Production by Hormones, Retinoids and the Testicular Paracrine Factor, PModS1

Transcription

1 BIOLOGY OF RPRODUTION 46, (1992) Developmentl Regultion of Sertoli ell Aromtse Activity nd Plsminogen Activtor Production by Hormones, Retinoids nd the Testiculr Prcrine Fctor, PModS1 MARINLLA ROSSLLI3 nd MIHAL K. SKINNR2 Reproductive ndocrinology enter,2 University of 4forni, Sn Frncisco Sn Frncisco, 4forni Deprtment of Phrmcology,3 Vnderbilt University School of Medicine, Nshville, Tennessee ABSTRAT Testiculr peritubulr cells produce prcrine fctor termed PMdS tht hs drmtic effects on Sertoli cell function in vitro. The current study ws designed to exmine the ctions of PModS nd hormones on Sertoli cell romtse ctivity nd plsminogen ctivtor production t vrious stges of pubertl development. Sertoli cells were isolted from 1-, 2-, nd 35. dy-old rts (ges correspond to prepubertl. midpubertl, nd lte-pubertl stges of development). Aromtse ctivity ws found to be high nd hormone-responsive in prepubertl Sertoli cells nd to decline nd be nonresponsive to hormones in lte-pubertl Sertoli cells. FSH ws the only hormone found to influence romtse ctivity nd estrogen production. PMdS lone ws not found to ffect romtse ctivity t ny of the developmentl stges exmined. Interestingly, PMOdS ws found to suppress the bility of FSH to stimulte romtse ctivity nd estrogen production in midpubertl Sertoli cells. Results imply tht PMdS my promote Sertoli cell differentition to more dult stge of development tht is less responsive to FSH in stimulting romtse ctivity. In contrst to romtse ctivity, plsminogen ctivtor production ws found to increse during pubertl development. Production of Sertoli cell tissue-type plsminogen ctivtor (tp) ws stimulted by FSH t ech of the developmentl stges exmined, wheres production of urokinse.type plsminogen ctivtor (up) ws influenced by FSH only in prepubertl Sertoli cells, Insulin lso stimulted up nd tp production by prepubertl Sertoli cells, nd retinol significntly suppressed up production nd the bility of FSH to stimulte tp production by midpubertl Sertoli cells. Purified PMdS ws found to hve no effect on plsminogen ctivtor production. Results imply tht PMOdS my not influence Sertoli cell functions tht pper in prt independent of cellulr differentition such s plsminogen ctivtor production. ombined results support the proposl tht PMOdS my ct s differentition fctor to promote nd mintin Sertoli cell differentition during pubertl development. In ddition, dt indicte tht the Sertoli cell hormone responsiveness nd the ction of hormones vry s the cell differentites during pubertl development. INTRODUTION Sertoli cells ply fundmentl role in the mintennce of spermtogenesis by creting the proper cytorchitecturl support nd unique microenvironment for developing germinl cells. Peritubulr cells surround the seminiferous tubule nd interct with Sertoli cells through the complex extrcellulr mtrix between the cells. In ddition, peritubulr cells nd Sertoli cells interct through the production of locl prcrine fctors [1]. Peritubulr cells produce fctor termed PModS [2] tht hs drmtic effects on Sertoli cell function in vitro [3,4]. PModS hs been purified nd exists in two functionlly relted forms, PModS(A) nd PModS(B) [3]. The production of PModS ppers to be under the control of ndrogens [2, 5]. The hypothesis hs developed tht the gondotropin LH stimultes Leydig cell ndrogen production, which cts on peritubulr cells to increse the secretion of PModS, which in turn modultes Sertoli cell functions importnt for the process of spermtogenesis [2,3, 6]. The purified prcrine fctor PModS stimultes midpubertl Sertoli cell production of trnsfer- Accepted November 12, Received July 24, This study ws supported by Swiss Ntionl Foundtion support of M.R nd n NIH (HD2583) grnt for M.K.S. who is PW Scholr. orrespondence: Michel K. Skinner, Reproductive ndocrinology enter, University of lifomi, Sn Frncisco, Sn Frncisco, A rin nd ndrogen-binding protein (ABP) to greter extent thn ny single regultory gent previously identified, including FSH [3,4,7]. The ctions of purified PModS on dditionl Sertoli cell functions t vrious stges of pubertl development hs not been thoroughly investigted nd is the focus of the present study. A cellulr function primrily ttributed to erly pubertl Sertoli cells is the romtiztion of ndrogens to estrogen by the P45-ssocited enzyme romtse [8-1]. FSH hs been shown to stimulte romtse ctivity in prepubertl Sertoli cells [8-11]; however, the bility of FSH to regulte romtse ctivity decreses in more dult stges of development [9]. Therefore, romtse ctivity is high nd responsive to hormones in the erly pubertl stges but decreses to low levels s the Sertoli cell differentites to more dult stge of development. Previously, crude mixture of peritubulr cell-secreted proteins (PSP) ws shown to decrese romtse ctivity in midpubertl Sertoli cells [121. The possibility tht this inhibitory ctivity is due to PModS is ddressed in the current study. Plsminogen ctivtor (P) is Sertoli cell product tht is postulted to be importnt for the proteolytic ctivity needed for tissue remodeling of the seminiferous tubule, prticulrly during the trnsloction of spermtogenic cells cross the blood-testis brrier [13, 14]. Through incresing the levels of camp, FSH stimultes P production by Sertoli 586

