Androgen-binding Protein Production Research Articles

Inhibitory effects of alkane sulphonates on the function of immature rat Leydig, Sertoli and peritubular cells cultured in vitro

Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40-160 micrograms/ml) blocked the conversion of C21 and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C21 steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.

Testicular Peritubular Cells Secrete a Protein under Androgen Control that Inhibits Induction of Aromatase Activity in Sertoli Cells*

We investigated the role of peritubular cell-Sertoli cell interactions in the control of Sertoli cell function by androgens. Decreased FSH-inducible aromatase activity and increased secretion of androgen-binding protein (ABP) were used as parameters of androgen action on Sertoli cells. It is demonstrated that coculturing Sertoli cells with limited amounts of peritubular cells (20%) has only marginal effects on inducible aromatase activity or ABP secretion, but markedly increases the response of these parameters to androgens. Conditioned media derived from peritubular cells pretreated with androgens mimick the effects observed in the coculture system. Evidence is presented that androgen action on peritubular cells is mediated by an androgen receptor and that the concentration of this receptor is increased up to 3-fold by androgens. Preliminary experiments suggest some analogy between the peritubular cell factors that stimulate ABP production and those that inhibit aromatase induction. The active principles responsible for both activities are thermolabile trypsine-sensitive proteins with a mol wt between 50,000-100,000, and androgen induction by both activities shows an identical time course, characterized by a 4-day latent period. Nonetheless, much higher concentrations of peritubular cell secretion products seem to be required to inhibit aromatase induction than to stimulate ABP production, indicating that the active principles are not necessarily identical. It is concluded that peritubular cell secretion products mimick and mediate not only stimulatory but also inhibitory effects of androgens on Sertoli cells.

Prolonged exposure to androgens suppresses follicle-stimulating hormone-induced aromatase activity in rat Sertoli cell cultures

We studied the effects of androgens on basal and FSH-stimulated aromatase activity in Sertoli cell-enriched monolayers. Daily exposure to androgens from the first day of culture results in a time- and dose-dependent decrease in inducible aromatase activity. The inhibition is observed whether FSH, l-isoproterenol or (Bu) 2cAMP is used as inducer of the aromatase activity. Basal activity is not affected by preincubation with androgens and the inhibitory effect is not observed after short-term exposure (24 h) to these hormones. The ability of different androgens to decrease inducible aromatase activity does not depend on their ability to serve as a substrate for the aromatase but parallels their ability to stimulate the production of androgen-binding protein. Moreover, the effect of testosterone is neutralized by the antiandrogen cyproterone acetate, suggesting that it is mediated by the androgen receptor. These data suggest that testicular androgens may be responsible for the decrease in FSH-inducible aromatase activity observed in intact rats between days 10 and 30 of life. Similarly, the removal of these androgens during the preparation of Sertoli cell cultures may explain the spontaneous increase in inducible aromatase activity observed when these cultures are maintained in the absence of androgens.

In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages.

Indirect (hypotonically treated culture) and direct (coculture) approaches were used to study the influence of germ cells on androgen-binding protein (ABP) and 17 beta-estradiol secretion by Sertoli cells prepared from 10-, 15-, 20-, and 45-day-old rats. Using these approaches, the mechanisms that govern Sertoli cell-germ cell membrane recognition were shown to be unaltered by the hypotonic treatment, to vary with the age of the Sertoli cell donors, and to be FSH/(Bu)2cAMP dependent in the younger animals (10-20 days old). Hypotonic treatment had no effect on estradiol levels at all ages studied, but resulted in a marked fall of ABP production by Sertoli cells from 20 days onward. However, the relative stimulation of ABP induced by FSH/(Bu)2cAMP (ABP levels in FSH/(Bu)2cAMP/hypotonically treated Sertoli cell cultures vs. ABP in hypotonically treated cultures) was markedly increased at 20 and 45 days, indicating that germ cells influence Sertoli cell responsiveness to FSH/(Bu)2cAMP. The addition of crude germ cell preparation to the Sertoli cell cultures stimulated ABP and inhibited estradiol levels at all ages studied. Moreover, the addition of germ cells to hypotonically treated cultures induced a decrease in the relative ABP response to FSH and (Bu)2cAMP, thus confirming (see above) that germ cells may be involved in the mechanism of Sertoli cell refractoriness to FSH that occurs with advancing age in the rat. Germ cell-stimulated ABP production was higher in adult Sertoli cells than in immature cells. That Sertoli cell responsiveness to germ cells varies with age is further supported by the data showing that whereas spermatocytes stimulated ABP and inhibited estradiol productions at all ages studied, early spermatids only influenced these parameters from 20 days onward. It is concluded that germ cell control over Sertoli cell function in vitro may be of important physiological significance during Sertoli cell maturation in mammals.

