Sertoli Cell-enriched Cultures Research Articles

Identification of stage-specific proteins synthesized by rat seminiferous tubules.

Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time.

Changes of sertoli cell glycoproteins induced by removal of the associated germ cells

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Regulation of inhibin secretion by Sertoli cell-enriched cultures.

Sertoli cells secrete a factor which has the same bioactivity as ovine testicular lymph inhibin: it selectively suppresses the secretion of FSH by cultured pituitary cells. We investigated the factors that acutely modulate the secretion of this inhibin by cultured Sertoli cells derived from immature rats. The secretion of inhibin was studied on day 7 of culture after a 24 h period of incubation in the presence or absence of steroids, gonadotrophins and foetal bovine serum, added alone or in various combinations. It could be demonstrated that aromatisable as well as non-aromatisable natural and synthetic androgens promote the secretion of inhibin in a dose-dependent way. FSH and pregnant mare serum gonadotrophin--at concentrations that clearly stimulate Sertoli cell aromatase activity--did not affect basal or androgen-stimulated production of inhibin. hCG was equally uneffective. The effect of androgens was not modified by the addition of an aromatase inhibitor but it was neutralized by the antiandrogen cyproterone acetate. Oestradiol-17 beta did not influence the secretion of inhibin whereas progesterone decreased it. Serum enhanced basal as well as androgen stimulated secretion of inhibin. It is concluded that androgens are the major factor which acutely stimulates the production of Sertoli cell inhibin.

ANDROGENS PROMOTE THE SECRETION OF FOLLICLE STIMULATING HORMONE INHIBITING FACTOR ‘INHIBIN’ BY SERTOLI CELL‐ENRICHED CULTURES

Annals of the New York Academy of SciencesVolume 383, Issue 1 p. 507-508 ANDROGENS PROMOTE THE SECRETION OF FOLLICLE STIMULATING HORMONE INHIBITING FACTOR ‘INHIBIN’ BY SERTOLI CELL-ENRICHED CULTURES G. Verhoeven, G. Verhoeven Rega Institute Laboratory for Experimental Medicine, Catholic University of Leuven 3000 Leuven, Belgium Institute of Medicine Radioimmunoassay Laboratory University of Liège BelgiumSearch for more papers by this authorP. Franchimont, P. Franchimont Rega Institute Laboratory for Experimental Medicine, Catholic University of Leuven 3000 Leuven, Belgium Institute of Medicine Radioimmunoassay Laboratory University of Liège BelgiumSearch for more papers by this author G. Verhoeven, G. Verhoeven Rega Institute Laboratory for Experimental Medicine, Catholic University of Leuven 3000 Leuven, Belgium Institute of Medicine Radioimmunoassay Laboratory University of Liège BelgiumSearch for more papers by this authorP. Franchimont, P. Franchimont Rega Institute Laboratory for Experimental Medicine, Catholic University of Leuven 3000 Leuven, Belgium Institute of Medicine Radioimmunoassay Laboratory University of Liège BelgiumSearch for more papers by this author First published: June 1982 https://doi.org/10.1111/j.1749-6632.1982.tb23223.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume383, Issue1The Cell Biology of the TestisJune 1982Pages 507-508 RelatedInformation

Improved in vitro bioassay of follitropin.

The FSH-dependent aromatase activity of Sertoli cell enriched cultures of testicular cells from immature rats was utilized as a sensitive and specific in vitro bioassay for FSH activity. The conversion of 19-hydroxyandrostenedione to estradiol was used as the end point of the assay. By the introduction of a phosphodiesterase inhibitor into the culture medium, the sensitivity was improved 5-10 times, so that 0.05 mIU of FSH showed a response significantly different from the blank. Other pituitary or placental hormones, such as hLH, hTSH and hCG exhibited less than 0.8% cross-reactivity, whereas hGH, hPRL, ACTH1-24, LH-RH, and prostaglandin E2 alpha did not show any cross-reaction at the doses tested.

Pure Sertoli Cell Cultures: A New Model for the Study of Somatic—Germ Cell Interactions

A new technique involving a brief hypotonic treatment was developed for obtaining pure rat Sertoli cell cultures. This method for the selective removal of the germ cells present in Sertoli cell enriched cultures (SCEC) Is based on the differential response of the two cell types to changes in osmolarity. It was found that the optimal conditions for germ cell detachment without Sertoli cell impairment consist of incubation for 2.5 minutes at 20 C in 20 m TRISHCI. When compared with SCEC, the Sertolicell‐only cultures (SCOC) thus obtained retain unaltered morphologic features and responsiveness to FSH stimulation (morphologic modifications and 17 β‐estradiol secretion). The availability of pure Sertoli cell cultures (Ie, free of contaminating germ cells) provides an advantage in the study of their metabolic activity. Moreover, with this technique it is feasible to compare Sertoli cell function in association with germ cells to function in the absence of germ cells.

