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.     

Ethanol-induced sleeping time in mice: age and sex differences

Ethanol-induced sleeping time was determined in mice of both sexes at ages ranging from 3 weeks to just over 1 year. A progressive increase in sleeping time was seen in both sexes up to 26 weeks of age; no subsequent changes were observed, except for a modest decrease in the oldest female group. In the majority of age groups, sleeping time values were higher in females, but few statistically significant sex differences were found.     

In vitro perfusion of isolated mouse testes: a model system for investigating testicular steroidogenesis

1. A procedure was developed to perfuse isolated mouse testes in vitro and investigate testosterone secretion. 2. The perfused testis preparation was validated by determining organ edema, glucose uptake, enzymatic and morphological indices of cellular integrity, and testosterone secretion in the absence and presence of LH and other pituitary peptides. 3. The results establish that this procedure can be exploited as a model system to investigate biochemical, physiological, and genetic aspects of steroidogenesis and Leydig cell function in the mouse, and presumably other small rodents as well.     

Lunar synchronization of testicular development and steroidogenesis in rabbitfish

Lunar synchronization of testicular development in the golden rabbitfish, Siganus guttatus, was assessed by measuring changes in sperm motility and conditions in the seminal plasma, and by in vitro production of steroid hormones in testicular fragments and sperm preparations. The duration and percentage of sperm motility was low 1 week before spawning (the new moon), but increased significantly on the day of spawning (the first lunar quarter). During the first lunar quarter, the osmolality decreased, but Ca(2+) concentration increased in the seminal plasma. These results suggest that spermiation occurs rapidly towards the specific lunar phase. Testicular fragments and sperm preparations were incubated with human chorionic gonadotropin (hCG) and two precursor steroid hormones, 17alpha-hydroxyprogesterone (17alpha-OHP) and testosterone (T), during the two lunar phases. The production of 11-ketotestosterone (11-KT) increased significantly when the testicular fragments were incubated with hCG at the first lunar quarter, while incubation of sperm preparations with 17alpha-OHP during the same moon phase resulted in a significant increase in 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) production in the medium. These results suggest that 11-KT is produced in the somatic cells of the testis under the influence of gonadotropin, and that sperm can convert 17alpha-OHP to DHP. Additionally, steroidogenic activity was considered to increase toward the specific lunar phase. The synchronous increase in testicular activity supports the hypothesis that lunar periodicity is a major factor for the testicular development of S. guttatus.     

Interspecies differences in testicular LH receptors and in vitro testosterone production among rodents

Testes from rats, mice and hamsters were incubated for 4 h with 0, 3.125 or 12.5 mIU hCG/ml. The LH receptor concentration in incubated testes of rats and mice was higher than that observed in hamsters. Testosterone levels in incubation media were significantly different among species (mice greater than rats greater than hamsters). During the incubation, hCG caused an increase in testosterone levels in all three species, but produced no significant changes in LH receptor concentration. Furthermore, a correlation between LH receptor concentration and testosterone only in hamsters is observed. The efficiency of the LH receptor-steroidogenesis interaction was estimated from the ratio of testosterone levels to receptor concentration under basal conditions and was found to differ among species (mice greater than hamster greater than rats). The levels of PGE and PGF in incubation media were higher in mice than in rats or hamsters, and hCG did not alter prostaglandin levels in any of the species. The present results indicate that acute in vitro hCG stimulation of testosterone synthesis does not involve appreciable changes in testicular LH receptor levels.     

Steroid profiling in the study of rat testicular steroidogenesis

Twenty authentic steroids, derivatized as O-methyl oximes (MO), trimethylsilyl (TMS) ethers or as MO-TMS ethers have been subjected to capillary gas chromatography using two different columns. Virtually all of the steroid derivatives have been resolved, one difficult pair to separate being 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol on the non-selective phase OV-1. Where syn and anti forms of MO derivatives arose, these were also resolved under the conditions utilised. This technique of 'steroid profiling' has been applied to the separation and quantification of metabolites of pregnenolone which were formed during incubations of the microsomal and cytosolic fractions from rat testes. The majority of the metabolites were found in the microsomal incubation. These compounds included some odorous 16-androstenes as well as other C21 and C19 steroids, the formation of which was consistent with the 5-ene and 4-ene pathways of testosterone biosynthesis being operative. In addition, evidence was obtained for 16 alpha-hydroxylation of C21 steroids. Very much less metabolic activity was found in the cytosolic fraction of rat testes. Metabolic pathways have been proposed which both confirm and extend earlier work. We conclude that the rat testis can only form some of the odorous, possibly pheromonal, 16-androstenes and that these are quantitatively less important than in the porcine testis.     

