Short term treatments with prolactin, HMG or testosterone fail to affect testicular inhibin content in rats

To investigate the effect of prolactin (PRL) on testicular function, especially on spermatogenesis, testicular inhibin content in male rats treated with PRL was compared with those treated with HMG and testosterone. Mature Wistar male rats were given 10 or 50 IU of ovine PRL, 10 IU of HMG and 5 mg of testosterone, i. m. for 5 consecutive days and testes were removed for assessing inhibin content. Inhibin content was measured by a FSH suppressing activity in cultured rat anterior pituitary cells using aquous extract of testes. Five days' treatment with PRL, HMG, or testosterone did not influence testicular inhibin content in male rats. The possibility that these treatments had transiently affected testicular inhibin content, or that inhibin content did not reflect inhibin production was not ruled out.     

Direct biphasic modulation of gonadotropin-stimulated testicular androgen biosynthesis by prolactin

Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Testosterone regulates 25-hydroxycholesterol production in testicular macrophages

Recently, we found that testicular macrophages produce 25-hydroxycholesterol (25-HC) and express 25-hydroxylase, the enzyme that converts cholesterol to 25-HC. In addition, 25-HC may be an important paracrine factor mediating the known interactions between macrophages and neighboring Leydig cells, because it is efficiently converted to testosterone by Leydig cells. The purpose of the present study was to determine if testosterone can regulate the production of 25-HC in rat testicular macrophages, representing a potential negative-feedback loop from Leydig cells. We found that expression of 25-hydroxylase mRNA and production of 25-HC by cultured testicular macrophages were significantly inhibited by testosterone at 10 micro g/ml. This dose of testosterone did not have an effect on cell viability and did not change the rate of mRNA degradation in the presence of actinomycin D. These studies indicate that production of 25-HC is negatively regulated by testosterone, which may be representative of a paracrine negative-feedback loop.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.     

Intercellular communication between Sertoli cells and Leydig cells in the absence of follicle-stimulating hormone-receptor signaling

Effective interactions among the various compartments of the testis are necessary to sustain efficiency of the spermatogenic process. To study the intercellular communication between the Sertoli and Leydig cells in the complete absence of FSH receptor signaling, we have examined several indices of Leydig cell function in FSH receptor knockout (FORKO) mice. The serum testosterone levels were reduced in the 3- to 4-mo-old adult FORKO males compared to wild-type mice despite no significant alteration in circulating LH levels. Treatment with ovine LH resulted in a dose-dependent increase in serum testosterone levels in all three genotypes (+/+, +/-, and -/-). However, the response in FORKO males was significantly reduced. Similarly, the total intratesticular testosterone per testis was also lower, but the intratesticular testosterone per milligram of testis was significantly elevated in the FORKO males. Western blot analysis revealed an apparent higher expression of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as LH-receptor density in the testis of FORKO males. Immunohistochemistry also showed an increase in the intensity of 3beta-HSD staining in the testicular sections of FORKO males. Although LH receptor binding increased per unit weight in FORKO mice, the total LH binding remained the same in all genotypes. Taken together, the results of the present study suggest that, in the absence of FSH receptor signaling, the testicular milieu is altered to affect Leydig cell response to LH such that circulating testosterone is reduced in the adult mutant. Studies are currently under way to understand the mechanisms underlying this phenomenon.     

In vitro studies of prolactin inhibition of luteinizing hormone action on Leydig cells of rats and mice

Previous in vivo studies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitro using both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro. NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose-response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose-response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitro interstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.     

Receptor binding, biological, and immunological properties of chemically deglycosylated pituitary lutropin.

