Estrogen synthesis by immature rat sertoli cells in vitro

Estrogen Synthesis by Immature Rat Sertoli Cells In Vitro CARLOS A. JOANNE SUAREZ-QUIAN, MARTIN BRUMBAUGH, of FSH-stimulated aromatization was Ever like immature Drs. Medical Sertoli cells, estrogen synthesis, cases have School-School and Dym’s present of Dentistry, address: Department Submitted for ceived September 21, 1982. publication 21, 1982; June accepted 11, 1982; revised for publication (Hunt and Teilum, in both The in men has 1939; evidence and greater and tradiol erous tial Medical 3900 re- tissue 203 for 1979) not Sertoli gained by et mg than al, protein in isolated More re- rat testes certainty of Andrology be stimu- as the source of testicusupport when 17f3-esrat et al, 1974). with can FSH. cells much established Society (Leach et al, 1976). found to increase of the mature (dejong cell Adincrease shown that aromatase activity et al, 1979) and mature (Val- from immature immunoreactive of estrogens 4:203-209) to cells Leydig per testes (Payne Sertoli cells to produce the identity © American 1983; but the estrogen in whole Payne, hCG was tubules de- Moulder, aromatization. caused an of estrogen tubules The case estrogens and supports production cently, it has been in immature (Canick lar Huggins site of testicular of hCG in men was by been used in the and Sertoli synthesis. observed in 1949). seminiferous Georgetown (J Androl CANICK (1934) first noted estrogentestes, the testicular cell aromatization testosterone 1956). of Anatomy, 0196-3635/83/0500/0203/$01.15 Budd, Experimental lated version September A. testicular tumors were believed from either Leydig or Sertoli as the major ministration aromatase, 3900 Reservoir Rd. N.W., Washington, D.C. 20007. Supported by grant no. 7R01 HD16260-01. Reprint requests: C. A. Suarez-Quian, Georgetown School-School of Dentistry, Department of Anatomy, Reservoir Rd, N.W., Washington, D.C. 20007. for wherein originated 1945; also testis. Suarez-Quian Zondek in stallion responsible ladares words: MAKRIS, JACOB bated. Indirect methods of observation past have implicated both the Leydig cells as likely candidates for estrogen For example, feminization of men was obtained by RIA. Neither control nor LH-treated cells, however, in the presence of substrate, produced detectable levels of labeled or immunoassayable estrogens. Aromatase activity was also studied in the presence of a cold estrogen trap and measured by direct tracer methodology. In this case, estrogen synthesis was more than doubled. These data may suggest the further metabolism of estrogen by Sertoli cells. Finally, our results are discussed with respect to the role of the immature Sertoli cell in the synthesis of testicular estrogens. Key AND the Departments of Anatomy and and Gynecology, Laboratory of Reproduction and Reproductive Harvard Medical School, Boston, Massachusetts since activity type ANASTASIA RYAN, From Obstetrics Human Biology, Past research has implicated both the Leydig and Sertoli cells as sources of testicular estrogens. To study this issue further, we investigated the ability of Sertoli cells isolated from the testes of immature (10-day-old) rats to synthesize estrogens in response to FSH in vitro. The activity of Sertoli cells was examined by aromatase comparing simultaneously two different methods: 1. the conversion of 19-(6,7-3H)-hydroxyandrostenedione to (3H)estrone and (3H)estradiol, measured by silica gel thin layer chromatography; and 2. the conversion of unlabeled 19-hydroxyandrostenedione to total estrogen (El + E2), measured by RIA. Sertoli cells, cultured in the presence of substrate, exaromatase activity in response to FSH. Values hibited obtained for total estrogen synthesis, measured by direct tracer methodology, at FSH dosages of 0.5 &g/ml and 1.0 &g/ml, during a 24-hour period of incubation, were 620 pg/mg and 580 pg/mg, respectively; and after a incubation, values obtained were 570 