In vivo and in vitro production of inhibin by cryptorchid testes from adult rats

Adult male rats were made bilaterally cryptorchid for 3, 7, 10, and 14 days, and the peripheral serum, testicular vein serum, and interstitial fluid levels of inhibin were measured by RIA, and compared with values obtained in intact animals. The levels of inhibin in peripheral serum, testicular vein, and interstitial fluid were significantly elevated (P less than 0.01) after 3 days of cryptorchidism but declined significantly thereafter. The secretion of inhibin was also studied in vitro using isolated segments of seminiferous tubules from scrotal and abdominal testis in adult rats made unilaterally cryptorchid for 3, 6, and 12 days. The basal inhibin secretion by 3-day cryptorchid seminiferous tubules incubated at 37 C was significantly greater when compared with the scrotal seminiferous tubules at 32 C. If seminiferous tubules from scrotal testes were incubated at 37 C, the secretion of inhibin was greatly increased to similar levels observed by the 3-day abdominal seminiferous tubule cultures. In addition inhibin secretion was significantly higher when tubules from 5-week hypophysectomized rats were cultured at 37 C compared to 32 C. The inhibin secretion by seminiferous tubules from 6-day abdominal testes had returned to scrotal seminiferous tubule levels but then decreased below scrotal seminiferous tubule levels after 12 days of cryptorchidism. Seminiferous tubules from cryptorchid testes remain responsive to FSH stimulation (500 ng/ml) up to 12 days of cryptorchidism. FSH-stimulated inhibin production was increased at 3 and 12 days after cryptorchidism, but similar at 6 days after cryptorchidism, compared to the response of tubules obtained from scrotal testes. Furthermore, using seminiferous tubules from normal adult rats, FSH-stimulated inhibin production was increased by raising the incubation temperature from 32 C to 37 C. These in vivo and in vitro data suggest a dual stimulatory and inhibitory effect of higher temperature on inhibin production with an initial rise in basal and FSH-stimulated inhibin secretion by the cryptorchid testis which seems to be due to a direct effect of temperature on Sertoli cells. The subsequent decline in inhibin secretion could be due to the disruption of the seminiferous epithelium.     

In vitro bioactive and immunoactive inhibin concentrations in bovine fetal ovaries and testes throughout gestation.

This study investigates the concentrations of inhibin in the bovine fetal ovary and testis throughout gestation (days 40 to 270/term) as determined by inhibin in vitro bioassay and RIA techniques. In addition, the expression of the inhibin alpha- and beta-subunits (beta A and beta B) in these tissues was evaluated by Northern blot analysis and in situ hybridization. Testicular concentrations of inhibin bioactivity and immunoactivity increased 2.5-fold (103 +/- 24.5 to 256 +/- 11.7 U/g wet weight; mean +/- SE) and 10.4-fold (139 +/- 56 to 1430 +/- 172) respectively, between 90 days and term. The corresponding ratio of inhibin biological: immunological activities (B/I ratio) decreased 8.3-fold (1.5 +/- 0.7 to 0.18 +/- 0.01). The concentration of ovarian bioactive inhibin increased significantly (P less than 0.05) 4.6-fold between 90 days and term (73.6 +/- 14.7 to 340 +/- 11.1 U/g wet weight), whereas the immunoactive inhibin concentration increased 10.3-fold between 120 and 210 days of gestation (7.2 +/- 1.9 to 40.0 +/- 8.7). The corresponding B/I ratio remained unchanged throughout gestation (8.3 +/- 2.4 to 12.5 +/- 4.0). Although the levels of alpha subunit mRNA in the testis and ovary increased over gestation, the levels of testicular beta A subunit mRNA remained low and unchanged. Ovarian levels of beta A subunit mRNA were also low but variable. Furthermore, no beta B subunit mRNA could be detected in gonadal tissue throughout gestation. alpha-Subunit mRNA was detected by in situ hybridization in the sex cords of the fetal testis and the granulosa cells of the fetal ovary while beta A subunit mRNA was detected only in the granulosa cells of the fetal ovary. It is concluded that inhibin is produced by the fetal testis and ovary and these tissue levels increase throughout gestation. The location of alpha- and beta A-subunit mRNA to the sex cords of the testis and granulosa cells of the ovary indicate that these cells are the primary source of inhibin production. The rapid fall in inhibin B/I ratio in testicular extracts over gestation is attributed to the production of an inhibin-related protein with limited or negligible biological activity.     

