular HCG binding in cryptorchid rats

Response of the cryptorchid testis to gonadotrophic stimulation was assessed by comparison of the androgen production capability in vivo and in vitro with that of the normal scrotal testis. Serum androgen concentrations in cryptorchid rats were similar to those in normal rats, and the incremental increase 60 min after 50 i.u. hCG (i.v.) was about 7-fold for both groups. Basal and hCG-stimulated androgen production in vitro was higher for abdominal testes (557 and 3286 ng/pair) than for scrotal tests (157 and 504 ng/pair). Specific binding of hCG by testicular homogenates was slightly higher (P < 0.05) for cryptorchid testes when expressed per unit weight, but Scatchard analysis indicated that although hCG binding affinities did not differ (Ka = 2 x 10(10) M-1), hCG binding capacity of cryptorchid testes was only 75 ng, compared to 219 ng for scrotal testes. These data indicate that a discrepancy exists between androgen production in vivo and in vitro by cryptorchid testes and that normal serum androgen concentrations are maintained in the presence of decreased numbers of testicular LH/hCG receptors.     

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins in vivo

The effect of $\text{in vivo}$ exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin $\text{in vivo}$ produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH $\text{in vitro}$. The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.     

The effects of hypophysectomy and human chorionic gonadotropin administration on the in vitro metabolism of progesterone by the rat testis

Changes in the $\text{in}$$\text{vitro}$ capacity to convert progesterone to its metabolites were studied in testes of adult rats hypophysectomized for varying lengths of time. After 30 days of hypophysectomy rats were injected for periods of 10 and 20 days with 100 i.u. of HCG daily to observe what changes could be induced in the testicular conversion of progesterone. Hypophysectomy increased the formation of 20α-hydroxy-4-pregnen-3-one and decreased the formation of testosterone. In hypophysectomized animals injected with HCG there was an immediate decrease in the 20α-hydroxy-4-pregnen-3-one formation, but no appreciable accumulation of testosterone, as the animals demonstrated an immature pattern of testicular function. The results indicate that 20α-hydroxy-4-pregnen-3-one may act as a positive feedback agent to prolong and heighten gonadotropin discharge, and confirm the importance of metabolites of testosterone prior to adulthood.     

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.     

Ontogeny of the gonadal receptor for luteinizing hormone in the pig

Homogenates of porcine ovaries and testes collected between 70 d post coitum and 42 d post partum were incubated with radiolabelled human chorionic gonadotropin (hCG) to determine the presence and relative amounts of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors. Specific binding of (125)I-hCG to ovaries and testes occurred at all stages of fetal and postnatal development. Ovarian tissue possessed relatively low affinity, high capacity LH/hCG binding sites that were most numerous at Day 80 of gestation and decreased thereafter. In contrast, high affinity, low capacity LH/hCG binding sites were found in the testes. In males, the total number of LH/hCG binding sites remained stable until near term and then increased with age, but the number of sites per gram of testicular tissue did not change (P>0.05). In summary, differential binding of LH/hCG in gonadal tissue occurred in male and female piglets during pre- and post-natal periods, and this binding reflected the known differential pattern of development of the male and female gonad.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.     

Stimulation of leydig cell testoterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone

Injection of a luteinizing hormone-releasing hormone (LHRH) agonist into 55-day-old male rats which had been hypophysectomized 3 days earlier resulted in a 10- to 30-fold increase in the levels of testosterone in serum and testicular interstitial fluid (IF) in the 4h following injection. The levels achieved were within or above the normal range for intact untreated rats of this age. In similar animals, injection of LHRH agonist also enhanced the serum testosterone response to injected hCG at $\text{1}\text{1}\text{2}\text{h}$, but not at later times after injection, and by 24h reduced IF levels of testosterone suggested that LHRH agonist had begun to inhibit stimulation by hCG. $\text{In vitro}$, dispersed Leydig cells from untreated hypophysectomized rats showed a 2-fold increase in testosterone responsiveness to LHRH agonist when compared to cells from intact rats, and this change was associated with an 80% increase in the number of Leydig cell LHRH-receptors.     

Ethanol-induced inhibitions of testicular steroidogenesis in the male rat: Mechanisms of actions

Ethanol markedly inhibits the biosynthesis of testosterone in the male of several species. Since several $\text{in vitro}$ studies have suggested that ethanol $\text{per se}$ is not a gonadal toxin and that it must be metabolized to exert its effects, we examined this possibility under $\text{in vivo}$ conditions in the present studies. We found that the administration of the alcohol dehydrogenase inhibitor, pyrazole, to adult male rats significantly elevated blood ethanol levels. However, rather than resulting in a potentiation of the effects of ethanol on testicular steroidogenesis, pyrazole-induced elevations in blood ethanol concentrations produced a significant attenuation of ethanol's effects. In view of these observations, it is difficult to maintain that ethanol itself is responsible for inhibiting the production of testosterone. On the contrary, our results may provide the first $\text{in vivo}$ support for the hypothesis that ethanol must be metabolized to exert its effects on testicular steroidogenesis.     