pg/ml and 48-hour 560 pg/mg, respectively. When measured by RIA, simiwere obtained for estrogen synthesis at idenlar results tical FSH dosages and times of incubation. A dose response DYM, J. KENNETH in isolated but not Moreover, seminifin intersticultured rats have been shown estrogens, although produced could not be by the methodology 204 Journal employed (Dorrington Rommerts et tioned, al, cells odology was These when aromatization examined (Welsh Steinberger, for measuring borated initial that capable and of by cells of tracer to estrogens in vitro of estrogens Sertoli are (Gore- cells, produced we compared the 19-hydroxyandrostene- as measured by RIA, with the of 19-(6,7-3H)hydroxyandrostenedione investigation by report, was support these respect to the direct tracer published in progress, findings role Materials methodwhile our the identities and are of estrogen and revealed experi- and containing testis. Methods less than 0.05 times NIH-LH-S1; and NIH bLH-4, potency 1.8 to 3.2 U/mg, ing less than 0.05 NIH-FSH-S1 U/mg were the National Pituitary Agency 3H)hydroxyandrostenedione England Nuclear, special droxyandrostenedione tase substrates. Preparation of Sertoli were C from culture one dish). for two cultured testis days in per dish DMEM + 4 (30 x 5% FCS CO2. After two days in culture, the medium was changed; unattached cells, consisting mostly of germ cells, were rinsed away with a Pasteur pipette and new medium, containing 2% FCS in DMEM, was added. 37 in an atmosphere of of Substrates and 95% air and 5% Addition Cell Hormones to Sertoli Cultures cells had been in culture for two days, the medium was removed and 19-(6,7-3H)hydroxyandrostene= 0.5 .tM with the addione, (1 MCi, final concentration dition of unlabeled 19-hydroxyandrostenedione) in DMEM + 2% FCS was added to each culture dish. In some experiments, cold estrone and estradiol (1 M) were added to each culture dish to establish an estrogen (RIA) studies, unlabeled trap. For radioimmunoassay 19-hydroxyandrostenedione, 0.5 tM final concentration, was used as substrate. Incubation times and dosages for gonadotropin treatments were as indicated in the legends for each experiment. discussed in the Ten-day-old male Sprague-Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Trypsin, collagenase (Worthington Biochemical Co.), soybean trypsin inhibitor, BSA, and deoxyribonuclease (Sigma Chemical Co.) were prepared in Dulbecco’s Modified Eagle’s Medium (DMEM) (Grand Island Biological Co.) Penicillin 10,000 units/mI, 10,000 Fungizone#{174} 25 mcg/ml, and streptomycin mcg/ml were added to DMEM with or without fetal calf serum (GIBCO). NIH oFSH (Batch #13), potency 15 U/mg, obtained Falcon mm, Vol. indirats of the immunoreactive estrogens were with certainty (Ritz#{233}n et al, 1981). Our ments and 1983 cells 15 Cells method corro- data, total at meth- immature #{149} May/June After unlabeled to estrogens as measured ology. In a very recent with quescultured Tcholakian estrogens 1980). the identity immature conversion own direct 1976; from synthesizing conversion dione by Wiebe, were by radioimmunoassay Sertoli Langton et al, To establish in vitro results 1975; 1979). Finally, an alternative estrogens, by tritium release, the cating Armstrong, 1978). however, Sertoli and of Andrology (NIAMDD). (SA, synthesis) (Steraloids), Cell and containprovided by 32CiJmmole) and were unlabeled used 19-(6,7- (New 19-hy- as aroma- Assay of Cells and Culture After appropriate incubation times, media were removed from each culture dish, extracted twice with under N2. The separation of diethyl ether, and dried (3H)estrone and (3H)estradiol from 19-hydroxylated androgens was carried out by silica gel thin layer chromatography (benzene-heptane, ethyl acetate, 50:30:20). RIA studies were performed as described previously (Canick and Ryan, 1976) using an antiserum (Abraham 310 #5) that cross reacts equally with estrone and estradiol. Cells were removed