Hormones and the differentiation of type A spermatogonia in mouse cryptorchid testes incubated in vitro

In order to study hormonal effects on testicular germ cell differentiation, especially on type A spermatogonia, artificially induced cryptorchid testes of adult mice were cultured in a medium containing testosterone, dihydrotestosterone, tri-iodothyronine, dibutyryl 3':5' cyclic adenosine monophosphate, human chorionic gonadotrophin, LH, FSH, insulin and transferrin. These substances, with the exception of FSH, showed no stimulatory effect on the differentiation of type A spermatogonia. However, FSH activated cell division in type A spermatogonia and stimulated them to differentiate, while LH showed neither the promotion of differentiation nor a synergistic effect on FSH-mediated germ cell differentiation.     

In vitro synthesis and release of inhibin in response to FSH stimulation by isolated segments of seminiferous tubules from normal adult male rats

The production of inhibin by isolated segments of seminiferous tubules from adult male rats cultured in vitro was investigated using a heterologous specific radioimmunoassay. Increasing lengths of tubules (5, 10, 20 and 40 cm) maintained in culture for 4 or 5 days produced increasing amounts of inhibin in vitro. A dose-dependent increase in inhibin production was observed after stimulation with ovine follicle-stimulating hormone (FSH)-s17 (0.1-1000 ng/ml). The tubule segments remain sensitive to FSH stimulation for up to 20 days of culture despite a progressive decline in basal inhibin production, resulting in an increase in the magnitude of the response to FSH stimulation between 0-5, 5-10 and 10-20 days of culture. In the presence of the protein synthesis inhibitor, cycloheximide (50 micrograms/ml), both basal and FSH-stimulated inhibin secretion are inhibited. Testosterone (10(-8)-10(-5) M) does not affect basal inhibin production, although inhibition of the FSH-induced production of inhibin occurred at only the highest dose of testosterone used (10(-5) M). These data demonstrate that the production of inhibin by segments of seminiferous tubules from adult male rats can be used to study the control of inhibin secretion.     

Effect of human chorionic gonadotropin on luteinizing hormone and prolactin binding by testes of bilaterally cryptorchid rats

This study investigates the ability of the testis in the cryptorchid state to bind luteinizing hormone (LH) and prolactin (Prl). For this purpose, rats were made bilaterally cryptorchid at 21 days of age; sham-operated rats were used as controls. At 56 days, the animals were injected with saline or with increasing doses of human chorionic gonadotropin (hCG: 1, 10 or 100 IU) and killed 8 or 24 h after injection. Cryptorchidism and injection of hCG did not alter plasma Prl levels. In cryptorchid rats, both LH and Prl binding expressed in pmol/testis were lower than in sham-operated controls (about 75% and 65%, respectively), but unchanged (LH binding) or increased (Prl binding) when expressed in pmol/g testis. In controls, 100 IU hCG induced a significant decrease in LH binding at 24 h. Prl binding was also significantly lower in controls injected with 100 IU hCG than in those injected with 1 or 10 IU hCG, at 8 h. In cryptorchid rats injected with 100 IU hCG, the LH binding fell 8 h and 24 h after injection; at 24 h, hCG reduced Prl binding. In conclusion, there was a considerable decrease in LH and Prl receptors in the abdominal testes. The negative regulation of these receptors in response to hCG was maintained, but at times and for doses which differed from those observed in scrotal testes.     

In vitro insulin action on erythrocyte glucose metabolism in normal and diabetic rats

Summary Alloxan-induced diabetes in rats significantly impaired the capacity of the erythrocytes to metabolise glucose in vitro to either lactic acid or CO₂. Both these metabolic activities were initially insensitive to insulin in normal as well as in diabetic animals; but became responsive when these cells were subjected to insulin and glucose starvation for 1 h through incubation in their absence. This action of insulin in starved cells showed concentration dependence and required preincubation with the hormone prior to addition of glucose.     

Characterisation of adult Sertoli cell cultures from cryptorchid rats: inhibin secretion in response to follicle-stimulating hormone stimulation.