Evidence From Sertoli Cell-Depleted Rats Indicates That Spermatid Number in Adults Depends on Numbers of Sertoli Cells Produced During Perinatal Development*

To probe the relationship between the size of the Sertoli cell population, established during perinatal development, and production of germ cells in the adult testis, a Sertoli cell-depleted rat model was developed. This was accomplished by delivering an antimitotic drug, cytosine arabinoside (araC), directly to the testis of newborn pups. Initial studies of these araC-treated neonates indicated that 1) the drug is cleared rapidly from the testis; 2) it substantially reduces the level of Sertoli cell proliferation; 3) Sertoli cell division ceases at a normal time in spite of the previous drug treatment; and 4) araC itself has no residual effect on germ cell proliferation, which begins several days after the injection. Pups given araC were allowed to reach maturity, and their testes were perfuse-fixed for light microscopic morphometry. When the numbers of Sertoli cells in adult rats given araC as were compared with those in normal littermates, a 54% decrease in the size of the Sertoli cell population was detected in treated rats, now referred to as Sertoli cell-depleted. Moreover, when round spermatids were quantified and compared in normal and Sertoli cell-depleted adults, testes of the latter were found to contain 55% fewer round spermatids. Since, in the araC-treated group, the decrease in Sertoli cell population size was paralleled by a reduction in spermatid production of equal magnitude, the number of round spermatids per Sertoli cell was essentially identical in normal and Sertoli cell-depleted animals. Measurements of serum androgen-binding protein (ABP) and FSH in both groups indicated that the circulating level of ABP in Sertoli cell-depleted rats was approximately half, and the concentration of FSH approximately twice, that in normal animals. Thus, even though FSH is elevated in Sertoli cell-depleted rats, the production of ABP per Sertoli cell is unchanged. In addition, collective volume of Leydig cells and ventral prostate weights were normal in the Sertoli cell-depleted group, suggesting that Leydig cell function in these rats is normal. In summary, a Sertoli cell-depleted rat model has been produced by interfering specifically with Sertoli cell proliferation early in postnatal life, before onset of germ cell division. Moreover, our findings with this model indicate that production of normal numbers of germ cells in adults depends, at least in part, on the size of the Sertoli cell population. Thus, our observations identify the perinatal period, when the Sertoli cell population is established, as critical for development of quantitatively normal spermatogenesis in the adult.

Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Hormonal regulation of β-endorphin in the testis

It is well established that β-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that β-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, β-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of β-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites ( K d in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic β-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic β-endorphin treatment does not affect testosterone production by Leydig cells in virto, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of β-endorphin. Also, fetal Leydig cells represented a preferred in virto system to study β-endorphin release since in adult cell culture a marked degradation of the peptide was detected (> 50%). β-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50 − 30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10–12-fold the β-endorphin secretion. The hormone stimulation of β-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated β-endorphin production (150–200%), suggesting autocrine negative modulation of Leydig cell β-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated β-endorphin production (by 50%). Altogether these findings indicate that β-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.

Triiodothyronine decreases the production of androgen binding protein by rat Sertoli cells

Triiodothyronine (T 3) effects on cultured Sertoli cells from immature rats were investigated by evaluating the production of androgen binding protein (ABP) a biochemical marker of Sertoli cell function. The results demonstrate that T 3 administration to the rat as well as T 3 addition to the culture medium specifically decreases ABP production by Sertoli cells, suggesting a direct regulatory role of thyroid hormone on male reproductive function.

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies.

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.