from the culture dish with 2% sulfate containing 1 mM EDTA and sodium dodecyl protein determinations were performed (Lowry et al, 1951). The cultured cells were processed for light microscopy or transmission electron microscopy as described by Brinkley et al (1967). The cells were fixed with 1.5% glutaraldehyde and 1.5% p-formaldehyde in 0.1 M cacodylate buffer in situ and post-fixed with 1% 0s04 in 1.5% potassium ferrocyanide (Karnovsky, 1971). Sections were cut parallel to the plane of growth and examined with a JEOL-100S electron microscope. Culture Sertoli cell cultures were prepared using the method of Dorrington and Fritz (1975) with the following modification: the enzyme sequence was reversed, collagenase treatment preceded trypsin digestion. The final cell suspension to be plated contained Sertoli cells in two forms, single cells and aggregates, and germ cells, also individually and in association with Sertoli cell aggregates. Since individual cells in aggregates are very difficult to count accurately, we seeded cells in culture dishes not as number of cells per dish, but rather as a ratio of Media Results toli Figure cells 1A is a light which were micrograph embedded into thick sections parallel The purity of Sertoli cells nique is readily apparent. this light micrograph type is present. Sertoli presence of irregularly of cultured Serin plastic and cut to the plane of growth. obtained by this techCareful inspection of illustrates that only cells are identified shaped nuclei, one cell by the con- each No. SERTOLI 3 taming only power electron ture. The homogenous one nucleolus. the Leydig cells micrographs, identity periments these constitute less of our and myoid although two than synthesis of time different of a low culan estab- types have 1% of total been cells (3H)estrone in ex- shown to in vitro. and and FSH, examined 48 hours, 0.5 levels g/ml and and 1.0 obtained FSH more effect was given time estrogen of by by The in media from harvested at two concentration required greater than before aromatase lated. a FSH pg/ml. after At level 24 hours dose of estrogen treated indicated control did The only small precursor. doubling appear quantities of the to further At time either of incubation stimulate estrogen syn- thesis. These data duced cells. were were suggested being the gens, whereas (3H)estradiol. gens accumulated FSH stimulation, those 3. As before, negligible FSH-stimulated measurable Sertoli Sertoli cells in the presence control levels cells amounts of synthesized (3H)estrone and Further, newly synthesized estroin the medium with time after reaching levels much higher obtained in the absence of an estrogen is shown described cells were Methods, cultured as no exoge- to the media, for different and cells time peri- nous were in Figure 4. Sertoli in Materials and estrogens stimulated were added with FSH ods as indicated LH-treated cells cant levels of in Figure synthesized estrogens aromatization to estrogen 4. Neither statistically when control compared of nor signifiwith FSH obof measured dishes were stimulation. 0.01 g/ml activity A of FSH was was stimu- than ase, was reversed. that a purer obtained terpretation 0.5 g/ml, the slightly. Synthesis Morphologic in et al, 1978; 1979). to constant specific radio further activity by comparing methods: 1. the using silica recrystallization conversion of tein in our experiments, methodologies, others (Dorrington is cells de1979; recently have emfrom the et al, 1980) (3H)estro- activity (Ritz#{233}n et the Sertoli simultaneously two conversion of to (3H)estrone 19-(6,7and gel thin of the dif- layer chromatoproducts, and 2. 