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 < 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.     

Endocrine correlates of meiosis in the male rat.

Changes in the proportion of cells within various DNA classes of dispersed testicular cells from the developing rat were monitored by microflow fluorometry and correlated with changes in the function of the pituitary (FSH), of the Leydig cells (androgens) and Sertoli cells (androgen-binding proteins, ABP). Peaks of androgens and of FSH appeared simultaneously and coincided with an accumulation of tetraploid cells and with the first appearance of haploid cells in the testis and ABP in the epididymis. Estrogen treatment (5 micrograms/day) of developing rats from day 7 completely prevented the appearance of haploid cells in the testis as well as ABP in the epididymis. In these animals the wave of tetraploid cells started and progressed normally, indicating that transformation and progression of germinal cells to the stage of the primary spermatocytes were taking place. A combined treatment with FSH and dihydrotestosterone propionate (DHTP) resulted in a premature start of Sertoli cell secretion of ABP into the epididymis, but in a normal appearance of haploid and tetraploid cells. The time correlation between peaks in FSH/androgens, the start of Sertoli cell secretion, and the occurrence of haploid cells in the testis stresses the importance of these two hormones for normal Sertoli cell function and the importance of a functional Sertoli cell for the completion of meiosis.     

Age-dependent Sertoli cell responsiveness to germ cells in vitro

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.     

Effects of experimental cryptorchidism and subsequent orchidopexy on seminiferous tubule functions in the lamb

The reversibility of damage caused by cryptorchidism to the seminiferous tubules of the lamb was investigated at various ages. Lambs were made bilaterally cryptorchid either at birth or at 2 months of age. Then orchidopexy was performed at either 2 or 4 months of age. In permanently cryptorchid lambs, spermatogenesis stopped completely, and Sertoli cell function, as measured by FSH receptors, androgen receptors and ABP, was much reduced (-96%, -86% and -81%, respectively). Orchidopexy allowed the cryptorchid seminiferous epithelium to grow again, but the more differentiated the germ cells, the less they were capable of restoration. Even in 0- to 2- and 0- to 4-month-old temporarily cryptorchid lambs that had recovered normal Sertoli cell function, 16 to 49% of the tubules still were empty. It was concluded that cryptorchidism irreversibly damages the seminiferous tubules at a level other than the hormone receptors.     

Changes in the secretion of ABP into testicular interstitial fluid with age and in situations of impaired spermatogenesis

Androgen-binding protein (ABP) was measured in serum and testicular interstitial fluid (IF) from rats during sexual maturation or in adult rats in which impairment of spermatogenesis had been induced by (i) testosterone withdrawal following Leydig cell destruction, (ii) local heating (43 degrees C) of the testes for 30 min or (iii) induction of unilateral cryptorchidism (UCD). The changes observed were related to the IF levels of testosterone and, in most instances, to the serum levels of FSH. The levels of ABP in serum and IF decreased together with age, being highest at 30 days, falling steeply by 40 days and then slowly but progressively up to 100 days of age. A similar pattern was observed for serum FSH, except that the initial fall occurred beyond 40 days of age. Treatment with EDS or exposure to local heating caused comparable reductions in testicular weight (25-30% by 7 days after treatment, 50% by 21-28 days) and raised the serum levels of FSH. In both groups the levels of ABP in IF were increased by two- to three-fold while the levels of testosterone were either reduced markedly (EDS-treatment) or remained unchanged (local heating). In rats made UCD for 60 days, the weight of the abdominal testis was reduced by 75%, compared with the contralateral scrotal testis, while the IF levels of ABP and testosterone were significantly increased (55%) and decreased (90%), respectively. Short-term (3 days) deprivation of testosterone in adult rats, following immunoneutralization of LH, was without significant effect on IF levels of ABP. It is concluded that ABP secretion into IF is increased in situations of subnormal (or sub-adult) numbers of germ cells and this is usually associated with high levels of FSH. Measurement of ABP levels in IF should prove of value for the monitoring of Sertoli cell function in vivo and may be of diagnostic use for the detection of changes in germ cell numbers.     

