Differential Steroidogenic Response of Subpopulations of Porcine Granulosa Cells to Insulin-Like Growth Factor-1 (IGF-1) or IGF-1 Analogs'

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1 BIOLOGY OF RPRODUCTION 5, 8-5 (994) Differentil Steroidogenic Response of Subpopultions of Porcine Grnulos Cells to Insulin-Like Growth Fctor- (IGF-) or IGF- Anlogs' HJ. HOWARD nd JJ. FORD 2 U.S. Deprtment of Agriculture, Agriculturl Reserch Service, Romn L. Hrusk U.S. Met Animl Reserch Center, Cly Center, Nebrsk ABSTRACT Two experiments were conducted to exmine responses of porcine grnulos cells to insulin-like growth fctor- (IGF-) or IGF-I nlogs (des [-3] nd Long R3) tht hve reduced ffinity for IGF-binding proteins (IGFBP). Both experiments evluted estrdiol nd IGFBP production by grnulos cells fter seprtion of cells into subpopultions tht mintin long-term estrdiol production in vitro (tightly bound) nd those tht do not (wekly ssocited). Grnulos cells were obtined from mediumsized follicles (4-6 mm) t rndom stges of the estrous cycle in experiment nd from the lrgest follicles per ovry t or 48 h fter wening in experiment 2. Follicle dimeter nd folliculr fluid estrdiol concentrtions incresed with time fter wening (p <.5). Tightly bound cells produced more estrdiol thn wekly ssocited cells t 24-2 h of culture in experiment nd from to 48 h in experiment 2 (p <.5). In tightly bound but not wekly ssocited cells, IGF- stimulted estrdiol production. The IGF nlogs were more potent stimultors thn IGF- in experiment (p <.5); nd in experiment 2, this response ws restricted to cells collected t 48 h fter wening. Conversely, tightly bound cells obtined t h fter wening responded similrly to IGF- nd des (-3). During the finl 48 h of culture, wekly ssocited cells produced greter quntities of 28-3-kD IGFBP thn did tightly bound cells in response to IGF- or nlogs (both experiments; p <.5). Concentrtion of 4-44-kD IGFBP ws influenced minimlly by IGF-I or its nlogs. We conclude tht ) tightly bound grnulos cells produce greter mounts of estrdiol in vitro thn wekly ssocited cells when smpled from medium-sized follicles; 2) tightly bound cells from follicles of sows t rndom stges of the cycle or t 48 h fter wening produce more estrdiol in response to IGF nlogs thn to IGF-; nd 3) tightly bound cells secrete less 2-3-kD IGFBP in long-term culture thn wekly ssocited cells. These results indicte tht IGFBP modulte secretion of estrdiol in porcine grnulos cells in vitro nd implicte their potentil for in vivo regultion. INTRODUCTION Folliculr development in swine represents complex process whereby during the folliculr phse of the estrous cycle, finite number of preovultory follicles re selected from much lrger pool of ntrl follicles, the mjority of which undergo tresi. While this process is under endocrine regultion by FSH nd LH, it is generlly ccepted tht locl regultory fctors in the ovry hve role in determining the emergence of the dominnt follicles t this time of the cycle. Much ttention hs been given to the introvrin insulin-like growth fctor system [, 2]. strdiol nd insulin-like growth fctor- (IGF-) increse in prllel with one nother ccording to some studies [3, 4], nd IGF- or insulin stimultes in vitro steroidogenesis in porcine grnulos [5-7] or thec intern cells [8]. Recently, reserch efforts hve lso ddressed whether IGF-binding proteins (IGFBP) hve n importnt role in folliculr development nd steroidogenesis. Porcine grnulos cells hve the bility to produce IGFBPs in vitro [9, ], nd IGFBPs of certin moleculr weights decrese in folliculr fluid with dvncing folliculr development [, 2]. This provides evidence Accepted Mrch 9, 994. Received June 23, 993. 