Identification of a non-mitogenic paracrine factor involved in mesenchymal-epithelial cell interactions between testicular peritubular cells and Sertoli cells

Seminiferous peritubular cells have previously been shown to secrete a protein termed P-Mod-S which modulates the functions of Sertoli cells. The present study provides an initial characterization of P-Mod-S and examines the actions of P-Mod-S on Sertoli cells. Gel filtration chromatography demonstrates that P-Mod-S has an apparent molecular weight of 70 000 that could not be dissociated to a lower molecular weight form. A 40- to 90-fold purification of P-Mod-S was obtained with a predicted half maximal effective concentration for Sertoli cells of less than 10 −9 M. Through an analysis of the actions of P-Mod-S on Sertoli cells it is demonstrated that P-Mod-S stimulates the Sertoli cell to a greater extent than any single hormone or vitamin known to influence the cell. P-Mod-S maximally stimulates testicular transferrin and androgen-binding protein production by Sertoli cells, but does not stimulate levels of plasminogen activator activity. P-Mod-S also appears to induce the synthesis of several proteins that are not detected in control non-treated Sertoli cell cultures. One such protein whose synthesis was stimulated by P-Mod-S treatment of Sertoli cells was a component having a molecular mass of 20 kDa. This 20 kDa Sertoli cell-secreted protein was specifically immunoprecipitated with an antibody against an epididymal lactalbumin-like protein. This implies that P-Mod-S can induce Sertoli cells to synthesize and secrete a lactalbumin-like protein. P-Mod-S was found not to contain mitogenic activity. Data presented indicate that testicular peritubular cells synthesize and secrete a 70 kDa non-mitogenic paracrine factor termed P-Mod-S which has a dramatic influence on Sertoli cell functions. Results are discussed with respect to modulation of epithelial (Sertoli) cell functions by components produced by mesenchymal (peritubular) cells.

Opiate receptors are present in the rat testis. Identification and localization in Sertoli cells.

We have characterized opioid binding sites in the Sertoli cells of adult and 18-day-old rat testes. Maximal specific etorphine binding was attained after 30 min at 4 C. The binding was reversible, with association and dissociation rate constants of 0.98 X 10(5) M-1 min-1 and 0.33 min-1, respectively. Scatchard analyses and saturation curves revealed a single class of high-affinity, low-capacity binding sites. No opioid binding was observed in Leydig cell cultures. Exposure to opioids for 3 days caused a significant increase in [3H]etorphine specifically bound to the Sertoli cells that was completely prevented by naloxone, demonstrating opioid up-regulation of its own receptor. Chronic opioid treatment of the cultures significantly inhibited androgen-binding protein production, and this effect was prevented by naloxone. Since the circulating concentrations of endorphins (10(-12) M) are lower than the Kd of testis opiate receptors, it is conceivable that opioids of Leydig cell origin act on the specific high-affinity receptors of the Sertoli cells, and may play a role in modulating their function.

Androgen stimulation of sertoli cell function is enhanced by peritubular cells

Rates of production of androgen binding protein (ABP) declined during culture of purified Sertoli cell-enriched aggregates in serum-free minimal essential medium (MEM). This decline was partially prevented by the addition of testosterone to the medium, but not by 17β-estradiol addition. The androgen effect was greater in Sertoli cell aggregates in co-culture with peritubular cells than in Sertoli cell preparations depleted of peritubular cells. Initial rates of ABP production by purified Sertoli cell-enriched aggregates were also sustained when preparations were cultured in MEM supplemented with conditioned medium in which peritubular cells had been previously maintained. We interpret data presented to indicate that the stimulatory effects of peritubular cells on functions of Sertoli cells in co-culture, specifically sustained rates of ABP production, are mediated at least partially by component(s) synthesized by peritubular cells and secreted into the medium. We offer the hypothesis that androgens may influence Sertoli cells at two levels: (1) a direct action on Sertoli cells; (2) and indirect influence on Sertoli cells mediated by a direct action on peritubular cells, resulting in the formation of product(s) which modulate the rates of ABP synthesis.

Testicular peritubular cells secrete a protein under androgen control that modulates Sertoli cell functions.