Temperature and germ cell regulation of Leydig cell proliferation stimulated by Sertoli cell-secreted mitogenic factor: a possible role in cryptorchidism

Summary. Local control of Leydig cell morphology and function by seminiferous tubules was suggested in previous in vivo studies, especially those that used experimental cryptorchid rat testis as a model. These studies reported changes in morphology, increases in cell number and mitotic index and decreases in testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels of Leydig cells. However, little is known about how these changes are mediated. We recently observed that a novel Sertoli cell-secreted mitogenic factor stimulated proliferation, decreased testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels, and dramatically altered the morphology of Leydig cells in culture. In the present studies, we demonstrate that an increase in coculture temperature from 33 to 37 °C increased [³H]-thymidine incorporation (5.6- vs. 19.2-fold) and labelling index (4.3% vs. 15.8%), and accelerated proliferation (2.1- vs. 3.9-fold) of cultured immature Leydig cells. In addition, testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels of Leydig cells cocultured with Sertoli cells were further decreased following a 4°C increase in coculture temperature. This elevation in culture temperature increased both the secretion of this factor by Sertoli cells and responsiveness of Leydig cells to this factor. In addition, the presence of germ cells, especially pachytene spermatocytes, inhibited the secretion of the mitogenic factor by Sertoli cells. These temperature- and germ cell-associated effects mimicked the morphological and functional changes of Leydig cells reported following experimental cryptorchidism. These observations suggest a possible role of this Sertoli cell-secreted mitogenic factor in explaining Leydig cell changes following experimental cryptorchidism.     

Effect of cryptorchidism and orchidopexy on inhibin secretion by rat Sertoli cells

The present study explored the effects of experimental bilateral cryptorchidism (of 21-, 28-, and 35-days duration) and orchidopexy (14 and 42 days) in the adult rat on the secretion of inhibin by cultures of isolated Sertoli cells. Changes in serum levels of gonadotropins, testis weight, and spermatogenesis also were assessed to verify the effectiveness of the surgical procedures. Cryptorchidy resulted in a progressive decline in testicular weight and a loss of germ cells, associated with increasing serum levels of FSH and LH. Inhibin secretion in vitro became nondetectable by 28 days after surgery. At 42 days after orchidopexy, spermatogenesis showed qualitative recovery, with a small increase in testes weight. Levels of LH in the circulation declined, but only to twice the intact control levels. However, inhibin secretion and serum FSH levels returned to nearly normal values. These results indicate that bilateral cryptorchidism severely impairs the secretion of inhibin and possibly other Sertoli cell functions which may account, at least partly, for the increase in circulating FSH levels and the arrest of spermatogenesis. The effects of cryptorchidism on these parameters can be reversed to a large degree by orchidopexy.     

Effect of germ cells on vectorial secretion of androgen binding protein and transferrin by immature rat Sertoli cells in vitro

The influence of germ cells (greater than 85% pachytene spermatocytes) on vectorial secretion of androgen binding protein (ABP) and transferrin by immature rat Sertoli cells was investigated using two-compartment culture chambers. The ratio of ABP secreted into the outer and inner compartment in control cultures of Sertoli cells alone was 1.9, and was not influenced by either FSH or testosterone. Co-culture of Sertoli cells in direct contact with germ cells in the presence of FSH decreased this ratio, the decrease being most pronounced (0.7) after 2 days of co-culture. This effect was not observed if the germ cells were not in direct contact with Sertoli cell monolayers. The outer to inner compartment ratio of transferrin in Sertoli cell-alone cultures was 1.6 and, in contrast to ABP, was not significantly influenced by the addition of germ cells, even in the presence of FSH. It is concluded that in immature rat Sertoli cells the polarity of ABP secretion, but not that of transferrin, may be regulated by pachytene spermatocytes (and possibly other germ cells), and that this process is FSH-dependent.     

支持细胞功能和分化的激素调控

支持细胞 (Sertolicell)是启动青春期发育和维持成年精子发生的上皮细胞 ,其功能和分化受多种因素调控 ,包括激素、细胞因子和细胞粘附分子等。卵泡刺激素 (follicle stimulatinghormone ,FSH)是调节支持细胞大部分分化功能的内分泌激素 ,刺激合成雄激素结合蛋白 (androgenbindingprotein ,ABP)、抑制素 (inhibin)和转铁蛋白 (transferrin ,Tf)等 ,主要信使为cAMP和Ca2 + 。另一个重要的调控激素是甲状腺素 (thyroidhormone ,T3 ) ,可调节支持细胞增殖过程、成熟支持细胞数量以及睾丸产生精子的能力。     

Interactions Between LHRH, Sex Steroids and "Inhibin" in the Control of LH and FSH Secretion

Using anterior pituitary cells obtained from adult male rats, 17β-estradiol stimulated both spontaneous and LHRH-induced LH and FSH secretion. After in vivo administration, estrogens showed inhibitory or stimulatory effects on gonadotropin secretion, the effect being dose- and time-dependent. Contrary to estrogens, androgens had differential effects at the anterior pituitary level: they inhibited LH but stimulated FSH secretion. These in vitro data were confirmed by in vivo experiments using anti-androgens. Sertoli cell culture medium was found to be a potent inhibitor of spontaneous FSH secretion while no effect was detected on LH release. When the gonadotropin response to LHRH was studied, Sertoli cell culture medium was a more potent inhibitor of FSH than LH release although clear inhibition of LHRH-induced LH release did occur.     