Chemical deglycosylation of ovine pituitary lutropin with anhydrous HF has been investigated. Treatment of the hormone for 75 min at 0 °C removed nearly two-thirds of the carbohydrate moiety. Deglycosylation altered the gel filtration and electrophoretic behavior of the hormone. Carbohydrate removal also resulted in dissociation into subunits to the extent of about 20%. In a rat ovarian radioreceptor assay, the deglycosylated hormone derivatives had approximately 35–40% of the binding activity of the native hormone. Immunological activity was fully retained as seen by the gel diffusion method and an α-subunit conformation oriented radioimmunoassay. In collagenase dispersed rat testicular interstitial cells, the derivatives had poor steroidogenic activity (less than 3%) and failed to elicit maximal testosterone production. The deglycosylated derivatives effectively antagonized the steroidogenic activity of the native hormone in rat testicular interstitial cells.     

Testicular GnRH-like factors: characterization of biologic activity

Observations that gonadotropin releasing hormone and its agonists directly inhibit gonadal function by binding to receptors on the Leydig cells had led to search for testicular GnRH-like peptide(s). This communication presents evidence that GnRH-like factors isolated from rat testis by immunoaffinity chromatography and previously characterized by radioimmunoassay and radioreceptor assay possess biologic activity. The partially purified material led to dose dependent inhibition of oLH stimulated testosterone production in a mixed Sertoli-Leydig cell monolayer culture. Pre-incubation of the cells with a potent GnRH antagonist prevented the inhibitory effects of the partially purified material suggesting that inhibition of oLH stimulated testosterone production may be receptor mediated.     

Factors affecting the entry of testosterone into the lumen of the cauda epididymidis of the anaesthetized rat.

When [3H]testosterone was infused into the general circulation of the rat, perfusion of a length of the cauda epididymidis (17 +/- 1.0 (s.e.m.) cm, n = 36) with perfusates of varied composition revealed a low entry of radioactivity (1--10% plasma levels; 10 exps) with protein-free perfusates, and a greater entry (15--48%; 10 exps) when the perfusate contained bovine serum albumin (38 mg/ml). When the perfusate contained ovine or rat testicular fluid, or rat epididymal fluid at protein concentrations of 3 mg/ml or less, the entry of radioactivity into the epididymis was greater than when the perfusate contained 3 mg BSA/ml. The addition of ovine rete testis fluid protein (3 mg/ml to BSA (38 mg/ml) in the perfusate increased the uptake of radioactivity (58--106%; 6 exps). Radioactivity in blood was principally associated with testosterone (90, 95% total blood activity, 2 rats), whereas both [3H]testosterone (37, 41% total perfusate activity) and [3H]dihydrotestosterone (42, 63% total perfusate activity) was present in BSA-containing perfusates. The proportion of dihydrotestosterone appeared to increase when the perfusate contained protein of testicular origin.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

Chronic corticosterone treatment impairs Leydig cell 11beta-hydroxysteroid dehydrogenase activity and LH-stimulated testosterone production.

The effects of excess corticosterone on luteinizing hormone (LH)-stimulated Leydig cell testosterone production and activity of 11beta-HSD was studied. Adult male rats (200-250 g body weight) were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days. Another set of rats was treated with corticosterone (dose as above) plus LH (ovine LH 100 microg/kg body weight, s.c., daily) for 15 days. Corticosterone administration significantly increased serum and testicular interstitial fluid (TIF) corticosterone but decreased testosterone levels. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone. The oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was significantly decreased in Leydig cells of rats treated with corticosterone alone and in combination with LH. The direct effect of corticosterone on Leydig cell steroidogenic potency was also studied in vitro. Addition of corticosterone to Leydig cell culture showed a dose dependent effect on LH-stimulated testosterone production. Corticosterone at 50 and 100 ng/ml did not alter LH-stimulated testosterone production, but at high doses (200-400 ng/ml), decreased basal and LH-stimulated testosterone production. Basal and LH-stimulated cAMP production was not altered by corticosterone in vitro. It is concluded from the present study that elevated levels of corticosterone decreased the oxidative activity of 11beta-HSD and thus resulting in impaired Leydig cell steroidogenesis and the inhibitory effects of corticosterone on testosterone production appear to be mediated through inhibition of LH signal transduction at post-cAMP level.     

Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement

This review centers around studies which have used ethane dimethane sulphonate (EDS) selectively to destroy all of the Leydig cells in the adult rat testis. With additional manipulations such as testosterone replacement and/or experimental induction of severe seminiferous tubule damage in EDS-injected rats, the following questions have been addressed: 1) What are the roles and relative importance of testosterone and other non-androgenic Leydig cell products in normal spermatogenesis and testicular function in general? 2) What are the factors controlling Leydig cell proliferation and maturation? 3) Is it the Leydig cells or the seminiferous tubules (or both) which control the testicular vasculature? The findings emphasize that in the normal adult rat testis there is a complex interaction between the Leydig cells, the Sertoli (and/or peritubular) cells, the germ cells, and the vasculature, and that testosterone, but not other Leydig cell products, plays a central role in many of these interactions. The Leydig cells drive spermatogenesis via the secretion of testosterone which acts on the Sertoli and/or peritubular cells to create an environment which enables normal progression of germ cells through stage VII of the spermatogenic cycle. In addition, testosterone is involved in the control of the vasculature, and hence the formation of testicular interstitial fluid, presumably again via effects on the Sertoli and/or peritubular cells. When Leydig cells regenerate and mature after their destruction by EDS, it can be shown that both the rate and the location of regenerating Leydig cells is determined by an interplay between endocrine (LH and perhaps FSH) and paracrine factors; the latter emanate from the seminiferous tubules and are determined by the germ cell complement. Taken together with other data on the paracrine control of Leydig cell testosterone secretion by the seminiferous tubules, these findings demonstrate that the functions of all of the cell types in the testis are interwoven in a highly organized manner. This has considerable implications with regard to the concentration of research effort on in vitro studies of the testis, and is discussed together with the need for a multidisciplinary approach if the complex control of spermatogenesis is ever to be properly understood.     

Intratesticular distribution of testosterone in rats and the relationship to the concentrations of a peptide that stimulates testosterone secretion

Methods have been established and validated for quantitative assessment of the distribution of testosterone in the testis, by measurement of testosterone concentrations in whole testis, in isolated seminiferous tubules and in testicular interstitial fluid. These measurements were made in individual rats injected 2-40 h previously with saline (0.9% NaCl) or a potent antiserum to ovine LH. Testosterone concentrations in interstitial fluid and seminiferous tubules were closely correlated (r = +0.98; n = 60) and their relationship was log linear over a 200-fold range. However, although the concentrations of testosterone in interstitial fluid and seminiferous tubules decreased progressively with time after LH antiserum injection, this decrease was far more pronounced for interstitial fluid. In association with this change there was a significant increase in the amounts of a locally-produced factor in interstitial fluid which stimulates basal and hCG-stimulated testosterone production by isolated purified Leydig cells. This increase was reversed by injection of hCG but not by peripheral injection of a dose (20 mg) of testosterone propionate which restored normal intratesticular concentrations of testosterone. It is concluded that the tubular 'conservation' of testosterone, which occurs as interstitial fluid levels of this steroid decrease, may be a consequence of restricted diffusion of testosterone out of the tubules, but is also associated with increased amounts of a peptide stimulator of testosterone production.     

Dissociation of lutropin-induced loss of testicular lutropin receptors and lutropin-induced desensitization of testosterone synthesis.

