Sertoli Cell Factor Research Articles

Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone suppression

Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.

Expression and phylogeny of candidate genes for sex differentiation in a primitive fish species, the Siberian sturgeon, Acipenser baerii

The molecular mechanisms underlying testis differentiation in basal actinopterygian fish remains poorly understood. The sex differentiation period was investigated in the Siberian sturgeon, Acipenser baerii, by expression profiling of Sertoli cell transcription factors (dmrt1, sox9) that control testis differentiation in vertebrates; Leydig cell factors (cyp17a1, star) affecting androgen production; the androgen receptor (ar); a growth factor controlling testis development (igf1); and a gene coding for a gonadotropin hormone (lh). Two genes were characterised for the first time in the Siberian sturgeon (dmrt1, cyp17a1), while the others came from public databases. Sturgeon gonad development is very slow, with a late sexual differentiation time during their juvenile stage, and are still immature at 3 years of age. Immature fish showed a sex-dimorphic pattern; all the genes studied displayed a higher expression level in male gonads. We took advantage of the presence of juvenile fish with pre- and post-differentiated gonads (16 and 18 months old) to characterise them at the molecular level. The post-differentiated fish displayed a sex dimorphism of gene expression in their gonads for all genes studied, with the exception of sox9. The trends in undifferentiated fish lead us to propose that sturgeons undergoing male differentiation express high levels of Sertoli cell factors (dmrt1, sox9) and of genes involved in the production and receptivity of androgens (cyp17a1, star and ar) together with lh. Expression profiles and phylogenetic studies suggest that these genes are potential regulators of testis development in the Siberian sturgeon.

A preliminary study of chemo- and cytokine responses in rabies vaccine recipients of intradermal and intramuscular regimens

Plasma from 10 patients who had received rabies vaccine either intradermally (ID) or intramuscularly (IM) was examined for 20 chemo- and cytokines. Plasma samples were withdrawn on days 0, 3 and 7 after vaccination. These chemo- and cytokines and sampling days were chosen based on data collected from a protein array analysis of 122 cytokines conducted on one recipient of vaccine administered IM and one recipient of vaccine administered ID. Although eotaxin, interleukin (IL)-5 in the ID and IL-1 beta in the IM group were the only chemo- and cytokines that reached statistical significance ( p < 0.05), the overall trends may suggest bias on Th1 or Th2 according to vaccination routes. IL-1 alpha, -2, and -6, hemofiltrate cysteine–cysteine chemokine (HCC-4), glucocorticoid induced tumor necrosis factor receptor (GITR), tumor necrosis factor (TNF) related apoptosis inducing ligand-receptor (TRAIL-R3) had some degree of elevation in the ID group. TNF-alpha, gamma-interferon, granulocytes/macrophages – colony stimulating factor (GM-CSF), transforming growth factor (TGF)-beta, lymphotactin and pulmonary and activation-regulated chemokine (PARC) were elevated, although not to a significant level, in the IM group. IL-12, interferon-inducible T cell alpha chemoattractrant (I-TAC) and sertoli cell factor (SCF) were not significantly elevated in both groups whereas IL-4 and -10 were unchanged. Further studies are required to determine whether the presence of specific chemokines, such as eotaxin, is responsible for the production of high levels of rabies virus neutralizing antibody after administration of the dose-sparing ID regimen.

Sperm characteristics and ultrastructure of testes of rats after long-term treatment with the methanol subfraction of Carica papaya seeds.