Peritubular cells of the seminiferous tubule synthesize component(s) that stimulate Sertoli cells in culture to increase the production of androgen-binding protein and testicular transferrin. The active peritubular cell component(s) are trypsin-sensitive, heat-sensitive, acid-stable molecule(s) having a molecular weight between 50,000 and 100,000. These specific factors(s) are referred to as P Mod-S to designate protein(s), produced by peritubular cells (P), that modulate the functions of Sertoli cells (S). The degree of stimulation by P Mod-S is comparable to that obtained by maximal hormonal stimulation of the synthesis of ABP and transferrin by Sertoli cells. Levels of P Mod-S secreted into the medium by primary cultures of peritubular cells are increased in the presence of testosterone. Comparable concentrations of 17 beta-estradiol do not stimulate peritubular cells to synthesize P Mod-S. Data are interpreted to indicate that androgens act on testicular peritubular cells to increase the formation of P Mod-S and that P Mod-S may modulate the properties of adjacent Sertoli cells. Findings are discussed in relation to the nature of mesenchymal-epithelial cell interactions in the seminiferous tubule and to the possible role of P Mod-S as a mediator of androgen actions of Sertoli cells.

A filter disc assay for the measurement and characterization of androgen-binding protein in unconcentrated media from Sertoli cell-enriched cultures

As easy, rapid and sensitive assay which permits measurements of androgen binding protein (ABP) in unconcentrated spent media from Sertoli cell cultures is described. The method is based on the adsorption of the 5α-dihydrotestosterone-ABP complex onto DEAE-cellulose filter paper discs at pH 8.5. It is demonstrated that this method permits the characterisation of ABP (ligand specificity, kinetic properties) and represents a useful tool for the study of the effects of various hormones on ABP production by cultured Sertoli cells. When added on day 5 of culture, FSH and other agents that raise intracellular cAMP increase ABP secretion up to 3 times. Natural and synthetic androgens provoke a 1.5–1.8-fold increase. The effects of androgens and FSH are additive and cyproterone acetate blocks the effects of androgens in the presence as well as in the absence of FSH.

Changes in testicular function induced by short-term exposure of the rat testis to heat: further evidence for interaction of germ cells, Sertoli cells and Leydig cells.

This study was designed to determine the effects of a short episode of testicular heating (43 degrees C for 15 min) on spermatogenesis and Sertoli and Leydig cell function. Rats killed at intervals up to 156 days after heating were assessed by histological examination, and by measurement of serum FSH and LH, and by tests of Sertoli cell function consisting of fluid production, androgen binding protein (ABP) content of the ligated and unligated tests, together with the binding of [125I]FSH. Leydig cell function was assessed by in vitro testosterone production, serum testosterone levels and [125I]hCG binding to testes homogenates. Testis weight declined 7 days after heating to 70% of control and remained lower until 82 days, whereas epididymal weight did not decrease significantly until 26 days and also recovered by 82 days. Fluid production was significantly lower in heated testes at 26 days and returned to normal at 56 days. ABP production measured as the difference between the ABP content of ligated and unligated testes was significantly reduced at 14 and 26 days, but subsequently recovered. Serum FSH levels were significantly elevated from 14-26 days in the heat treated group and the binding of [125I]FSH was reduced at 26 days post-heating. Basal and stimulated in vitro T production was significantly increased in the heat-treated testes at 14 days and subsequently returned to normal whilst [125I]hCG binding was significantly lower in the heat-treated testes from 7-26 days. Serum T and LH did not alter significantly during the study. Primary spermatocytes and young spermatids were the most heat sensitive germ cell type and a reduction in spermatogenesis was noted from 7 to 26 days, although recovery appeared complete by 56 days and thereafter. These results demonstrate that the transient spermatogenic disruption induced by heating is accompanied by significant alterations in Sertoli and Leydig cell function which are identical to those produced in other models of spermatogenic dysfunction. The results suggest that the duration of these changes appears to correlate closely with alterations occurring in the germ cell compartment.

Reinitiation of sperm production in gonadotropin-suppressed normal men by administration of follicle-stimulating hormone.

