Purification of “colony-forming” cells from immature rat testis

Follicle stimulating hormone (FSH) enhances colony formation (as a result of reaggregation) by dissociated 10-day-old rat testis cells in primary culture. The purpose of this study was to examine various cytological characteristics of the FSH-responsive cells and develop techniques for their purification. The ability of testis cells to form colonies in response to FSH (5 μg/ml) was tested at various ages and was found to be maximal at 15 days of age. No colony formation occurred at ages greater than 20 days. Using colony formation as an assay for the FSH-responsive cells, techniques were developed for their purification. Colony cells were purified on a continuous bovine serum albumin (BSA) step gradient (1 ml/step). In fractions purified in this manner and subsequently cultured 24 h with FSH (5 μg/ml) 99.4% of attached cells were colony cells. Light and electron microscopy indicated that the colony cells (1) were one cell type; (2) were not germinal cells; (3) ultrastructurally resembled in situ Sertoli cell of the immature rat testis; and (4) contained a nucleolus with satellite karyosomes, structures which are characteristic of rat Sertoli cells. The mitotic index of the purified cells was 0.014% following 24 h in 10⁻⁵ M colchicine. Based on these data, it was concluded that the FSH responsive cells in culture are Sertoli cells.     

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.     

Stimulatory effect of Sertoli cell culture medium on the FSH release by pituitary halves in vitro

The influence of the medium collected from cultured rat Sertoli cells on the spontaneous and LHRH-stimulated release of gonadotropins by incubated rat pituitary halves was examined. The homogeneity of the cultured population of Sertoli cells taken from 20-day-old rats ranged up to 98%. The cells in culture responded to FSH stimulation with characteristic morphological changes and with increased secretion of estradiol-17 beta. The hemi-pituitaries obtained from sexually mature male rats were incubated for 5 hours in the presence of Sertoli cell culture medium (SCCM) or its fractions obtained by use of ultrafiltration. The SCCM fraction deprived of MW less than 10 kD compounds exhibited a typical inhibin-like activity, whereas crude SCCM as well as its low-molecular-weight fraction stimulated the basal FSH release to about 150% and 175% of the control values, respectively. These fractions exerted an inhibitory effect on the LHRH-stimulated secretion of both LH and FSH. It is concluded that Sertoli cells cultured in chemically defined medium release, apart from inhibin, a non-steroidal, heat-labile substance of MW less than 10 kD which stimulates the basal secretion of FSH and LH and inhibits the LHRH-stimulated secretion of both gonadotropins from incubated rat hemi-pituitaries.     

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP

The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.     

Rat Sertoli cell aromatase cytochrome P450: Regulation by cell culture conditions and relationship to the state of cell differentiation

Summary Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the³H₂O release method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cells is associated with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

Paracrine role of Sertoli cells

Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Levels of binding proteins for retinoids in cultured Sertoli cells: effect of medium composition

The levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) have been measured in Sertoli cells maintained under different cultural conditions. Sertoli cells were isolated from prepubertal rats and cultured in a chemically defined medium without or with follicle-stimulating hormone (FSH), insulin, retinol or testosterone added individually or in combinations. The additions were made at the beginning of the culture or 24 h before the cells were subjected to determinations of CRBP and CRABP by radioimmunoassay. No differences were observed either after 1 or 4 days of treatment. The results obtained indicated that the levels of the two retinoid-binding proteins were unchanged in Sertoli cells in response to hormone and/or retinol administration. To rule out the possibility that the Sertoli cells used in our study were unresponsive to the hormones, lactate production by the cells cultured in the presence of FSH or insulin was measured. The amount of lactate produced under hormonal stimulation was significantly higher than the amount produced in absence of the hormones, thus indicating the ability of our Sertoli cells to respond to the hormonal stimulation.     

Regulation by FSH and dibutyryl cyclic AMP of the formation of androgen-binding protein in Sertoli cell-enriched cultures.