The contraceptive efficacy of Carica papaya seeds after short-term evaluation has been well established. We have examined the safety and mechanism of contraception in rats after long-term treatment with the methanol subfraction (MSF) of C. papaya seeds. The test substance was administered orally to the male albino rats (n = 40) at 50 mg per kg body weight each day for 360 days. Control animals (n = 40) received olive oil as a vehicle. Recovery was assessed up to 120 days after treatment withdrawal. Sperm parameters, serum testosterone levels, fertility, histology and ultrastructure of the testis, haematology and serum clinical chemistry were evaluated to establish the safety and efficacy of the test substance. Safety of long-term treatment was evidenced by unaltered health status, organ weight, haematology and clinical chemistry, and by an increase in body weight. The mechanism of contraception was shown by reduction in nuclear and cytoplasmic volume, normal nuclear characteristics and vacuolization in the cytoplasmic organelles of the Sertoli cells, as well as nuclear degeneration in spermatocytes and spermatids indicating disturbed spermatogenesis. Leydig cells were normal. Initial effects were observed in Sertoli cells at 60 days of treatment. Spermatocytes and spermatids were affected after 120-240 days of treatment. A significant decline in sperm count and viability, total inhibition of sperm motility, increased numbers of sperm abnormalities, normal serum testosterone levels and 100% sterility were evident after 60 days of treatment. All the altered parameters, including percent fertility, were restored to control level 120 days after treatment withdrawal. It is concluded that the MSF is safe for long-term treatment and the mechanism of contraception is shown by its effect on spermatid differentiation in the testis, possibly mediated by the Sertoli cell factors.

Androgens and Postmeiotic Germ Cells Regulate Claudin-11 Expression in Rat Sertoli Cells

In the present study we investigated whether fetal exposure to flutamide affected messenger and protein levels of claudin-11, a key Sertoli cell factor in the establishment of the hemotesticular barrier, at the time of two key events of postnatal testis development: 1) before puberty (postnatal d 14) during the establishment of the hemotesticular barrier, and 2) at the adult age (postnatal d 90) at the time of full spermatogenesis. The data obtained show that claudin-11 expression was inhibited in prepubertal rat testes exposed in utero to 2 and 10 mg/kg x d flutamide. However, in adult testes, the inhibition was observed only with 2, and not with 10, mg/kg x d of the antiandrogen. It is shown here that these differences between prepubertal and adult testes could be related to dual and opposed regulation of claudin-11 expression resulting from positive control by androgens and an inhibitory effect of postmeiotic germ cells. Indeed, testosterone is shown to stimulate claudin-11 expression in cultured Sertoli cells in a dose- and time-dependent manner (maximum effect with 0.06 microm after 72 h of treatment). In contrast, postmeiotic germ cells potentially exert a negative effect on claudin-11 expression, because adult rat testes depleted in spermatids (after local irradiation) displayed increased claudin-11 expression, whereas in a model of cocultured Sertoli and germ cells, spermatids, but not spermatocytes, inhibited claudin-11 expression. The apparent absence of claudin-11 expression changes in adult rat testes exposed to 10 mg/kg x d flutamide therefore could result from the antagonistic effects of 1) the inhibitory action of the antiandrogen and 2) the stimulatory effect of the apoptotic germ cells on claudin-11 expression. Together, due to the key role of claudin-11 in the hemotesticular barrier, the present findings suggest that such regulatory mechanisms may potentially affect this barrier (re)modeling during spermatogenesis.

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Mullerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values).

MRNAs Encoding a von Ebner's-like Protein and the Huntington Disease Protein Are Induced in Rat Male Germ Cells by Sertoli Cells

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

Expression and regulation of neuropeptide Y messenger ribonucleic acid in cultured immature rat Leydig and Sertoli cells.

Neuropeptide Y (NPY) potentiates the release of gonadotropins from the pituitary in response to GnRH in the hypothalamus and modulates reproductive function. In the present study, we showed that 1) specific organs in the male rat reproductive tract express NPY messenger RNA (mRNA); 2) the multifactorial regulation of NPY mRNA in rat Leydig and Sertoli cells is temporally and hormonally regulated in vitro; 3) both Sertoli cell factor(s) and germ cell factor(s) potentiated to stimulate NPY gene levels in Leydig cells; and 4) intense NPY immunoreactivity was detected in cultured Leydig cells. Using the RT-PCR method, we found that Leydig cells, Sertoli cells, epididymis, and vas deferens expressed NPY mRNA, whereas germ cells, seminal vesicle, and prostate did not. Northern blot analyses showed that NPY mRNA was not expressed in freshly isolated immature Leydig cells, but that NPY mRNA levels were increased by the addition of LH, cytokines such as interleukin-1 alpha and -1 beta, forskolin, or phorbol 13-myristate 12-acetate. Npy mRNA levels in immature Sertoli cells were also increased by FSH. In addition, a germ cell factor(s) secreted from pachytene spermatocytes or round spermatids purified by centrifugal elutriation as well as a Sertoli cell factor(s) stimulated by FSH increased NPY gene levels in Leydig cells. Immunocytochemical analyses showed that the immunostaining was more marked in Leydig cells than in Sertoli cells in vitro. These findings indicate that testicular NPY gene expression is induced in Leydig cells or Sertoli cells by gonadotropins or cytokines within the testes, and that factors secreted from Sertoli cells or germ cells affect NPY gene expression in Leydig cells in vitro. Our findings suggest that NPY expressed in the reproductive system may modulate reproductive function as well as that in the nervous system.