The specific roles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in controlling human spermatogenesis are poorly understood. We studied the effect of an experimentally induced, selective LH deficiency on sperm production in normal men. After a 3-mo control period, five men received 200 mg testosterone enanthate (T) i.m./wk to suppress LH, FSH, and sperm counts. Then, while continuing T at the same dosage, human FSH (hFSH) was administered simultaneously to replace FSH activity, leaving LH activity suppressed. Four men received 100 IU hFSH s.c. daily plus T (high dosage hFSH) for 13-14 wk, while one man received 50 IU hFSH s.c. daily plus T (low dosage hFSH) for 5 mo. The effect on sperm production of the selective LH deficiency produced by hFSH plus T administration was assessed. In the four men who received the high dosage hFSH regimen, sperm counts were markedly suppressed during T administration alone (0.3+/-0.2 million/cm(3), mean+/-SE, compared with 94+/-12 million/cm(3) during the control period). Serum LH bioactivity (determined by in vitro mouse Leydig cell assay) was suppressd (140+/-7 ng/ml compared with 375+/-65 ng/ml during control period) and FSH levels (by radioimmunoassay) were reduced to undetectable levels (<25 ng/ml, compared with 98+/-21 ng/ml during control period) during T alone. With the addition of 100 IU hFSH s.c. daily to T, sperm counts increased significantly in all subjects (33+/-7 million/cm(3), P < 0.02 compared with T alone). However, no subject consistently achieved sperm counts within his control range. Sperm morphology and motility were normal in all four men and in vitro sperm penetration of hamster ova was normal in the two men tested during the hFSH-plus-T period. During high-dosage hFSH administration, serum FSH levels increased to 273+/-44 ng/ml (just above the normal range for FSH, 30-230 ng/ml). Serum LH bioactivity was not significantly changed compared with the T-alone period (147+/-9 ng/ml). After the hFSH-plus-T period, all four men continued to receive T alone after hFSH was stopped. Sperm counts were again severely suppressed (0.2+/-0.1 million/cm(3)), demonstrating the dependence of sperm production on hFSH administration. Serum T and estradiol (E(2)) levels increased two- to threefold during T administration alone compared with the control period. Both T and E(2) levels remained unchanged with the addition of hFSH to T, confirming the lack of significant LH activity in the hFSH preparation. In the one man who received low dosage hFSH treatment, sperm counts were reduced to severely oligospermic levels, serum FSH was suppressed to undetectable levels, and serum LH bioactivity was markedly lowered during the T-alone period. With the addition of 50 IU hFSH s.c. daily to T, sperm counts increased, to a mean of 11+/-3 million/cm(3). During this period, serum FSH levels increased to a mean of 105+/-11 ng/ml (slightly above this man's control range and within the normal adult range), while LH bioactivity remain suppressed. After hFSH was stopped and T alone was continued, sperm counts were again severely reduced to azoospermic levels. We conclude that FSH alone is sufficient to reinitiate sperm production in man during gonadotropin suppression induced by exogenous T administration. FSH may stimulate sperm production in this setting by increasing intratesticular T through androgenbinding protein production or by increasing the sensitivity of the spermatogenic response to the intratesticular T present during exogenous T administration.

Interactions of sertoli cells with myoid cells in vitro.

Preparations of Sertoli cells and peritubular myoid cells from testes of 20-day-old rats were cultured alone or together, and cellular interactions were assessed. In the co-cultured system, Sertoli cells within plaques became elevated, forming macroscopically visible mounds and protrusions. The morphologic characteristics of these structures were determined with scanning and transmission electron microscopy. The formation of a limiting membrane observed between individual Sertoli cell nodules and surrounding myoid cells resembled some aspects of the morphology of seminiferous tubules. Long-term co-cultures of Sertoli cells and myoid cells, stimulated by follicle stimulating hormone (FSI-l), continued to secrete androgen binding protein (ABP) under conditions in which Sertoli cell monocultures ceased production of ABP. The specificity of these cellular interactions was investigated. Bladder smooth muscle cells could substitute for peritubular myoid cells in eliciting mound formation of co-cultured Sertoli cells, but embryonic fibroblasts could not. However, embryonic fibroblasts, which did not secrete ABP, permitted FSIl-stimulated Sertoli cells in long-term co-culture to maintain ABP production. In contrast, fibroblasts from the tunica albuginea, when co-cultured with Sertoli cells, neither elicited mound formation nor supported ABP production. We conclude that while unique specificity of cellular interactions between Sertoli cells and peritubular myoid cells is not apparent in cultured preparations from testes of 20-day-old rats, these cells do retain some of the properties required for aggregation and tubule formation characteristic of tubule morphogenesis.