19-hydroxyandros(El + E2) using RIA. produced per mg prousing similar and Sertoli in- initially Armstrong, demonstrates in immature unlabeled tenedione to total estrogen The amount of estrogen is the et al, data (Gore-Langton of synthesized Our report of aromatase in vitro alternative methods loss of hydrogen of testosterone during 3H)hydroxyandrostenedione the by was and Verhoeven These aromatization reaction and by recrystallization (3H)estradiol graphy and vitro immature animals RIA (Dorrington in indicated cells of et al, in vitro collagen- thus facilitating studies. Rommerts al, 1981). presence 1975) and studies of Sertoli estrogens cell pub- Fritz, trypsin this method, of biochemical from by Damme Sertoli a previously and sequence, been corroborated by ploying the stereospecific the lB and 2B positions gens to obtain from population by obtained termined used (Dorrington enzyme 1975; have procedure ferent the of as decreased slightly the cells trap. Measurement by RIA of unlabeled 19-hydroxyandrostenedione we varied that van and LHof estro- method lished pro- by consisting of cold 1 M estrone The results of this experiment are shown in Figure treated cells produced easily estrogens further To examine this hypothesis, incubated with (3H)substrate of an estrogen trap and 1 M estradiol. than that metabolized were Discussion LHcultures of FSH, not and of es- produced 3H)hydroxyandrostenedione dose cells On the other hand, FSH-stimulated Serdid synthesize substantial amounts of and (3H)estradiol from the 19-(6,7- cells trogens. toli cells (3H)estrone that culture higher a of total of FSH-stim- cells all produced of accumudose and methods methodology. response Sertoli an levels cells different tracer a dose In our before values FSH-treated two distinctly RIA and direct potential. results maximal at two Untreated control cells, in the presence of precursor alone, as well as LH-treated (5 jg/ml) cells, were examined with respect to their aromatization Our once similar aromatization RIA. elapsed stimulation, 5 illustrates ulated cells. 12 hours took place, no further was noted. For a given synthesis Figure by 2. FSH-stimulated than observed; tained using measurement, 205 et al with experiment, (3H)estradiol to FSH, as measured is shown in Figure was Sudrez-Quian estrogen synthesis lation with time Occasion- cells are observed from numerous cell 24 hours doses cells. SYNTHESIS in exhibiting further cultured of aromatization intervals, is cells nuclei, pattern, by Sertoli cells in response direct tracer methodology, Stimulation lB of Sertoli irregularly shaped euchromatin ally, light ESTROGEN Figure micrograph lish The CELL to Armstrong, either that of reported 1975; the two by Rom- 206 Fig. Thick shaped Original Typical peripheral containing Journal 1. A. Light micrograph section was cut parallel nuclei, each containing magnification x820. of Andrology #{149} May/June of rat Sertoli cell monolayer after three days in to the plane of growth. Asterisk (*) indicates area only one nucleolus (n), establishes the identity B. Electren micmgraph of thin section (uranyl acetate Sertoli cell characteristics are observed: heterochromatin; 2. large lipochrome foliate cristae generally organized 1983 culture (embedded of culture dish not of these cells. Note a,,, Reynold’s stain) Vol. 4 in plastic, toluidine blue stain). invaded by Sertoli cells. Irregular that only one cell type is present. cut parallel to the plane of grewth. 1. irregularly shaped nuclei with abundance of euchromatin, pigment granules (L); 3. small, round lipid granules (1); and transversely (m). Original magnification x6000. lacking 4. slender large clumps mitochondria of No. SERTOLI 3 CELL ESTROGEN SYNTHESIS Sudrez-Quian 207 et al 5, I 0 Ii. \ F 0, 24 48 24 48 24 48 24 48 hours TIME Fig. 2. Measurement of aromatase activity of immature rat Sertoli cells by direct tracer methodology using 19-(6,7-3H)hydroxyandrostenedione as substrate. Cells were cultured for two days prior to gonadotropin treatment. Media were harvested and assayed at times indicated in figure. (Values reported are mean ± SE; n = 4). merts Damme et al, et 1978; Verhoeven al, 1979; Gore-Langton Ritz#{233}net al, dione, trone ments. 