Follicle - stimulating hormone stimulates estradiol - 17 f synthesis in cultured

  • H . DORRINGTON, DAVID T . ARMSTRONGt
  • Published 2003

Abstract

Sertoli cells isolated from testes of 20-dayold rats and maintained in primary culture synthesized estradiol-17i, [1,3,5(10)estratriene-3,17IB-diolI (measured by specific radioimmunoassay) when testosterone (17fl-hydroxy-4-androsten-3-one) 0.5 IMM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3f,-hydroxypregn5-en-20-one), 0.5 AM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S1O, 5 Mg/ml) stimulated estradiol synthesis 12to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 Mg/ml of medium, whereas luteinizing hormone (NIH-LH-SIJ8, 5 Mtg/ml) caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17,3 production by Sertoli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate. The secretion of estrogens is a well-recognized function of the mammalian testes (1, 2), although the cellular source of this class of steroids has not been established. Hunt and Budd (3) implicated the Leydig cell as the site of testicular estrogen synthesis on the basis of evidence of feminization in men with interstitial cell tumors. Support for this conclusion came from the observations of Maddock and Nelson (4) that prolonged administration of human chorionic gonadotropin (HCG) to normal and hypogonadal men resulted in increased urinary estrogen excretion. Upon histological examination the Leydig cells appeared to be stimulated, whereas the germinal epithelium regressed and the Sertoli cells underwent changes described as "degenerative" (4). On the other hand, feminization of male dogs (5) and humans (6) with Sertoli cell tumors indicated that the Sertoli cell may be involved in estrogen synthesis. Further evidence was obtained by the demonstration that canine Sertoli cell tumors contained substantial amounts of estrogenic material (7, 8). Recently, methods have been reported for the isolation and maintenance in primary cultures of Sertoli cells obtained from immature rat testes (9, 10). These cultures have been shown to respond to added follicle-stimulating hormone (FSH) and dibutyryl-adenosine 3':5' cyclic monophosphate (Bt2cAMP) (11-13). The development of a method for the isolation of Sertoli cells provided us with a direct means of determining whether Sertoli cells are capable of estrogen biosynthesis. The present studies were undertaken with this goal in mind, as well as to examine possible gonadotropic requirements for estrogen biosynthesis by Sertoli cells. MATERIALS AND METHODS Materials and Animals. Ovine luteinizing hormone (LH) (NIH-LH-S18; potency 1.03 NIH-LH-S1 units/mg and containing less than 0.050 NIH-FSH-S1 units/mg) and ovine FSH (NIH-FSH-S1O; potency 1.10 NIH-FSH-S1 units/mg and containing less than 0.010 NIH-LH-S1 units/mg) were provided through the Hormone Distribution Office, National Institutes of Health, Bethesda, Md. Highly purified FSH (G4-150C; having an activity of 50 NIH-FSH-S1 units/mg in the Steelman-Pohley assay and 0.02 NIH-LH-S1 units/mg in the ovarian ascorbic depletion test) was a gift from Dr. H. Papkoff. N6,02'-Dibutyryl-adenosine 3':5' cyclic monophosphate (Bt2cAMP) was obtained from Sigma Chemical Co. Testosterone (17fl-hydroxy-4-androsten-3-one), pregnenolone (3f3-hydroxypregn-5-en-20-one), collagenase type 1 (125 units/mg of solid), deoxyribonuclease 1 (crude, from beef pancreas, 780 Kunitz units/mg), and soybean trypsin inhibitor (type IS) were purchased from Sigma Chemical Co.; Hanks' solution was obtained from Difco Laboratories. Eagle's minimum essential medium, nonessential aminoacid solution, L-glutamine, penicillin, streptomycin and Fungizone were obtained from Grand Island Biological Co. [2,4,6,7-3H]Estradiol-17fl [1,3,5(10)-estratriene-3,17f3-diol], specific activity 106 Ci/mmol, was obtained from New England Nuclear. Normal male Wistar rats (14 days of age) were purchased from Canadian Breeding Laboratories, Montreal. They were maintained with mothers in a lightand temperature(240) controlled room until 18-20 days of age. Preparation of Sertoli Cell Aggregates. The method used for the preparation of Sertoli cell aggregates was based on that described in detail by Dorrington and Fritz (10). Briefly, the procedure for each experiment was as follows: The testes were removed from ten 18to 20-day-old rats and decapsulated, and the testicular tissue was chopped twice, at right angles, with a Mickel mechanical tissue slicer, set at 0.5 mm. The testis fragments were digested for 30 min at 320 in 50 ml of 0.25% trypsin in Hanks' balanced salt solution (ESS) containing 0.5 mg of deoxyribonuclease. Soybean trypsin inhibitor was added and the suspension was passed through a 2677 Abbreviations: FSH, follicle-stimulating hormone; LH, luteinizing hormone; Bt2cAMP, N6, 02'-dibutyryl-adenosine 3':5' cyclic monophosphate; BSS, Hanks' balanced salt solution. 