'This mteril is bsed on work supported by the Coopertive Stte Reserch Service, U.S. Deprtment of Agriculture, under Agreement No Mention of trde nme does not constitute gurntee or wrrnty of the product by the U.S. Deprtment of Agriculture. ZCorrespondence. FAX: (42) for physiologicl, possibly inhibitory, role of IGFBP in the regultion of IGF- ctions in folliculr development nd steroidogenesis. Wheres long-term secretion of estrdiol hs been mintined in cultures of rt [3] or humn [4] grnulos cells, mintennce of estrdiol production by cultured porcine grnulos cells hs been very limited. Generlly, porcine grnulos cells lose considerble romtse ctivity during the first h in culture [5], becoming primrily progesterone-secreting cells. Recently, using cells from smll (-3 mm) follicles, we reported [6, 7] greter success t mintennce of in vitro estrdiol biosynthesis by porcine grnulos cells for up to 8 dys in culture. Seprtion of grnulos cells into two subpopultions-tightly bound nd wekly ssocited-provided cells tht differ drmticlly in estrdiol production. Wekly ssocited grnulos cells lose FSH-stimulted romtse ctivity very rpidly, much s reported previously [5]. Tightly bound grnulos cells, on the other hnd, mintin higher estrdiol production in long-term culture. The objective of the present work ws to exmine how these two grnulos cell subpopultions, obtined from medium (4-6 mm) follicles (experiment ) or from the lrgest follicles of wened sows (experiment 2), differ in their production of estrdiol nd IGFBP in response to IGF- or nlogs of IGF- tht hve reduced ffinity to IGFBPs [8, 9]. Lctting sows remin nestrous until fter removl of the litter; then rpid folliculr growth occurs in ssocition with incresed estrdiol secretion cul- 8

2 2 AND IGFBP FROM SUBSTS OF PORCIN GRANULOSA CLLS 9 minting in ovultion, bout 6 dys fter removl of the litter. This llowed exmintion of interreltionships between estrdiol secretion in vitro nd components of the introvrin system in cells from follicles t specific stges of development (experiment 2). MATRIALS AND MTHODS Tissue Collection nd Cell Culture Porcine ovries from cyclic femles (see description of nimls in specific experiments) were collected t slughter. Grnulos cells were isolted by the method of My nd Schomberg [5]. Wekly ssocited nd tightly bound grnulos cells were seprted by filtrtion on.72-plm nylon mesh filter [6,7], with wekly ssocited cells pssing through the filter nd tightly bound cells remining on the filter. Tightly bound grnulos cells were dissocited enzymticlly [6], nd cells were wshed in Dulbecco's PBS with % BSA. Grnulos cells were plted t density of 5 x 5 vible (trypn blue exclusion) cells/cm 2 in 48-well culture dishes tht were coted with Peptite 2 ( g/ cm 2 ; Telios Phrmceuticls, Sn Diego, CA). Serum-free culture medium consisted of : Dulbecco's Modified gle's Medium:Hm's F-2 (Gibco Lbs., Grnd Islnd, NY) supplemented with penicillin ( U/ml), streptomycin (. mg/ml), mphotericin B (.25 pig/ml), ndrostenedione ( - 6 M), Albumx II (lipid-rich BSA,.%; Gibco), nd Pentex x-cyte VL (lipid supplement,.25%; Miles Lbs, lkrt, IN). Concentrtions of Albumx II nd x-cyte were bsed on recommendtions of the mnufcturers for ttchment-dependent cells. No IGFBP ctivity ws detected in unconditioned medium. Cultures were crried out in 5% 2, 5% CO 2 environment t 39 C. Concentrtions of CO 2 nd 2 were ech mintined t 5% vi mixture of ir with CO 2 nd N 2 gses. xperiment Grnulos cells from medium (4-6 mm) follicles were collected from crossbred sows killed t rndom stges of the estrous cycle. Pools of cells were mde from -5 sows per replicte (five replictes totl). Smpling in this fshion should result in collection of grnulos cells both from follicles destined to ovulte nd from those destined to undergo tresi. No ssessment of folliculr helth sttus ws conducted other thn exclusion of opque follicles. Cells were treted, in triplicte wells, with or ng/ml FSH (USDApFSH-B, potency:.69-strength NIH-FSH-S; LH potency: <.