Identification of estradiol produced by sertoli cell enriched cultures during incubation with testosterone

The FSH dependent aromatase activity in Sertoli cell enriched cultures from 10-day-old rats has been used as a sensitive and specific bioassay for FSH. The product is measured by a rapid radioimmuno-assay procedure using specific estradiol antibodies. The identity of this product has been questioned by others. In the present study, 0.5–1% of the [ 3H]-testosterone used as a substrate was aromatized, and approximately 25% of the phenolic material could be accounted for as authentic 17β-estradiol by crystallization to constant specific activity.

Structural response of adult rat sertoli cells to peritubular fibroblasts in vitro

Sertoli cells were harvested from sexually mature rats and maintained in vitro for up to ten days (Sertoli cell-enriched cultures) or co-cultured with rat peritubular fibroblasts. Cultures were examined by differential light microscopy and by scanning or transmission electron microscopy. The presence of peritubular fibroblasts in co-culture greatly enhanced plating efficiency and viability of adult Sertoli cells. Sertoli cell aggregates preferentially adhered to peritubular cells, and by ten days had flattened and spread across these cells. Sertoli cells retained their characteristic ultrastructural features. Early in co-culture a collagen-like extracellular material was seen bridging Sertoli and peritubular cells, and its appearance was coincident with the presence of swollen rough endoplasmic reticulum in peritubular cells. Interperitubular cell spaces became engorged with a fibrillar material morphologically similar to the basal lamina of the seminiferous tubule wall. In the absence of peritubular cells, in culture medium "conditioned" by prior incubation with peritubular cells but not containing them, or when cultured with other fibroblastic cells, plating efficiency was low; Sertoli cells never flattened and cell ultrastructure progressively degenerated. The results indicated that peritubular cells support adult Sertoli cells in culture, possibly via extracellular material derived from peritubular cells, and suggest that Sertoli cells have an inductive effect on peritubular cell secretory activity.

Desensitization of cultured rat sertoli cells by follicle-stimulating hormone and by l-isoproterenol

When Sertoli cell-enriched cultures derived from 19-day-old Wistar rats are exposed to FSH or L-isoproterenol, cAMP accumlates in the medium. Previous contact of cultured cells with either one of these agents results in a reversible state of desensitization. During secondary stimulation up to 20 times less cAMP accumulates in the medium and in the cells. The extent of desensitization depends on the concentration of FSH or L-isoproterenol to which the cells have been exposed and on the duration of this previous contact. Pre-incubation with FSH results in cross-desensitization for L-isoproterenol. Comparable heterologous desensitization is not observed after pre-incubation with L-isoproterenol. This suggests that down-regulation of the respective receptors cannot be the only explanation. Refractoriness can also be induced by dbcAMP. This opens the possibility that cAMP itself contributes to the homologous and heterologous desensitization process.

Effects of serum components on the morphology of Sertoli cells in culture.

Studies of Sertoli cell structure, maturation, and function have been aided by the use of in vitro systems. Although numerous papers have appeared that utilize the Sertoli cell culture model, few papers have dealt with the characterization of these cells under various culture environments. Recently, it has been reported that the addition of serum to the culture medium prevents induction of long cytoplasmic appendages in cultured Sertoli cells that have been treated with FSH, TSH, or c-AMP. The purpose of this investigation was to determine which serum components, obtained by gel filtration, are capable of inhibiting the morphological response induced by FSH, TSH, or c-AMP. Sertoli cell-enriched cultures were prepared using collagenase and trypsin digestion, each followed by gravity sedimentation. Untreated cells grown on plastic or glass substrates assumed an epithelioid appearance after several days. Cells treated with FSH, TSH, or c-AMP formed long cytoplasmic appendages after 1-2 days. This response was prevented or reversed by the addition of fetal calf serum (10%), crystallized bovine serum albumin (0.25%-2%), or purified albumin obtained by gel filtration of whole serum (0.25%). It was also found that fractions that elute between the void volume and the initial albumin fractions (molecular weights of approximately 50,000 and greater) mimic the hormone-induced response after only 10-12 hours. The results of this investigation indicate that albumin is the primary serum component responsible for inhibiting morphological alterations induced by FSH, TSH, and c-AMP. Furthermore, it is apparent that the production of long filamentous cytoplasmic appendages in Sertoli cells can be induced by a wide variety of substances.