2 PModS AND PUBRTAL SRTOLI LL DVLOPMNT 587 cells [13, 15]. Although the developmentl regultion of P production hs not been thoroughly exmined, P production ppers to be somewht independent of Sertoli cell differentition nd primrily requires elevtion of camp. Previously crude preprtion of PSP tht contined PModS ws not found to influence P production by Sertoli cells [16]. Peritubulr cells, however, hve been shown to produce n inhibitor of P [14] tht my hve msked the effects of PModS in crude secreted protein preprtions [16]. In the study reported here, we exmined the ctions of purified PModS on P production. ell Preprtion nd ulture MATRIALS AND MTHODS Sertoli cells nd peritubulr cells were isolted from the testis of rts 1, 2, nd 35 dys of ge. Sertoli cells were isolted by sequentil enzymtic digestion [17], including trvpsin, coligense, nd hvluronidse, with modified procedure previously described [18]. With 1-dy-old rts, short, very low-speed centrifugtions (2 X g) were required to sediment Sertoli cells between enzymtic tretments nd wshes. ells were plted t pproximtely 5 x 1 cells per well. Peritubulr cells were obtined from the collgense digestion superntnt of the testiculr cell preprtion s previously described [3]. The cells were collected by low-speed centrifugtion, resuspended, nd plted t pproximtely 1 x i5 cells per (1.7 cm2) 1-mi well of 24-multi-well plte. ells were mintined t 32#{176} in 5% O2 tmosphere with medium chnges every 48 h. Sertoli cells were mintined in Hm s F-12 medium supplemented with.1% BSA, nd peritubulr cells were mintined in medium with 1% newborn clf serum. In some experiments, the cells were cultured with one or more regultory gents, including FSH (Ntionl Pituitry Agency, Bltimore, MD), 1 ng/ml; insulin, 5 p.g/ml; retinol,.35 i.lm; nd testosterone 1 p.m. Where indicted, cells were treted with PSP t concentrtion of 5 p.g protein/ml, or PMdS(A) or PMdS(B) t miniml concentrtion of 25 ng protein/mi. Tretments were initited t the time of plting nd mintined until the times stted in Results. Histochemicl nlysis of the cultured cells from ech ge group ws performed to estblish the purity of the cell popultions utilized. Germinl cell contmintion ws determined with the method of Berg [19], nd peritubulr cell contmintion ws determined with the immunohistochemicl locliztion of desmin [2] nd -smooth muscle isoctin [21] s previously described. The germ cell contmintion fter 5 dys of culture ws not detectble in Sertoli cell preprtions from 1-dy-old rts nd ws 4% nd 9% of the cell popultions from 2- nd 35-dy-old rts, respectively. The peritubulr cell contmintion ws less thn 2% in the Sertoli cell cultures from 2- nd 35-dy-old rts nd ws 4% in cell preprtions from 1-dy-old rts. PModS Preprtion PModS ws purified from concentrted peritubulr cell serum-free conditioned medium s previously described [3]. Briefly, freshly collected peritubulr cell serum-free conditioned medium ws concentrted 1-fold by ultrfiltrtion with n Amicon system (Amicon orp., Lexington, MA) using membrne with 3 Mr exclusion limit. An mmonium sulfte precipitte of this concentrted conditioned medium ws pplied to size-exclusion HPL column. The ctive pek, determined by biossy of Sertoli cell trnsferrin production, ws collected nd pplied to heprin-sephrose ffinity column. Bound proteins were eluted with high-slt buffer nd pplied to two successive 4 reverse-phse columns nd eluted with cetonitrile. Purified forms of PModS were stored t -7#{176}before use, in the presence of 1 mg/ml BSA. The purity of PModS preprtions ws determined chromtogrphicllv nd electrophoreticlly s previously described [3]. The mss of PModS used ws determined with combined procedures including bsorbnce t 214 nm, biochemicl protein ssys, nd mino cid Aromtse nlysis. Activity Aromtse ctivity ws ssessed by the relese of 3H2 from romtiztion of [l-3h]ndro-4-ene-3,1 7-dione or [1,2ct- 3H]testosterone (New nglnd Nucler, Boston, MA) s previously described [22] with the following modifiction: 5 p.1 medium ws removed from the 1-ml cell culture on the dy of the ssy nd replced with 5 p.1 medium contining 1 p.i/ml rdiolbel. After 17-h incubtion t 32#{176} in 5% O2 tmosphere, the cultures