Testes from adult (90-120-day-old) rats, which had been made cryptorchid 28 days previously, were dispersed by successive treatment with trypsin, collagenase and hyaluronidase. The resulting crude cell suspension was fractionated on discontinuous Percoll density gradients to yield five distinct cell bands (1-5), at the interface between successive layers of Percoll. Crude cells and purified fractions were cultured for up to 7 days, and inhibin was subsequently measured in the media by radioimmunoassay and in vitro bioassay. Sertoli cells from density gradient bands 2 (1.03-1.04 g/ml) and 3 (1.04-1.05 g/ml) showed minimal germ cell or peritubular cell contamination, as determined by morphological and histochemical techniques. Cells from these bands secreted significantly higher levels of immunoactive inhibin/microgram DNA/48 h under both basal and either follicle-stimulating hormone (FSH)- (100 ng/ml) or dibutyryl cAMP-stimulated (100 micrograms/ml) conditions than did cells from the other bands. While there was a decline in basal secretion of inhibin with increasing duration of culture, the capacity of the purified Sertoli cells (bands 2 and 3) to respond to both FSH and dibutyryl cAMP increased over the culture period. The addition of dibutyryl cAMP (31.25-500 micrograms/ml) to the purified Sertoli cells also caused a stimulation of bioactive inhibin. Immunoactive inhibin production by purified Sertoli cells was unaffected by the addition of either rat LH (8 ng/ml) or testosterone (10(-6) M). The data describe a method for the isolation of adult Sertoli cells from cryptorchid testes, and demonstrate their responsiveness to both FSH and dibutyryl cAMP in vitro using the measurement of immunoactive inhibin as a marker of Sertoli cell function.     

Differences in the regulation of steroidogenesis and tropic hormone receptors between the scrotal and abdominal testes of unilaterally cryptorchid adult rats

Rats were made unilaterally cryptorchid by cutting the gubernaculum testis at birth. At 100 days age, the rats were injected with 600 IU/kg hCG. A biphasic testosterone response was seen in the scrotal (Scr) testis in response to hCG, with maxima at 1 h and 3 days after injection. The acute peak of testosterone was of similar magnitude in the abdominal (Abd) testis, but the secondary peak was not present. The response of testicular progesterone concentration to hCG stimulation showed a maximum at 1 day in both gonads, but it was 10- to 20-fold higher (P less than 0.01) in the Abd testis. The content of LH, FSH, and PRL receptors per testis was decreased on the Abd side. After hCG injection, the loss of available LH receptors was faster in the Abd testis. Likewise, the recovery of binding was faster in the Abd testes; at day 10 of the experiment, it was 102 +/- 5% of the starting levels compared to 52 +/- 4% on the Scr side (P less than 0.01). hCG did not affect FSH binding of the Scr testes, but induced a transient drop of 25-35% on day 1 on the Abd side (P less than 0.05). Thereafter, on days 3-10, the FSH binding of the Abd testes was 20-40% higher than on the Scr side (P less than 0.05-0.01). In PRL binding, similar heterologous down-regulation of 50-80% was found in both testes between 12-24 h. Thereafter, the Abd testis PRL receptors showed a transient elevation of 25-70% (P less than 0.05-0.01) on day 3, which was not seen in the Scr testes. In conclusion, the Abd testis displays a dramatically enhanced blockade of C21 steroid side-chain cleavage upon gonadotropin stimulation. The kinetics of changes in testicular LH receptors after hCG stimulation is faster in the Abd testis. Only Abd testes displayed hCG-induced changes in FSH binding and transient up-regulation of PRL receptors. The altered tropic regulation of Leydig and Sertoli cells of the Abd testis are indicative of direct functional changes in these cells in the elevated intra-Abd temperature and/or of changes in the paracrine component of testicular regulation.     

Prolactin binding by testes of unilaterally cryptorchid rats: the effect of hCG, testosterone, prolactin and orchiopexy

The effect of unilateral cryptorchidism on prolactin binding to the testes was studied in the rat. Cryptorchidism was rendered surgically for 3 weeks and 3, 6 and 9 weeks later prolactin binding was measured in testicular homogenates. Prolactin binding to the cryptorchid testes decreased significantly at 3 weeks with a further decrease at 6 and 9 weeks. Binding by the contralateral testes decreased at 3 weeks and increased at 6 and 9 weeks. To examine the possible mechanism of these changes one group of rats was treated for 5 weeks with testosterone and another with hCG. Testosterone treatment resulted in a significant fall in prolactin binding to normal, cryptorchid and contralateral testes. hCG also produced a slight but significant reduction in prolactin binding. To study the effect of surgical relocation of the testes into the scrotum, orchiopexy was performed in another group of rats. Orchiopexy increased prolactin binding only if performed 3 weeks after cryptorchidism. At 6 and 9 weeks after cryptorchidism orchiopexy did not increase prolactin binding. Treatment of cryptorchid rats with prolactin for 5 weeks induced an increase in prolactin binding to control, cryptorchid and contralateral testes. It is concluded that testicular atrophy follows upon placement of a testis within the peritoneal cavity. This atrophy lowers the total amount of prolactin binding and increases binding to the contralateral testes. Intratesticular concentration of testosterone may play a major role in the decrease of prolactin binding. Orchiopexy improves prolactin binding only if performed before 6 weeks of age. Administration of prolactin augments prolactin binding to the testes, irrespective of their location.     

