Chronic propranolol treatment causes desensitization of the steroidogenic response in testicular interstitial cells but does not alter protein kinase C

We investigated effects of chronic propranolol treatment on the secretory response of rat testicular interstitial cells (testosterone secretion) to subsequent in vitro stimulation with activators of protein kinase-C (PK-C) (L-propranolol, phorbol 12, 13-dibutyrate (PDBu), LHRH) or activators of protein kinase A (PK-A), (hCG or dibutyryl cAMP (dbcAMP)). We determined [3H]PDBu binding and PK-C activity in these cells. Treatment of rats with propranolol (Inderal 500 mg/L of water for 5 weeks) reduced by 48%, 50% and 29% the L-propranolol-, LHRH- or PDBu-induced testosterone secretion, respectively, when compared to cells from controls. This desensitization in testosterone secretion in vitro was also present when the testicular interstitial cells were stimulated with hCG or dbcAMP (secretion decreased by 65%/57%, respectively, when compared to cells from control rats). Challenging the cells originated from rats that received propranolol chronically with the addition in vitro of propranolol resulted in an additional reduction of the hCG/dbcAMP-stimulated testosterone secretion. Chronic propranolol-induced desensitization was not associated with a loss in [3H]PDBu binding or a decrease in PK-C activity. Chronic propranolol-induced desensitization can be uncoupled from down-regulation of protein kinase C. The effector responsible for the desensitization could be distal to the protein kinase C and protein kinase A.     

Effects of bromocriptine on pituitary-testicular function in the rat: possible inhibition of in vitro production of androgen by Leydig cells

The effect of bromocriptine (BR) on pituitary-testicular function has been investigated in vivo and in vitro in adult male rats. Testosterone production in vitro by collagenase- dispersed Leydig cells from 84-day-old rats was evaluated in the presence and absence of hCG and/or different doses of BR. In the presence of 1.5 X 10^-5 M BR, both basal and hCG-stimulated testosterone production were decreased whereas at lower doses BR was ineffective. In vivo 60-day-old rats were injected sc with BR (150 μg/rat or 750 μg/rat twice daily) or vehicle for 24 days. This treatment reduced the plasma level and pituitary content of prolactin, slightly increased the plasma levels of LH and FSH but did not affect pituitary gonadotrophin content. Irrespective of the dose of BR injected, plasma levels of androgen did not change, but with the large dose of BR a decrease in testicular content of testosterone (P = 0.05) was observed. In the same animals the number of LH/hCG receptors was significantly reduced, and the sensitivity of the isolated Leydig cells to hCG stimulation in vitro was reduced; however, both the basal secretion and the maximum testosterone response to hCG were unaffected. These results show impairment of pituitary-testicular function in BR-treated rats, either as a result of BR-induced hypoprolactinaemia or as a consequence of direct effects of BR on the Leydig cells.     

Effects of short- and long-term administration of tamoxifen on hCG-induced testicular steroidogenesis in man: no evidence for an oestradiol-induced steroidogenic lesion

hCG-induced testicular desensitization is characterized by inhibition at the level of the C-17,20-lyase enzyme. This defect has been attributed to an early rise in oestradiol (E2) following hCG administration. To test this hypothesis the E2-receptor antagonist, tamoxifen, was employed. From in vitro studies the evidence suggests that tamoxifen depletes the E2-receptor within 24 h. In this in vivo study, short-term (36 h) administration of tamoxifen (to 6 eugonadal men) did not affect basal plasma levels of LH, FSH, 17 alpha-hydroxyprogesterone (17-OHP), testosterone (T) and E2, whereas long-term (3 months) tamoxifen with treatment of 6 normogonadotrophic oligozoospermic men increased LH and T levels, indicating a biological effect of tamoxifen. The response of 17-OHP, T, E2 and the 17-OHP/T ratio to hCG was similar in short-term and long-term tamoxifen-treated men as well as in 6 untreated eugonadal male controls. These results do not suggest a role for endogenous E2 in the hCG-induced testicular steroidogenic block.     