were sonicted nd n liquot ws removed for DNA nlysis. The remining smple ws trnsferred to tube nd incubted with dextrn-coted chrcol (.2% [wt/vol] 7K dextrn [Sigm hemicl o., St. Louis, MO], ctivted 2% [wt/vol] with Norit- A chrcol, 1 mm Tris, nd 1 mm DTA, ph 7.5) for 2 h t 4#{176}, nd then centrifuged t 13 X g for 15 mm. Auquots of the supemtnt contining 3H2O were then counted. Blnk vlues were estblished from identicl incubtions in the bsence of cells. Aromtse ctivity (3H2 relesed) ws expressed s counts per minute (cpm) nd ws normlized per microgrms of DNA. To confirm the production of estrogen by the cells, n estrogen RIA ws used with direct ssy of Sertoli cell-conditioned medium. P Assy P ctivity ws mesured with n indirect spectrophotometric ssy [23]. A 1-p.l smple of Sertoli cell-conditioned medium ws incubted t 37#{176} in 4 p.! Tris.1 M, ph 7.5, contining.13 p.m plsmmnogen (bovine plsm, Sigm);.1% (v/v) Tween 8; nd.3 mm S-2251 (D-Vl L-Leu L-Lys, p-nitronilide, Sigm). When required,.6 mg/ ml fibrinogen frgment ws dded for the ctivtion of the tissue-type (tp) form of the enzyme. The bsorbnce chnge

3 588 ROSLLI AND SKINNR -..4) -, - U -, U L ug DNA U.U / nmol substrte (S-2251) FIG. 1. Linerity of romtse ctivity nd P ctivity ssys. Aromtse ctivity in Sertoli cells isolted from 2-dy-old rts, with freshly isolted cells plted t incresing cell number (p.g DNA/mI) in the presence of.5.i/ml 3H-ndro-4-ene-3,17-dione fter 17-h incubtion. P ctivity ws ssyed with incresing concentrtions of hydrolyzed substrte (nmol substrte, S-2251) s detected with n bsorbnce t 45 nm..,.4j - 4- i ).6.4 #{149}.2 fter 6-h incubtion ws mesured ginst blnks without conditioned medi. The P-independent protese ws detected b omitting plsminogen from the ssy mixture. The results of the ctivity ssy were clculted in regrd to the nnomole converted substrte (S-2251) nd normlized to the microgrms of DNA present in the culture well. A stndrd curve of the mount of rected substrte ws obtined by hydrolyzing the substrte S-2251 non-enzymticlly with 1 M NOH. For the preprtion of the fibrinogen frgment, 1 mg of fibrinogen (humn plsm, Sigm) ws treted with 13 mg of cynogen bromide in 1 ml 7% formic cid overnight t room temperture. The solution ws diluted 1:1 with wter nd dilyzed ginst wter overnight. The fibrinogen frgment ws lyophilized nd stored t -2#{176}. The protein concentrtion ws mesured ccording to the method of Brdford [24]. DNA Assy DNA ws mesured fluorometriclly with ethidium bromide [25] s previously described [3]. An liquot of the sonicted cell suspension ws dded to n equl volume of ethidium bromide solution nd llowed to incubte t room temperture for 3 mm. Fluorescent emission t 585 nm with 35 nm excittion ws then monitored. A stndrd curve with clf thymus DNA ws used to quntify DNA levels in the culture wells. This ssy hs Sensitivity of pproximtely.1 pg DNA. ) RSULTS For the vlidtion of the ctivity ssys, the linerity of both the romtse nd P ssys re shown in Figure 1. The romtse ctivity ssy ws bsed on the relese of l-i2o from 3H-ndrogen upon romtiztion to estrogen [22). U (, - -I -, U- -..4). -, -, - ),) U 4-, (U - - TABL 1. P ctivity (nmol converted substrte). Plsminogren Fibrinogren - - up (16 I NDt tp (.12 I ND ND 54.4 SSP (1 g) ND 246 ± ± 46 *Stndrd preprtions (l of tp (Boehringer, Indinpolis, IN) nd up (Sigm, St. Louis. MO) were compred to Sertoli cell-secreted proteins (SSP) in the P enzymtic ssy in the bsence or presence of plsminogen nd fibrinogen frgment. tnd denotes nondetectble levels of P ctivity. Sertoli cells from midpubertl nimls were plted t vrious cell densities nd then incubted with 3H-ndrogen to ssess romtse ctivity. The romtse ctivity (relese of 3H2) ws found to be liner to the microgrms of DNA present in the culture well (Fig. 1). The cell densities generlly used were between 1 nd 2 p.g DNA/well nd re within the liner rnge of the ssy. The P ssy ws bsed on the hydrolysis of chemiclly rective peptide (S-2251) tht cn be detected in colorometric ssy [23). This P spectrophotometric ssy is liner over brod rnge of substrte (S-2251) concentrtions with regrd to the bsorbnce t 45 nm generted from the hydrolyzed product (Fig. 1). The bsence of plsminogen in the ssy is used to determine the bckground non-p proteolytic ctivity, which ws negligible. Fibrmnogen frgments hve previously been shown to specificlly ctivte tp ctivity [23]. The presence of plsminogen nd fibrinogen frgment ws used to specificlly detect tp. The presence of plsminogen nd the bsence of fibrmnogen frgments ws used to detect urokinse-type plsminogen ctivtor (up). The use of tp nd up stndrds in the ssy is shown in Tble Dy of ulture , -.. o Q 4-) #{149} (U. - -) 4-) -) (U -. - FIG. 2. Time course of romtse ctivity nd P production in Sertoli cells from 2-dy-old rts with smples/cells collected on Dys 3, 5, nd 7 of culture. ells were incubted in the bsence () or presence (S) of 1 ng/ml FSH t the time of plting. The results re presented for romtse ctivity s fold increse of ctivity (cpm/pg DNA) present in control cells on Dy 5 of culture. The results re presented for P ctivity s the level of converted substrte s ssessed with bsorbnce t 45 nm. The men ± SM is presented from three different experiments done in triplicte. D.. (U Dy of ulture

