Testosterone production by collagenase-dispersed cells from baboon fetal testis

We determined whether dehydroepiandrosterone (DHA) and androstenedione (A) were converted to testosterone (T) by the midgestation primate fetal testis in the absence of gonadotropins. Testes from six baboon (Papio anubis) fetuses, obtained by cesarean section at Day 100-107 of gestation (term = Day 184) were dispersed with 0.2% collagenase. Cells (1.1 X 10(6)) were suspended in 4 ml Eagle's Minimum Essential Medium containing penicillin/streptomycin (MEM) and incubated for 20 h (37 degrees C) with or without DHA, A, pregnenolone (P5), 17 alpha-hydroxypregnenolone (17OH-P5), progesterone (P4) or 17 alpha-hydroxyprogesterone (17OH-P4). Concentrations of T, A, P4, and 17OH-P4 in the medium and cells were measured by radioimmunoassay. Mean secretions of T and A, in the absence of exogenous substrates, were 0.5 +/- 0.2 and 0.8 +/- 0.3 ng/mg testis, respectively, and were not elevated by human chorionic gonadotropin (hCG). Addition of DHA at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) the production of T to 6 +/- 0.6, 33 +/- 10, and 64 +/- 26 ng/mg testis and the production of A to 13 +/- 5.5, 54 +/- 10, and 67 +/- 22 ng/mg testis, respectively. Similarly, addition of A at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) production of T to 27 +/- 5.3, 155 +/- 29, and 254 +/- 79 ng/mg testis, respectively. In contrast, production of T and A remained near baseline concentrations when cells were incubated with 1000 ng/4 ml of P5, P4, 17OH-P5, or 17OH-P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

Assay system for simultaneous measurement of three steroidogenic enzyme activities in rat and human testis--effect of human chorionic gonadotropin

An assay system that measures the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4 pathway of testosterone biosynthesis using rat and human testicular homogenate was examined. This system involves the simultaneous separation of the steroid intermediates by a three-step TLC procedure. The observed Rf values were 0.78 for progesterone (P), 0.59 for 17 alpha-hydroxyprogesterone (17 alpha-HP), 0.70 for androstenedione (A), 0.5 for testosterone, 0.64 for dihydrotestosterone, and 0.45 for 3 alpha, 17 beta-androstanediol. The identification of these steroid intermediates was further accomplished by acetylation and rechromatography of the representative samples along with the authentic standards and by recrystallization to constant specific activity until three consecutive crystallizations were within +/- 5% of the mean value. Incubation time up to 30 min and increasing protein concentrations showed a linear relationship with respect to these three enzymatic activities. The optimum temperature for these enzymatic activities varied from 32 to 34 degrees C, with a sharp decline between 37 and 40 degrees C. The Michaelis constants (Km) for the rat testis homogenate samples were 0.17 microM for P, 0.22 microM for 17 alpha-HP, and 2.5 microM for A, while for the human testis the Km values were 1.2, 2.2, and 2.3 microM, respectively, for these substrates. The concentrations of the endogenous steroid substrates present in these homogenate samples did not alter the Km or Vmax values. The effect of human chorionic gonadotropin (hCG) in vitro on these steroidogenic enzyme activities was also studied. In the rat testis, 10 IU of hCG produced a significant rise in all the three enzyme activities whereas in the human testis 10 and 30 IU of hCG showed no significant change in any of these enzymatic activities. However, 100 IU of hCG resulted in a significant increase in 17 alpha-hydroxylase and 17,20-desmolase activities in the human testis. These studies suggest that this assay system for the measurement of these enzymatic activities using a testicular homogenate sample provides consistent and reproducible results. Based on the sensitivities of the measurements and our experience with testicular biopsy technique, we conclude that a routine testicular biopsy in the human should provide sufficient tissue to run these enzymatic assays.     