c-jun对离体睾丸间质细胞睾酮分泌作用的机理研究

目的本研究拟通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节hCG促进睾丸间质细胞(leydigcells,LC)睾酮分泌中的作用机制。方法用c-junASODNs拮抗c-jun,再加用cAMP观察其对睾酮分泌的影响,用放射免疫方法检测睾酮水平。结果hCG可刺激LC睾酮分泌,是LC功能研究的有用模型。c-junASODNs呈剂量依赖性地抑制hCG诱导下的离体LC的睾酮分泌(P<0.01)。加用cAMP后睾酮分泌增加。结论c-jun促进hCG诱导的大鼠LC的睾酮分泌,c-jun表达可能与cAMP相关。     

Modulation of responsiveness of the adenylate cyclase system in avian chondroprogenitor cells by pertussis toxin, PTH, and PGE2

Chondroprogenitor cells, derived from avian tibia epiphyseal growth plate, were cultured in vitro. Incubation of these cells with pertussis toxin augmented their cAMP response to parathyroid hormone (PTH), attenuated the response to forskolin, but did not modify the response to PGE2. Pertussis toxin modulation of the cAMP response was accompanied by ADP ribosylation of two proteins with molecular weights of 39 and 40 kD. Using specific antibodies, the 39 kD protein was identified as the inhibitory guanine nucleotide binding protein (Gi) of the adenylate cyclase system. The other ADP-ribosylated protein has not been identified. Preincubation of the chondroprogenitor cells with PTH or PGE2 resulted in time-dependent heterologous desensitization of the cAMP response to a second challenge of either hormone. The cells did not recover from the densitization for at least 18 h after removal of the hormones. PTH and PGE2 treatment did not affect the cAMP response to forskolin and cholera toxin. The PTH-dependent cAMP production was also not altered by forskolin treatment. PTH homologous desensitization was not affected by pertussis toxin treatment, but the heterologous desensitization due to PGE2 was significantly attenuated. These results suggest that exposure of chondroprogenitor cells to PTH and PGE2 results in heterologous desensitization of the cAMP response. The desensitization is not due to changes in the adenylate cyclase activity. The pertussis toxin-sensitive G proteins are involved in the PTH heterologous rather than homologous desensitization of the cAMP response.     

Does gonadotropin receptor complex have an amplifying role in cAMP/testosterone production in Leydig cells?

The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.     

Heterogeneity of adult mouse Leydig cells with different buoyant densities.

The authors investigated the morphologic characteristics and human chorionic gonadotrophin (hCG)-stimulated testosterone production of adult mouse Leydig cells in vitro, which have different buoyant densities. Leydig cells from five testes of Swiss outbred male mice (15 weeks old) were isolated and purified by mechanical dispersion followed by density gradient centrifugation using Percoll. Two groups of Leydig cells were obtained with different buoyant densities: group 1 had densities of 1.0667 to 1.0515 g/cm3 and group 2 had densities of 1.0514 to 1.0366 g/cm3. In vitro testosterone production of these Leydig cells, in response to different doses of hCG (0, 5, 25, 125, 625, and 3125 pg/mL), was determined by radioimmunoassay. Leydig cells were fixed and processed for electron microscopic stereology to quantify the organelles by volumes and surface area. In Leydig cells of Group 1, testosterone production per cell in vitro in response to 0 and 5 pg/mL hCG was not significantly different (P greater than 0.05). Increases in the dose up to 25 pg/mL produced a significant increase (P less than 0.05) in testosterone production, although hCG doses of 125 and 625 pg/mL did not produce further increases in testosterone levels. However, 3125 pg/mL hCG further elevated the testosterone production by those Leydig cells with high buoyant density. In Leydig cells in group 2, the patterns of testosterone production in response to hCG doses of 0, 5, and 25 pg/mL were similar to those of Leydig cells in group 1. Those Leydig cells with low buoyant density, however, were unable to stimulate further testosterone production by an hCG dose of 3125 pg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased steroidogenesis and cAMP production in vitro by leydig cells isolated from rats made hypothyroid during adulthood