4 PMocIS AND PUBRTAL SRTOLI LL DVLOPMNT ) , > -1_J -I-I > -I-f 3 -), D 4-s -. 2 Age (dys) FIG. 3. Aromtse ctivity in Sertoli cells from vrious stges of pubertl development. Sertoli cells were isolted from 1-, 2-, nd 35-dyold rts nd cultured for 5 dys in the bsence () or presence l#{149}) of 1 ng/mi FSH. Sertoli cells were incubted in the presence of.5 p.i/ml 3Hndro-4-ene-3,17-dione for 17 h nd subsequently were sonicted; the mount of 3H2 relese (cpm) ws detected with the chrcol ssy. Results re presented s cpm/g DNA nd re the men ± SM from minimum of three different experiments done in triplicte. U.. O. - -,, D o o Age (dysl U. -,.. -, U. u - U D o 4 #{176} Age (dys) FIG. 4. P production by Sertoli cells from vrious stges of pubertl development. Sertoli cells were isolted from , nd 35-dy-old rts. nd 72-h-medium collection ws obtined on Dy 5 of culture with cells cultured in the bsence () or presence (#{149}) of 1 ng/ml FSH. Both up nd tp ctivity ws exmined. Results re presented s nmol converted substrte/ij.g Sertoli DNA nd re the men of representtive experiment. to demonstrte the specificity of the P ssy. Herefter, P ctivity will denote tp nd up ctivity combined. The romtse ctivity in midpubertl Sertoli cells nd P ctivity present in conditioned medium t vrious durtions of cell culture re shown in Figure 2. ells were incubted in the bsence or presence of FSH for 3, 5, nd 7 dys of culture. Bsl levels of romtse were found to remin reltively constnt, nd optiml FSH stimultion ws observed on Dy 5 of the culture (Fig. 2). Bsl levels of P production were found to increse during culture, nd FSH stimultion ws similr t ech time point exmined (Fig. 2). A more thorough nlysis with sttistics is shown in subsequent figures. Dt indicted tht optiml nlysis of romtse ctivity ws on Dy 5 of culture nd for P production on either Dy 5 or Dy 7 of culture. Preliminry nlysis indicted tht similr dt were obtined with prepubertl nd lte-pubertl Sertoli cells. For subsequent experiments, dt were obtined on Dy 5 of Sertoli cell culture. The bsl nd FSH-stimulted levels of romtse nd P production by Sertoli cells from 1-, 2-, nd 35-dy-old rts re shown in Figures 3 nd 4. For romtse ctivity, the bsl levels remined reltively constnt between the prepubertl, midpubertl, nd lte-pubertl Sertoli cells (Fig. 3). FSH-stimulted romtse ctivity ws highest with pre- 4 pubertl Sertoli cells, reduced with midpubertl cells, nd bsent with lte-pubertl cells. Therefore, s the Sertoli cells reched more dult stge of differentition, romtse ctivity becme nonresponsive to FSH. The P production ws seprted into up nd tp ctivities (Fig. 4). With pubertl development, up ws found to increse slightly, nd it ws responsive to FSH only t the prepubertl stge of development Bsl levels of tp production were lower thn those of up nd incresed slightly with pubertl development. FSH ws found to stimulte tp production by prepubertl, midpubertl, nd lte-pubertl Sertoli cells. As previously shown [26], tp is the form of P tht is hormone responsive, wheres up is nonresponsive to FSH in midpubertl nd lte pubertl Sertoli cells. The hormone stimultion of tp ws found to be independent of the differentited stge of Sertoli cells. A more detiled nlysis of the regultion of P production is shown in Figures 5 nd 6. Both midpubertl nd lte-pubertl Sertoli cells were nonresponsive to FSH in regrd to up production; interestingly, however, FSH significntly stimulted up production by prepubertl Sertoli cells (Fig. 5). As indicted bove, FSH stimulted tp production from Sertoli cells t ll developmentl stges exmined (Fig. 4 6). Insulin t high concentrtions (5 p.g/ml/ml), probbly cting t the insulin-like growth fctor-i (IGF-I) receptor, stimulted both up nd tp production from prepubertl Sertoli cells. This ws in contrst to the lck of insulin effects observed with midpubertl nd lte-pubertl Sertoli cells. Retinol lone hd no effect on prepubertl up production, but it did significntly inhibit bsl up production