Bioconversion of the androgen precursors by pre- and post-pubertal rat testes with special reference to local irradiation and gonadotropin stimulation

Pubertal changes in the testicular steroid enzyme activities, responsible for the androgen production, were studied in rats in relation to the effects of testicular irradiation, followed by gonadotropin stimulation and cyproterone suppression. Five groups of pro-pubertal and adult rats were used in this study. The $\text{in vitro}$ bioconversion from progesterone-4-14C and 17-hydroxyprogesterone-44C to testosterone, androstenedione, androstanediol, dihydrotestosterone and androsterone, demonstrated the effect of age in all cases of drug response investigations. The sexually immature animals in the control group had higher levels of androstenedione than testosterone, in contrast to the findings in the adults. With irradiation, androgen biosynthesis was suppressed in both age groups, which did not recover, under gonadotropin stimulation, in spite of the generation of new cells caused by the treatment. The irradiated adult testes demonstrated ‘pre-pubertal’ type bioconversion by catabolizing the substrates more towards 5α-reduced androgens, like androstanediol (5α-androstane-3α 17β-diol) and androsterone. With cyproterone the 17α-hydroxylase activities were found to be diminished.     

Treatment of cryptorchidism with human chorionic gonadotropin or gonadotropin releasing hormone. A double-blind controlled study of 243 boys

We have conducted a modified double-blind study on the effect of human chorionic gonadotropin (hCG), gonadotropin releasing hormone (GnRH) and placebo on bilateral and unilateral maldescended testes. One hundred and fifty-five boys with bilateral and 88 boys with unilateral cryptorchidism fulfilled the inclusion criteria and completed the treatment protocol. The boys were between 1 and 13 years of age. hCG was administered as intramuscular injections twice weekly for 3 weeks. GnRH and placebo were given intranasally. hCG was superior to GnRH and placebo in the treatment of bilateral maldescended testes (p = 0.0009). Both testes descended in 25% of the boys following treatment with hCG, and improvement in the position of the testes was obtained in a further 25% of the cases. hCG administration resulted in complete testicular descent in 14% of boys with unilateral cryptorchidism compared with 3 and 0% after placebo and GnRH, respectively (p = 0.07). The testis had moved to a more distal position in 46% of the boys treated with hCG. There was no significant difference between the treatment groups with regard to age or initial position of the testes. We conclude that a success rate of 25% justifies the use of hCG in the treatment of maldescended testes, whereas the study did not support a general use of GnRH administered intranasally.     

The aging leydig cell: VII. Cytoplasmic estradiol receptors

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2–3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 ± 0.3 fmol/mg protein ($\text{x}\text{±}\text{SE, n}\text{=12}$), which was comparable to that of the old rats, 5.7 ± 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 × 10¹⁰M^?1. Plasma estradiol levels were also similar in both groups-19.6 ± 2.8 pg/ ml (n=14) for the young and 19.2 ± 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.     

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.     

Tetrahydropapaveroline: formation in vivo and in vitro in rat brain

Gas chromatographic-mass spectrometric techniques have been used for the measurement of tetrahydropapaveroline (THP) from brain. The formation of THP from 1-DOPA or dopamine $\text{in vitro}$ has been confirmed by obtaining a complete mass spectrum of the product as its trifluoroacetylated derivative. Following chronic administration of 1-DOPA or 1-DOPA in combination with ethanol to rats, THP formed $\text{in vivo}$ in in brain could be detected in small quantities although it could not be detected when ethanol alone was administered.     

Effect of hypophysectomy and gonadotropin treatment on metabolism of 3H-progesterone by rat testicular tissue

