Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

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1 Brzilin Journl of Medicl nd Biologicl Reserch (1997) 30: FSH nd insulin in lipogenesis in Sertoli cells ISSN X 591 Effect of FSH nd insulin on lipogenesis in cultures of Sertoli cells from immture rts F.C.R. Gum 1, M. Wgner 2, L.H. Mrtini 1 nd E.A. Bernrd 1 1 Deprtmento de Bioquímic nd 2 CPG Fisiologi, Instituto de Biociêncis, Universidde Federl do Rio Grnde do Sul, Porto Alegre, RS, Brsil Abstrct Correspondence F.C.R. Gum Deprtmento de Bioquímic IB, UFRGS Ru Srmento Leite, 500/ Porto Alegre, RS Brsil Fx: 55 (051) E-mil: fgum@vortex.ufrgs.br Reserch supported by FINEP, CNPq, nd FAPERGS. Received June 27, 1996 Accepted Jnury 29, 1997 Follicle-stimulting hormone (FSH) nd insulin regulte glycide metbolism in Sertoli cells, thus stimulting lctte production. These stimultory effects of FSH nd insulin do not require protein synthesis, suggesting modultion of enzyme ctivity nd/or regultion of glucose trnsport. The present investigtion ws performed to chrcterize the hormonl control of lipid metbolism in Sertoli cells. The dt indicte tht FSH nd insulin hve regultory effect on lipid metbolism in Sertoli cells. After 8 h of preincubtion with insulin (5 µg/ml), the ctivity of the enzyme ATP-citrte lyse in cultured Sertoli cells ws incresed from 0.19 to 0.34 nmol NAD + formed µg protein -1 min -1. FSH (100 ng/ml) hd no effect on this enzyme. Glycerol phosphte dehydrogense ctivity ws not ffected by ny of the hormones tested. When Sertoli cells from 19-dy old rts were incubted with [1,2-14 C]cette for 90 or 360 min, the [ 14 C] lbel ws present predominntly in triglyceride nd phospholipid frctions with minor mounts in other lipids. In Sertoli cells pretreted for 16 h with insulin nd FSH, n increse in cette incorportion into lipids ws observed. Most of the lbel ws in esterified lipids nd this percentge incresed with the time of tretment; this increse ws remrkble in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides prticipte in the control of spermtogenesis, the present dt suggest tht the hormonl control of lipid metbolism in Sertoli cells is importnt not only for mintining the energy of the cell itself, but lso for the control of the spermtogenesis process. Key words Sertoli cells Lipids FSH Insulin Introduction Testiculr lipids hve n ctive metbolism, nd re formed both from dietry sources nd s the result of processes of synthesis, elongtion, desturtion, interconversion, esterifiction nd oxidtion by testiculr tissue. Testiculr tissue hs the full complement of cytosolic enzymes required for de novo synthesis of ftty cids. Vrious fctors hve been shown to lter the lipid composition of the testis. Testiculr lipids generlly increse in concentrtion with development. Nutritionl fctors such s innition, vitmin deficiency, essentil ftty cid deficiency nd certin minerl deficiencies ffect the testiculr lipid composition (1). Tretment with thyroid hormone, prolctin or progesterone cuses decrese in Brz J Med Biol Res 30(5) 1997