5 59 ROSLLI AND SKINNR 1 Dy 2 Dy 35 Dy ( >- ) 6 5 I 6 5 -I-) l) o I 4 3jj T ( - 1 F I ATM - -_ Fl ATM AB F IA TM AB FIG. 5. ffect of regultory gents on up production by Sertoli cells isolted from 1-, 2-, nd 35-dy-old rts. ells were cultured in the bsence (ontrol, ) or presence of too ng/mi FSH (F); 5.g/ml insulin (I),.35 M retinol (R); 1 M testosterone (T); mixture of FSH, insulin, retinol, nd testosterone (M); nd 25 ng/mi PModS(A) (A) or PModSIB) (B). Medium collected on Dy 5 of culture ws ssessed for ctivity nd expressed s nmol converted substrte/g Sertoli DNA. Results re presented s the men ± SM from minimum of three different experiments done in triplicte. Sttisticlly different from control, with p <.5 using Student s t-test. by niidpubertl Sertoli cells nd tended to reduce up production by lte-pubertl cells, lthough the reduction ws not sttisticlly significnt (Fig. 5). Retinol lone hd no effect on bsl tp production, but inhibited the bility of FSH to stimulte tp production by midpubertl Sertoli cells (Fig. 6). Therefore, retinol inhibited bsl levels of up production nd FSH-stimulted tp production by midpubertl Sertoli cells. The minimum nd mximum effective retinol concentrtions for this inhibitory effect were 1 nm nd 1 p.m, respectively (Fig. 7). The I for the inhibitory effects of retinol on midpubertl Sertoli cell P production ws pproximtely 25 nm. Testosterone ws not found to influence Sertoli cell P production t ny of the developmentl stges exmined (Figs. 5 nd 6). The purffied forms of PModS, PMdS(A) nd PModS(B), were not found to influence P production by prepubertl, midpubertl, or ltepubertl Sertoli cells (Figs. 5 nd 6). PModS lso filed to influence the ctions of FSH on Sertoli cell P production (dt not shown). A detiled nlysis of the regultion of romtse ctivity is shown in Figure 8. FSH ws the only regultory gent found to stimulte prepubertl nd midpubertl Sertoli cell 1 Dy 2 Dy 35 Dy ( -4-) -4., ) l,.1-i O. - - r FIRTP4AB FIRTMAB FIRTMA FIG. 6. ffect of regultory gents on tp production by Sertoli cells isolted from 1-, 2-, nd 35-dy-old rts. ells were cultured nd treted nd the dt re presented s described in the legend to Figure 5.

6 PModS AND PUBRTAL SRTOLI LL DVLOPMNT 591 > -I-, -I-, 4-, -) - -s-i O ) ) c D I-I c -I-I. #{149}1I II 1 2: Retinol (um) FIG. 7. Inhibitory effects of retinol on P production by Sertoli cells isolted from 2-dy-old rts. ells were incubted in the presence of FSH nd incresing concentrtions of retinol (ij.m), nd 72-h-medium collection ws obtined on Dy 5 of culture to determine P ctivity for both up () nd tp (#{149}). Results re presented s the percentge of inhibition nd re the men from representtive experiment. All tretments were found to be sttisticlly different (p <.5) from control non-retinol treted cells with Students t-test. romtse ctivity. Insulin, retinol, nd testosterone hd no effect on romtse ctivity t ny of the developmentl stges exmined (Fig. 8). A combintion of FSH, insulin, retinol, nd testosterone hd similr effects s FSH lone. None of the regultory gents including FSH hd ny effect on romtse ctivity in lte-pubertl Sertoli cells. The concentrtions of regultory gents used hve previously been shown to be optiml for the regultion of number of Sertoli cell functions [27]. Anlysis of the ctions of PModS on romtse ctivity is lso shown in Figure 8. Neither PMdS(A) nor PModS(B) lone ws found to ffect bsl levels of romtse ctivity in prepubertl, midpubertl, or lte-pubertl Sertoli cells. The sme preprtions of PModS t the sme concentrtions were found to optimlly stimulte trnsferrin production by midpubertl Sertoli cells (dt not shown). To extend the nlysis of PModS ctions on romtse ctivity, cells were treted with FSH in the bsence or presence of PMOdS (Fig. 9). Neither of the forms of PModS ws found to influence the ctions of FSH on prepubertl or lte-pubertl Sertoli cells. Interestingly, PModS(B), but not PMdS(A), inhibited the ctions of FSH on midpubertl Sertoli cells (Fig. 9). Similr effects were observed with 2- nd 5-ng/ml concentrtions of PMdS(A) nd PMdS(B). To confirm this inhibitory ctivity of PMdS(B), 3H-testosterone ws lso used s substrte in the romtse ctivity ssy (Fig. 1). A similr inhibition of FSH-stimulted romtse ctivity ws observed with PModS(B). strogen ccumultion ws ssessed in midpubertl Sertoli cell-conditioned medium from Dys 2-5 of culture with the sme tretment conditions (Fig. 1). PModS(B) lso inhibited the bility of FSH to stimulte estrogen production by midpubertl Sertoli cells. PMdS(A) hd no sttisticlly significnt 1 Dy 2 Dy 35 Dy 8u, ,.. W V I,. -I, ) : I * * I ( L - #{241}#{241}#{241}#{241}#{241 II 2: nflm T. II II F IA TM AB 2n #{241}#{241}#{241} F I A T N AS F IA TM AS FIG. 8. ffect of regultory gents on romtse ctivity in Sertoli cells isolted from 1-, 2-, nd 35-dy-old rts. Aromtse ctivity ws determined on Dy 5 of culture fter 17-h incubtion with H-ndro-4-ene-3,17-dione nd presented s cpm/p.g Sertoli DNA. ells were cultured nd treted nd the dt re presented s described in the legend to Figure 5.