-strength NIH-LH-S) nd,,, or ng/ml IGF- (GroPep LTD. or Bchem CA, Inc., Torrnce, CA), des (- 3), or Long R3 (GroPep LTD). Both des (-3) nd Long R3 re IGF- nlogs tht hve reduced ffinity for IGFBP [8,9]. Tretments commenced t the time cells were plted. Medium ws collected nd replenished t 24 nd 72 h, nd tretments were repplied; cultures were terminted t 2 h. xperiment 2 Results of experiment from medium follicles of unknown developmentl sttus indicted tht estrdiol production in distinct subpopultions of grnulos cells differed in response to IGF- vs. the two IGF- nlogs tested. This provided impetus to exmine these interreltionships in follicles of more defined stges of preovultory development. To this end, first-prity sows were utilized t or 48 h fter wening of their litters. The objective ws to smple t h, when ovrin ctivity ws repressed, nd t 48 h postwening, when follicles were undergoing rpid growth. Grnulos cells from the lrgest follicles per ovry were collected, nd cells from two sows t ech time postwening were pooled (n = 2 sows; 5 replictes/time). Grnulos cells were cultured for 96 h with medium chnged t 48 h. Cells were treted with or ng/ml FSH nd,,, or ng/ml IGF- or des (-3). Since des (- 3) nd Long R3 gve similr steroidogenic responses in experiment, only des (-3) ws utilized for comprison with IGF- in experiment 2. Tretments were dministered when cells were plted nd repplied when medium ws chnged t 48 h of culture; cultures were terminted t 96 h. Assy of Hormones, Totl Cellulr DNA, nd IGFBPs Unextrcted liquots (--Rl equivlent) of culture medium were nlyzed for estrdiol nd progesterone by RIA [2] utilizing ' 25 I-lbeled steroids. The cross-rectivity of the estrdiol ntiserum to ndrostenedione ws <.% [2]. The interssy coefficients of vrition were 6 nd 7% for the estrdiol nd progesterone ssys, respectively. Totl cellulr DNA per culture well t the end of the culture period ws determined by the methods of Lbrc nd Pigen [2]. Grnulos cells were disrupted with % cholic cid (sodium slt) nd.% SDS nd were frozen until ssyed. Western lignd blotting ws utilized to exmine in vitro IGFBP production [22]. Culture medium (25,ul) ws concentrted by precipittion with two volumes of bsolute ethnol followed by centrifugtion t 8 x g for 2 min, reconstituted in smple buffer, nd resolved vi 2.5% SDS- PAG [2]. Scnning densitometry ws utilized to chrcterize chnges in bnd intensity. Sttisticl Anlysis Dt were nlyzed by lest-squres nlysis of vrince using the Generl Liner Models procedure of the Sttisticl Anlysis System [23]. All studies were treted s fctoril designs, nd nlysis of covrince ws used with cellulr DNA s the covrite to correct steroid concentrtions nd IGFBP bnd intensities for differences in cell plting. When dt were expressed on per microgrm DNA bsis, the conclusions drwn were similr to those from the nlyses of covrince included in the present report. In experiment 2, replicte nested within time postwening ws utilized s error to test effect of time postwening. Or-

3 HOWARD AND FORD - - UI TB - IGF- -M- TB - des (-3) TB - Long R3 U 2' 8' 6' 4' WA - IGF- -- WA - des (-3) _ WA - Long R3 I= = FIG.. Representtive Western lignd blot developed during experiment 2; culture medium (concentrtion of 25 Il) from wekly ssocited nd tightly bound grnulos cells treted with dosges of to ng/ml IGF- (top utordiogrph) or des (-3) (IGF- nlog, bottom utordiogrph). A single pool of porcine folliculr fluid (pff) served s reference for ech lignd blot. thogonl polynomil contrsts were utilized to exmine the effect of dose of IGF- or nlog, s well s the interctions with the other min effects. Min effects or interctions were not significnt unless so stted. Generl Results RSULTS In both experiments, FSH ws without significnt effect on the secretion of estrdiol, lthough there ws numericl dvntge for FSH-treted cells. During the initil period of culture, tightly bound grnulos cells produced greter estrdiol concentrtions thn wekly ssocited cells (p <.5), lthough no effect of FSH or IGF ws noted. Becuse there ws not significnt interction of FSH by dosge of IGF- or nlog, dt presented for both experiments were verged cross the nd ng/ml dosges of FSH. In both experiments, tretment of cells with FSH incresed (p <.5) progesterone production t termintion of cultures (.5-fold in experiment ; 2.