Differential effects of follicle-stimulating and luteinizing hormones on testosterone production by mouse testes

In adult mice, direct intratesticular injection of ovine follicle-stimulating hormone (o-FSH-13; AFP 2846-C, from NIAMDD, less than 1% LH contamination) at 10, 100 or 1000 ng significantly elevated concentrations of testosterone (T) within the testis. These effects were rapid, with peak values attained by 15 min, and transient, with return to values comparable to that in the contralateral, saline-injected testis within 90 min. Intratesticular injection of FSH (1 microgram) significantly increased testicular T levels in 15- and 60-day old mice. This contrasted with the effects of intratesticular administration of human chorionic gonadotropin (hCG), which stimulated T production significantly at 30 days of age through adulthood. In adult mice, the equivalent LH to the possible contamination in the FSH preparation (1 ng) had no effect. Intratesticular injection of 10 ng LH produced comparable stimulation to that by 100 ng FSH (approximately 7-fold). Systemic pre-treatment with a charcoal-treated porcine follicular fluid (PFF) extract for 2 days reduced plasma FSH levels [86 +/- 17 (5) vs 700 +/- 8 (6); P less than 0.05], but had no effect on plasma LH. Twenty-four hours after the last treatment, the response to intratesticular injection of hCG (2.5 mIU), FSH (100 ng) or LH (10 ng) was also significantly attenuated in these mice. Intratesticular injection of PFF had no direct effect on testicular T levels. In vitro T production in the presence of hCG, LH or FSH were differentially affected by the concentrations of calcium (Ca2+) or magnesium (Mg2+) in the incubation media. The stimulatory effects of FSH were apparent at significantly lower levels of Ca2+ or Mg2+, than were those of LH or hCG. The results of these studies indicate that FSH is capable of stimulating testicular T production. Furthermore, the responsiveness to FSH is qualitatively different than that to LH/hCG in terms of the age pattern, as well as the dependence on Ca2+ or Mg2+. In addition, plasma FSH levels appear to influence testicular responsiveness to direct exogenous administration of gonadotropins. These studies indicate that FSH stimulation of T production can be differentiated from those of LH, and that these effects of FSH can be observed under physiological conditions.     

Effect of transforming growth factor-beta on human chorionic gonadotropin induced testosterone production by cultured rat testicular cells

The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.     

Inhibition of testicular testosterone biosynthesis following experimental varicocele in rats

In an attempt to determine whether defective testicular testosterone (T) biosynthesis may be associated with a varicocele, an experimental study was performed in adult rats whereby a unilateral left varicocele was surgically created. At 2, 4, 8, and 12 wk following the creation of the varicocele, intratesticular T as well as the activities of three (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) of the five enzymes in the delta 4 pathway of testicular T biosynthesis were measured. Intratesticular T (ng/g testis +/- SEM) in the left testis decreased significantly from 121 +/- 21 in the control group to 59 +/- 8 in the two-wk varicocele group (p less than 0.01), and remained significantly suppressed throughout the experimental period. The T concentrations in the right testis paralleled those in the left in both the control and varicocele animals. At 2 wk following the creation of the varicocele, the activity (nmol/min/testis +/- SEM) of the 17,20-desmolase enzyme decreased significantly, from 115 +/- 8 in the left testis of control rats to 87 +/- 6 in the left testis of the varicocele animals (p less than 0.025), and remained low throughout the 12 weeks of the study. The activity of the 17 alpha-hydroxylase enzyme was significantly decreased at the 8th and 12th weeks of the study, while the 17 beta-hydroxysteroid dehydrogenase activity did not show any significant change during the study period. The enzyme activities in the right testis paralleled those in the left testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of season on seminal characteristics and endocrine status of adult free-ranging African buffalo (Syncerus caffer)

Pituitary, gonadal and adrenal activity were compared in free-living, adult African buffalo bulls during the breeding and nonbreeding seasons. Frequent blood samples were collected for 2 h from anaesthetized bulls treated intravenously with saline, gonadotrophin-releasing hormone (GnRH, 200 micrograms), human chorionic gonadotrophin (hCG, 10,000 i.u.) or adrenocorticotrophic hormone (ACTH, 1.5 mg). Electroejaculates also were collected from anaesthetized bulls during the breeding and nonbreeding seasons. Pretreatment testosterone concentrations among bulls varied more during the breeding (0.17-23.0 ng/ml) than the nonbreeding (0.15-2.21 ng/ml) season. The variation within the breeding season was attributed to 8 of 25 bulls producing higher (P less than 0.05) serum testosterone (High-T; 16.28 +/- 2.03 ng/ml) and testicular LH receptor (1.53 +/- 0.22 fmol/mg testis) concentrations compared with their seasonal counterparts (Low-T; 0.95 +/- 0.26 ng/ml; 0.38 +/- 0.04 fmol/mg) or with all bulls during the nonbreeding season (0.90 +/- 0.27 ng/ml; 0.31 +/- 0.04 fmol/mg). The magnitude of GnRH- and hCG-induced increases in serum testosterone was similar (P greater than 0.05) between Low-T bulls and bulls during the nonbreeding season. In the High-T animals treated with GnRH or hCG, serum testosterone did not increase, suggesting that secretion was already maximal. Peak serum LH concentrations after GnRH were greater (P less than 0.05) in bulls during the nonbreeding than the breeding season; FSH responses were similar (P greater than 0.05). ACTH treatment did not increase serum cortisol concentrations above the 2-fold increase measured in bulls treated with saline, hCG and GnRH (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of spermatocyte development in prepubertal rats by the Sertoli cell steroid, 3 alpha-hydroxy-4-pregnen-20-one

