Immature Sertoli Cells Research Articles

Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein.

Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.

Anti-müllerian hormone and Sertoli cell function.

Anti-müllerian hormone (AMH) is a glycoprotein that is produced by immature Sertoli cells and is responsible for the regression of müllerian ducts in male fetuses. It is a useful marker of testicular function in children, when anorchia is suspected and in intersex states. In the persistent müllerian duct syndrome, both AMH-positive and AMH-negative forms have been reported. In three cases of the latter, a mutation of the AMH gene is thought to have been responsible for the condition.

A partial characterization of a Sertoli cell-secreted protein stimulating Leydig cell testosterone production.

To examine whether immature rat Sertoli cells in culture secrete a factor(s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase. Sertoli cells were plated at an initial density of 3-5 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cadmium chloride on transepithelial electrical resistance of sertoli cell monolayers in two-compartment cultures—A new model for toxicological investigations of the “Blood-testis” barrier in vitro

The effect of various doses (0.75–24 μ m) of cadmium chloride (CdCl 2) on the development of intercellular tight junctions by immature rat Sertoli cells (Sc) was investigated in vitro using the two-compartment culture system. The status of tight junctions was monitored by repeated measurements of the transepithelial electrical resistance (TER). For defining the specificity of CdCl 2 effects, the TER changes were correlated with Sc secretory activity (immunoactive inhibin), the cell number (DNA content), and viability (MTT test). The effects of CdCl 2 depended on the concentration of the toxicant as well as on the onset and duration of exposure (4 and 18 hr on Day 1 or 5 of culture). The observed effects could be divided into four categories: (1) At highest doses employed, the TER values decreased significantly and irreversibly during 13 days of culture, and the decrease was accompanied by a significant and irreversible drop in inhibin secretion, cell viability, and cell number. (2) Within a narrow range of doses, the irreversible, or only partially reversible, decrease of TER was accompanied by a transient decrease, or no change, of the secretory activity and no significant changes in Sc cell number and/or viability. (3) With still lower doses, the TER values rapidly decreased and then returned to control level within 3–4 days. In this group, no changes in either inhibin secretion or cell viability were observed. (4) Exposure to the lowest doses of CdCl 2 caused a delayed, but significant increase in TER. This increase was not accompanied by noticeable changes in other parameters evaluated. These data suggest that CdCl 2 may selectively compromise, at least in vitro, the development and maintenance of the inter-Sc tight junctions, without affecting the secretory activity or the cell number and viability. However, increasing cumulative doses of CdCl 2 (concentration multiplied by the time of exposure) led to decreased inhibin secretion and cell viability and then, finally, to irreversible cell damage and death. We believe that the experimental model and approach reported in this paper should be very useful for investigating the mechanism of action of known or potential testicular toxicants, particularly those suspected to compromise the integrity of the “blood-testis” barrier.

Inhibin and inhibin alpha-chain precursors are produced by immature rat Sertoli cells in culture.

Stimulation of Sertoli cells from immature rats with dibutyryl cyclic (dbc) AMP resulted in a decrease in the ratio of inhibin biological (B):immunological (I) activities in vitro. To establish the basis for this decrease, culture medium from Sertoli cells stimulated with dbcAMP was fractionated by dye-affinity chromatography, reverse-phase HPLC, and preparative PAGE. Two peaks of inhibin activity were identified: a predominantly bioactive 29-kDa peak I material (B:I ratio = 5.0) and a bio-inactive, immunoactive 27-kDa peak II material (B:I ratio = 0.1). Evidence of a subunit structure was established by iodination and immunopurification using an inhibin alpha-subunit antiserum. On reduction, peak I (29-kDa) material showed bands of 19 kDa and 14 kDa, whereas peak II (27-kDa) material showed a single 20-kDa band. On the basis of HPLC retention position, molecular mass, evidence of subunit structures and their molecular masses, and inhibin in vitro bio- and immunoactivities, peak I and II materials were most likely inhibin and the alpha-subunit precursor protein pro-alpha C. Western blotting of Sertoli cell culture medium using antiserum directed against the NH2 terminal region (alpha N) of the alpha-subunit precursor also indicated the presence of 24-kDa alpha N. It is concluded that after dbcAMP stimulation, Sertoli cells produce 29-kDa inhibin and the alpha-subunit precursor proteins pro-alpha C and alpha N. The production of the alpha-subunit precursor in addition to inhibin provides an explanation for the decrease in the inhibin B:I ratio following dbcAMP stimulation of Sertoli cells in culture.