Sertoli cell-enriched preparations from testes of 20-day-old rats were cultured in a defined medium in the presence and absence of FSH or dibutyryl cyclic AMP (dcAMP). Androgen-binding activity was assayed in the culture medium, and related to testicular androgen-binding protein (ABP). The production and secretion of ABP by the Sertoli cell-enriched preparation was increased after FSH or dcAMP treatment of the primary culture. It is concluded that ABP is produced by Sertoli cells. The possibility of involvement of other cell types in the testis in ABP production is discussed.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

Human Sertoli cells in vitro: morphological features and androgen-binding protein secretion.

Sertoli cells play a pivotal role in the regulation of spermatogenesis as they provide the anatomical basis of the blood-testis barrier. In the present paper we report some results of our studies on the ultrastructural features, the responsiveness to FSH, and the ability to secrete androgen-binding protein (ABP) of human Sertoli cells in vitro. The nucleus showed the characteristic foldings of the nuclear membrane, scattered chromatin, and a fibrillar nucleolus. In the cytoplasm Charcot-Boettcher crystals were present and active phagocytic activity was documented by the presence of vacuoles containing lipids and cellular debris. Human Sertoli cells in culture responded to FSH with a maximal rise in cAMP that was approx. 3-fold. This response to FSH is comparable to that reported for the adult rat but lower than that of the immature rat, and suggests that human as well as rat Sertoli cells could have a reduced response to FSH since sexual maturation was achieved. As no evidence has been reported on ABP secretion by human Sertoli cells in culture we evaluated the concentration of this protein in the Sertoli cell spent media. Human Sertoli cells in culture produced ABP and the response to FSH was dose-related. The Kd value of human ABP (hABP) was approx. 7.5 nM, being slightly higher than that of the rat ABP and an order of magnitude different from that of sex hormone-binding globulin (SHBG) present in human plasma. We also measured the association and dissociation rates of dihydrotestosterone-hABP complexes and the Kd/Ka ratio was very close to the value of Kd of the Scatchard analysis. The differences between hABP and SHBG may open the way to the selective measurement of ABP in many conditions of male infertility.     

Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development.

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell- to-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle-stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, syndecan-1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan-4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG-specific and depends on the Sertoli cell developmental stage.     

Increase by FSH hormone and depression by testosterone of the diffusional coupling between Sertoli cells from immature rat testis in primary culture

Previous studies have suggested that FSH promotes the intercellular coupling of Sertoli cells from immature rat testis in primary culture ([1], [2]). In order to test this hypothesis, we have investigated the diffusional coupling between Sertoli cells in primary culture with the FRAP technique. The coupling is low in unstimulated cells but increases in the presence of FSH. This effect is not reversed by returning to the control medium. Testosterone decreases this coupling, an effect which is reversed by a new exposure to FSH. Taken together these data show that FSH initiates diffusional coupling in Sertoli cells and that testosterone antagonizes this effect.     

Membrane molecules involved in adhesion properties of cultured sertoli cells

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody-induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS-PAGE), M_r 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.     

Paracrine control of Leydig cell activity by FSH dependent proteins from Sertoli cells: an in vitro study

The regulating effect of follicle-stimulating hormone (FSH) on Leydig cell function was studied using a model of immature porcine Leydig and Sertoli cells cultured in a hormone supplemented defined medium. FSH pretreatment for 2 days of Leydig cells cultured alone was with no effect. FSH pretreatment of Leydig cells cocultured with Sertoli cells increases Leydig cell activity in an FSH dose-dependent manner with a maximal effect observed at 50 ng/ml porcine FSH (pFSH). Leydig cells cultured for 2 days in conditioned medium (CM) by FSH stimulated (FSH-CM) Sertoli cells, as compared to CM by unstimulated (control) (C-CM) Sertoli cells show an increase of their activity with a maximal effect observed at 50 ng/ml pFSH. Leydig cells cultured in CM as compared to non CM, show a marked development of organelles (smooth endoplasmic reticulum and mitochondria) involved in the steroidogenic activity. The activity of FSH-CM as compared to C-CM on Leydig cell function was non dialyzable and trypsin sensitive. These data suggest that Sertoli cells exert a regulatory action on Leydig cell steroidogenic activity via FSH dependent secreted proteins.