Effect of temperature and the role of testicular descent on post-natal testicular androgen production in the mouse.

Testes from mice aged 3, 15, 25, 30 or 60 days were incubated under basal conditions or in the presence of hCG. One testis from each animal was incubated at 37 degrees C while the contralateral testis was incubated at 32 or 34 degrees C. During development total androgen production in response to hCG (at 32 degrees C) showed a marked increase between 15 and 30 days. The major androgens secreted at this time were testosterone and 5 alpha-androstane-3 alpha,17 beta-diol. There was little change in total androgen production between 30 and 60 days but by 60 days testosterone was the dominant androgen. Both basal and hCG-stimulated androgen production were temperature sensitive. These effects were most pronounced at 30 and 60 days with androgen production significantly inhibited at 37 degrees C. To examine the role of testicular descent in regulating steroidogenesis animals were rendered unilaterally cryptorchid at 19 days of age. At 25 days, when descent is normally completed in the mouse, there was no significant difference in steroidogenesis between scrotal and abdominal testes. By 30 days, however, the steroidogenic potential of the abdominal testis was significantly lower than that of the scrotal testis. These results show that testicular steroidogenesis is sensitive to temperature changes around the time of testicular descent, although descent itself is not required to achieve an adult level of steroidogenesis. The results also show, however, that testicular descent is required to maintain the adult level of steroidogenesis.     

Progesterone and androgen secretion by isolated cultured bovine corpus luteum cells: effect of LH, hCG, PRL, estradiol 17beta and testosterone

Primary cell cultures of bovine corpora lutea were used in order to examine their morphology and secretion of progesterone and androgen in vitro. The cells were grown as monolayers up to 6 days at 37 degrees C medium 199 supplemented with 10% calf serum. The concentration of progesterone and androgen was measured using appropriate radioimmunoassays [1,3] respectively. Luteal cells were cultured with addition of the following amounts of hormones: 100 ng LH, 10 i.u. hCG, 100 ng PRL, 150 ng Estradiol 17 beta and 150 ng Testosterone/ml of culture medium. The luteal cells also created considerable amounts of androgens. It was found that only estradiol added to the culture medium caused an increase in the level of testosterone. Progesterone secretion following the addition of hormones increased under the influence of LH, T, and E2 in statistically significant manner while hCG and PRL had no statistically significant effects.     

Photoperiod-induced changes in the testicular metabolism of [4-14C]17 alpha-hydroxyprogesterone in the bank vole (Clethrionomys glareolus)

Minces of the testes of bank voles, born and reared in a long (18L:6D) photoperiod until weaning (18-22 days of age) and subjected thereafter to a short (6L:18D, Group S) or a long (18L:6D, Group L) photoperiod for 6-9 weeks, were incubated with [4-14C]17 alpha-hydroxyprogesterone in the presence of cofactors (NADP/NADPH, 1.3 mmol/1) for 1 h at 37 degrees C. The radioactive metabolites were characterized and identified by thin-layer chromatography with derivative formation and chromatography to constant specific activity and isotope ratio. In Group L virtually all of the substrate was utilized and it was readily converted to androgens (48% of the radioactivity recovered) such as androstenedione and testosterone. The only pregnane metabolite identified was 17 alpha-hydroxy,20 alpha-dihydroxyprogesterone (43.3%). In Group S there was a decreased production of 17 alpha-hydroxy,20 alpha-dihydroprogesterone and androgens (25.4% and 10.4% respectively) and a substantial portion of the substrate was not metabolized (38.8%). The main androgen metabolites identified, androst-4-ene-3 beta,17 beta-diol and 5 alpha-androstane-3,17-dione are hormonally quite inert steroids. No androstenedione or testosterone was found. The results indicate that exposure to short photoperiod induces a decrease in the testicular C17-C20 lyase and 20 alpha-hydroxysteroid dehydrogenase.     

Effects of androgens, progesterone and their antagonists on the developmental competence of in vitro matured bovine oocytes