7 592 ROSLLI AND SKINNR 1 Dy 2 Dy 35 Dy )..) S.) -, ( 4-) ( - - F FA FB F FA FB F FA FB FIG. 9. ffect of PMdS on FSH regultion of romtse ctivity in Sertoli cells. ells were isolted form 1-, 2- nd 35-dy-old rts nd cultured for 5 dys in the bsence (control, ) or presence of FSH (F); FSH plus PModS(A) (FA); or FSH plus PModS(B( (FBI. Aromtse ctivity ws determined fter 17-h incubtion with H-ndro-4-ene- 3,17-dione nd presented s cpm/p.g Sertoli DNA. Results re presented s the men ± SM from minimum of three different experiments done in triplicte. The letter inset within brs denotes the results of sttisticl nlysis of vrince, with difference in letter indicting sttisticl difference (p <.5). effect on FSH ctions with romtse ctivity or estrogen production, but did hve slight inhibitory ctions (Figs. 9 nd lob). Dt indicte tht PModS(B), but not PMdS(A), cn reduce the bility of FSH to stimulte romtse ctivity nd estrogen production by midpubertl Sertoli cells. DISUSSION The testiculr prcrine fctor PModS hs been shown to hve drmtic effects on Sertoli cell trnsferrin nd ABP production in vitro [3, 4, 7). The ctions of PModS on other Sertoli cell functions hve not been exmined nd were investigted in the study reported here to help elucidte the potentil physiologicl role of PModS. Two previously identified Sertoli cell functions re the bility of the cells to romtize ndrogen to estrogen with the P45 romtse enzyme complex [8,9) nd the production of P [13]. The procedures for ssessing the levels of romtse nd P ctivities were estblished nd vlidted s previously described, nd optiml culture conditions for exmining these ctivities were determined. An experimentl vrible to consider, however, is the purity of the cell popultions used. Peritubulr cells produce PModS nd re the primry somtic cell contminnt of Sertoli cell preprtions. As previously described [7], both midpubertl nd lte-pubertl Sertoli cell cultures contin low level (less thn 2%) of peritubulr contminnt. However, prepubertl Sertoli cells from 1-dy-old rts contin higher level of contminnt (pproximtely 4%), which must be considered [7]. The presence of peritubulr cells interferes with dt normliztion nd hs been shown to influence Sertoli cell function nd hormone responsiveness [2, 28, 29]. The potentil production of PModS by this peritubulr cell contminnt in the prepubertl Sertoli cell preprtion my msk the effects of dded PModS. In ddition, the effects of extrcellulr mtrix derived from peritubulr cells cn lso influence Sertoli cell hormone responsiveness [3]. Therefore, this experimentl limittion needs to be considered in subsequent nlysis of dt on prepubertl Sertoli cell preprtions. Sertoli cells hve been shown to produce both up nd tp [14]. The proteolytic ctivity ssocited with P nd its bility to ctivte plsminogen re postulted to be importnt for the continuous tissue remodeling required for spermtogenesis. Therefore, the expression of P is regrded s requirement throughout pubertl development nd in the dult for processes such s the trnsloction of spermtogenic cells cross the blood-testis brrier [13]. The primry regultory gent controlling production of P ctivity is FSH through the ctions of camp [13, 15, 26]. The form of P ctivity influenced by FSH is tp, wheres up hs been shown to be reltively nonresponsive to FSH [26]. Results presented in the current study, however, indicte tht FSH cn significntly stimulte up production by the prepubertl Sertoli cell preprtion. Therefore, s the Sertoli cell becomes differentited, the bility of FSH to regulte up expression is lost; in contrst, tp is regulted by FSH throughout pubertl development. Previous reports hve indicted tht FSH is the primry regultor of P production [13, 15]; however, results of the current study indicte tht insulin cn stimulte the production of both up nd tp by prepubertl Sertoli cells to the sme degree s FSH. These ctions of insulin, probbly cting t the IGF-I re-

8 PModS AND PUBRTAL SRTOLI LL DVLOPMNT 593 o. -) , -, - U U ( - - A IOU t,_ b F FA FB b FIG. 1. ffect of PMdS on FSH regultion of (A) romtse ctivity nd (B) estrogen production by Sertoli cells isolted from 2-dy-old rts. ells were cultured in the bsence (ontrol, ) or presence of FSH (F); FSH plus PMdS(A) (FA); or FSH plus PModS(B) (FB). Aromtse ctivity ws determined on Dy 5 of culture fter 17-h incubtion with 3H-testosterone nd dt re presented s cpm/g Sertoli DNA. strogen production ws ssessed with 72-h-medium collection on Dy 5 of culture nd is expressed s pg/pg Sertoli DNA. Results re presented s the men ± SM from minimum of three different experiments. The letter inset within the brs denotes the results of n nlysis of vrince, with difference in letter indicting sttisticl difference (p <.5). ceptor, re lost