The Leydig cell function of adult male rats made hypothyroid with 6-propyl-2-thiouracil (6-PT, 0.1% w/v in drinking water for 1 month) was studied and compared with that of age-matched controls. After 6-PT treatment, a slight, non-significant decrease in serum testosterone was observed, but no changes in testis weight or number of Leydig cells were noted. The in vitro function of Leydig cells was therefore investigated during incubation for 3 h in the presence or absence of several stimuli: LH (30 mIU/mL), forskolin (FK 1 microM), isobutylmethylxanthine (IBMX, 100 microM), GnRH (100 nM) or FK 1 microM + IBMX 100 microM. Irrespective of the stimulus, cells from hypothyroid rats secreted less cyclic AMP, 17-hydroxyprogesterone, androstenedione and testosterone. No differences in LH receptors were noted between the groups. Prolonged incubation with triiodothyronine (5-250 ng/mL) or thyroxine (5-250 ng/mL) for 3, 16, 24 or 48 h did not affect testosterone secretion in either group; however, administration of IGF-I (8 ng/mL for 24 h) resulted in increased spontaneous and stimulated testosterone production in both groups. However, when hypothyroid animals were supplemented in vivo with thyroxine a full recovery of Leydig cell function in vitro was noted. In conclusion: (1) Leydig cells from rats made hypothyroid during adulthood produce less testosterone in vitro, both spontaneously and in response to cAMP and non-cAMP-mediated stimuli; (2) this is due to a reduction in cAMP production and in the activity of the enzymes in the androgen biosynthetic pathway, and not to changes in LH receptors; (3) direct administration of thyroid hormones did not improve testosterone secretion in either group, while incubation with IGF-I did.     

The Influence of Sexual Maturation and Immunization Against LH-RH on Testicular Sensitivity to Gonadotrophin Stimulation In Vitro

Binding of ¹²⁵[-labelled hCG to testicular homogenates from 60- and 120-day-old rats was between 3 and 4 times higher than to tissue from 25-day-old rats. However, testes from the latter showed maximal testosterone production (35 ng/testis) in vitro in the presence of 5 pM hCG whereas testes from 60- and 120-day-old rats responded maximally (560 ng and 400 ng/testis respectively) with an hCG concentration of 45 pM; the minimum effective dose of hCG was 0.5 pM hCG in 25-day-old rats and 5–11 pM in 60- and 120-day-old rats.
In adult rats actively immunized against LH-RH, the testes had regressed to less than 15 % of their normal weight and showed maximal testosterone production (4 ng/testis) in the presence of 10 pM hCG whereas testes from adult control rats required 45 pM hCG to elicit this response (600 ng/testis); the minimum effective dose of hCG was 2 pM in LH-RH immunized rats compared with 11 pM for controls.
It is concluded that the maturational increase in the testicular capacity to secrete testosterone is associated with a decrease in 'sensitivity' to hCG-stimulation in vitro ; the latter change appears to be related to the level of gonadotrophin exposure in vivo .     

hCG-treatment alone is insufficient for restitution of spermatogenesis in a state with arrest at the spermatogonial level

In an infertile man with azoospermia and arrest at the spermatogonial stage, hCG treatment alone improved the spermatogenesis but not beyond the primary spermatocyte stage. During hCG treatment steroid conversion in vitro in testicular biopsy material, as well as serum testosterone concentrations increased dramatically. When hMG treatment was added, spermatogenesis was complete. Combined hCG/hMG treatment seems to be an efficient therapy in well-selected infertile men, whereas increased testosterone production induced by hCG-treatment may be insufficient for restitution of spermatogenesis.     

Diminished response of ovarian cAMP to luteinizing hormone in experimental uremia

In prepuberal female rats with acute bilateral nephrectomy or chronic subtotal nephrectomy, the increase of ovarian cAMP concentration in response to submaximal doses of luteinizing hormone (LH 10 micrograms) and human chorionic gonadotropine (hCG 2.5 IU) was diminished (CO + 2.5 IU hCG 488 +/- 49 pmoles cAMP/mg protein; NX + 2.5 IU hCG 366 +/- 56. P less than 0.05). The cAMP response to follicle stimulating hormone (FSH) was unchanged. The abnormality was found both after administration of LH in vivo and incubation of ovaries with LH in vitro. Similarly, plasma estradiol concentrations in response to submaximal hCG stimulation were diminished. Basal cAMP concentrations and cAMP concentrations after maximal stimulation were unchanged. The defect was observed both in ovaries of untreated prepuberal rats, of pregnant mare serum (PMS)-treated rats (follicular phase) and PMS/hCG-treated rats (luteal phase). Diminished ovarian cAMP response to LH was observed both in parathyroid intact and in parathyroidectomized rats. Administration of 1,25(OH)2D3 in physiological doses (60 ng/kg) to acutely uremic rats restored diminished ovarian cAMP response to submaximal LH stimulation irrespective of parathyroid status. The effect of 1,25(OH)2D3 could not be reproduced by hypercalcemia resulting from intraperitoneal calcium injection. In vivo administration of indomethacin further diminished ovarian cAMP response in uremic animals and had no effect in control animals. Incubation of ovaries with PGE1 and PGE2 increased basal and stimulated cAMP concentrations and abolished the difference between control and uremic animals. The diminished response of ovarian cAMP content to submaximal doses of hCG was not corrected by bromocriptine (1 mg/kg) despite normalization of hyperprolactinemia. The present study shows diminished ovarian cAMP and plasma estradiol response to LH in experimental uremia. It documents a role of 1,25(OH)2D3 and prostaglandins in the genesis of this abnormality.     

