D. Marić

Effects of acute and chronic immobilization stress on rat Leydig cell steroidogenesis

In rats, acute immobilization (IMO) stress (2 h) induced a fall in the serum androgen concentrations (T+DHT) without detectable changes in serum luteinizing hormone (LH) values. In vitro studies, using a suspension of Leydig cells from adult rat testis, demonstrated that acute stress inhibited conversion of progesterone (P) or 17hydroxyprogesterone (17OHP) to T while conversion of androstendione (delta 4 A) was not affected. Acute IMO reduced activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and decreased basal and hCG-stimulated progesterone and androgen production. Chronic IMO stress (2 h daily for 10 days) induced a decrease in serum androgen level with decline in serum LH values. In vitro, hCG-stimulated progesterone and androgen production by suspension of Leydig cells, as well as conversion of P and 17OHP to T were not significantly altered. Our data demonstrates that acute IMO stress impaired testicular steroidogenesis primarily at the testicular level (decreasing the activity of certain enzymes), while chronic IMO stress exerts the effect mainly on the hypothalamic-pituitary axis; reduced serum LH levels elicit a decrease in serum androgen levels.

Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis

The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2x2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N(omega)-nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2x600 microg/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes.

The involvement of nitric oxide in stress-impaired testicular steroidogenesis

The participation of the nitric oxide (NO) pathway in downregulation of testicular steroidogenesis in normal and stressed rats was investigated both in vivo and in vitro. In Leydig cells from normal animals, isosorbide dinitrate, an NO donor, decreased the human chorionic gonadotropin (CG)-stimulated and progesterone-derived androgen production. Also, the intratesticular injection of a precursor of NO, arginine (10 mg/testis), transiently decreased serum androgen levels and inhibited human CG-stimulated androgen production in acute testicular cultures. These effects were eliminated in rats cotreated with Nomega-nitro-L-arginine methyl ester (L-NAME) (2 X 600 microg/each testis). Acute immobilization stress (2 h) decreased serum androgen levels and inhibited human CG-stimulated androgen production in vitro. These effects were accompanied by a significant increase in nitrite, a stable oxidation product of NO, in testicular cultures. Bilateral intratesticular injection of L-NAME prevented the stress-induced decrease of human CG-stimulated androgen production, and significantly reduced the nitrite levels. These results implicate NO in normal and stress-impaired testicular steroidogenesis.

The effect of opioid antagonists in local regulation of testicular response to acute stress in adult rats

The present study examined the effects of naloxone (N) and naltrexone-methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) on testicular steroidogenesis during acute immobilization stress (IMO; 2 h) in adult rats. Unstressed rats as well as IMO rats were treated by unilateral intratesticular injection of N (20 micrograms/testis), NMB (36 micrograms/testis), or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats serum T levels were significantly reduced, while serum luteinizing hormone levels were not affected. N and NMB normalized serum T levels in IMO rats and had no effects in controls. In IMO rats the activities of 3 beta-hydroxysteroid dehydrogenase (HSD) and P450(17 alpha, lyase) were significantly reduced, while the activity of 17 beta-HSD was not affected. N and NMB antagonized the inhibitory effect of IMO on 3 beta-HSD and P450(17 alpha, lyase) but did not alter enzyme activity in freely moving rats. Acute IMO decreased basal and human chorionic gonadotropin-stimulated androgen production by hemitestis preparation, but N (10(-4) M) added directly to the incubation medium blocked the decrease and had no effect on testes from freely moving control rats. These results support the conclusion that endogenous opioid peptides are potentially important paracrine regulators of testicular steroidogenesis under stress conditions.

The adaptive response of adult rat Leydig cells to repeated immobilization stress: The role of protein kinase A and steroidogenic acute regulatory protein

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study, we investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Δ4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells

Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 microg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was significantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data confirmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition.