Species-specific effect of proteins secreted by cultured pre-pubertal rat Sertoli cells on natural killer cell activity

The eflect of proteins secreted by cultured pre-pubertal rat Sertoli cells (pSCP) on natural killer (NK) cell activity of rat, mice and guinea pig splenocytes and human peripheral blood lymphocytes was estimated. The results indicate that pSCP inhibited, in a dose-dependent manner, NK cell activity of mice, guinea pig and human lymphocytes but did not suppress lysis of YAC-1 target cells by rat NK cells. Species-specific differences in the efiect of pSCP on NK cell activity probably result from differences in the binding of proteins within the effector cells. These data indicate that in the pre-pubertal period of gonadal development immature Sertoli cells synthesize a factor/s which might contribute to the maintenance of specific testis immunological environment.

Testicular damage caused by inhalation of ethylene oxide in rats: light and electron microscopic studies.

Although testicular damage caused by ethylene oxide vapor (EtO) has been previously reported, the morphological changes occurring in seminiferous tubules remain unclear. We examined the time course of the testicular lesion induced by EtO in order to clarify its morphogenesis. Wistar rats were exposed to 500 ppm EtO for 6 hr per day, 3 times per week for 2, 4, 6, or 13 weeks through inhalation. In the 2-week exposure group, Sertoli cells often showed condensation and retraction of the cytoplasm, and dilatation of the endoplasmic reticulum (ER). In apical Sertoli cells, processes which encapsulated the heads of elongate spermatids, ectoplasmic specializations, and tubulobulbar complexes were often deformed and many elongate spermatids were degenerated. In the 4- and 6-week exposure groups, many degenerated Sertoli cells were present, and deformed germ cells, sometimes with multinucleation, appeared to make direct contact with each other without interlocation of Sertoli cell lateral processes. A few scattered immature Sertoli cells were evident in the 6-week exposure group. In the 13-week exposure group, seminiferous tubules containing almost all types of germ cells reappeared, mixed with atrophic tubules containing Sertoli cells only. In the former tubules, Sertoli cells often possessed regularly regenerated lateral processes, which were interposed between germ cells. These results indicate that the germ cell damage may be associated with damage to Sertoli cells. In spite of the intermittent exposure, focal regeneration of Sertoli cells appeared after 6 weeks of exposure to EtO and preceded patchy recovery of germ cells. Therefore, the data suggest that Sertoli cell regeneration may permit regeneration of germ cells.

Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture

The effects of dibutyryl cyclic AMP ((Bu) 2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 Ωcm 2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 Ωcm 2 after a transient (24–36 h) delay. The initial delay was prolonged to 3–4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 μM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 μM) mimicked these FSH effects. (Bu) 2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100–500 μM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu) 2cAMP (4–20 μM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu) 2cAMP. These results demonstrate, for the first time, a concentration-dependent, dual effect of exogenous cAMP on the Sc function. Moreover, our data suggest that, at least in vitro, FSH regulates the formation of inter-Sc tight junctions via a cAMP-mediated mechanism and that protein kinase C may modulate the testosterone-, but not FSH-dependent development of tight junctions by immature Sc.

Serum Bioactive Follicle-Stimulating Hormone-Like Activity in Human Pregnancy Is a Methodological Artifact*

Serum from pregnant women has been shown to contain both FSH-like and FSH antagonistic activities when measured by an in vitro bioassay based upon FSH-dependent aromatase activity of immature rat Sertoli cells. In the present study we further tested the hypothesis that the FSH-like bioactivity of pregnancy serum was due to an authentic aromatase stimulator. A potent inhibitor of aromatase which completely blocked the FSH action on Sertoli cells had no effect on the bioactivity of pregnancy serum. Experiments using conversion of tritiated testosterone to estradiol showed that the factor did not stimulate aromatase activity in rat Sertoli cells. After incubation with pregnancy serum, equally high amounts of estradiol were measurable in the medium in both the absence and presence of Sertoli cells. The activity was almost completely lost after charcoal treatment or ether extraction of the serum and was shown to probably be due to the release of endogenous estrogens by the carrier proteins in the incubation medium of the Sertoli cell assay. These data suggest that the FSH-like bioactivity in serum from pregnant women is an artifact due to nonspecific interference(s) in the bioassay.