The temporal response of rat testes to hCG stimulation during sexual maturation

Serum testosterone responses to a single sc injection of hCG (25 IU/100 g body weight) were monitored for 5 days in rats throughout sexual maturation (22-70 days). Two hours after hCG injection serum testosterone levels rose in 22, 37 and 53 day-old animals and remained elevated for 2 days, returning to control levels on day 3. This response differed markedly from the biphasic secretion of testosterone reported for adult animals. In 70 day-old animals the serum testosterone response approached that seen in adult animals. Testosterone levels were elevated 2 h after hCG injection (25.4 +/- 2.5 ng/ml) and declined significantly at 12 and 24 h to 17.1 +/- 1.0 and 16.1 +/- 3.4 ng/ml, respectively. Testosterone levels tended to increase again on days 2 and 3 (19.9 +/- 2.8 and 21.1 +/- 3.5 ng/ml, respectively) but the increase was not statistically significant. This response differed markedly to the biphasic secretion of testosterone reported for adult animals. In vitro patterns of basal and hCG-stimulated testosterone secretion by decapsulated testes following a single hCG injection also changed during sexual maturation. In 22 day-old animals the testes exhibited refractoriness to in vitro hCG stimulation at 12 h, but testes from 37 day old rats were refractory from 2 to 24 h. In vitro testosterone responses of testes from 53 and 70 day-old rats were similar to that reported for adult rats with a period of refractoriness from 12 h to 2 days. This study demonstrates that during sexual maturation in the rat alterations occur in the temporal patterns of testosterone secretion in vivo and in vitro following hCG stimulation.     

In vitro metabolism of 3H-pregnenolone and 3H-progesterone by adult and prepubertal human testicular tissue before and during gonadotrophic treatment

The in vitro conversion of tritiated pregnenolone and progesterone was studied in testicular tissue from three infertile adult males before and during 25-30 weeks of therapy with hCG alone or combined with hMG. Furthermore, the in vitro conversion of pregnenolone was studied in testicular tissue from five prepubertal boys with undescended testes, two of whom had been subjected to hCG treatment for 5 weeks. The gonadotrophic treatment appeared to augment the steroid conversion mediated by the enzymes 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase in adult as well as prepubertal testicular tissue. The conversion mediated by C17-20-lyase along the delta 4 metabolic pathway was not increased, causing a "trap" along the delta 4 metabolic pathway. The increased production of testosterone in vitro from tritiated pregnenolone, which was observed during gonadotrophic treatment, probably took place along the delta 5 metabolic pathway through the C17-20-lyase step, whereas C21 steroids converted to the delta 4 metabolic pathway were found to be "trapped" as 17 alpha-hydroxyprogesterone.     

Influence of hCG treatment on the metabolism of progesterone and pregnenolone in vitro by the human undescended prepubertal testis

Testicular biopsies were taken from 56 prepubertal and pubertal boys, aged 3-17 years, with undescended testes. The biopsies were incubated in vitro with 3H-progesterone or 3H-pregnenolone. Fifteen of the boys had been treated with human chorionic gonadotrophin (hCG) prior to operation. Nine boys were operated on within 1 week of the last injection of hCG while the others were operated on between 12 days and 2 years after hCG treatment. In non-treated prepubertal boys very small amounts of substrate were converted by means of the enzymes 3 beta-hydroxysteroid-dehydrogenase and 17 alpha-hydroxylase and virtually no testosterone was produced. Immediately after treatment with hCG there was a large increase in the conversion of substrate by 17 alpha-hydroxylase and 3 beta-hydroxysteroid-dehydrogenase. Considerable amounts of testosterone were formed, especially from pregnenolone, while smaller amounts were formed from progesterone. This suggests that testosterone production occurred primarily via the delta 5 pathway, at least beyond the 17 alpha-hydroxylase-step. Within 2 weeks of the last hCG injection, steroidogenic activity had decreased towards levels similar to those found in prepubertal testis from untreated boys. These observations indicate that hCG treatment of boys with undescended testes does not result in irreversible or even long-lasting stimulation of their steroidogenic function.     