Potentiation by epidermal growth factor of the in vitro HCG stimulation of testicular steroidogenesis in hamsters.

Epidermal Growth Factor (EGF) has been reported to stimulate or inhibit steroidogenesis in murid Leydig cells depending on the experimental conditions used. In the present study, testicular fragments from an adult cricetid rodent, the Syrian hamster, were incubated with various doses of mouse EGF (0-2.0 micrograms/ml media), in the presence or absence of HCG (0-12.5 mlU/ml media). Although EGF alone did not affect in vitro testicular steroidogenesis, it significantly potentiated the HCG-induced elevation of the accumulation of testosterone and 17-hydroxyprogesterone in the media. In contrast, the effect of HCG on media progesterone concentration was not affected by EGF. Since in the Syrian hamster intracellular calcium loading functions as a gonadotropic stimulus, the present results could be a consequence of the EGF-induced increase in cellular calcium levels.     

Reduced testicular steroidogenesis in tumor necrosis factor-alpha knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.     

R1881 regulation of steroidogenesis in cultured testicular cells

The influence of a synthetic androgen R1881 upon hCG stimulated steroidogenesis in cultured rat testicular cells was investigated. Testicular cells were cultured for 8 days in medium alone and thereafter reincubated for 48 h with appropriate treatments before the collection of media for steroid RIA. Addition of R1881 (10(-6) M) resulted in an overall decrease of hCG (0.3-10 ng/ml) stimulated pregnenolone and progesterone production by cultured cells. The conversion of exogenous steroids of the delta 4 pathway (progesterone,17 alpha-OH-P and delta 4-A) was also studied in cultures supplemented with cyanoketone (10(-5) M) and/or spironolactone (10(-5) M) to prevent endogenous testosterone production. R1881 inhibited progesterone and 17 alpha-OH-P conversion to testosterone (T) and was ineffective when delta 4-A served as precursor for T biosynthesis. The inhibitory effect of R1881 upon Testosterone production was prevented by concomitant treatment with CPA. These observations suggest that R1881 decreases the hCG stimulated testosterone production via inhibition of CSCCE,3 beta-HSD,C17-20 Lyase and likely 17 alpha-Hydroxylase, whereas no effect on 17 beta-HSD could be observed.     

Effect of surgery and efferent duct ligation on testicular blood flow and testicular steroidogenesis in the rat

A moderate reduction in testicular blood flow was observed in both testes 24 h after unilateral efferent duct ligation without any corresponding change in testosterone secretion as indicated in the peripheral blood, in testicular venous blood, or in testicular tissue fluid. At 21 days a pronounced unilateral decrease in blood flow was associated with the extensive degeneration of tubules in the testis on the ligated side. These changes were also associated with decreased testosterone secretion by the testis on the ligated side, although Leydig cell function was not abolished since testosterone in the tissue increased rather than decreased. It is therefore concluded that testicular blood flow may play an important role in the changes of testosterone secretion that follow unilateral efferent duct ligation.     

Superoxide dismutase as a regulatory switch in mammalian testicular steroidogenesis

The delta 4-pathway of testosterone biosynthesis in leydig cells, widely believed to proceed through pregnenolone--greater than pregnenedione--greater than progesterone route catalyzed by 5 delta-3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase respectively is shown to pass through an alternate pathway mediated by superoxide dismutase and peroxidase. A built-in regulatory switch is incorporated in this route, with the superoxide dismutase inducible upon LH-stimulation of the leydig cells.     

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.     

Inhibition of rat testicular microsomal steroidogenesis by oxytocin and metyrapone

Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.     