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: modulation by calcium.

We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/l) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091 +/- 322 to 1480 +/- 84 pmol/l (p less than 0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360 +/- 45 to 1242 +/- 133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 mumol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 mumol/l) and A23187 (2 mumol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by greater than 80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 mumol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 mumol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.

Inhibition of protein kinase C by staurosporine increases estrogen secretion by rat Sertoli cells

We have examined the effect of inhibition of protein kinase C activity by staurosporine on estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Staurosporine lead to a dose-related increase in estradiol secretion independent of FSH, such that with 100 nmol/l staurosporine basal estradiol levels increased 10-fold. The maximal response seen with staurosporine alone (100 nmol/l) or in combination with FSH (0.4-8 IU/l) was similar to that seen with a saturating dose of FSH (8 IU/l). There was no evidence of synergy between FSH and staurosporine. Activation of protein kinase C by phorbol 12,13 dibutyrate (10(-7) mol/l) resulted in a 53-74% inhibition of estradiol production provoked by FSH (8 IU/l), staurosporine (5-100 nmol/l) or staurosporine in combination with FSH. Staurosporine (5-100 nmol/l), in the absence or presence of FSH, was unable to overcome inhibition of estradiol secretion by phorbol ester, indicating the presence of at least two independent binding sites on protein kinase C for these molecules. Forskolin (1 mumol/l)- and dibutyryl cAMP (1 mmol/l)-stimulated estradiol secretion was inhibited by 31 +/- 5% and 64 +/- 5% respectively, by phorbol 12,13 dibutyrate (10(-7) mol/l). We conclude that FSH-induced estradiol secretion in immature rat Sertoli cells is affected by protein kinase C activity.

In-vitro proliferation of germ cells and supporting cells in the neonatal mouse testis

Testicular cells were prepared from neonatal (48 h after birth) mice by enzymatic dissociation and were cultured in serum-supplemented medium to investigate cell proliferation in vitro. The cultured cells were composed mostly of germ cells, identified by immunocytochemistry using a germ cell-specific antiserum, and supporting (immature Sertoli) cells. After 36 h in culture, the cells were pulse-labeled with 3H-thymidine and fixed at 2-h intervals for 36 h after labeling. Numbers of labeled and unlabeled metaphases of germ cells and supporting cells were counted, and percent labeled metaphases for both cell types were determined for cell-cycle analysis. The results indicate that germ cells, as well as supporting cells, incorporate 3H-thymidine and progress through the cell cycle in vitro. From the curve of the percent labeled metaphases for the supporting cells, the total cell cycle and intervals of DNA synthesis were estimated to be 27.2 h and 13.2 h, respectively.

Follicle-stimulating hormone signal transduction: Role of carbohydrate in aromatase induction in immature rat Sertoli cells