s the Sertoli cells re differentited to the midpubertl nd lte-pubertl stges of development. Results imply tht the regultion of P production by prepubertl Sertoli cells is distinct from the predominnt FSH regultion of tp production observed t lter stges of pubertl development. A limittion of this interprettion of dt, however, is the presence of peritubulr cells in the prepubertl Sertoli cell preprtion. Although the ctions of insulin on peritubulr cells hve not been documented, the possibility tht insulin my medite its effects indirectly through the peritubulr cell requires further investigtion. As previously reported, ndrogens do not influence P production with the culture procedures utilized; however, retinol inhibited bsl up production nd FSH-stimulted tp production by midpubertl Sertoli cells. The ctions of retinoids on other Sertoli cell functions hve previously been demonstrted [27], but this study ppers to be the first to report inhibitory ctivity ssocited with retinol. The inverse ctions of FSH nd retinol provide potentilly efficient mechnism for regulting P production. Further investigtion of the ctions of retinoids on P production is required to ddress questions such s why the inhibitory response ws primrily isolted to midpubertl Sertoli cells. P production ws found to increse slightly with the more dvnced developmentl stges of cultured Sertoli cells, nd FSH incresed tp production by prepubertl, midpubertl, nd lte-pubertl Sertoli cells. Therefore the production nd regultion of P production ppers somewht independent of the differentition stge of the Sertoli cells. Previously, crude preprtion of PModS ws found to hve no influence on P production by midpubertl Sertoli D. - -) -. U D -.4-, LU B cells [16]. Recently, however, peritubulr cells hve been shown to produce P inhibitor [14] tht my msk the ctions of PModS in the crude preprtion of PSP [161. The current study demonstrted tht the purified forms of PMOdS, PMdS(A) nd PMdS(B), hd no effect on P production by cultured Sertoli cells from ny of the developmentl stges exmined. This is the first Sertoli cell function observed tht is not influenced by the ctions of PModS. The lck of P regultion by PModS suggests tht PModS is not generl ctivtor of ll Sertoli cell functions, but insted my be primrily involved in influencing the differentition of the cell. This possibility is more directly ddressed with the F FA FB other Sertoli cell function exmined, romtse ctivity. The bility of freshly isolted [1] nd cultured [8, 9] Sertoli cells to synthesize estrogen hs been shown to be due to the romtse enzyme complex ssocited with the endoplsmic reticulum of the cell. The initil function proposed for estrogen production by Sertoli cells ws regultion of Leydig cell steroidogenesis [8-11]; however, Leydig cells hve lso been shown to produce estrogen [11,31]. The physiologicl role for estrogen production by Sertoli cells remins to be elucidted. FSH hs been shown to be the primry regultor of estrogen production nd romtse ctivity [8-1, 22]. strogen production is Sertoli cell function tht is predominnt t the erly stges of puberty [9]. Aromtse ctivity ws found to be high nd hormone responsive in prepubertl Sertoli cells but declined nd ws not responsive to hormones t the lte-pubertl stges of development. Therefore, romtse ctivity is suppressed s the Sertoli cell differentites. The purified forms of PModS, PMdS(A) nd PModS(B), lone were found to hve no influence on romtse ctivity t ny of the developmentl stges exmined. A limittion of this interprettion of the dt, however, is the presence of peritubulr cells in the prepubertl Sertoli cell preprtion tht produced PModS. Although the levels of PModS present in unconcentrted conditioned medi from these preprtions were negligible, the possibility tht endogenous PModS my msk effects of exogenous PModS remins to be investigted. The inbility of PModS to directly influence romtse ctivity probbly correltes with the Lck of effect of PMOdS on camp levels. PModS hs been shown to hve no effect on camp levels, but insted stimultes cgmp levels [4]. Aromt.se ctivity in severl cell types, including Sertoli [9, 22], Leydig [1,31], nd humn dipose cells [32], hs been shown to be regulted by ltertions in camp levels nd camp-dependent protein kinse ctivity. Since PModS hs no effect on camp, PModS lone my not hve the bility to directly influence romtse ctivity. xmintion of the combined effects of PModS nd FSH demonstrted tht PModS(B) inhibited the bility of FSH to stimulte romt.se ctivity in midpubertl Sertoli cells. PModS(A) cused suppression tht ws not sttisticlly significnt. Optiml concentrtions of both PMdS(A) nd PModS(B) from vrious preprtions were used in the nlysis, s previously demonstrted [3].