The aim of the present study was to determine whether androgens and progesterone influence the in vitro maturation of bovine oocytes as assessed by cleavage rates and competence to form blastocysts after in vitro fertilization. Bovine cumulus-oocyte complexes were cultured (n = 20 per drop) for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, eCG (2.5 iu ml(-1)) and a range of treatments that included aromatizable (testosterone; 100 nmol l(-1)) and non-aromatizable (dihydrotestosterone; 100 nmol l(-1)) androgens, an androgen antagonist (flutamide; 36 micromol l(-1)), progesterone (300 nmol l(-1)) and a progesterone antagonist (mifeprisone, RU486; 100 nmol l(-1)). Production of inhibin A, total alpha-subunit, activin A and follistatin by each group of cumulus-oocyte complexes was also measured, since inhibin-related peptides have been implicated as modulators of oocyte maturation and their production may be influenced by steroids and anti-steroids. Both testosterone and dihydrotestosterone increased oocyte cleavage rate (25%; P < 0.01) and dihydrotestosterone also increased (24%; P < 0.05) the proportion of oocytes that reached the >/= eight-cell stage. However, neither androgen affected blastocyst yield, or the proportion of blastocysts that hatched. The stimulatory effect of dihydrotestosterone on cleavage rate was reduced by flutamide but the anti-androgen had no effect when tested alone. Treatment with testosterone, but not dihydrotestosterone, decreased (P < 0.05) endogenous follistatin and increased (P < 0.05) the activin A:follistatin ratio in maturation medium. Concentrations of inhibin A, total alpha-subunit and activin A were not affected significantly by androgen or flutamide. Addition of progesterone or the anti-progestin mifepristone to cumulus-oocyte complexes had no effect on cleavage rate. However, progesterone reduced by approximately 40% (P < 0.05) the proportions of both total oocytes and cleaved oocytes that formed blastocysts. This effect was partially reversed by mifepristone. Neither progesterone nor mifepristone affected inhibin A, activin A or follistatin production. However, total alpha-subunit concentration was significantly greater in the progesterone-treated group than in the controls (50%; P < 0.05), indicating that the negative effect of progesterone on blastocyst yield may be mediated by increased inhibin alpha-subunit expression by cumulus cells.     

Effect of injection of gonadotrophin-releasing hormone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Hypogonadal (hpg) mice were injected once daily with 10 ng, 50 ng or 1 microgram GnRH for 5, 10 or 20 days or 12 times daily with 4.2 ng GnRH for 5 days. Basal and hCG-stimulated production in vitro of androstenedione, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol (androstanediol) were measured by radioimmunoassay. All doses of GnRH increased testicular weight and in-vitro androgen production although seminal vesicle weights were unchanged and serum testosterone concentrations remained undetectable. After 5 days' treatment androstenedione and androstanediol were the dominant androgens produced, the latter indicating the presence of high levels of 5 alpha-reductase. By 20 days testosterone production was predominant after treatment with higher doses of GnRH. Total androgen production (androstenedione + testosterone + androstanediol) after 5 and 10 days was similar at all concentrations of GnRH used. After 20 days' treatment total androgen production was significantly greater with 50 ng GnRH/day than with 10 or 1000 ng/day. Multiple daily injections of 4.2 ng GnRH (total dose 50 ng/day) had no greater effect on androgen production in vitro compared to single daily injections of 50 ng. This suggests that under the conditions used in this study the testis does not require pulsatile release of the gonadotrophins. The pattern of [3H]pregnenolone metabolism was measured after 5 days injection of 50 ng GnRH/day. Compared to control hpg animals there was a significant increase in formation of C19 steroids, synthesis being solely through the 4-ene pathway. These results show that GnRH treatment of hpg mice will induce testicular steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental change in the ability of estradiol to suppress testosterone secretion by the testis of the rat

An intratesticular site of action has been proposed for the ability of estradiol (E2) to suppress testosterone secretion. Because testicular testosterone and E2 secretion as well as E2 receptors change during development, a physiologic role for E2 is possible. The present experiments compared the testes from 12-day-old and adult rats for the capacity of in vivo estradiol treatment to change in vitro androgen secretion in response to luteinizing hormone (LH) and dibutyryl cyclic AMP (Bt2cAMP). After 5 days in vivo treatment, in vitro responsiveness was estimated by radioimmunoassay (RIA) measurement of androgen secretion elicited by various doses of NIAMDD-LH-24 or 1.0 mM Bt2cAMP. Five days of E2 alone (500 ng/g BW s.c. once daily) markedly inhibited basal, LH-stimulated and Bt2cAMP-stimulated androgen production at both ages. Similar treatment of infant rats with LH (100 ng NIAMDD-LH-24/g BW) caused an increase in basal and LH-stimulated androgen secretion in vitro, but had no effect on the response to Bt2cAMP. The same pretreatment of adults with LH had no effect on basal, but inhibited LH- or Bt2cAMP-stimulated androgen secretion. Combined treatment of infants with E2 and LH for 5 days had no effect on basal or maximally stimulated androgen production; the in vitro response to submaximal stimulation with LH was significantly inhibited. Combined E2/LH treatment of adults significantly decreased the basal production of androgens and the response to LH or Bt2cAMP. These results suggest a major difference between the response to E2 of the Leydig cells from the rats of the two ages tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

Characterization of the androgen receptors in the hypothalamus, preoptic area and brain cortex of the rat.