The effect of 3 alpha-hydroxy-4-pregnen-3-one (3HP), a Sertoli cell steroid, on spermatogenesis was examined in normal and gonadotropin-suppressed rats, through s.c. as well as direct intratesticular injections. Early experiments employing normal prepubertal male rats indicated that 3HP, when administered at 65 micrograms/100 g BW daily for 15 days, was capable of stimulating pachytene spermatocyte number to 149% of untreated control numbers. It was of interest to determine if this effect could be amplified in gonadotropin-suppressed animals. Neonatally estrogenized rats (500 micrograms estradiol benzoate in 0.1 ml oil at 2 days) were treated on alternate days with 3HP (100 micrograms/100 g BW) for 3 wk, starting at 7 days of age. This treatment significantly increased the number of spermatocytes per tubule cross section from 17.3 +/- 1.9 (in estradiol benzoate-only animals) to 47.1 +/- 7.9 (p less than 0.01). In a similar study, 100 micrograms/100 g BW of testosterone propionate could stimulate spermatocyte number to only 15.1 +/- 2.2 cells per tubule cross section versus estradiol benzoate-only numbers. A single intratesticular injection of 3HP (2 ng in 2.0 microliters oil) in Methallibure-treated rats resulted in a significant increase in late pachytene spermatocyte numbers from 0.77 +/- 0.12 in Methallibure-only-treated rats to 1.70 +/- 0.10 (p less than 0.001) cells per tubule cross section in 28-day-old rats. However, in this study, no other progesterone metabolite or androgenic steroid (testosterone, 5 alpha-dihydrotestosterone, or 5 alpha-androstan-3 alpha, 17 beta-diol) tested was capable of this level of germ cell stimulation. In conclusion, 3HP appears to have a direct effect on germ cell development within the testis at levels much lower than those shown to be effective for androgens. It does not appear that this effect is mediated through the conversion of 3HP to any C21 or C19 steroids, and appears to be the first report of a Sertoli cell steroid with a possible role in the process of mammalian spermatogenesis.     

Testicular responsiveness to a single hCG dose in patients with testicular feminization

The suggestion that androgens may regulate testosterone (T) production in rat Leydig cells by a receptor-mediated feed-back mechanism, led us to investigate whether in vivo the absence of testicular androgen receptors, as it occurs in testicular feminization (TF), may modify the characteristic testicular response observed in men and prepubertal children after a single dose of hCG. Subjects consist of: 1) six normal men, 2) two adult patients with the complete form of androgen insensitivity syndrome (TF), 3) 12 normal prepubertal boys, 4) one prepubertal boy with the same form of TF. Each subject received i.m. a single dose of hCG 3500 IU/m2 b.s. and blood samples were collected basally and 2, 4, 24, 48, 72 and 96 hours after the hormonal stimulus. Serum levels of T, 17 alpha hydroxyprogesterone (17OHP) and 17 beta estradiol (E2) were measured at each collection time. In normal men a significant increase in T (M +/- SE) was observed at 4 h (758.6 +/- 135 ng/dl, P less than 0.05) and a more significant increase at 48 h (1082 +/- 60.3 ng/dl, P less than 0.001). E2 and 17OHP peaked significantly at 24 h (81.5 +/- 9.6 pg/ml and 460.7 +/- 90.9 ng/dl respectively). This response pattern is characteristic of the testicular desensitization which occurs in normal man after a single hCG dose. The same response pattern has been observed in the two TF adult patients suggesting that human testicular desensitization in vivo does not depend on androgen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Oogenesis and plasma levels of sex steroids in cultured females of brown trout (Salmo trutta linnaeus, 1758) in Chile