2 592 F.C.R. Gum et l. rt testiculr lipids. Both protein nd vitmin A deficiencies in 21-dy old rts resulted in decresed testis weight long with decresed mounts of phospholipid frction contining phosphtidylserine nd inositol (1). The metbolism of [ 14 C]plmitic cid in primry cultures of rt Sertoli cells isolted from testes of 19-dy old rts hs been reported (2). The oxidtion of plmitte ws concentrtion dependent. About 65% of plmitte oxidtion over 5-h period ws recovered s crbon dioxide. Almost ll rdioctive soluble compounds secreted by Sertoli cells were either ß-OH butyrte or cetocette. The ddition of glucose hd no effect on plmitte oxidtion but cused 9- fold increse in esterifiction into tricylglycerols. The uthors concluded tht ftty cids pper to be mjor energy substrte for Sertoli cells (2). The metbolism of olete in isolted rt testiculr cells ws investigted by Yount nd Hrris (3). The rtes of oxidtion of olete to crbon dioxide were higher in cells preprred from immture rts thn in cells from dults. In cells from young but not from dult rts, olete inhibited the oxidtion of glucose to crbon dioxide. The uthors suggested tht lipids my be n importnt source of energy for the prepubertl rt testis, but the mjor source of energy for the dult rt testis ppered to be glucose. The metbolism of linoleic, rchidonic nd 22:5n-6 cids in Sertoli cells nd germinl cells ws compred by Beckmn nd Coniglio (4). Following n intrperitonel injection of lbeled 22:5n-6 ftty cids greter proportion of the recovered [ 14 C] in Sertoli cells, rther thn in germinl cells, ws present in 20-crbon ftty cids, indicting tht the metbolism of the pentene ws more ctive in Sertoli cells thn in germinl cells. In both cell types more of the recovered [ 14 C] ws in tricylglycerols during the erly periods nd in phospholipids fter 24 h, suggesting the possibility of the trnsfer of the biosynthesized pentene from Sertoli to germinl cells using tricylglycerols s trnsfer vehicle (5,6). The metbolism of Sertoli cells is under the complex control of hormones, growth fctors nd even prcrine, utocrine nd juxtcrine rections occurring between the vrious cell types in the testis. The hormonl control of glycide metbolism in Sertoli cells is well known. In immture rts, folliclestimulting hormone (FSH) regultes the production of lctte, preferentil energy substrte for pchytene spermtocytes nd round spermtids (7,8). Oonk et l. (9) showed tht insulin increses the rte of lctte production in vitro. This effect is obtined with low insulin concentrtions nd is medited by insulin-specific receptors existing on Sertoli cells (10). In comprtive study, Oonk et l. (9) showed tht the stimultory effects of FSH nd insulin on lctte production do not require the synthesis of new proteins, suggesting modultion t the level of the ctivity of enzymes involved in crbohydrte metbolism nd/or in glucose trnsport regultion (9,11). In view of the importnce of lipids in spermtogenesis nd the role of Sertoli cells in the seminiferous epithelium, the objective of the present study ws to investigte how the hormones FSH nd insulin ct on lipid metbolism in cultures of Sertoli cells from immture rts. Mteril nd Methods FSH (ovine, NIADDK-oFSH-17, specific ctivity 20 U/mg) ws gift from NIH, Bethesd, Mrylnd. Insulin (bovine pncres) ws purchsed from Sigm Chemicl Co. nd [1,2-14 C]cette (specific ctivity 54.7 mci/ mmol) ws from New Englnd Nucler (Wilmington, DE). All other chemicls were regent grde. Culture of Sertoli cells Sertoli cells were prepred from 19-dy old Wistr rts ccording to the procedure of Brz J Med Biol Res 30(5) 1997