The specificity of inhibin bioassay using cultured pituitary cells

It has been established that the inhibition of the cellular content of FSH in cultured pituitary cells can be used as a sensitive and precise bioassay for inhibin. During studies on the inhibin content of human plasma, FSH suppression was noted to occur together with LH suppression. Further studies revealed that where combined FSH and LH suppression occurred, cytological changes in the pituitary cells suggestive of toxicity were found, indicating non-specific effects of these substances. Charcoal treatment or gel filtration of seminal plasma removed or separated the toxic substances from the inhibin activity, the latter characterized by FSH suppression parallel to the standard preparation, no LH suppression and no light-microscopic changes in pituitary cells. It is recommended that careful evaluation of all inhibin bioassay systems should be undertaken to detect substances producing non-specific effects and additional guidelines for the assessment of specificity are suggested.     

Decreased testicular response to acute LH-stimulation and increased intratesticular concentration of oestradiol-17 beta in the abdominal testes in cryptorchid rats

Rats were made cryptorchid, bi- or unilaterally, by cutting gubernaculum testis at birth. At 100 days of age the basal plasma testosterone concentration was smaller in bilateral cryptorchid animals as compared to unilateral cryptorchid and control rats. After acute LH-stimulation plasma testosterone concentration increased in all rats, where the highest concentrations were found in the control group. The amount of testosterone in the testis after a 30 min LH-stimulation was lower in both kinds of cryptorchid testes but higher in the scrotal testis of the unilateral cryptorchid rats, all as compared to control testis. This decreased effect of LH observed in cryptorchid animals indicates an impaired Leydig cell function in cryptorchid testis. The results also show a compensatory hyperfunction of Leydig cells in the scrotal testes of unilateral cryptorchid rats. It was also found that the intratesticular concentration of oestradiol-17 beta was three-fold increased in unilateral cryptorchid testes and even more in bilateral cryptorchid ones.     

Measurement of exogenous and endogenous inhibin in sheep serum using a new and extremely sensitive bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro

An extremely sensitive and reliable bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro was developed and used to measure exogenous and endogenous inhibin activity in the ewe. The sheep inhibin bioassay is 30- to 40-fold more sensitive than conventional rat inhibin bioassays. The minimum sensitivity of each bioassay in the measurement of inhibin activity in 1 ml of sheep serum is 220 mu. and 4080 mu. in the sheep and rat bioassays respectively. This sensitive inhibin bioassay has permitted, for the first time, the measurement of endogenous inhibin in the peripheral and ovarian vein blood of the sheep, as well as exogenously administered inhibin. The half-life of exogenously administered ovine inhibin (in follicular fluid) in the sheep was calculated as two components (18-24 and 50-60 min) from the inhibin profiles of six ewes. Inhibin contained in the ovine follicular fluid, given as a bolus i.v. injection, increased to maximum levels after 5 min and then remained increased for 10-32 min depending upon the dose administered, before exponentially decaying. The time for inhibin to exert its effect ranged from 3 to 6 h after injection and appeared to be dose-related. The bolus injection of inhibin, apart from causing suppression of FSH, evoked a large rebound increase of FSH up to 400% of preinjection levels. The development of the sheep bioassay will allow the measurement of biologically active inhibin in the peripheral circulation and ovarian vein blood of sheep with the possibility of extending this to man.     

Differential effects of epidermal growth factor on the differentiation of type A spermatogonia in adult mouse cryptorchid testes in vitro.

The effect of epidermal growth factor (EGF) on testicular germ cell differentiation was investigated. Testicular fragments from surgically prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of EGF. Histological sections of testis were examined under a light microscope and each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. EGF at concentrations ranging from 100 to 200 ng/ml induced differentiation of type A spermatogonia. The observed maximal stimulatory activity of EGF at a concentration of 100 ng/ml was 30% of the positive control cultures treated with calf serum. EGF at concentrations ranging from 1 to 100 ng/ml significantly inhibited the mitotic activity of FSH, FSH plus retinol, or FSH plus fetuin on type A spermatogonia and their differentiation. The number of type A spermatogonia in testes cultured with FSH, FSH plus retinol, or FSH plus fetuin decreased when EGF was added. On the other hand, EGF stimulated the differentiation of type A spermatogonia induced with fetuin but did not influence retinol-induced differentiation. It is proposed that EGF inhibits testicular germ cell differentiation by blocking the proliferation of type A spermatogonia stimulated by FSH.