1981). and The substrate as further in the produced by of estrone and experiments metabolism has of been 1979; et al, obtained direct RIA Fig. 4. Aromatase by RIA. Unlabeled concentration, was van 1980; is probably our cross-reaction our direct estrogens an by previously added to immature Media were (Each column and Armstrong, harvested least a es- with tracer 12 stimulation by FSH (1 gJml) as measured 19-hydroxyandrostenedione, 0.5 m final used as substrate. FSH and substrate were rat Sertoli cells after two days in culture. and assayed at times indicated in figure. represents mean 1975) planned. In a time also since suggest observed (3H)es- tracer experifrom the same estradiol, gives equal In addition, methodology This we measured antisera estrogens. al, 24 TIME 19-hydroxyandrostene- substrate, (3H)estradiol estrogen combination trogen both Using a 17-keto et 12 course hours ± SE; and of estrogens were to FSH stimulation. has reported estrogen synthesis 48 24 48 24 48 immature rats beled substrate ported are mean were cultured in the presence ± SE; n = Sertoli of cold seen in the A similar for (Armstrong of at measurable media in lag phase FSH-dependent et al, 1975; Dor- cells. (Dorrington 24 estrogens. (Values Injq/mI Fig. 5. Dose response of aromatase stimulation by FSH during a 24-hour period as measured by RIA. Sertoli cells from immature rats were cultured for two days before addition of FSH 48 hours activity measured in the presence 1 j.m; E2, 1 j.m). Sertoli cells from for two days prior to adding la4). a lag are apparent TIME Fig. 3. Result of aromatase of a cold estrogen trap (El, 4), before previously FSH 24 studies (Fig. observed amounts response been additional experiment was n = 4). re- and unlabeled nadotropins. substrate. Line drawn Control between 0.5 g/ml FSH represents a best tween 0.5 gJml and 1.0 jg/ml response was observed at P < mean ± SE; n = 4). cultures dosages did not of 0.01 fit. Downward FSH 0.05. line receive g/ml drawn indicates that a different (Each point constitutes goand bea 208 of Andrology Journal rington et al, binding 1978) protein al, 1979). As the presence and by for secretion Sertoli cells of androgen in vitro the stimulation of aromatase to FSH (Dorrington et al, tema (Welsh suggested by others, these of numerous intracellular et data imply events for activity in 1976; Dorrington response et al, 1978). Moreover, the served (Fig. stimulation 1978). 48 hours previously and Armstrong, 1975; Rommerts In our experiments, of FSH others stimulation, (Rommerts Based in contrast al, 1978; our results not the with tures, strong and the evidence reports that mentioned estrogens Sertoli cells of immature animals. of al, tracer may antibody. cell cul- The response immature vivo to in vivo animals. the ability testosterone tive importance synthesis in the examined. zation in though (dejong of adult data paper has tubules that FSH reported of adult adult stimulated regard, aromatization hCG stimulation whereas intact been crease ick et Payne, estrogen synthesis in both al, 1979) and mature rats 1979) and in men (Payne recent paper, hCG aromatization immature or mature 1981). It is important also in purified rats to note lost during strong and maturation Dorrington, Sertoli originate cells has from (Valladares aromatase the In shown Leydig of the 1977). significantly testes. observed this to in- immature (Can(Valladares and et al, 1976). In a been that did or hypophy- hCG in has aromatirats, al- in vivo in intact rats, in- significant same paper administered aromatization of in also to estrogen must be re- the data are not statistically et al, 1974). In addition, the stimulate in Sertoli cells to aro1979). The rela- the Sertoli cell testis, however, sectomized late results by Pomerantz of immature (Pomerantz, Only one seminiferous presented not the conducted Sertoli Since same Moreover, seminiferous derived canine cryptorchid to stimucells and from gonadal has from been synthesis