Sertoli cell conditioned media modulate the androgen biosynthetic pathways in rat Leydig cell primary cultures.

Increasing evidence suggests the presence of local modulators that contribute to testicular function regulation in rats. The seminiferous tubules and Sertoli cell factors influence the steroidogenic activity of Leydig cells. Addition of Sertoli cell conditioned media to primary cultures of adult rat Leydig cells produces in a dose-dependent manner an inhibition of testosterone production with a concomitant decrease in the C21 delta 4-intermediates of the steroidogenic pathway, modifying the C21/C19 ratios in normal cells. The nature and mechanisms of action of this putative modulating activity are currently under study.

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro.

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.

Effects of Steroid-Free Rete Testis Fluid on Testosterone Concentration and Flow Rate of Ram Testicular Lymph.

The effects of intravenously injected steroid-free rete testis fluid (RTF) on the testosterone concentrations of testicular lymph and its flow rates have been studied. Lymphatic ducts of 5 rams were cannulated at the top of the vascular cone and the total output of testicular lymph was collected in serial samples for 3 censecutive days. Peripheral blood was obtained through an indwelling catheter in the jugular vein. Ten ml of RTF was injected intravenously once or 3 times. Testicular lymph testosterone levels, peripheral blood follicle stimulating hormone (FSH) and leuteinizing hormone (LH) were determined by radioimmunoassay. Injection of RTF suppressed FSH levels, but its effect on LH was variable. Testosterone profiles during 24h preceding and subsequent to the treatment period (24h) were characterized by 3 to 4 distinct testosterone spikes per day. Administration of RTF suppressed the amplitude of these testosterone spikes and prolonged the intervals between the spikes by up to 24h. The flow of testicular lymph was significantly reduced following injection of RTF. These results indicate that a non-steroidal Sertoli cell factor(s) in RTF may possess both paracrine and long-loop feedback modes of action, affecting both the endocrine and exocrine functions of the testis.

Cellular regulation of basal and FSH‐stimulated cyclic AMP production in irradiated rat testes

Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.

The adult rat leydig cell aromatase activity is stimulated by a sertoli cell factor [Postor 38

The adult rat Sertoli cell secretes a factor that stimulates the Leydig cell aromatase activity via protein synthesis, at a step beyond the adenylate cyclase. This factor is of proteic nature, species and tissue specific, different from the Sertoli cell LHRH-like compound, and the germ cells exert a negative control on its synthesis.

Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis.

Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an alpha beta heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the alpha beta heterodimer, the beta beta homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrating the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, we have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the alpha beta heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the beta beta homodimer suppresses androgen production. Furthermore, similar modulatory actions of inhibin-related proteins were found in cultured ovarian theca-interstitial cells and theca explants treated with LH. In contrast, treatment with the inhibin-related proteins alone did not affect gonadal steroidogenesis. Our data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops.

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity.

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.

Specificity and partial purification of a factor in spent media from sertoli cell-enriched cultures that stimulates steroidogenesis in leydig cells