Receptor activated adenylate cyclase acts as a major transmembrane signalling system. It is widely accepted that upon binding to its receptor, follicle-stimulating hormone (FSH) activates the cAMP-dependent pathway which in turn mediates FSH-induced estradiol production in Sertoli cells. Studies utilizing several chemically derived variants of FSH have demonstrated that these variants bind to the FSH receptors with equal avidity but differ in their ability to activate cAMP-dependent pathways. Since cAMP is believed to be the second messenger responsible for FSH signal transduction, we tested two hypotheses: (1) that the effects of different oFSH variants on cAMP production and aromatase induction (as measured by estradiol production) would be in parallel; and (2) that deglycosylated ovine FSH (DG-oFSH) would antagonize the ability of intact oFSH to stimulate aromatase induction, similar to its reported antagonistic effect on cAMP production. Immature rat (7- to 10-day-old) Sertoli cells were cultured and the effects of several different oFSH variants on cAMP production and/or aromatase induction were tested. The variants tested were native oFSH, DG-oFSH, asialo oFSH (AS-oFSH), a recombinant of intact LHα and FSHβ (α + β) and a recombinant of deglycosylated LHα and intact FSHβ (DGα + β). Both native oFSH and α + β recombinant at relatively large doses (10 ng) elicited a significant increase in extracellular cAMP accumulation as well as total cAMP production. In contrast, DG-oFSH did not produce an increase in cAMP even at 10-fold higher doses than native oFSH. Intracellular cAMP concentrations did not increase following stimulation with native oFSH, DG-oFSH or DGα + β. In contrast to the divergent effects of oFSH and DG-oFSH on cAMP production all variants of oFSH stimulated estradiol production from Sertoli cells albeit with varying potencies. The sensitivity (minimal effective dose) and ED 50 (dose at which half maximal response is achieved) of the estradiol (E 2) response curve to increasing concentrations of native oFSH were 0.025 ± 0.01 and 0.33 ± 0.05 ng, respectively. Asialo-oFSH (AS-oFSH) increased E 2 production with a potency (comparative dose required for effect) similar to that of native oFSH. In contrast, there was a 10-fold reduction in potency of DG-oFSH (ED 50 of 3.27 ± 1.7 ng). The efficacy (maximum effect on aromatase induction) of native, DG and AS-oFSH were equipotent. Coincubation of DG-oFSH (0.005–100 ng) with oFSH (0.01–0.625 ng) demonstrated the inductive effects to be additive. In contrast to the reduced potency of DG-oFSH, DGα — β recombinants (0.156–160 ng) increased E 2 production with a potency similar to that of the native oFSH. Our data suggest: (1) the effects of oFSH variants on cAMP production and aromatase induction are not always parallel; (2) DG-oFSH is a weak agonist and not an antagonist of oFSH-mediated aromatase induction; (3) the reduced potency of DG-oFSH must involve oligosaccharides internal to sialic acid; and (4) depleting the carbohydrate moiety on the α subunit alone may not be adequate to reduce the potency of oFSH to induce aromatase. Since DG-oFSH does not increase cAMP accumulation, but is able to induce aromatase, the FSH-mediated induction of aromatase may be transduced through other signal pathways. Alternatively, the maximal steroidogenic response may be induced by small, but undetectable, increases in intracellular cAMP concentrations or availability of segregated cAMP pools.

Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis

The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW > 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

Evaluation of changes in the secretion of immunoactive inhibin by adult rat seminiferous tubules in vitro as an indicator of early toxicant action on spermatogenesis

A method for culturing isolated seminiferous tubules (ST) from adult rats for 1–3 days has been developed and optimized rigorously on the basis of the secretion of immunoactive inhibin under basal conditions and after maximal stimulation with rat FSH or dibutyryl cyclic AMP. The effect on these cultures of three known testicular toxicants was assessed. Of these, two are though to act on the Sertoli cell, meta-dinitrobenzene (mDNB) and nitrobenzene (NB), while the third, methoxy acetic acid (MAA), is thought to act on pachytene spermatocytes. In addition, the effect of a possible testicular toxicant, 3-mononitrotoluene (3-MNT), was investigated. These data were compared with those obtained using cultures of immature rat Sertoli cells (SC) or SC + germ cells and with data on the effect of equivalent doses of the compounds on the secretion of immunoactive inhibin in vivo. In studies designed to optimize conditions for the secretion of immunoactive inhibin by ST in culture, significant effects were found of the type of culture medium used, the duration of culture, the total and individual length of tubules used, etc. All subsequent studies with toxicants utilized optimal conditions. Addition of either mDNB or NB to ST cultures at 10 −5 or 10 −3 m, or MAA at 10 −4 m, stimulated basal secretion of immunoactive inhibin by two- to fourfold on Days 1, 2, or 3 of culture while FSH or dibutyryl cyclic AMP-stimulated secretion of immunoactive inhibin was either unaffected or was enhanced to a small extent. At the same doses, mDNB or NB also enhanced secretion of immunoactive inhibin by SC cultures, although these effects were more variable and of smaller magnitude than the effects on ST cultures. In contrast, addition of up to 10 −3 m MAA to cocultures of SC + germ cells had no effect on the secretion of immunoactive inhibin. Exposure of rats in vivo to levels of mDNB, NB, or MAA similar to those which stimulated secretion of immunoactive inhibin in vitro resulted in a two- to fourfold increase in the levels of immunoactive inhibin in testicular interstitial fluid (IF) at 1 and 3 days post-treatment, and this was associated with early impairment of spermatogenesis (as judged by testis weight). In contrast to these effects, addition of 3-MNT to ST or SC cultures had no effect except at 10 −3 m, when the secretion of immunoactive inhibin was increased marginally. Treatment of rats with an equivalent dose of 3-MNT in vivo resulted in death, but exposure to the highest nonlethal dose (1 g/kg) had no significant effect either on spermatogenesis or on the levels of immunoactive inhibin in testicular IF. In view of these findings, it is concluded that modulation of the secretion of immunoactive inhibin by isolated ST from adult rats has considerable potential as an in vitro screening method for investigating potential adverse effects of chemicals on spermatogenesis, and thus merits more detailed evaluation. Moreover, because of the agreement between in vitro and in vivo findings, measurement of the levels of immunoactive inhibin levels in vivo in testicular IF (or blood) may also be useful in the detection of early adverse effects of chemicals on spermatogenesis.