Effects of vitamin D and parathyroid hormone on cyclic AMP production by bone cells isolated from rat calvariae

Studies presented here were designed to investigate further the basis for an impaired cAMP response to parathyroid hormone (PTH) in osteoblastlike calvarial bone cells isolated from vitamin D-deficient rat pups. The goal was to perturb Ca, PTH, and vitamin D in vivo in order to see which factors might be responsible for the impaired in vitro bone cell cAMP response. Pups either were parathyroidectomized (PTX) 3-5 days, implanted with osmotic minipumps delivering high doses of PTH, given repeated, high doses of 1,25(OH)2D3, or were D-deficient (-D, i.e., born and suckled by D-deficient mothers). Osteoblastlike bone cells, isolated by sequential enzyme digestion and centrifugation, were exposed to PTH for 5 min in the presence of a phosphodiesterase inhibitor. In bone cells isolated from -D rat pups, both basal and PTH-induced cAMP accumulation were significantly lower than in +D bone cells. Earlier, we had shown that two daily injections of -D pups with 50 ng 1,25(OH)2D3 restores this reduced bone cAMP response of -D pups toward normal. In the present study, neither basal nor PTH-induced bone cell cAMP accumulation was affected by subjecting D-replete pups to PTX, PTH infusion, or repeated high doses of 1,25(OH)2D3 despite the fact that each treatment markedly changed serum Ca or serum immunoreactive PTH. The results indicate that the impaired bone cell cAMP response seen in -D pups is not a direct result of chronic hypocalcemia and that the "heterologous desensitization" seen in vitro with added 1,25(OH)2D3 could not be duplicated by in vivo treatment of +D pups with supraphysiologic doses of 1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of transmembrane signaling in the fetal rat Leydig cell

Gonadotropin binding to the adult Leydig cell activates a GTP binding protein that interacts with adenylate cyclase to increase cAMP production within the cell. The increased production of cAMP stimulates steroidogenesis and leads to an increase in testosterone production and secretion. The fetal Leydig cell responds to LH with an increase in cAMP and testosterone production as early as 15.5 days of gestation, although the specific mechanism of transmembrane signaling has not been characterized. Fetal rat testis cells from 13.5-20.5 days of gestation were treated with dibutyryl cAMP (dbcAMP), cholera toxin, and hCG to determine the onset of steroidogenesis stimulation by activation of each moiety in the transmembrane signaling system of the fetal Leydig cell. Maximal stimulation at each age from 14.5 through 20.5 days of gestation was achieved with 1 mM dbcAMP, 500 ng/ml cholera toxin, or 10 ng/ml hCG. At 13.5 days of gestation, fetal testes did not produce any testosterone. These findings indicate that a cholera toxin-sensitive, stimulatory guanine-nucleotide regulatory protein is functional in the fetal Leydig cell as early as 14.5 days of gestation. The LH receptor becomes functional in the transmembrane signaling system of the fetal Leydig cell at 14.5 days of gestation.     

Effect of an LHRH Analogue on Testicular Function in the Immature Monkey (Macaca mulatta)

Response of the immature monkey testis to an LHRH analogue, D-Ser-(TBu)⁶-EA¹⁰-LHRH, has been assessed by measuring testicular androgen production in the immature rhesus monkey. Administration of the analogue (2.5 μg x 2 daily) for 5 days induced a significant increase in testicular testosterone compared with untreated animals. The response of the control and the LHRH-agonist treated monkey testis to gonadotrophin (hCG) was assessed by in vitro androgen production. Whereas the testicular tissue from LHRH analogue primed monkeys responded to in vitro stimulation of hCG, no significant increase in androgen production was observed when testicular tissue from untreated animals was incubated with the gonadotrophin. The present data suggest a functional differentiation of the immature monkey testis following the LHRH analogue treatment.     