Naturalized brown trout populations in Chile are a valuable genetic resource with aquaculture potential. The oogenesis of a three-year-old brown trout cultured population was studied in southern Chile. Gonadosomatic index (GSI), oocyte growth, gonadal microscopic characteristics, and plasma levels of estradiol-17beta (E2), testosterone (T), and 17alpha-hydroxyprogesterone (17alpha-HP) were measured bimonthly for a nine-month period before spawning. The maximum GSI level (22%) was similar to that described for other salmonids, although it was reached in May, more than one month before the population started spawning. Oocyte growth increases strongly from January when diameter reaches more than 1 mm. The vitellogenic period (six-seven months) is consistent with the long vitellogenesis, described for salmonid females maturing at three years old. E2 shows a slow increase from November, reaching its peak value in March (65.2+/-0.7 ng/ml), during maximal vitellogenic activity. T increases as oogenesis progresses, reaching a maximum of 90+/-20 ng/ml during May, and falling considerably during ovulation. Following a typical pattern of progestogens in salmonid oogenesis, 17alpha-HP stays at basal levels during most of oogenesis, but experiences a strong surge (2.0+/-0.4 ng/ml) just before ovulation.     

Progesterone production by luteal cells isolated from cynomolgus monkeys: effects of gonadotropin and prolactin during acute incubation and cell culture

Corpus luteum function in cynomolgus monkeys (Macaca fascicularis) during the menstrual cycle and immediately following parturition was evaluated through in vitro studies on progesterone production by dispersed luteal cells in the presence and absence of human chorionic gonadotropin (hCG) or human prolactin (hPRL). Luteal cells isolated between days 17-20 of the menstrual cycle secreted progesterone (P) during short-term incubation (21.6 +/- 1.2 ngP/ml/5 X 10(4) cells/3 hr, X +/- S.E., n = 7) and responded to the addition of 1-100 ng hCG with a significant (p less than 0.05) increase in P secretion. Cells removed the day of delivery secreted large, but variable (27.9-222 ng/ml, n = 4) amounts of P during short-term incubation. Moreover, hCG (100 ng/ml) stimulation of P production by cells at delivery (176 +/- 19% of control) was less than that of cells from the cycle of (336 +/- 65%). The presence of hPRL (2.5-5000 ng/ml) failed to influence P secretion by luteal cells during short-term incubation in the presence or absence of hCG. P production by luteal cells obtained following delivery declined markedly during 8 days of culture in Ham's F10 medium: 10% fetal calf serum. Continual exposure to 100 ng/ml of hCG or hPRL failed to influence P secretion through Day 2 of culture. Thereafter hCG progressively enhanced (p less than 0.05) P secretion to 613% of control levels at Day 8 of culture. In contrast, hPRL significantly increased P secretion (163% of control levels, p less than 0.05) between Day 2-4 of culture, but the stimulatory effect diminished thereafter. The data indicate that dispersed luteal cells from the cynomolgus monkey provide a suitable model for in vitro studies on the primate corpus luteum during the menstrual cycle, pregnancy, and the puerperium, including further investigation of the possible roles of gonadotropin and PRL in the regulation of luteal function in primates.     

Reproductive hormone secretion and testicular growth in bull calves actively immunized against testosterone and oestradiol-17 beta

Groups of bull calves received a primary immunization against testosterone (Group T; N = 7) or oestradiol-17 beta (Group E; N = 9) at 3 months of age and booster injections on four occasions at approximately 2 month intervals. Controls (Group C, N = 7) were immunized against human serum albumin alone using the same protocol. Immunity was achieved against both steroids as judged by the secondary antisteroid antibody titres in Group T (730 +/- 231; reciprocal of titre) and Group E (12,205 +/- 4366) bulls; however, peak antibody titres generally declined with successive booster injections. Mean plasma concentrations of LH, FSH and testosterone during the period from 3 to 10 months of age were higher (P less than 0.05) in Group T bulls than in Groups C and E. Group T bulls had larger testes compared with controls from 6 months of age onwards. At castration at 14 months of age, testes of Group T bulls were heavier (P less than 0.05) than those of Groups C and E (179 +/- 13, 145 +/- 8 and 147 +/- 6 g, respectively). At 10 months of age, there were no differences among treatment groups in LH responses to LHRH, but the testosterone responses were greater (P less than 0.05) in bulls in Group T (26.2 +/- 4.9 ng/ml) and Group E (16.6 +/- 1.8 ng/ml) compared with those in Group C (6.9 +/- 0.6 ng/ml). Testosterone responses to hCG determined at 13 months of age were also greater (P less than 0.05) in Groups T and E relative to controls. At 14 months of age daily sperm production rates per bull (X 10(-9)) were higher (P less than 0.10) in Group T bulls (2.2 +/- 0.1) than those in Groups C (1.6 +/- 0.2) and E (1.6 +/- 0.1). These results indicate that early immunity against testosterone is associated with increased gonadotrophin secretion and accelerated growth of the testes in prepubertal bulls. Also, chronic immunity against testosterone or oestradiol-17 beta enhances the steroidogenic response of bull testes to gonadotrophic stimulation. If the above responses observed in young bulls are shown to be sustained, then immunity against gonadal steroids early in life may confer some reproductive advantage in mature animals.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of cortisol and progesterone to mediate the stimulatory effect of adrenocorticotropic hormone upon testosterone production by the porcine testis