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [ 125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the M r 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the M r 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32 67 laminin binding proteins.

Pachytene spermatocyte protein(s) stimulate sertoli cells grown in bicameral chambers: Dose-dependent secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin

Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [35S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [35S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 micrograms/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100 micrograms/ml of pachytene spermatocyte protein. These results suggest that pachytene spermatocytes modulate Sertoli cell secretory function of at least four proteins in the regulation of spermatogenesis.

Interleukin-1 inhibits follitropin-induced aromatase activity in immature rat Sertoli cells in vitro

The effects of recombinant preparations of human interleukin-1 (IL-1) on basal and follitropin (FSH)-stimulated aromatase activity of immature rat Sertoli cells in vitro were studied. Sertoli cells were isolated from 7- to 10-day-old rats and cultured for 72 h at 32 ° C in the presence or absence of test materials. The cells were then washed and cultured for a further 24 h with different doses of FSH in the presence or absence of IL-1. Neither IL-1α nor IL-1β had any effect on basal aromatase activity. IL-1β inhibited FSH-stimulated aromatase activity in a dose-dependent manner while IL-1α had no significant effect. The inhibitory influence of IL-1β on FSH-stimulated aromatase activity was greater when IL-1β was present in low concentrations (0.5 and 1.0 U) during the initial 72 h culture period and a further 24 h incubation with IL-1β did not cause a further inhibition. When the cells were cultured for 72 h in the presence of both IL-lα and IL-1β, the inhibition of FSH-stimulated aromatase was higher than that obtained with IL-1β alone. The inhibitory activity of IL-1β was blocked by a specific IL-1β antiserum. Furthermore, IL-1β caused a significant inhibition of cAMP production in Sertoli cells and did not influence dibutyryl-cAMP-stimulated aromatase activity under identical experimental conditions. It is concluded that IL-1β specifically inhibits FSH-induced aromatase activity in immature rat Sertoli cells and this inhibition is caused by impaired cAMP production in these cells. IL-lα alone had no effect on Sertoli cell aromatase activity but enhanced the inhibitory effect of IL-1β.

Evaluation of Changes in the Secretion of Immunoactive Inhibin by Adult Rat Seminiferous Tubules in Vitro as an Indicator of Early Toxicant Action on Spermatogenesis