Temporal effects of diethylstilbestrol administration in vivo on testosterone production in Leydig cells

The temporal nature of estrogenic suppression of Leydig cell testosterone production was investigated. Adult rats were injected SC with 50 micrograms/100 g BW of DES or vehicle. Animals were sacrificed at 4, 8, or 12 h following a single injection or at 12 h following the latter of two daily injections for 1 or 2 days. Collagenase-dispersed interstitial cells were obtained, and population I and II Leydig cells were subsequently isolated on metrizamide gradients. Population I and II Leydig cells produced in vitro testosterone levels of 7.19 +/- 0.86 and 12.84 +/- 1.86 ng/10(6) cells/3 h, respectively. These levels were increased to 10- to 13-fold in the presence of hCG of dbcAMP. No significant difference was noted in the responsiveness of these two populations to the in vitro additions. DES administration in vivo for 8-48 h resulted in dramatic and significant decreases in basal and stimulated testosterone production in vitro in both populations. However, DES treatment for 4 h was relatively ineffective in blocking testosterone production in vitro. The inhibitory patterns exhibited by the two populations differed considerably. Population I displayed a uniform degree of inhibition throughout the treatment, whereas population II exhibited a more transient suppression by estrogen. Thus, population II appeared to be less sensitive to the estrogenic effects than population I at 48, 24, and 12 h of treatment. These data indicate that both population I and population II Leydig cells become sensitive to the inhibitory effects of estrogens between 4 and 8 h of in vivo treatment and suggest that certain differences exist between the two populations with respect to the temporal action of estrogens.     

Endocrinological profile of human spermatogenic impairment--evaluation of function of Leydig and Sertoli cells in vivo or in vitro studies

In vivo and in vitro studies were performed to determine the function of Leydig and Sertoli cells of the human testis with various degrees of spermatogenic impairment. The increases in basal and peak serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) after LH-RH administration correlated with the degree of impairment of spermatogenesis, and the basal peripheral blood levels of testosterone and that after human chorionic gonadotropin (hCG) administration for patients with moderate or severe impairment were significantly lower than the values for those with mild impairment. The concentration of testosterone in the internal spermatic vein of varicocele patients with or without hCG treatment did not differ between in mild and moderate impairment. In studies on cultured Sertoli cells, the production rate of plasminogen activator in patients with severe impairment was significantly lower than that in patients with moderate or mild impairment. The decrease in testicular high-affinity binding site for FSH correlated with the degree of hypospermatogenesis found in idiopathic male infertility, but, on the contrary, the hCG (LH) receptors showed no correlation with the degree of impairment of spermatogenesis. In the investigation of the relationship between testicular FSH receptors and the effectiveness of human menopausal gonadotropin (hMG)-hCG treatment on idiopathic male infertility, the presence or absence of testicular FSH receptors predicted the responsiveness to the treatment.     

Characterization of functional Leydig cells after purification on a continuous gradient of percoll

Two human chorionic gonadotropin (hCG) responsive cells from rat testicular interstitium were previously isolated on a discontinuous gradient of Percoll. The light cells were non-steroidogenic and bound 125I-labeled hCG with high affinity (Kd 3.0 x 10(-10) mol/L), whereas the steroidogenic heavier cells (Leydig cells) produced cyclic adenosine monophosphate (cAMP) and testosterone in response to hCG stimulation with very little hCG binding. In that study, the heavier cell fraction was contaminated with germ cells, red blood cells, and other cells. These cells have now been further purified on a continuous gradient of Percoll (20 to 60%, v/v), and have resolved into three visible bands. The cells in subfraction I, predominantly damaged Leydig cells, germ cells, and/or residual light cells, bind 125I-labeled hCG with high affinity (Kd 4.09 x 10(-10) mol/L) without producing cAMP and testosterone in response to hCG. Subfraction III consists mainly of red blood cells. The cells in subfraction II, identified as typical Leydig cells by electron microscopy, produce cAMP and testosterone in response to hCG but, again, bind only a small amount of hCG (4.5 +/- 0.3 fmol/2 x 10(6) cells/250 microliters/per hour at 37 degrees C). Thus, further purification of the heavier cell fraction from a discontinuous gradient of Percoll on a continuous gradient of Percoll yields Leydig cells, free of contaminating germ cells and red blood cells, which actively produce cAMP and testosterone with a very low level of hCG binding, the affinity of which is undetectable by current binding techniques.