3 FSH nd insulin in lipogenesis in Sertoli cells 593 Dorrington nd Fritz (12). A smll percentge (3-4%) of contminting peritubulr cells, indicted by the histochemicl demonstrtion of lkline phosphtse ctivity (13), ws present in these Sertoli cell preprtions. Cells were plted (2.4 x 10 5 cells/cm 2, 6 x 10 6 cells/25 cm 2 culture flsk) onto 199 medium supplemented with 1% fetl clf serum nd knmycin (100 mg/l) under n tmosphere of 5% CO 2 in ir t 34 o C. After 24 h the cellulr monolyer ws wshed nd fresh medium ws dded. On the 3rd dy fter plting, Sertoli cell cultures were incubted in the presence or bsence of FSH (100 ng/ml) or insulin (5 µg/ml). Cell lbeling nd lipid extrction After the exposure of the cells to hormones the medium ws chnged nd the incubtions were continued for min in the presence of [1,2-14 C]cette (1 µci/ ml). At the end of the incubtion the cells were wshed with PBS, scrped in PBS nd sonicted (2 x 30 s, 40 ma). An liquot ws used for protein determintion (14) nd the rest for lipid extrction ccording to the procedure of Folch et l. (15). An liquot of the extrct ws counted for rdioctivity. The rest ws pplied to silic gel pltes nd chromtogrphed in hexne:diethyl ether: cetic cid (80:20:2, by volume). After development, the pltes were subjected to utordiogrphy (X-OMAT, Kodk) for locliztion of rdiolbeled res. The lipid bnds were scrped directly from the pltes into liquid scintilltion vils nd counted. The stndrd lipids were locted by exposing the pltes to iodine vpor. Enzyme ssy Sertoli cells were incubted for 8 or 24 h with FSH (100 ng/ml) or insulin (5 µg/ml) for the determintion of ATP-citrte lyse ctivity by the method of Mckll et l. (16). Briefly, fter hormonl tretment the cells were collected nd sonicted (2 x 30 s, 40 ma) in 20 mm Tris-HCl buffer, ph 8.0, contining 1 mm EDTA, 1 mm dithiothreitol nd 0.25 mm sucrose. Enzyme ctivity ws determined in the 20,000 g superntnt solution. The incubtion system contined potssium citrte, NADH nd mlte dehydrogense. ATP-citrte lyse ctivity ws determined by coupling with the mlte dehydrogense rection. Gycerol phosphte dehydrogense ctivity ws determined by the method of Wise nd Green (17). As lso done for ATP-citrte lyse, the cells were collected nd sonicted fter hormonl tretment using 50 mm Tris-HCl buffer, ph 7.5, contining 1 mm EDTA nd 1 mm mercptoethnol. The ctivity ws lso determined in the 20,000 g superntnt solution using dihydroxycetone phosphte nd NADH s substrtes. An liquot of ech superntnt ws used for protein determintion (14). Sttisticl nlysis Differences mong the experimentl groups were nlyzed by two-wy nlysis of vrince. Mens were compred by the Newmn-Keuls test. Results Determintion of the effect of FSH nd insulin on ATP-citrte lyse nd glycerol phosphte dehydrogense ctivity As the first pproch to the study of lipid metbolism in Sertoli cells, we determined the effect of FSH nd insulin on two enzymes tht ct on lipid synthesis. The results listed in Tble 1 show tht ATP-citrte lyse ws stimulted when the cells were treted for 8 h with insulin. A more prolonged tretment (24 h) with insulin did not lter the ctivity of the enzyme, but FSH hd no effect t ny of the times tested. Glycerol phosphte dehydrogense ctivity ws not modified by either hormone. ATP-citrte lyse Brz J Med Biol Res 30(5) 1997

4 594 F.C.R. Gum et l. Tble 1 - Effect of FSH nd insulin on ATP-citrte lyse nd glycerol phosphte dehydrogense specific ctivity of Sertoli cells. Dt re reported s specific ctivity (nmol NAD + formed µg protein -1 min -1 ). Sertoli cells from 19-dy old rts were treted with medium contining FSH (100 ng/ml) or insulin (5 µg/ml) for 8 nd 24 h. The cultures used in the 8- nd 24-h experiments were from different preprtions. Enzyme ctivity ws mesured for 15 min t 37 o C. Dt re reported s the men ± SEM of 6 determintions. *P<0.05 compred to control nd FSH (Newmn-Keuls test). ATP-citrte lyse Glycerol phosphte dehydrogense 8 h 24 h 8 h 24 h Control 0.19 ± ± ± ± 0.04 FSH 0.23 ± ± ± ± 