Effects of acute graded reductions in testicular blood flow on testicular morphology in the adult rat

The effect of moderate reductions in testicular blood flow has not been studied systematically. The aim of this study was, therefore, to examine the effects of different degrees of blood flow reduction on testicular morphology and to determine how much flow can be reduced before damage occurs. The subcapsular testicular artery was partially ligated in the left testes of adult rats. Testicular blood flow was measured before, immediately after, and 5 h after the ligation using laser Doppler flowmetry. After 5 h of partial ligation, the testes were removed, and their morphology was examined and related to the degree of blood flow reduction. The number of in situ end-labeled- or TUNEL-positive (i.e., dying) germ cells and the volume density of intravascular polymorphonuclear (PMN) leukocytes were measured. When flow was reduced to approximately 70% or less of its pretreatment value, a dose-related increase in the number of dying spermatogonia and early spermatocytes was seen. The PMN leukocytes accumulated in testicular blood vessels after partial ligation, and the maximum number was observed in testes where flow was reduced by approximately 50% of the pretreatment value. In conclusion, early stages of spermatogenesis are sensitive to a moderate, acute reduction in blood flow. Discrete reductions in flow may, therefore, have a large impact on sperm production.     

The adrenal gland and progesterone stimulates testicular steroidogenesis in the rat in vivo

Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.     

Granulosa cell steroidogenesis before in vitro fertilization

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with over-ripeness and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.     

A simple technique for collecting blood of testicular origin: application to in vivo studies on testicular steroidogenesis in rats and Macaca fascicularis

A technique for rapidly collecting blood of testicular origin is described, one which can provide sufficient plasma amounts to investigate some steps of testicular steroid biogenesis in vivo in 2 species. In adult male rats, testosterone (T), androstenedione (4A) and 5-androstenediol (5AD) were determined in pampiniform plexus testicular venous blood (PPTV) and peripheral (PV) blood samples before and 2 h after human Chorionic Gonadotropin (hCG). PPTV concentration of 5AD was 0.83 +/- 0.1 ng/ml (mean +/- SEM) with a PPTV/PV ratio of 7.0 +/- 1.0, comparable to a PPTV/PV ratio for 4A of 5.8 +/- 1.8. After hCG, PPTV concentration of 5AD significantly increased to 1.28 +/- 0.15 ng/ml (P less than 0.05). Those data are in favor of a participation of 5-ene pathway to testicular biogenesis of T associated to a 4-ene pathway which is predominant. In adult male Macaca fascicularis, spermatic vein (SV) concentrations of 5AD and 4A were comparable (3.0 +/- 1.2 vs 4.3 +/- 1.0 ng/ml) as well as SV/PV ratios under basal conditions (3.5 +/- 0.9 vs 5.1 +/- 0.1), as well as 48 h after hCG, confirming in vivo that both 5-ene and 4-ene pathways are involved in testicular T biogenesis. Testicular production of estradiol (E2), estrone (E1) and their sulfates E2S and E1S showed a SV/PV ratio significantly higher than 1 (3.4 +/- 0.6; 2.4 +/- 0.1; 1.7 +/- 0.2 and 1.6 +/- 0.2, respectively).     

The role of fat body in testicular spermatogenesis and steroidogenesis inRana hexadactyla lesson

The fat body appears to contribute cholesterol for testicular steroidogenesis. It also appears to provide prostaglandins and cyclic AMP for testicular steroidogenesis since fatectomy impairs this process which is corrected by the addition of prostaglandins and cyclic AMP. Of the two, prostaglandins have a more important role in spermatogenesis and cyclic AMP functions in steroidogenesis These functions of the fat body suggests that it constitutes a link in the hypothalamohypophysial-gonadal axis.     

Ethanol-induced inhibition of pinocytosis and proteolysis in rat yolk sac in vitro

Pinocytic capture of 125I-labelled polyvinylpyrrolidone and of formaldehyde-denatured 125I-labelled bovine serum albumin by 17.5-day rat visceral yolk sacs incubated in vitro was rapidly and strongly inhibited by low concentrations (0.01 and 0.05%, v/v) of ethanol. The induced inhibition of pinocytosis was readily reversible, but a marked lag was observed before ethanol-exposed tissue regained its full proteolytic capacity towards the exogenous protein. These observations suggest that the acute administration of ethanol to a pregnant rat may give rise to concentrations of ethanol in the maternal blood and/or uterine fluid that induce dysfunction of the yolk sac. In late gestation such inhibition of yolk-sac function may interfere with the transfer of passive immunity across the yolk sac. If similar dysfunction is induced earlier in gestation, in the period before the chorioallantoic placenta is functional, this could cause a transient period of inhibition of histiotrophic nutrition that may be important to the pathogenic mechanism of action of ethanol as a teratogen.