Evaluation of Changes in the Secretion of Immunoactive Inhibin by Adult Rat Seminiferous Tubules in Vitro as an Indicator of Early Toxicant Action on Spermatogenesis. Allenby, G., Foster, P. M. D., and Sharpe, R. M. (1991). Fundam. Appl. Toxicol16, 710–724. A method for culturing isolated seminiferous tubules (ST) from adult rats for 1–3 days has been developed and optimized rigorously on the basis of the secretion of immunoactive inhibin under basal conditions and after maximal stimulation with rat FSH or dibutyryl cyclic AMP. The effect on these cultures of three known testicular toxicants was assessed. Of these, two are thought to act on the Sertoli cell, meta-dinitrobenzene (mDNB) and nitrobenzene (NB), while the third, methoxy acetic acid (MAA), is thought to act on pachytene spermatocytes. In addition, the effect of a possible testicular toxicant, 3-mononitrotoluene (3-MNT), was investigated. These data were compared with those obtained using cultures of immature rat Sertoli cells (SC) or SC + germ cells and with data on the effect of equivalent doses of the compounds on the secretion of immunoactive inhibin in vivo. In studies designed to optimize conditions for the secretion of immunoactive inhibin by ST in culture, significant effects were found of the type of culture medium used, the duration of culture, the total and individual length of tubules used, etc. All subsequent studies with toxicants utilized optimal conditions. Addition of either mDNB or NB to ST cultures at 10−5 or 10−3 m, or MAA at 10−4 m, stimulated basal secretion of immunoactive inhibin by two- to fourfold on Days 1, 2, or 3 of culture while FSH or dibutyryl cyclic AMP-stimulated secretion of immunoactive inhibin was either unaffected or was enhanced to a small extent. At the same doses, mDNB or NB also enhanced secretion of immunoactive inhibin by SC cultures, although these effects were more variable and of smaller magnitude than the effects on ST cultures. In contrast, addition of up to 10−3 m MAA to cocultures of SC + germ cells had no effect on the secretion of immunoactive inhibin. Exposure of rats in vivo to levels of mDNB, NB, or MAA similar to those which stimulated secretion of immunoactive inhibin in vitro resulted in a two-to fourfold increase in the levels of immunoactive inhibin in testicular interstitial fluid (IF) at 1 and 3 days post-treatment, and this was associated with early impairment of spermatogenesis (as judged by testis weight). In contrast to these effects, addition of 3-MNT to ST or SC cultures had no effect except at 10−3 m, when the secretion of immunoactive inhibin was increased marginally. Treatment of rats with an equivalent dose of 3-MNT in vivo resulted in death, but exposure to the highest nonlethal dose (1 g/kg) had no significant effect either on spermatogenesis or on the levels of immunoactive inhibin in testicular IF. In view of these findings, it is concluded that modulation of the secretion of immunoactive inhibin by isolated ST from adult rats has considerable potential as an in vitro screening method for investigating potential adverse effects of chemicals on spermatogenesis, and thus merits more detailed evaluation. Moreover, because of the agreement between in vitro and in vivo findings, measurement of the levels of immunoactive inhibin levels in vivo in testicular IF (or blood) may also be useful in the detection of early adverse effects of chemicals on spermatogenesis.

Follicle-stimulating hormone-stimulated secretion of an immunoreactive 29 kDa inhibin α-subunit complex, but not of 32 kDa bioactive inhibin, from cultured immature rat Sertoli cells

The medium of cultured Sertoli cells from immature rat testes contains 29 and 32 kDa proteins, which are recognized by an antiserum against the 22 N-terminal amino acids of the inhibin α-subunit. These proteins were detected by immunoprecipitation of labelled proteins after incubation of Sertoli cells with [ 35S]methionine, and by Western blotting. The amount of the 32 kDa protein was not affected by the addition of follicle-stimulating hormone (FSH) to the culture medium of the Sertoli cells, whereas FSH induced a large increase of the amount of the 29 kDa protein. Finally, the 29 and 32 kDa proteins in the medium from control and FSH-stimulated Sertoli cells were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis, and inhibin bio- and immunoactivity were determined in eluates of the slices of the gel. Equal amounts of bioactivity were found in control and FSH-stimulated samples at 32 kDa, while the amount of immunoactivity at 29 kDa was increased; no bioactivity was detected in the eluates of these slices. It is concluded that FSH stimulates the secretion of a 29 kDa inhibin-like protein, which does not contain inhibin bioactivity. This indicates that results of experiments, in which antibodies against N-terminal peptides of the inhibin α-subunit are used to detect inhibin, do not necessarily reflect the amount of bioactive inhibin produced.