Stimulation effect of neurotransmitters on the aromatization of testosterone by Sertoli cell-enriched cultures

The aromatization of testosterone has been studied in Sertoli cell-enriched cultures derived from 19-day-old rats. It was found that, besides dbcAMP and FSH, several neurotransmitters have a stimulatory effect. At a concentration of 10 −5 M, the strongest effects were noted with L-isoproterenol, L-norepinephrine, L-epinephrine and D,L-synephrine. Some stimulation was observed with dopamine, whereas histamine, melatonin and serotonin as well as acetylcholine and pilocarpine were ineffective. Experiments with α- and β-adrenergic antagonists suggest that ( β 2- and maybe also α-receptors are involved. The same maximal level of stimulation was generally observed for dbcAMP, FSH and isoproterenol. No further increase in activity was obtained with combinations of these compounds. It is concluded that adrenergic agonists may play a role in testicular development and function.

Metabolic responses of Sertoli cells in culture to various concentrations of follicle stimulating hormone and cholera toxin

The concentration of cholera toxin required for half-maximal stimulation of cAMP production by Sertoli cell enriched cultures (4.48 X 10(2) microgram/ml) is greater than that required for half-maximal stimulation of 17beta-estradiol synthesis from testosterone (2.34 X 10(-4) microgram/ml), [3H]thymidine incorporation into DNA (1.48 X 10(-5) microgram/ml), or androgen binding protein production (2.43 X 10(-6) microgram/ml). The same relative dose response hierarchy was obtained with respect to stimulation of Sertoli cells with follicle stimulating hormone (FSH) preparations. Again, highest concentrations were required to elicit maximal cAMP production. The data are discussed in relation to an apparent paradox: If cAMP is the mediating 'second messenger' following stimulation by FSH or cholera toxin, why should highest concentrations of these agents be required to elicit 50% of maximal cAMP levels?

Secretion of proteins by sertoli cell enriched cultures: Effects of follicle stimulating hormone, dibutyryl camp and testosterone and correlation with secretion of oestradiol and androgen binding protein

Sertoli cells from 21–23-day-old rats were maintained in a chemically defined medium with or without hormones for 7 days with daily renewal of medium. It was found that in addition to secretion of oestradiol-17β and androgen binding protein (ABP), radioactive proteins were present in the culture medium after incubation of the cells with [ 3H]leucine. These labelled proteins are secreted into the medium by the Sertoli cells. Lactate dehydrogenase was also present in culture medium as a secretion product from Sertoli cells. Stimulation of the secretion of the labelled proteins, ABP and oestradiol-17β at the 6th or 7th day could be observed when cells had been cultured in the presence of either testosterone, follicle stimulating hormone (FSH) or dibutyryl cyclic adenosine monophosphate (DcAMP) for 5 or 6 days. Addition of luteinizing hormone (LH) had no effect. One peak in the electrophoretic profile of labelled proteins decreased when cells had been cultured in the presence of FSH or DcAMP, but testosterone had no effect. No other effects of hormones on the profiles were observed. Germinal cells, interstitial cells or testicular fibroblasts did not contribute significantly to the quantity of the radioactive proteins. During the culture period and even in the presence of FSH or testosterone, decreasing daily production of ABP but a constant or increasing daily production of oestradiol-17β from testosterone was observed.

Secretion of plasminogen activator by Sertoli cell enriched cultures

Conditions were established to assay plasminogen activator (PA) levels in tissue culture medium in which Sertoli cell aggregates, prepared from testes of immature rats, had been cultured. PA activity was measured by determining 125I fibrinolysis dependent upon added plasminogen. The PA levels were higher in medium taken from Sertoli cell-enriched cultures treated with follicle-stimulating hormone (FSH) or dibutyryl cAMP. In addition, the fibrin-agar overlay technique was employed to detect plasminogen-dependent lysis zones around Sertoli cell aggregates in culture. Lytic zones appeared earlier and developed faster around FSH-treated Sertoli cell aggregates. Peritubular myoid cells had no detectable PA activity when tested by either technique. The possible physiological role of PA in the testis is discussed.

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.

Hormonal control of mammalian spermatogenesis.

Studies of the hormonal control of the spermatogenic process are reviewed. Most of the investigations used the techniques of ablation and replacement therapy and were performed on the rat. Results from the studies indicate that changes in the metabolism of proteins lipids and carbohydrates take place in the testes under the control of hormones. However it may be that the observed biochemical changes which occur are a reflection of the changes in cellular composition of testicular tissue rather than the result of metabolic changes within a specific cell type in response to the effect of a hormone. A hypothesis concerned with the qualitative aspects of hormonal control of spermatogenesis in the rat has been formed. It is proposed that the multiplication and growth of gonocytes prenatally and in the first few days after birth and formation of type A spermatogonia are under the control of testosterone. The formation of type A and B spermatogonia and primary spermatocytes and progression of the meiotic prophase may be influenced by growth hormone. Reduction division is under control of testosterone. The early steps of spermatid formation may be under no hormonal control or may require testosterone while the late stages of spermatid maturation require the presence of FSH.