This study was conducted to define the pattern of $\text{in}$$\text{vitro}$ metabolism of ³H-progesterone in incubates of rat testicular tissue at various time intervals after hypophysectomy and to determine the effect of $\text{in}$$\text{vivo}$ gonadotropin treatment on the metabolism of ³H-progesterone in posthypophysectomy regressed testes. Formation of tritium labeled testosterone, androstenedione, 5α-androstanediol and androsterone was markedly diminished within two weeks and only traces of these substances were formed between the 23rd and 54th day after hypophysectomy. The major metabolite throughout this time period was ³H-20α-dihydroprogesterone. These data demonstrate that in posthypophysectomy-regressed testes ³H-progesterone metabolism does not revert to that observed in fetal testes or testes from immature animals. Treatment with HCG, commencing on the 33rd day after hypophysectomy resulted first in formation of 5α-reduced androgens and marked decrease in 20α-dihydroprogesterone. Additional treatment produced increased formation of radiolabeled testosterone and androstenedione and diminution of 5α-reduced androgens. This metabolic pattern is reminiscent of that observed in normally developing testes. Treatment with PMS commencing on the 33rd day after hypophysectomy resulted in formation of large amounts of androstenedione and testosterone and decrease of 20α-dihydroprogesterone to trace amounts within 10 days of initiation of treatment. After additional 10 days of treatment the formation of androstenedione diminished, testosterone remained unchanged. The possibility is suggested that FSH activity in PMS may be responsible for the different pattern of progesterone metabolism. The data of an three experiments suggest that the 20α-hydroxysteroid oxidoreductase activity may be influenced by gonadotropins.     

Genetic Differences in Androgen Receptors and in Autoregulation of Testicular Human Chorionic Gonadotropin Binding Sites in the Mouse

Abstract

The autoregulation of testicular human chorionic gonadotropin (hCG) binding sites was studied in two strains of mice known to differ in their endocrine and reproductive characteristics (C57BL/10J and DBA/2J), and in their F₁ progeny (B10D2F₁). Basal hCG binding levels were higher in C57BL/10J than in DBA/2J mice, while B10D2F₁ mice had intermediate levels. Twenty-four h after injection, hCG produced dose-related changes in hCG binding in C57BL/10J and B10D2F₁ mice not observed in DBA/2J mice. However, 72 h after treatment with hCG there was a decrease in hCG binding in all the strains studied. These results suggest the participation of genetic factors in determining basal levels, dose-related changes and temporal response of testicular hCG binding sites to hCG administration. Androgen receptor levels were measured in the same strains of mice. DBA/2J mice had higher receptor levels in the kidney and coagulating gland, and lower levels in the hypothalamus and seminal vesicle when compred to C57BL/10J mice. B10D2F₁ mice had androgen receptor levels similar to those measured in C57BL/10J mice in all tissues studied, with the exception of the coagulating gland, where levels were similar to those observed in DBA/ZJ mice. These observations may indicate the existence of several loci coding for androgen receptors, with only one being expressed per tissue     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Influence of a prolonged tamoxifen administration on steroidogenesis by incubated rat testes

Tamoxifen was administered i.m. for 9 days to adult male rats in a daily dose of 100 micrograms or 1 mg. The treatment resulted in a significant reduction of the plasma levels of testosterone and LH, without modification of the plasma levels of FSH and of the testes weight. Upon incubation, the testes from the tamoxifen-treated rats produced less testosterone and 7 alpha-hydroxytestosterone, but metabolized [4-14C]testosterone in the same way as the control animals. Small doses of hCG (0.5 i.u. for 9 days) were unable to modify the tamoxifen effect on the testicular function, while tamoxifen significantly inhibited the increase of the plasma levels of testosterone induced by the administration of moderate doses of hCG (1.5 i.u. or 2.5 i.u. for 9 days) to hypophysectomized rats. Tamoxifen treatment, however, did not modify significantly the reactivity of the testes towards high doses of hCG (10 i.u.), administered either 2 h before sacrifice or for 9 days. It is concluded that a prolonged administration of tamoxifen in the rat has, besides an indirect effect resulting from a decrease of the LH levels, a direct inhibitory influence on the testicular testosterone formation, which can be reversed by high doses of hCG.     

On the mechanism of ethanol-induced accumulation of triglycerides in the liver

The possibility that ethanol or acetaldehyde has a direct effect on the activity of acyl-CoA-ligases or $\text{sn}$-glycerophosphate acyltransferases or on the biosynthesis of phosphatidic acid and triglycerides from free fatty acids was studied with subcellular preparations from rat liver. No stimulatory effect of ethanol or acetaldehyde could be observed in any case. It was further shown that the microsomal fraction of homogenate of livers of rats treated with ethanol (single peroral dose of 4.5 g of ethanol per kg body weight) did not have an increased capacity to biosynthesize phosphatidic acid. The possibility was excluded that excess cofactors necessary for formation of phosphatidic acid are responsible for the higher accumulation of triglycerides in livers of rats treated with ethanol.The results indicate that the increased formation of triglycerides in liver of rats treated with ethanol is not due to increased activity of acyl-CoA-ligase or $\text{sn}$-glycerophosphate acyltransferase or due to increased availability of $\text{sn}$-glycerophosphate, ATP or CoA-SH. It is suggested that increased availability of fatty acids is the major explanation for the increased accumulation of triglycerides in the liver after ethanol administration.