2678 Cell Biology: Dorrington and Armstrong wire grid (approximately 2 X 2 mm) to remove undigested clumps of tissue. The tubule fragments were washed in BSS and allowed to sediment under gravity. The supernatant fractions containing interstitial cells were discarded. Treatment of the tubules with collagenase (1 mg/ml) in BSS for 30 min at 32' followed by agitation using a Pasteur pipette effectively removed the peritubular layers (12). The tubule fragments were obtained by low-speed centrifugation and were washed in 25 ml of 10 mg/ml of bovine serum albumin in BSS. The cell aggregates (Sertoli cells and germinal cells) were further dispersed by drawing the suspension through a Pasteur pipette. The cell pellet obtained by low-speed centrifugation was washed three times in 10 ml of BSS. Culture of Sertoli Cells. The cell pellet was resuspended in an appropriate amount of Hanks' BSS so that 0.2 ml of the suspension contained approximately 1.5 mg of protein. This amount of cell protein was added to 5 ml of culture medium in Falcon plastic culture flasks. The medium used was similar to that described by Steinberger and Steinberger (14) and consisted of Eagle's minimum essential medium (minus phenol red), supplemented with 0.1 mM of the following amino acids: L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and glycine. In addition, glutamine (to give a final concentration of 4 mM), Fungizone (2.5 ,ug/ ml), penicillin (100 units/ml), and streptomycin (100 Ag/ml) were added. The cell aggregates attached to the surface of the culture flask; in contrast, single germinal cells remained free-floating in the medium and were discarded when the medium was changed. Thus, the cells which were attached to the culture flasks after one change of medium consisted of approximately 85% Sertoli cells, 10% degenerating cells and 5% germinal cells. The identity and morphological and biochemical characterization of these cells have been described previously (10-13). The cells were maintained in the standard culture medium in an atmosphere of 95% air and 5% CO2 at 32'. After 24 hr the medium was discarded and replaced by 2 ml of fresh medium. Substrates (testosterone or pregnenolone, 0.5 MiM) and test substances (5 Aig of NIH-FSH-S10/ml, 0.25 Mig of Papkoff's FSH/ml, 5 Mig of LH/ml, or 0.1 mM Bt2cAMP) were added. Since testosterone is metabolized rapidly by Sertoli cells to dihydrotestosterone and 5a-androstanediols (10), additional substrate (0.5MuM of either testosterone or pregnenolone) and also gonadotropins were added after a further 24 hr (i.e., 48 hr in culture). After 72 hr in culture the medium was removed and was frozen imm~ediately in dry ice and acetone. Extraction and Assay of Culture Media and Cells. The cells, after incubation, were extracted twice with 1 ml of absolute ethanol. In each experiment, cells that had been in culture for 24 hr only were extracted twice with1 ml of absolute ethanol, in order to determine the estradiol-17fl content before the addition of exogenous substrate and hormones. In addition, culture flasks containing culture medium, substrate, and test substances, but not cells, were included in every experiment. The ethanolic extracts and the media were stored at-20° until they were assayed for estradiol-17,B. The cells, after extraction with ethanol, were scraped from the surface of the culture flask, 1 M sodium hydroxide was added, and aliquots were assayed for protein content (15). Aliquots of culture media were extracted twice with 5 volumes of diethyl ether, and the ether extracts were transferred to 12 X 75 mm disposable culture tubes and evaporated to dryness under nitrogen. Aliquots of the ethanolic ex2-

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Cite this paper

@inproceedings{DORRINGTON2003FollicleS, title={Follicle - stimulating hormone stimulates estradiol - 17 f synthesis in cultured}, author={H . DORRINGTON and DAVID T . ARMSTRONGt}, year={2003} }