0.04 Insulin 0.34 ± 0.01* 0.34 ± 0.01* 1.33 ± ± 0.05 Tble 2 - Effect of FSH nd insulin on [1,2-14 C] cette incorportion into Sertoli cell lipids. Sertoli cells were pretreted for 16 h with 100 ng/ ml FSH or 5 µg/ml insulin nd incubted for 90 min with 1 µci/ml nd for 360 min with 0.5 µci/ml [1,2-14 C]cette. Dt re reported s the men ± SEM of 6 determintions. *P<0.05 compred to control; + P<0.05 compred to control nd FSH (Newmn-Keuls test). [1,2-14 C]cette (cpm/µg protein) 90 min 360 min Control 23.2 ± ± 3.4 FSH 26.7 ± 1.3* 77.1 ± 6.5* Insulin 28.3 ± 0.9* 52.8 ± ctivity in Sertoli cell cultures ws very low (bout 0.20 nmol NAD + formed µg protein -1 min -1 ), i.e., pproximtely 10 times lower thn tht demonstrted in the epididymis of rts of the sme ge (2.3 ± 0.05 nmol NAD + formed µg protein -1 min -1 ). Activity levels s low s those reported here for Sertoli cells hve been reported by Brown et l. (18) in the rt testis. [ 14 C]cette incorportion The next step ws to determine the kinetics of rdioctive cette incorportion into lipids nd to investigte the effects of FSH nd insulin on this incorportion. Tble 2 shows tht both FSH nd insulin significntly stimulted cette incorportion into Sertoli cells nd tht this effect ws relted to time of incubtion. We then determined the distribution of the rdioctive lbel mong the different lipid clsses using two prmeters, i.e., time nd hormone effect. Tble 3 compres two different times of incubtion with cette in ech experimentl sitution, nd Figure 1 presents comprison of the ction of the two hormones. Anlysis of Tble 3 shows tht, s erly s fter 90 min of incubtion with cette, pproximtely 70% of the lbel ws distributed mong triglycerides (TG) nd phospholipids (PL), nd the remining 30% ws distributed mong free ftty cids (FA), monoglycerides (MG), diglycerides (DG) nd cholesterol (C). In 360-min incubtions, the sme rdioctive compounds ppered with slight percent ltertions, showing possible migrtion of the rdioctive lbel from FA, MG nd DG to TG. Incorportion into cholesterol ws lwys very low nd decresed with time, nd the incorportion into cholesterol ester ws negligible. Action of FSH nd insulin on [ 14 C]cette incorportion Besides determining how the distribution of rdioctivity incorported into lipids is ffected by time of incubtion with [ 14 C] cette, we compred the ction of FSH nd insulin. The cells were preincubted with FSH or insulin for 16 h nd incubted with cette for the sme times s described bove. Anlysis of Figure 1A nd 1B shows tht tretment with the hormones did not chnge the distribution profile of rdioctivity described bove for the controls (Tble 3). In cells treted with insulin, fter 90 to 360 min of incubtion with the precursor, the trnsfer of the rdioctive lbel from FA, MG nd DG to TG ws similr to tht observed in the Brz J Med Biol Res 30(5) 1997

5 FSH nd insulin in lipogenesis in Sertoli cells 595 Tble 3 - Effect of time of incubtion with [1,2-14 C]cette (90 or 360 min) on Sertoli cells in culture pretreted with FSH or insulin for 16 h nd on control cells. The results re reported s percent totl incorportion of rdioctivity into lipids in Sertoli cell cultures. Sertoli cells were pretreted for 16 h with FSH or insulin nd incubted for 90 min with 1 µci/ml nd for 360 min with 0.5 µci/ml [1,2-14 C]cette. The lipid extrct ws chromtogrphed nd the rdioctivity in lipid bnds counted. Dt re reported s the men ± SEM of 6 determintions. *P<0.05 compred to 90-min incubtion time (Newmn-Keuls test). Control FSH Insulin 90 min 360 min 90 min 360 min 90 min 360 min