Rapid Naloxone-Induced Alterations of Androgen Variables in the Growing Male Rat

Contrary to earlier views on the inability of naloxone to affect androgen variables by way of general circulation, systemically applied naloxone (2.5 mg/kg body weight, single i.p. or i.v. injection) has been shown to rapidly induce (within an hour) a significant fall (-35.7% on the average) of the concentration of serum androgen (testosterone and dihydrotestosterone; (T + DHT) in peripubertal rats (51-58 days old). Such a response to the opiate antagonist was absent, however, in low-androgen prepubertal animals (37-44 days old) and in those among peripubertal rats which still showed subcritical initial levels of androgen in circulation (less than 1.5 ng/ml; experiments with repeated blood sampling in catheterized animals). In peripubertal rats naloxone was also shown to induce a significant decrease (-36%) in basal in vitro androgen production by testes removed 15 or 30 min following the intraperitoneal administration of the opiate antagonist. Such an inhibitory effect on basal steroidogenesis has not been observed in control multiple-dose experiments in which incubated testes from naloxone-naive rats have been directly challenged with naloxone; on the contrary, enhancing direct effects were recorded, but only with the highest concentration of naloxone tested (10(-4) M). The possibility thus remains open that indirect inhibitory effects of injected naloxone may be operational in intact animals. Hypoprolactinemia, known to interfere in an age-dependent manner with the responsiveness of Leyding cells to luteinizing hormone (LH), may be of particular relevance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of short-term and long-term hyperprolactinemia on the developmental pattern of androgen and LH levels in the immature male rat.

Developmental patterns of serum levels of androgens and of the luteinizing hormone (LH) were studied comparatively in: i) long-term hyperprolactinemic (LT) immature male rats bearing ectopic pituitary grafts since the age of 21 days and examined at weekly intervals thereafter; and ii) in short-term hyperprolactinemic (ST) males grafted at several postnatal intervals and sacrificed 7 days postoperatively. In both ST and LT rats serum LH was markedly reduced, but only during the early prepubertal period (30 to 37days), characterized in normal rats by conspicuously high LH levels. During the next pubertal phase of development (51 to 58 days), normally characterized by a steep rise of serum androgens in the presence of relatively low LH, the androgen surge was significantly attenuated in LT, but not in ST animals. This suggests that elevated PRL, if maintained long enough prior to the pubertal age, may significantly attenuate or delay the intensified secretion of androgens characteristic of puberty, presumably by suppressing the intensive prepubertal LH secretion. In LT rats the testicular growth was moderately retarded, but the growth of the dorsal prostate was enhanced, suggesting a PRL-induced increase of responsiveness to androgen.

The Influence of the Gonads on the Functional Development of the Hypothalamo-Hypophysial System of the Male Rat

The relationship between the neonataltestes and the functional development of the hypothalamo-hypophysial system of the male rat was studied. Males were castrated on day 3, 4, 5, 8 or 10 of life. At 40 days of age one-half of an ovary from a littermate was grafted under the kidney capsule. 20 days after transplantation into males castrated on days 3 to 8, the histology of the ovarian grafts showed a relative abundance of corpora lutea, indicating the development of the female-specific functional pattern of the hypothalamo-hypophysial system. After 10 days, in spite of castration, the hypothalamo-hypophysial system was of the male-specific type. Castration and the simultaneous transplantation of ovaries in the abdominal region caused a significant increase in the weights of the anterior pituitary and the seminal vesicles.

Effects of Bromocriptine-Induced Hypoprolactinaemia on the Developmental Pattern of Androgen and LH Levels in the Male Rat

In immature male rats, receiving daily injections of bromocriptine (3 mg/kg bw), serum prolactin (Prl) remains low throughout development. In such hypoprolactinaemic males androgen variables are affected: a) the normally low pre-pubertal serum androgen (testosterone, dihydrotestosterone-T, DHT) is considerably increased, in correlation with a precocious development of the testicular Leydig cell population; b) the peri-pubertal rise of androgen is not prevented, but is is followed by significantly lower post-pubertal T, DHT levels, in correlation with moderately reduced Leydig cell counts and with strongly attenuated growth rates of sex accessory organs. Observed alterations of the post-natal androgen pattern cannot be related to LH changes since developmental LH values remained essentially unaltered. Results are in concordance with a marked age-dependent sensibility of androgen variables to a lack of Prl in the developing male.