cAMP response of cultured Sertoli cells from immature and adult hamsters. Effect of hypophysectomy and cryptorchidism

The ability of FSH to stimulate cAMP accumulation in Sertoli cells cultured from either hamsters or rats declines with the age of the animal. Hypophysectomy or bilateral cryptorchidism of immature rats prevents this normal age-related decline in Sertoli cell response to FSH. However, neither hypophysectomy nor cryptorchidism of adult rats can restore the Sertoli cell response to that of the immature animal. In contrast, the Sertoli cell response to FSH in the adult golden hamster, a photoperiodic animal, can be restored to that of an immature animal during short photoperiod-induced testicular regression. Thus, the purpose of these experiments was to determine if the Sertoli cell response to FSH would also be restored to that of an immature animal when testicular regression was induced by other means, namely, hypophysectomy or bilateral cryptorchidism. As in the rat, hypophysectomy or cryptorchidy of immature hamsters resulted in decreased testicular growth and a cAMP response to FSH in cultured Sertoli cells, which remained at the high level of an immature animal. However, in contrast to the rat, hypophysectomy or bilateral cryptorchidism of the adult hamster resulted in not only testicular regression, but also a restored ability of FSH to stimulate cAMP accumulation in Sertoli cells cultured from these animals. Indeed, the magnitude and specificity of the Sertoli cell cAMP response to agents was the same in Sertoli cells cultured from testes undergoing testicular regression induced by either short photoperiod (ie, natural means), hypophysectomy, or cryptorchidy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites of Action of Cyproterone and Cyproterone Acetate in the Immature Male Rat

Plasma testosterone (Leydig cell function), LH and FSH (pituitary function), the epididymal content of androgen binding protein (ABP) (Sertoli cell function) and plasma corticosterone (adrenal cortical function) were determined after 10 daily injections of varying doses of cyproterone and cyproterone acetate, beginning at 21 days of age. Daily doses of 0.5 mg or greater of either antiandrogen resulted in a marked depression in the levels of plasma testosterone and intra-testicular testosterone (measured only in the cyproterone group) with a dose-dependent decrease in testis and epididymal weights; both effects occurring with only minor changes in the levels of circulating gonadotrophins. In addition, these compounds caused a marked decrease in Sertoli cell secretion as reflected in a significant fall in the epididymal content of ABP.
A more detailed examination of the apparently direct effects of cyproterone on Leydig cell function revealed: 1) no major effects of in vivo treatment for 6 days (5 mg/day) on [I¹²⁵]hLH binding to testis membrane particles or on the in vitro response of enriched Leydig cell suspensions to hCG, 2) a dose-dependent inhibition of the hCG responsiveness of normal Leydig cells in the presence of the drug in vitro . The inhibitory effects on steroidogenesis in vivo can well be explained by a direct inhibition of the 3β-steroid-dehydrogenase-isomerase exerted by both antiandrogens.     

Intratesticular steroids and gonadotrophin receptor concentrations in the testes of immature unilaterally cryptorchid rats

Testicular descent was prevented unilaterally in newborn rats. In this experimental model of cryptorchidism the first morphological changes are noted in the abdominal testis at 16 days of age. Testicular weight was increased in the abdominal testis at 16 days of age, was unaffected at 20 days but was decreased at 30 days of age. The concentrations of LH and FSH receptors, and the hCG-stimulated progesterone and testosterone secretion in vivo were analysed in scrotal and abdominal testes at different ages. LH- and FSH-receptor content per testis were unaffected by cryptorchidism at 12, 16 and 20 days of age but were depressed markedly at 30 days of age. At 20 days of age (but not earlier) hCG-stimulated progesterone was reduced while testosterone secretion remained unchanged. It is concluded that the early functional changes in abdominal testes are not related to changes in gonadotrophin receptor content.     