9 594 ROSLLI AND SKINNR This difference in the ctions of PMdS(B) on romtse is the first functionl difference observed between the two forms of PModS. Further chrcteriztion of the two forms is required to understnd the significnce of this difference in bioctivity. The bility of PModS(B) to suppress the ctions of FSH on midpubertl cells suggests n ltertion in Sertoli cell differentition to more dult stge of development. Results imply tht PModS my promote Sertoli cell differentition to lte-stge pubertl stte tht is less hormone responsive. Previously crude preprtion of PSP ws shown to inhibit the ctions of FSH on romtse ctivity with midpubertl Sertoli cells [12], nd it ws suggested tht this inhibitory ctivity my be due to the presence of PModS. The ctions of purified PModS(B) observed in the current study support this previous specultion. The current study provides thorough nlysis of the hormonl regultion of romtse ctivity nd P production t vrious stges of pubertl development. P production ppers in prt to be independent of Sertoli cell differentition wheres romtse ctivity is n erly pubertl cell function tht decreses in lte-pubertl Sertoli cells. Both ctivities pper to be primrily regulted by FSH. Anlysis of PMOdS ctions suggests tht potentil function of PMOdS my be to promote nd mintin Sertoli cell differentition. This is supported by the observtion tht PModS hd no effect on P production nd suppressed romtse ctivity in response to FSH. hrcteriztion nd moleculr cloning of PModS re required to further our understnding of the functions nd ctions of this testiculr prcrine fctor. In ddition, the observtions mde with PModS in the current study utilized n in vitro nlysis of Sertoli function; elucidtion of the physiologicl importnce nd function of PModS will ultimtely require in vivo physiologicl studies. AKNOWLDGMNTS We thnk Byron Glenn. Lis H!burnt, Jeff Prrott. nd Dr. therine T. Anthony for expert technicl ssistnce nd Mrgret Pech nd ludi Schumnn for ssistnce in preprtion of this mnuscript. We cknowledge the ssistnce of the Vnderbilt Reproductive Biology Reserch enter. RFRNS 1. Skinner MK. ell.cell interctions in the testiv ndocr Rev 1991: 12: Skinner MK. Fritz lb. Testiculr perituhulr cells secrete protein under ndrogen control tht modultes Sertoli cell functions. Proc NtI Acd Sci USA 1985: 82: Skinner MR. Fetterolf PM. Anthony F. Purifiction of the prcrine fctor PMod- S. produced hy testiculr peritubulr cells tht modulte Sertoli cell function. l3iol hem 1988; 263:28&i Norton JN, Skinner MR. Regultion of Sertoli cell differentition through the ctions of testiculr prcrine fctor P-Mod-S. ndocrinology 1989: 124: S. Skinner MR. Fritz lb. Androgen stimultion of Senoli cell function is enhnced hy perituhulr cells. Mol ell ndocrinol 1985: :1l Skinner 51K. ell-cell interctions in the testis. Ann NY Acd Sci 1987; 513:158- l l. 7. Anthony T, Rosselli M, Skinner MK. Actions of the testiculr prcrine fctor (PModS) on Sertoli cell trrisferrin secretion throughout pubertl development. ndocrinology 1991; 129: Dorrington JH, Armstrong DT. Follicle-stimulting hormone stimultes estrdiol- 17 synthesis in cultured Sertoli cells. PNAS 1975: 72: Dorrington JH, Fritz lb. Armstrong DT. ontrol of testiculr estrogen synthesis. Biol Reprod 1978; 18: Tsi-Morris, Aquilno DR. Dufu ML. ellulr locliztion of rt testiculr romtse ctivity during development. ndocrinology 1985; 116: Mlle L, Mchdo AJ, Nvroli F, Rommerts FFG, Modultion of stimultorv ction of follicle-stimulting hormone (FSH) nd inhibitory ction of epiderml growth fctor (GF) on romtse ctivity in Sertoli cells by clcium. FB 1987; 218: Verhoeven G, ille J. Testiculr peritubulr cells secrete protein under ndrogen control tht inhibits induction of romtse ctivity in Sertoli-cells. ndocrinologv 1988; 123: Lcroix M, Smith F. Fritz lb. Secretion of plsminogen ctivtor by Sertoli cell enriched cultures. Mol ell ndocrinol 1977: 9: Hettle AJ, Blek(in, lung PS, Fritz tb. Rt testiculr peritubulr cells in culture secrete n inhibitor of plsminogen ctivtor ctivity. Biol Reprod 1988; 38: Fritz LB. Krmllv l( Hormonl influences on formtion of plsminogen ctivtor by cultured testis tubl segments t defined stges of the cycle of the seminiferous epithelium. n J Biochem ell Biol 1983; 61: Skinner MK, Fritz lb. Identifiction of non-mitogenic prcrine fctor involved in mesenchvml-epithelil cell interctions between testiculr peritubulr cells nd Sertoli cells. Mol ell ndocrinol 1986; 44: Dorrington JH, Roller NF, Fritz LB. ffects of follicle stimulting hormone on cultures of Sertoli cell preprtions. Mol ell ndocrinol 1975; 3: lung PS, Skinner MK, Fritz LB. Fibronectin synthesis is mrker for peritubulr cell contminnts in Sertoli cell enriched cultures. Biol Reprod 1984; 3: Berg JW. Differentil stining of spermtozo in sections of testis. 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