Phospholipds 50.8 ± ± ± ± ± ± 1.4* Cholesterol 3.8 ± ± 0.1* 1.6 ± ± 0.01* 2.9 ± ± 0.01 Ftty cids 10.3 ± ± 0.1* 5.4 ± ± 0.01* 5.3 ± ± 0.01* Monoglycerides 4.2 ± ± 0.1* 5.6 ± ± 1.4* 2.8 ± ± 0.01* Diglycerides 8.2 ± ± 0.1* 10.7 ± ± 0.7* 12.9 ± ± 0.7* Triglycerides 18.8 ± ± 1.7* 24.6 ± ± 1.4* 24.9 ± ± 0.7* controls. Nevertheless, there ws significnt increse in TG lbeling fter FSH tretment. The two hormones lso provoked decrese in the mount of rdioctivity incorported into PL compred to the controls (Figure 1B). These effects re more clerly visulized in Tble 3 which shows the distribution of the rdioctive lbel mong cells treted with insulin nd FSH t the two times of incubtion with cette. FSH induced n increse in the trnsfer of rdioctivity only to TG, nd insulin hd the sme effect, lthough of lower mgnitude, on TG nd PL. FSH lso induced slight increse in the levels of cholesterol. Discussion The present dt show tht both FSH nd insulin significntly stimulted cette incorportion into Sertoli cells from prepubertl rts nd tht this effect ws relted to time of incubtion (Tble 2). The effect of insulin on lipid synthesis ws obtined with concentrtions bove physiologicl rnge (the norml insulin concentrtion in peripherl circultion is 0.5 ng/ml (100 pm) in fsted nimls), nd my reflect binding of insulin to insulin receptors or to receptors for insulin-like growth fctor I (IGF-I). Borlnd et l. (19) lso reported tht micromolr concentrtions of insulin were required to obtin smll stimultory effect on lctte production, nd DNA nd protein synthesis by Sertoli cells from 2-week old rts. Tble 3 shows tht fter 90 min of incubtion with [ 14 C]cette, 70% of the rdioctivity incorported into lipids ws simultneously present in TG nd PL. This percentge ws still higher fter 360 min of incubtion with the precursor (Tble 3). On the bsis of the comprison of the rte of cette incorportion with the low levels of ATPcitrte lyse ctivity (Tble 1) detected in Sertoli cells, we suggest tht these cells utilize cette for lipid synthesis without the involvement of mitochondri. This my indicte tht the testes tke up circulting cette in vivo nd utilize it directly vi cytoplsmic cetyl-coa synthetse for ftty cid synthesis. This is possible becuse of the considerble mounts of cette existing in rt blood, most of it produced by the intestinl microbil flor (20). Aveldño et l. (6) incubted seminiferous tubules from 6-week old rts with [ 14 C] cette nd observed tht, fter 1 h, rdioctivity ppered minly in TG nd PL. Between 1 to 4 h there ws decrese in the rdioctivity detected in TG (49 to 41%). Simi- Brz J Med Biol Res 30(5) 1997

6 596 F.C.R. Gum et l. % of Totl lipid incorportion % of Totl lipid incorportion b lr results obtined by Fisher nd Coniglio (21) in cultures of Sertoli cells from 35-dy old rts showed tht fter 24 h of incubtion with [ 14 C]cette most of the rdioctivity ws in esterified lipids (TG nd PL). The role of Sertoli cells in the seminiferous epithelium requires crefully controlled metbolism. Sertoli nd germ cells interct in environmentl, nutritionl nd regultory terms. Some investigtors hve shown tht glucose my not be energeticlly importnt for Sertoli cells. The mount of glucose oxidized to CO 2 is only smll frction of the metbolized glucose, while most of it is trnsformed into pyruvte nd lctte (22), which Control Insulin FSH PL C FA MG DG TG PL C FA MG DG TG Figure 1- Effects of FSH nd insulin on [1,2-14 C]cette incorportion into clsses of lipids of Sertoli cells. Sertoli cells were pretreted for 16 h with 100 ng/ml FSH or 5 µg/ml