The relationship between changes in testicular lutropin receptors, as measured by specific binding of 125I-labeled human chorionic gonadotropin, and testosterone synthesis in response to lutropin (testicular responsiveness) was studied in intact and hypophysectomized rats. Administration of a single 200-microgram dose of ovine lutropin to intact rats results at 3 days in a 58% decrease in lutropin receptors associated with a parallel decrease in testicular responsiveness. A single 30-microgram dose of lutropin to intact rats resulted in a comparable decrease in lutropin receptors with a transient increase in testicular responsiveness. Rats receiving twice-daily injections of 15 microgram lutropin for 10 days exhibited a 48% decrease in lutropin receptors by day 3 which persisted during the 10-day treatment period, but was accompanied by a progressive increase in testicular responsiveness to lutropin. Hypophysectomy resulted in an 80% loss of receptors and a 72% loss in responsiveness 7 days after surgery. Daily treatment with lutropin initiated immediately following surgery resulted in a further dose-dependent decrease in lutropin receptors and a dose-dependent increase in testicular responsiveness. Loss of lutropin receptors was not due to occupancy of the receptor by exogenous lutropin. These studies demonstrate a dissociation between the negative regulation of lutropin receptors and testicular responsiveness to lutropin. Furthermore, the studies in hypophysectomized rats indicate that lutropin is the only hormone essential for maintenance of steroidogenesis and that this is independent of lutropin receptor concentration.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.     

Evaluation of the possible direct effects of gonadotrophin-releasing hormone analogues on the monkey (Macaca mulatta) testis

In Exp. 1, the effect of treatment with a GnRH agonist on basal concentrations of serum testosterone and peak values of serum testosterone after administration of hCG was determined. One group of adult male monkeys was treated with a low dose (5-10 micrograms/day) and a second group with a high dose (25 micrograms/day) of a GnRH agonist for 44 weeks. Basal and peak testosterone concentrations were both significantly reduced by GnRH agonist treatment in all groups compared to untreated control animals, but the % rise in serum testosterone above basal values in response to hCG administration was unchanged by agonist treatment. In Exp. 2, the GnRH agonist (100 or 400 ng) or a GnRH antagonist (4 micrograms) was infused into the testicular arteries of adult monkeys. The agonist did not alter testosterone concentrations in the testicular vein or testosterone and LH values in the femoral vein. In Exp. 3, testicular interstitial cells from monkeys were incubated with three concentrations (10(-9), 10(-7) and 10(-5)M) of the GnRH agonist or a GnRH antagonist with and without hCG. After 24 h, neither basal nor hCG-stimulated testosterone production was affected by the presence of the GnRH agonist or antagonist. The results from all 3 experiments clearly suggest that GnRH agonist treatment does not directly alter steroid production by the monkey testis.     

Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas?

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.     

Insulin augmentation of testosterone production in a primary culture of rat testicular cells

The direct effects of insulin on basal and human chorionic gonadotropin (hCG)-stimulated accumulation of testosterone were investigated in vitro using a primary culture system of rat testicular cells from adult hypophysectomized male rats. The basal accumulation of testosterone was low throughout the 10-day incubation period. Treatment of testicular cells with insulin (10 micrograms/ml) by itself was without effect on the basal accumulation of testosterone, while treatment with increasing concentrations (0.1--10 ng/ml) of hCG resulted in dose-dependent increases in the accumulation of testosterone. Furthermore, concomitant treatment with increasing concentrations (0.01--10 micrograms/ml) of insulin led to a dose-dependent augmentation (up to 116% on Day 10) in the hCG-stimulated accumulation of testosterone, as well as a 1.6-fold increase in the testicular responsiveness to hCG. In contrast, treatment with desoctapeptide insulin (10 micrograms/ml), a trypsin degraded insulin, was without effect on the hCG-stimulated accumulation of testosterone. Increasing duration (12--72 h) of treatment with insulin resulted in time-dependent increases in the hCG-stimulated accumulation of testosterone achieving statistical significance (P less than 0.05) by 36 h. In addition, pretreatment with insulin (10 micrograms/ml) brought about significant (P less than 0.01) increases in the choleragen and Bt2cAMP-stimulated accumulation of testosterone. The augmenting effect of insulin was equally effective upon culturing in a glucose-free medium and was not associated with significant alterations in testicular cell number or cellular DNA or protein content. It is concluded that diminished testicular steroidogenesis in the diabetic rats may represent, at least in part, a direct consequence of insulin deficiency at the testicular level and that insulin may play an important role in the augmentation of testicular androgen production.