The specific androgen receptors for testosterone (T) (1) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the hypothalamus, preoptic area and brain cortex of the rat have been characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fractions in vivo and in vitro we were able to demonstrate androgen-receptor complexes moving with an electrophoretic mobility (R(f) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. Polyacrylamide gel electrophoresis was used as a quantitative assay for androgen receptors in the tissues. The hypothalamus, preoptic area and brain cortex were found to possess a single class of high affinity binding sites for androgens and the dissociation constants (K(D) were estimated to be 3.4, 4.3 and 2.6 X 10 (-10M) respectively. The binding capacities were 3.7 (hypothalamus), 3.5 (preoptic area) and 1.8 X 10 (-15) (brain cortex) moles of high affinity binding sites per mg protein. Like other androgen-receptor complexes, the testosterone-receptor complexes of the hypothalamus, preoptic area and brain cortex were temperature labile, sulfhydryl dependent and revealed a very slow rate of dissociation at o degrees C (t1/2 greater than 36 hr). The receptors in all the tissues had an isoelectric point of 5.8. The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for (3H)-testosterone binding. In the three tissues in investigation the following order of affinity was found: DHT greater than T greater than Cyproterone acetate greater than progesterone greater than androstenedione greater than 17beta-estradiol. Cortisol did not effect androgen binding significantly. Thus, the physiochemical characteristics of the cytoplasmic androgen receptors of the hypothalamus, preoptic area and brain cortex are very similar, if not identical, to those of the androgen receptors described in the anterior pituitary, ventral prostate, epididymis and testis.     

CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures

Cultured rat epididymal tissue explants formed >90% pure, adherent growing epithelial cell monolayers. Despite their flattened and apparently androgen receptor-negative phenotype, these cells for a short period kept characteristics of the epididymal duct epithelium, i.e., expression of the tissue-specific marker CD52 and responsiveness of its mRNA toward temperature elevation and androgen withdrawal. When cells were grown on permeable supports at 33 degrees C, androgen supplementation or withdrawal specifically modulated the levels as well as the length of the CD52 mRNA. Elevation of the culture temperature to a quasi abdominal milieu of 37 degrees C selectively reduced the CD52 mRNA levels under all culture conditions. This reduction was not affected by the presence of androgens and was not accompanied by changes in length, suggesting that the modulation of CD52 mRNA in epididymal cells by androgens and by temperature is synergic, but may involve different molecular mechanisms. CD52 mRNA levels, however, were not stable in the primary cultures but decreased rapidly to undetectable levels after 4-5 days at all culture conditions. GAPDH mRNA levels, on the other hand, were stable throughout the culture period.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish, Clarias gariepinus

The present report focuses on the mechanism(s) involved in the steroid-induced decrease of androgen production in immature African catfish testes that was observed in previous studies. Juvenile animals were implanted with Silastic pellets containing different 11-oxygenated androgens (11-ketotestosterone, KT; 11 beta-hydroxyandrostenedione, OHA; 11-ketoandrostenedione, KA), testosterone (T) or estradiol-17 beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were either incubated with increasing concentrations of catfish luteinizing hormone (LH), or incubated with [3H]-pregnenolone ([3H]-P5) or [3H]-androstenedione ([3H]-A). Tissue fragments were also prepared for the quantitative assessment of Leydig cell morphology. Most of the parameters studied were not affected significantly by implantation of E2. Implantation of all androgens inhibited both the basal and the LH-stimulated androgen secretory capacity in vitro. This was associated with a reduced size of the Leydig cells and loss of half of their mitochondria. The studies on the metabolism of tritiated steroid hormones indicated that steroidogenic steps prior to 11 beta-hydroxylation, probably C17-20 lyase activity, were affected by all androgens. Although the effects of 11-oxygenated androgens and T on Leydig cells were mostly similar, previous work showed that only the 11-oxygenated androgens stimulated spermatogenesis, suggesting that distinct mechanisms of action are used by 11-oxygenated androgens and T. These mechanisms, however, seem to merge on the same target(s) to impair Leydig cell androgen production. Such a negative feedback mechanism may be of relevance in the context of the decline in androgen secretion per milligram testis tissue that accompanies the first wave of spermatogenesis in pubertal African catfish.     

Seasonal variation in steroid biosynthesis by the testis of the lizard Tiliqua Rugosa.

1. Seasonal change in the biosynthesis of androgens by testicular tissue from a scincid lizard Tiliqua rugosa has been studied in vitro using [7-3H]pregnenolone and [4-14C]progesterone as substrates. 2. Evidence is presented for the synthesis of epitestosterone, testosterone, androstenedione, 17 alpha-hydroxyprogesterone, 20 beta-hydroxypregn-4-en-3-one, and 17 alpha, 20 beta-dihydroxypregn-4-en-3-one. 3. Epitestosterone was the major conversion product. Its yield was highest (68%) during summer and lowest (14%) during spring. The yield of testosterone was maximal (10%) during spring (mating season). The production of 20 beta-hydroxypregn-4-en-3-one was highest during spring and undetectable during summer. 4. Histological evidence and changes in testicular weight indicated that spermatogenic activity was greatest during spring. Pronounced regression of the testis occurred immediately after the breeding season and lasted until autumn.     