insulin nd incubted for 90 min with 1 µci/ml nd for 360 min with 0.5 µci/ml [1,2-14 C]cette. A, 90 min of cette tretment; B, 360 min of cette tretment. Dt re reported s the men ± SEM of 6 determintions. P<0.05 compred to control; b P<0.05 compred to control nd FSH; c P<0.05 compred to control nd insulin. (Newmn-Keuls test). PL, Phospholipids; C, cholesterol; FA, ftty cids; MG, monoglycerides; DG, diglycerides; TG, triglycerides. b b c A B re the preferred energy substrtes for spermtocytes nd spermtids (8). FSH stimultes the production of these compounds. Oonk et l. (9) showed tht fter 3 h of incubtion FSH or insulin stimultes lctte production in cultures of Sertoli cells. Jutte et l. (7) demonstrted tht Sertoli cells survive for s long s 24 h in glucose-free medium wheres germ cells degenerte. An indiction tht lipids my be more importnt energy substrte thn glucose for Sertoli cells ws provided by Jutte et l. (2) who showed tht Sertoli cells oxidize plmitte in concentrtion-dependent mnner nd tht the ddition of glucose to the incubtion system does not chnge plmitte oxidtion but cuses 9-fold increse in plmitte esterifiction to TG, probbly due to n increse in glycerol-3-phosphte levels. Fisher nd Coniglio (21) demonstrted tht the mount of rdioctivity in the TG of Sertoli cells ws constnt between 24 nd 48 h but ws decresed in PL, suggesting higher FA turnover in PL thn in TG. In the present study we showed tht high incorportion of rdioctivity into complex lipids (TG nd PL) occurred in ll experimentl situtions with incresing time of incubtion with cette (Tble 1). Figure 1 shows tht percent increse of cette incorportion into PL occurred from 90 to 360 min while the incresed incorportion into TG ws proportionlly higher only in control cells. Morphologic studies of Sertoli cells hve shown tht the mount of ft droplets contining TG chnges ccording to the different stges of the spermtogenesis cycle nd with different hormonl regimens in vivo (23). Aveldño et l. (6) reported tht the triglycerides of Sertoli cells hve high level of polyunsturted long-chin ftty cids, nd Beckmn nd Coniglio (4) demonstrted tht Sertoli cells re more ctive thn germ cells in the synthesis of these compounds. These uthors lso proposed tht these polyunsturted cids my be trnsferred to germ cells nd tht TG my ct s trnsfer vehicle. Brz J Med Biol Res 30(5) 1997

7 FSH nd insulin in lipogenesis in Sertoli cells 597 As erly s 1969, Collins nd Lcy (23) reported tht the mount of lipid droplets in Sertoli cells incresed in the presence of dmge to the seminiferous epithelium becuse the germ cells no longer consumed lipids s they usully did. These fcts indicte tht, in ddition to hving function s n energy substrte, the TG of Sertoli cells my be involved in the mintennce of spermtogenesis. This hypothesis is supported by the results of Beckmn nd Coniglio (4) who demonstrted tht polyunsturted ftty cid deficiency provokes the interruption of spermtogenesis. Our results showing tht FSH nd insulin stimulte the process of lipid esterifiction in Sertoli cells permit us to stte tht the two hormones modulte lipid metbolism in Sertoli cells. On the bsis of our results nd of those reported by others, we suggest tht the control of lipid metbolism in Sertoli cells is importnt not only for mintining the energy of the cell itself, but lso for the control of the spermtogenesis process. Acknowledgment We re grteful to NIH for the generous gift of FSH. References 1. Coniglio JG (1994). Testiculr lipids. Progress in Lipid Reserch, 33: Jutte NHPM, Eikvr