The influence of corticosteroids and progesterone upon porcine testicular testosterone production was investigated by administration of exogenous adrenocorticotropic hormone (ACTH), cortisol and progesterone, and by applying a specific stressor. Synthetic ACTH (10 micrograms/kg BW) increased (P less than 0.01) peripheral concentrations of testosterone to peak levels of 5.58 +/- 0.74 ng/ml by 90 min but had no effect upon levels of luteinizing hormone (LH). Concentrations of corticosteroids and progesterone also increased (P less than 0.01) to peak levels of 162.26 +/- 25.61 and 8.49 +/- 1.00 ng/ml by 135 and 90 min, respectively. Exogenous cortisol (1.5 mg X three doses every 5 min) had no effect upon circulating levels of either testosterone or LH, although peripheral concentrations of corticosteroids were elevated (P less than 0.01) to peak levels of 263.57 +/- 35.03 ng/ml by 10 min after first injection. Exogenous progesterone (50 micrograms X three doses every 5 min) had no effect upon circulating levels of either testosterone or LH, although concentrations of progesterone were elevated (P less than 0.01) to peak levels of 17.17 +/- 1.5 ng/ml by 15 min after first injection. Application of an acute stressor for 5 min increased (P less than 0.05) concentrations of corticosteroids and progesterone to peak levels of 121.32 +/- 12.63 and 1.87 +/- 0.29 ng/ml by 10 and 15 min, respectively. However, concentrations of testosterone were not significantly affected (P greater than 0.10). These results indicate that the increase in testicular testosterone production which occurs in boars following ACTH administration is not mediated by either cortisol or progesterone.     

The effect of testosterone withdrawal and subsequent germ cell depletion on transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the adult rat testis.

We have examined the effects of decreasing intratesticular testosterone concentration and of decreasing germ cell number on levels of transferrin mRNA and sulfated glycoprotein (SGP)-2 mRNA in the adult rat testis. Intact rats received implants of testosterone- and estradiol-filled capsules to suppress LH secretion from the pituitary, thereby suppressing Leydig cell testosterone production. The levels of intratesticular testosterone declined 70% to 20 ng/ml within 3 days, were reduced further to approximately 15 ng/ml by 14 days, and subsequently reached a minimum of about 10 ng/ml. In contrast, the number of elongated spermatids per testis remained unchanged through 14 days, then declined to fewer than 20% of normal between 14 and 28 days, and reached zero by 56 days postimplantation. Likewise, both pachytene spermatocytes and round spermatids declined only after 14 days postimplantation. Northern blots of testicular RNA showed that Sertoli cell transferrin mRNA per testis decreased markedly between 14 and 28 days postimplantation. However, SGP-2 mRNA per testis was unchanged over the time course of the experiment. The decrease in transferrin mRNA, concomitant with germ cell loss, suggests that this mRNA is regulated by the number of germ cells in the testis and not directly by testosterone. In contrast, the constant level of SGP-2 mRNA in the face of reduced intratesticular testosterone and the subsequent loss of germ cells suggests that this mRNA is constitutively maintained in the adult rat testis.     

Testicular responsiveness to hCG of the ovine fetus in the last third of gestation

The in vivo and in vitro testicular responsiveness to hCG of hemicastrated lamb fetuses 95-99, 110-118 and 130-141 days of gestational age was studied. Basal plasma testosterone (T) levels were similar at all ages (less than 0.25 ng/ml), while the mean testicular concentrations of dehydroepiandrosterone sulfate (DHA-S), 17 alpha-hydroxyprogesterone (17-OHP) and T were higher in 95- to 99-day-fold fetuses. Plasma T levels and the concentration of T, DHA-S, 17-OHP, androstenedione (A) and cyclic adenosine 3'5'-monophosphate (cAMP) were increased by hCG in the hemicastrated animal at all ages. cAMP and T production by enriched preparations of dispersed interstitial cells from control testes was increased by hCG in all groups. In fetuses pretreated with hCG in vivo the addition of hCG in vitro failed to modify cAMP and T production. 100 micrograms of LHRH to a 130-day-old fetus increased plasma LH and T levels. From these experiments, it is suggested that the low plasma LH and T levels found throughout the last trimester of fetal life reflect a relative lack of endogenous LHRH synthesis and/or release, rather than reduced testicular steroidogenic capacity.     