Age Related Effects on Androgen Binding Protein (ABP) in Sheep Testis and Epididymis1

An androgen binding protein (ABP) has been demonstrated in testis and epididymis (caput, corpus, cauda) supernatants of sheep aged 50, 120 and 200 days. This protein has a high affinity for 5α‐dihydrotestosterone (Ka = 0.184 ± 0.023 × 109 M‐1), an electrophoretic mobility (RF) of 0.52–0.56 on PAGE and a molecular weight of about 70 000 daltons. In 50 day‐old animals (infantile) ABP is only present in the testis (392 fmoles/mg protein) with no dihydrotestosterone binding detectable in the epididymis. This is the first evidence of production of ABP by the testis some time before initiation of spermatogenesis. In the 120 day‐old animals (prepubertal), ABP is observed both in testis (142 fmoles/mg) and epididymis: caput (90 fmoles/mg), corpus (41 fmoles/mg) and cauda (165 fmoles mg). In the 200 day‐old rams (postpubertal) testis ABP is less concentrated than in younger animals (22 fmoles/mg), but in the epididymis the levels of ABP are twice those encountered in 120 day‐old animals.

Follicle-stimulating hormone and testosterone independently increase the production of androgen-binding protein by Sertoli cells in culture.

Dose-response curves were obtained for the production of androgen-binding protein (ABP) by Sertoli cells prepared from testes of 20-day-old rats and treated in culture with either FSH or testosterone (T). FSH stimulated ABP production by up to 3.5 times control levels. For NIH-FSH-S11, the ED50 was 3 ng/ml, and for highly purified ovine FSH, the ED50 was 0.066 ng/ml. Addition of T produced a stimulation of up to 3 times control levels; half-maximal response was obtained at a dose of 4 nM. The presence of small numbers of contaminating Leydig cells in some preparations resulted in production of endogenous T, especially when high doses of NIH-FSH, which contains some LH, were employed. A modified preparation method involving exposure of the cells to distilled water reduced the endogenous T production to low levels. In cultures of cells prepared in this way, addition of the antiandrogen cyproterone acetate at a concentration high enough to reduce fractional occupancy of androgen receptors by endogenous T to 0.014 or less had no effect on the stimulation by FSH of ABP production in the cultures. In contrast, cyproterone acetate inhibited stimulation by T of ABP production. We conclude that FSH and T act independently on Sertoli cells from immature rats to increase the secretion of ABP.

Sertoli Cell Function in the Androgen Insensitive (TFM) Rat

The Sertoli cells of the androgen insensitive (tfm) rat are capable of producing androgen binding protein (ABP) both in vivo and in vitro. ABP levels in the tfm testis are significantly higher (P &lt; 0.01) than that in normal littermates (NL); 2.9 ± 0.5 (SD) pmoles/mg protein and 63.7 ± 15 (SD) pmoles/testis in the tfm compared to 0.41 ± 0.07 pmoles/mg protein and 34.3 ± 6.7 pmoles/testis in the NL. However, ABP secretion rate in vitro is significantly lower in the tfm testis than that in the NL testis; 0.12 ± 0.02 (SD) pmol/testis per h and 0.31 ± 0.06 pmol/testis per h respectively. Thus, the increased levels of ABP in the tfm testis does not reflect increased ABP secretion, but is due to the atresia of the excurrent ducts and accumulation of seminiterous tubular secretion. The tfm testes show a higher concentration of FSH receptors than that in NL (171 fmoles/mg protein and 57 fmoles/mg protein respectively). However, when FSH binding is calculated per testis, binding capacities in tfm and NL testis are very similar (632 fmoles/testis and 798 fmoles/testis respectively). The present study shows that normal induction of FSH receptors is not dependent on the presence of an androgen receptor and that production and secretion of ABP is taking place in a situation where the target cells are insensitive to physiological doses of androgen.The reduction in ABP secretion rate in vitro in the tfm rat testis supports previous studies showing that both FSH and androgens are important for normal Sertoli cell function.