Temperature-dependent expression of a 95 kD protein in rat testis

Summary.  Testicular tissue from mature male Sprague-Dawley rats was maintained in culture at 33 °C or 37 °C. Detergent-extracted proteins were separated by polyacrylamide gel electrophoresis (PAGE) followed by staining or fluorography. Unilateral surgical cryptorchidism was performed as an in vivo model for testicular exposure to abdominal temperature. Testes were harvested at various time points, followed by protein analysis as performed for in vitro studies. Tissue incubated in vitro for 48 h at 37 °C demonstrated loss of a prominent actively synthesized 95 kD protein (p95) seen at 33 °C. No other temperature-dependent protein changes were observed. Liver, kidney, spleen and thymus failed to reveal p95 or any other temperature-sensitive proteins when incubated at 33 °C and 37 °C. Surgical cryptorchidism resulted in loss of p95 by 1 week in abdominal testes and normal p95 expression in sham-operated scrotal testes. p95 appears to be a temperature-sensitive protein in rat testis, with increased degradation accounting for its loss at abdominal temperature. The failure to identify similar protein changes in other tissues suggests a role for p95 in the temperature-dependent function of the testis.     

On the morphology of the human Sertoli cells under normal conditions and in patients with impaired fertility.

Sertoli cells in testicular biopsies from 7 patients with Sertoli-cell only syndrome, 8 patients who had been treated with cyclophosphamide for testicular neoplasia, 8 patients with oligozoospermia, 7 patients with cryptorchidism, and 8 patients with seminoma were examined under the electron microscope and compared with Sertoli cells of normal tissues. The investigations reveal that each of these conditions of impaired fertility is characterized of a special type of Sertoli cell. The cell pattern is either restricted to one or two of the normally occurring cell types, or modified cells predominate that might be typical of the underlying disturbances of spermatogenesis. It is suggested that the cell type which prevails in the Sertoli-cell only syndrome is capable of maintaining a basic production of a substance that inhibits FSH secretion.     

Thyroid hormones: their role in testicular steroidogenesis

Thyroid hormones are important for growth and development of many tissues. Altered thyroid hormone status causes testicular abnormalities. For instance, juvenile hypothyroidism/neonatal transient hypothyroidism induces macroorchidism, increases testicular cell number (Sertoli, Leydig, and germ cells) and daily sperm production. Triiodothyronine (T3) receptors have been identified in sperm, developing germ cells, Sertoli, Leydig, and peritubular cells. T3 stimulates Sertoli cell lactate secretion as well as mRNA expression of inhibin-alpha, androgen receptor, IGF-I, and IGFBP-4. It also inhibits Sertoli cell mRNA expression of Müllerian inhibiting substance (MIS), aromatase, estradiol receptor, and androgen binding protein (ABP) and ABP secretion. T3 directly increases Leydig cell LH receptor numbers and mRNA levels of steroidogenic enzymes and steroidogenic acute regulatory protein. It stimulates basal and LH-induced secretion of progesterone, testosterone, and estradiol by Leydig cells. Steroidogenic factor-1 acts as a mediator for T3-induced Leydig cell steroidogenesis. Although the role of T3 on sperm, germ, and peritubular cells has not yet been completely studied, it is clear that T3 directly regulates Sertoli and Leydig cell functions. Further studies are required to elucidate the direct effect of T3 on sperm, germ, and peritubular cells.     

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.     

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.     

Recovery of testicular functions after surgical treatment of experimental cryptorchidism in the rat

When rats were made unilaterally cryptorchid at 17 days of age (before spontaneous descensus), the further maturation of the testis was prevented. At 34 days of age, the abdominal testis was smaller than the scrotal testis and showed less secretion of the Sertoli cell specific androgen binding protein (ABP). In 120–130 days old rats that were made bilaterally cryptorchid at 17 days of age, testicular weight, histology, secretion of fluid and ABP were restored and testosterone secretion and fertility were normal if orchidopexy was performed at 33 days of age. If the orchidopexy was delayed until 59 days of age, the recovery of testicular function and morphology was only partial. The results show that in the rat, the testicular damage caused by cryptorchidism is reversible, if the abdominal testis is surgically descended during early sexual maturation.     

Recovery of testicular functions after surgical treatment of experimental cryptorchidism in the rat

When rats were made unilaterally cryptorchid at 17 days of age (before spontaneous descensus), the further maturation of the testis was prevented. At 34 days of age, the abdominal testis was smaller than the scrotal testis and showed less secretion of the Sertoli cell specific androgen binding protein (ABP). In 120-130 days old rats that were made bilaterally cryptorchid at 17 days of age, testicular weight, histology, secretion of fluid and ABP were restored and testosterone secretion and fertility were normal if orchidopexy was performed at 33 days of age. If the orchidopexy was delayed until 59 days of age, the recovery of testicular function and morphology was only partial. The results show that in the rat, the testicular damage caused by cryptorchidism is reversible, if the abdominal testis is surgically descended during early sexual maturation.