Gonadotropin stimulated androgen secretion of rainbow trout (Salmo gairdneri Richardson) testis in vitro

1. The secretion of five androgens was quantified from trout testes under GTH-stimulation in vitro before and after the onset of milt production, and a general increase of basal and GTH stimulated androgen secretion was recorded during this period. 2. 11-Ketotestosterone, testosterone, and in spermiating males, 11 beta-hydroxytestosterone as well showed GTH dependent concentration increases, while androstenetrione and 11 beta-hydroxyandrostendione were found in highly variable amounts. 3. 17 beta-Hydroxyandrogen glucuronides in the medium (with the exception of testosterone) and tissue androgens were by far exceeded by the free androgens in the medium.     

In vitro effect of temperature on phagocytic and cytotoxic activities of splenic phagocytes of the wall lizard, Hemidactylus flaviviridis.

The in vitro effect of temperature on phagocytosis, nitric oxide production and interleukin-1 (IL-1) secretion by splenic phagocytes isolated from the wall lizard (Hemidactylus flaviviridis) demonstrated that changes in temperature altered non-specific defenses. The LPS-induced percentage phagocytosis and phagocytic index were recorded maximum at 25 degrees C. The phagocytic activity declined considerably when the phagocytes were incubated at low (7 and 15 degrees C) or high (37 degrees C) temperatures. The presence of bacterial lipopolysaccharide (LPS) in the incubation medium could considerably enhance the phagocytic activity of splenic phagocytes. A similar temperature-related effect was also observed on LPS-induced cytotoxic activity of phagocytes. LPS could stimulate the nitrite release indicating nitric oxide production only at 25 degrees C. Likewise, the proliferative responses of immature rat's thymocytes to LPS-induced phagocyte-conditioned medium suggest that IL-1 secretion was enhanced when phagocytes were cultured at 25 degrees C. This suggests that 25 degrees C is the optimal temperature for phagocyte functions in H. flaviviridis. The decrease or increase in temperature other than at 25 degrees C dramatically suppressed the phagocyte activities.     

Androgen-induced nuclear accumulation of the estrogen receptor.

The effect of androgens on the nuclear uptake of both tritiated estradiol (3H-E2) and the estrogen receptor was studied in immature rat uteri. It was demonstrated that in vitro preincubation of immature rat uteri with various androgens (1 muM to 50 muM) followed by incubation with 3H-E2 (20 nM) resulted in a greatly decreased specific nuclear uptake of 3H-E2. Non-androgenic steroids had no effect. It was also confirmed that 5alpha-dihydrotestosterone (DHT) causes the accumulation of the estrogen receptor in the nuclei of uterine tissue. In vitro incubations of rat uteri with DHT (1muM and 50muM) were found to cause maximal nuclear estrogen receptor accumulation to the same degree as caused by estradiol, i.e. the nuclear uptake of approximately 100% of the estrogen receptor. Antiandrogens, which block the binding of androgens to the testosterone receptor in various tissues, did not inhibit the DHT - induced decrease in the 3H-E2 uptake by the uterine nuclei or the DHT - caused accumulation of the estrogen receptor in nuclei. These results seem to indicate that the uterine testosterone receptor has no role in the androgen - induced nuclear uptake of the estrogen receptor. However, the non-steroidal antiestrogens inhibited the DHT - induced nuclear accumulation of the estrogen receptor. This would seem to indicate that the estrogen - and androgen - induced nuclear accumulation of the estrogen receptor share a common mechanism.     

Effect of long term diazepam administration on testicular benzodiazepine receptors and steroidogenesis.

We evaluated the effect of acute and chronic diazepam administration on testicular peripheral type benzodiazepine receptors (PBZD-R), serum testosterone and LH levels and the "in vitro" androgen production in response to Ro 5-4864, a PBZD-R agonist. The chronic diazepam treatment induced a significant fall in plasma testosterone concentration while LH levels remained unchanged. The number of PBZD-R was reduced by 37% and low concentrations (10(-8)-10(-6) M) of Ro 5-4864 failed to stimulate "in vitro" androgen production. The acute diazepam administration caused a significant increase in plasma testosterone levels while no changes were observed in LH concentrations and testicular PBZD-R. These results further suggest a modulatory role of PBZD-R on testicular steroidogenic activity.