in adult reported only in cells of transformed testes Sertoli (Huggins and et al, 1949). In this case, Sertoli cells dedifferentiate the type, immature regaining ex- activity in as a vestig- 1945; Berthrong ceivable that the Moulder, it is conback to ability to aroma- tize. role ing Consequently, in promoting Leydig cell dejong et al, if Sertoli cell estrogens play a spermatogenesis or in modulatfunction (Dorrington et al, 1978; 1974; it is likely animals. for in estrogen these If there Leydig it appears by Armstrong that in immature the that Leydig cell such cell and effects Dorrington, take place only is a physiologic function in estrogen role the must itself, or adult be syn- is of serum origin. blas- Ritz#{233}nEM, matogenesis. Nayfeh New SN, eds. Hormonal York: Plenum Press, JH. Synthesis stimulation of by FS, Hansson V, regulation of sper1975; 85-96. Armstrong DT, Dorrington JH. Estrogen biosynthesis in the ovaries and testes. In: Thomas JA, Singhal RL, eds. Regulatory mechanisms affecting gonadal hormone action. Baltimore: Berthrong M, University Goodwin Park WE, Press, Scott J Clin Endocnnol by the testis. Brinkley BR, Murphy P, Richardson 1977; WW. 217-258. Estrogen 1949; production 9:579-592. LC. Procedure for embedding in situ selected cells cultured in vitro. J Cell Biol 1967; 35:279-283. Canick JA, Ryan KJ. Cytochrome P-450 and the aromatization of 16-hydroxytestosterone and androstenedione by human placental microsomes. Mo! Cell Endocrinol 1976; 6:105115. Canick JA, Makris A, Gunsalus GL, Ryan KJ. Testicular aromatization in immature rats: localization and stimulation after gonadotropin administration in vivo. Endocrinology 1979; 104:285-288. Canick JA, Brumbaugh JM, Makris A, Ryan KJ. The effect of hCG, FSH and PMSG on testicular aromatization in immature Wistar rats. Biol Reprod 1980; 22:77a. dejong FH, Hey AH, van der Molen HJ. Oestradiol-1713 and testosterone in rat testis tissue: effect of gonadotropins, loJ Endocrinol 1974; calization and production in vitro. 60:409-419. Dorrington JH, Armstrong DT. Follicle-stimulating hormone Proc Dorrington is (Armand Armstrong DT, Moon YS, Fritz lB. Dorrington, estradiol-1713 by Sertoli cells in culture: FSH and dibutyryl cAMP. In: French stimulates Payne, activity cell Leydig a speculative estrogen tubules tumors 1980), 4 References of FSH Furthermore, experiments dicated matize administration Dym, presence of aromatase cells is that it remains experiments of Canick et al (1980) are important in this respect, as they noticed a small, yet measurable aromatization ial function. thesized above, provide are produced by and for the Sertoli animal, in Sertoli Vol. (Pelliniemi 1977). suggest 1983 planation immature did to a metabolite our of aromatization al, time direct data of estrogen cross-react et increasing with studies FSH (Dor- with reports Verhoeven et our metabolism obof estrogens with experiments, does Our media et on further that however, in the methodology the synthesis the first described accumulate 1979). of estrogen during been rington not pattern 4) has #{149} May/June and erous 889. Dorrington ulates estradiol-17/3 Nat synthesis Acad Sci USA 1975; JH, Fritz lB. Cellular 3/3-hydroxysteroid tubule of the rat in cultured 72:2677-2681. localization dehydrogenase testis. Endocrinology JH, Fritz lB. Armstrong estradiol-17/3 biosynthesis Sertoli cells. of 5a-reductase in the 1975; seminif96:879- DT. Site at which FSH regin Sertoli cell prepara- No. 3 SERTOLI tions Dorrington trogen Gore-Langton in CELL ESTROGEN SYNTHESIS by Res 1945; Sertoli 1979; Teilum with interstitial cell tumor of the testicle. J Urol 1939; 42:1242-1250. Karnovsky MJ. Use of ferrocyanide-reduced osmium tetroxide J Cell Biol 1971; Abstract 284. in electron microscopy. Leach RB, Maddock WO, Tokuyama I, Paulsen CA, with the Folin phenol J reagent. Biol Valladares LE, aromatization Nelson Chem G. by luteinizing hormone 1979; 105:431-436. WO. Clinical studies of testicular hormone production. Recent Prog Horm Res 1956; 12:377-403. Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement van 1951; AH, Kelch RP, Musich SS, Halpern ME. Intratesticular 1983 The 1983 Testis Workshop mature rats. Biol MP, Robertson Reprod Acta mature rats. 25:752-758. 1981; also poster Catt, For be a section sessions. Richard further Kevin on new The Sherins, de Moor P. Stimulation effect of the aromatization of testosterone by cultures. Mol Cell Endocrinol 1979; 13:241-253. Welsh MJ, Wiebe stimulation Welsh JP. Sertoli cells of steroid Commun 1976; from immature metabolism. Biochem Raven Press, 1979; information, Catt, J. Endocrinology National 8C-407, National Bethesda, Telephone please “Hormonal Regulation Building Institutes in reproduction committee Dufau. of Tes- research, members are contact: M.D. and Institute Reproduction of Child 10 of Health MD 20205 (301) 496-2136 Research Health Res 89-98. of oestrogenic hormone 1934; 133:209-210. Workshop topic in vitro MJ, Van Sickle M, Means AR. Possible involvement of cyclic AMP, calcium and cytoskeleton in control of protein secretion by Sertoli cells. In: Steinberger A., Steinberger E, eds. Testicular development, structure, and function. New York: the rats: Biophys 69:936-941. Zondek B. Mass excretion of the stallion. Nature techniques local organizing and Maria Diczfamethod activ- P, on ticular Function” will be held at the National Institutes of Health on October 14-17, 1983. An introductory lecture on the evening of Friday, October 14th will be followed by 2-1/2 days of sessions on the following topics: peptide and steroid hormone action, sperm motility; and immunological, developmental and clinical aspects of testis function. There will Endocrinology OM, Marana R, Ritz#{233}nEM, and specific in vitro bioassay of follicle-stimulating hormone 1979; 9:224-237. Endocrinol Verhoeven G, Dierickx neurotransmitters Sertoli cell-enriched Testis on in Damme ity. site of aromatization in the human. J Clin Endocrinol Metab 1976; 42:1081-1087. Pelliniemi LI, Dym M. The fetal gonad and sexual differentiation. In: Tulchinsky D, Ryan KJ, eds. Maternal-fetal endocrinology. Philadelphia: WB Saunders, 1980; 252-280. Pomerantz DK. Effects of in vivo gonadotropin treatment on estrogen levels in the testis of the immature rat. Biol Reprod 1979; 21:1247-1255. Ritz#{233}nEM, van Damme MP, Froysat B, Reuter C, de la Torre B, Diczfalusy E. Identification of estradiol produced by Sertoli cell enriched cultures during incubation with testosterone. J Steroid Biochem 1981; 14:533-535. Rommerts FFG, Kruger-Sewnarain B Ch, van Woerkom-Blik A, Grootegoed JA, van der Molen HJ. Secretion of proteins by Sertoli cell enriched cultures: effects of follicle stimulating in AH. Effects of hCG and cyclic AMP on purified Leydig cells of immature and Payne lusy E. A sensitive for the measurement 193:265-275. Payne 33:495-526. Estrogen-producing Sertoli cell tumors (androblastoma tubulare lipoides) of the human testis and ovary: homologous ovarian and testicular tumors. J Clin Endocrinol 1949; 9:301-318. Valladares LE, Payne AH. Induction of testicular aromatization cell 5:510-514. associated 209 dibutyryl cAMP and testosterone and correlation with secretion of oestradiol and androgen binding protein. Mol Cel Endocrinol 1978; 10:39-55. Tcholakian RK, Steinberger A. In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH. Steroids es- method for the study of follicle-stimulating hormone effects on aromatase activity in Sertoli cell cultures. Endocrinology 1980; 107:464-471. production et al hormone, culture. Mol Cell Endocrinol 1976; 6:117-122. JH, Fritz lB. Armstrong DT. Control of testicular synthesis. Biol Reprod 1978; 18:55-64. R, McKeracher H, Dorrington J. An alternative Huggins C, Moulder PV. Estrogen tumors of the testes. Cancer Hunt VC, Budd JW. Gynecomastia Sudrez-Quian and Branch Human Development and Drs. two Kevin in the urine