L, Levy FO & Hnsson V (1985). Metbolism of plmitte in cultured rt Sertoli cells. Journl of Reproduction nd Fertility, 73: Yount EA & Hrris RA (1982). Ketone body nd cette formtion from olete by isolted rt testiculr cells. Archives of Biochemistry nd Biophysics, 217: Beckmn JK & Coniglio JG (1980). The metbolism of polyunsturted ftty cids in rt Sertoli nd germinl cells. Lipids, 15: Beckmn JK & Coniglio JG (1979). A comprtive study of the lipid composition of isolted rt Sertoli nd germinl cells. Lipids, 14: Aveldño MI, Robinson BS, Johnson DW & Poulos A (1993). Long nd very long chin polyunsturted ftty cids of the n- 6 series in rt seminiferous tubules. Journl of Biologicl Chemistry, 268: Jutte NHPM, Jnsen R, Grootegoed JA, Rommerts FFG & vn der Molen HJ (1982). Regultion of survivl of rt pchytene spermtocytes by lctte supply from Sertoli cells. Journl of Reproduction nd Fertility, 65: Jutte NHPM, Jnsen R, Grootegoed JA, Rommerts FFG & vn der Molen HJ (1983). FSH stimultion of the production of pyruvte nd lctte cells my be involved in hormonl regultion of spermtogenesis. Journl of Reproduction nd Fertility, 68: Oonk RB, Grootegoed JA & vn der Molen HJ (1985). Comprison of the effects of insulin nd follitropin on glucose metbolism by Sertoli cells from immture rts. Moleculr nd Cellulr Endocrinology, 42: Oonk RB & Grootegoed JA (1987). Identifiction of insulin receptors on rt Sertoli cells. Moleculr nd Cellulr Endocrinology, 49: Hll PF & Mit M (1984). Influence of follicle-stimulting hormone on glucose trnsport by cultured Sertoli cells. Biology of Reproduction, 31: Dorrington JH & Fritz MR (1975). Cellulr locliztion of 5α-reductse nd 3α-hydroxy-steroid dehydrogense in the seminiferous tubule of the rt testes. Endocrinology, 96: Chpin RE, Phelps JL, Miller BE & Gry TJB (1987). Alkline phosphtse histochemistry discrimintes peritubulr cells in primry culture. Journl of Andrology, 8: Lowry OH, Rosebrough NJ, Frr AL & Rndll RJ (1951). Protein mesurement with the Folin phenol regent. Journl of Biologicl Chemistry, 193: Folch J, Lees M & Sloone-Stnley GH (1957). A simple method for the isoltion nd purifiction of totl lipids from niml tissue. Journl of Biologicl Chemistry, 226: Mckll JC, Student AK, Polkis SE & Lne D (1976). Induction of lipogenesis during differentition in predipocyte cell line. Journl of Biologicl Chemistry, 251: Wise LS & Green H (1979). Prticiption of one isozyme of cytosolic glycerophosphte dehydrogense in the dipose conversion of 3T3-cells. Journl of Biologicl Chemistry, 254: Brown J, McLen P & Greenbum AL (1966). Influence of thyroxine nd luteinizing hormone on some enzymes concerned with lipogenesis in dipose tissue, testis nd drenl glnd. Biochemicl Journl, 101: Borlnd K, Mit M, Oppenheimer CL, Blindermn LA, Mssgué J, Hll PF & Czech MP (1984). The ctions of insulinlike growth fctors I nd II on cultured Sertoli cells. Endocrinology, 114: Buckley BM & Willimson DH (1977). Origins of blood cette in the rt. Biochemicl Journl, 166: Fisher DM & Coniglio JG (1983). Composition of, nd [ 14 C] cette incorportion into, lipids of rt Sertoli cells in culture. Biochimic et Biophysic Act, 751: Robinson R & Fritz IB (1981). Metbolism of glucose by Sertoli cells in culture. Biology of Reproduction, 24: Collins P & Lcy D (1969). Studies on the structure nd function of mmmlin testis. II. Cytologicl nd histochemicl observtions on the testis of the rt fter single exposure to het pplied for different lengths of time. Proceedings of the Royl Society of London B, 172: Brz J Med Biol Res 30(5) 1997