There is increasing evidence that factors derived from the seminiferous tubules influence Leydig cell function in a paracrine way. In previous experiments we demonstrated that conditioned media from Sertoli cell-enriched cultures contain a protein with stimulatory activity on prepubertal rat Leydig cells. In this paper we further studied the specificity of this factor. In addition we describe a simple but efficient partial purification procedure. It is demonstrated that Sertoli cell conditioned media contain a factor that stimulates the testosterone ouput from prepubertal and adult Leydig cells. The effects are evident within the first hour of incubation and can be observed in the presence as well as in the absence of LH. Peritubular cells do not produce a similar factor but enhance the production of the Leydig cell stimulating factor when cocultured with Sertoli cells. The Sertoli cell factor acts on rat as well as on mouse Leydig cells. It barely influences the adrenostenedione output of ovarian stromal cells or the corticosterone output of adrenal cells. The production of this factor is enhanced by dbcAMP, FSH, l-isoproterenol and glucagon but is not affected by androgens. The characteristics of the Sertoli cell factor have been compared with those of a Leydig cell stimulating factor in the medium from an established rabbit kidney cell line: RK 13. It is shown that the active principle in RK 13 conditioned medium is also a thermolabile trypsin-sensitive protein with a mol. wt of more than 10,000. Nonetheless, the RK 13 and Sertoli cell derived factors act by different mechanisms since at maximally effective concentrations their effects are additive. Finally it is demonstrated that molecular weight fractionation of Sertoli cell conditioned medium using an Amicon ultrafiltration system results in a 50- to 130-fold increase in Sertoli cell factor activity in a fraction corresponding to a mol. wt of 10,000 up to 30,000.

Effect of hypotonic treatment on sertoli cell purity and function in culture

Commonly used enzymic methods for the isolation of rat Sertoli cells yield populations containing approximately 15% germ cells. Although the germ cells become eliminated after several media changes, they could interfere with the use of Sertoli cells for critical studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4) was found to eliminate most of the residual contaminating germ cells. The duration of this treatment varied from 1.0 to 10 min, depending on cell density in the culture, the degree of germ cell contamination, and the age of animals used for Sertoli cell isolation. In a study of 95% pure, 7-d Sertoli cell cultures, the hypotonic treatment did not alter the DNA or RNA content per dish or the incorporation of [3H]uridine into total and poly A+ RNA. Also, the hypotonic treatment did not alter specific Sertoli cell functions, i. e., secretion of Sertoli cell factor (inhibin) and stimulation of cAMP levels by follicle stimulating hormone in 2-d cultures. Androgen receptor concentration per dish was also not changed. Changes in several general metabolic parameters observed after hypotonic treatment of 2-d cultures were attributed primarily to loss of contaminating germ cells. Consequently, hypotonic treatment can be used to eliminate contaminating germ cells from the Sertoli cell cultures without apparent detrimental effects on a number of Sertoli cell biochemical parameters. This may be of considerable importance when the purity of Sertoli cells is critical for the interpretation of experimental data.

A factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells

We investigated whether Sertoli cell spent media (SCM) contain a factor (or factors) which influences steroidogenesis in Leydig cells. Freshly prepared prepubertal interstitial cells or Percoll-purified Leydig cells and similar cells cultured in the presence or absence of LH were incubated for 24 h in the presence of a 5α-reductase inhibitor and in the presence or absence of SCM. The accumulation of 19-steroids (Testosterone and androstenedione), C21-steroids (progesterone, 17α-hydroxyprogesterone and 20α-hydroxypregn-4-en-3-one) and cyclic AMP was measured by radioimmunoassay. It could be demonstrated that SCM contains a factor that stimulates an early step in the steroidogenic pathway but at the same time hampers the conversion of C21-precursors into androgens. In freshly prepared Leydig cells the final effect is a stimulation of the androgen output. In Leydig cells cultured in the absence of LH, mainly C21-steroid output in increased. These biological effects resemble those observed with LHRH and its agonists. The activity of the Sertoli cell factor is not affected by an LHRH antagonist, however, and maximally effective concentrations of the factor and LHRH have additive effects, suggesting that they act by distinct receptor systems. Preliminary characterization shows that the factor in SCM is a thermolabile protein with a MW > 10 000. The production of the factor decreases during prolonged culture in serum-free medium. Addition of feta; calf serum causes a marked and dose-dependent increase in the production or activity of the factor. Several permanent cell lines (B16, Bowes, BHK, Ratec, RK13, Vero) produce a factor with comparable biological effects on Leydig cells. Nonetheless, the observation that the production of this factor by Sertoli cell cultures is stimulated by FSH and dbcAMP suggests that, in the testis, it may play a role in the paracrine control of Leydig cell function.