Antigonadal activity of the neurohypophysial hormones: in vivo regulation of testicular function of hypophysectomized rats

The "antigonadal" potential of the neurohypophysial hormones, previously demonstrated in vitro, was evaluated in vivo using hypophysectomized male rats. This approach minimizes the likelihood that the in vivo "antigonadal" effect of the neurohypophysial hormones may be due to their ability to attenuate the release of pituitary gonadotropins. Given that the identity of the putative endogenous occupant of testicular pressor-selective neurohypophysial receptors remains uncertain, use was made of a substitute probe, arginine vasotocin (AVT), the utility of which has been demonstrated in vitro. Concurrent in vivo treatment of follicle-stimulating hormone (FSH; 5 micrograms/rat/day)-maintained immature hypophysectomized rats with increasing doses of AVT (0.25-25 microgram/rat/day) produced significant (P less than 0.05) dose-dependent inhibition of the testicular luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor binding capacity (but not affinity; Kd = 1.8 X 10(-10) M) from 8.8 +/- (standard error; SE) 0.4 ng/testis to a level (3.2 +/- 0.2 ng/testis) lower than that of controls (64% reduction). This AVT-induced decrease in the testicular LH/hCG receptor content of FSH-maintained immature hypophysectomized rats was associated with significant (P less than 0.05) decrements in the hCG- and N6, 2'-O-dibutyryladeosine cyclic 3',5'-monophosphate [( Bu]2cAMP)-stimulated accumulation of 3 alpha-hydroxy-5 alpha-androstan-17-one (androsterone; 52% and 42% inhibition, respectively), with virtual elimination (98% inhibition) of the forskolin-stimulated accumulation of extracellular cAMP by testicular incubates in vitro, as well as with profound suppression of spermatogenesis. Taken together, these observations indicate that the "antigonadal" effect of the neurohypophysial hormones previously demonstrated in vitro, can be fully reproduced in vivo, and that the "antigonadal" activity of the neurohypophysial hormones may be accounted for, in large part, by decreased testicular LH/hCG binding capacity, stimulable adenylate cyclase activity, and cAMP-supported androgen biosynthesis.     

Total and free testosterone in plasma of hypo- and agonadal men.

Plasma total testosterone (T), apparently free T and testosterone binding globulin (TeBG) capacity determined in 14 normal men aged 30-40 years were 461 +/- 100 ng/100 ml, 9.4 +/- 3.0 ng/100 ml and 5.7 +/- 1.9 X 10(-8) M, respectively, whereas in 16 hypogonadal men the corresponding values were 38.6 +/- 27.2 ng/100 ml, 0.47 +/- 0.41 ng/100 ml and 10.4 +/- 3.4 X 10(-8) M showing the TeBG capacity significantly higher (p less than 0.001) in hypogonadal than in normal men. Treatment of 5 hypogonadal subjects with 250 mg testosterone enanthate plus 50 mg testosterone propionate decreased (p less than 0.001) the TeBG level from 14.7 +/- 2.5 X 10(-8YM to 8.3 +/- 1.4 X 10(-8) M on day 8 after a single injection. According to this difference in TeBG, the free T fraction in plasma rose from 0.94% to 1.9% of the total T concentration. These results suggest that alteration of total plasma T affected the TeBG capacity. Decreased T levels raised and increased T concentrations suppressed TeBG, but with a delayed response to the changed T concentrations. The initial mean values in 12 patients with prostatic cancer aged 60-74 years were 397 +/- 165 ng/100 ml, 4.05 +/- 1.8 ng/100 ml and 11.9 +/- 3.3 X 10(-8) M, respectively. The TeBG capacity in these patients was significantly higher and the free T concentration significantly lower (p less than 0.001) than those of the younger normal males. After treatment with 12 g diethylstilbestrol diphosphate and orchidectomy, the TeBG increased to 33.3 +/- 13.1 X 10(-8) M and the plasma free T concentration decreased to the minimal value of 0.053 +/- 0.04 ng/100 ml.