-fold in experiment 2). In both experiments, mjor bnds of IGFBPs (Fig. ) were detected in conditioned medium t pproximtely 28, 3, 4, nd 44 kd. A minor bnd of 34 kd ws lso detected; this ppered to be regulted coordintely with the 28-3-kD IGFBP. When the 28-3-kD bnds were mximlly stimulted, the 34-kD IGFBP could not be v Dosge of IGF- or Anlog (ng/ml) FIG. 2. Concentrtion of estrdiol in culture medium (24-72 h of culture) from tightly bound (TB, top pnel) nd wekly ssocited (WA, bottom pnel) porcine grnulos cells from 4-6-mm follicles. Cells were treted with dosges of - ng/ml IGF- or the IGF- nlogs, des (-3) nd Long R3. SM:.5 ng/ml. Probbility: interction of cell type with IGF tretment with dosge, p =.7. distinguished by densitometry from the 3-kD bnd. It ppered tht the contribution of the 34-kD IGFBP ws miniml, so the summry of densitometric quntifiction included ll bnds from 28 to 34 kd. xperiment At termintion of cultures, totl cellulr DNA for wekly ssocited grnulos cells incresed from 2.7 ±.7 l.g/ well in the bsence of IGF- or its nlogs to pproximtely 6-7,g/well t - ng/ml of des (-3), Long R3, or IGF- (p <.). For tightly bound cells, DNA remined t Lg/well nd ws unffected by dosge of IGF; no differences were seen in cellulr DNA in reltion to tretment with IGF- or IGF- nlogs. Production of progesterone from both 24 to 72 h nd 72 to 2 h of culture ws similr between subpopultions, ws incresed in culture medium with incresing dosges of IGF- or nlogs (p <.5), nd ws.9-fold greter for the nlogs thn for IGF- t the ng/ml dose (p <.). From 24 to 72 h of culture, tightly bound grnulos cells hd greter estrdiol secretion thn wekly ssocited cells, nd the tightly bound cells exhibited lrger differentil between IGF- nd its nlogs, mnifested primrily t the

4 2 AND IGFBP FROM SUBSTS OF PORCIN GRANULOSA CLLS C '5to * w c.5 ul I- I - -- V- WA - IGF- -- WA - des (-3) ; WA - Long R3 o Dosge of IGF- or Anlog (ng/ml) FIG. 3. Concentrtion of estrdiol in culture medium (72-2 h of culture) from tightly bound (TB, top pnel) nd wekly ssocited (WA, bottom pnel) porcine grnulos cells from 4-6-mm follicles. Cells were treted with dosges of - ng/ml of IGF- or the IGF- nlogs, des (-3) nd Long R3. SM:.2 ng/ml. Probbility: interction of cell type with IGF tretment with dosge, p <.. nd ng/ml dosges (p =.7, Fig. 2). From 72 to 2 h of culture, tightly bound grnulos cells produced concentrtions of estrdiol tht were pproximtely 4-fold higher thn those from wekly ssocited cells t the nd ng/ml dosges of des (-3), Long R3, or IGF- (Fig. 3, p <.). In tightly bound grnulos cells, the nlogs t ng/ml provided greter stimulus for estrdiol production thn did IGF- (p <.5, Fig. 3). Wekly ssocited but not tightly bound grnulos cells produced greter mounts of the IGFBP doublet t 4-44 kd in response to IGF- or nlogs, nd this increse with wekly ssocited cells ws greter for IGF- thn for des (-3) or Long R3 (Fig. 4, p =.). The IGFBP of 28-3 kd incresed with dosge of IGF- to lrger degree thn with IGF- nlogs; gin this differentil ws greter for wekly ssocited thn for tightly bound grnulos cells (p <.5). xperiment 2 From the results of experiment, it ws pprent tht IGF- nlogs were more potent thn IGF- in the stimultion of estrdiol from tightly bound grnulos cells in cul- :P O v -..O.- WA - IGF kd IGFBP "'"m' WA AnlO9S, - TB -IGF- -- TB - Anlogs...(7 TOr TT J I 'U ~~~~~~~~~~~~~~~~~4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ C 3 x O 6 n c t O-- WA - IGF WA - Anlogs -- TB - IGF- Z--- TB -Anlogs Dosge of IGF- or Anlogs (ng/ml) 28-3 kd IGFBP FIG. 4. Chnges in IGFBP (4-44 kd, top pnel; 28-3 kd, bottom pnel) from 72 to 2 h of culture for wekly ssocited nd tightly bound grnulos cells collected from 4-6-mm follicles from ovries of sows t rndom stges of the estrous cycle. Cells were treted with dosges of to ng/ml of IGF- or IGF- nlog (dt pooled for des [-3] nd Long R3). SM: 4-44 kd,.7; 28-3 kd,.2. Probbilities: 4-44 kd: interction of cell type with IGF tretment with dosge, p =.; 28-3 kd: interction of cell type with IGF tretment with dosge, p <.5. ture nd tht these nlogs differed from IGF- in ffecting in vitro production of IGFBP. Thus, experiment 2 ws conducted to compre the effects of IGF- vs. des (-3) on grnulos cells from follicles t two specific stges of development in wened sows. Folliculr dimeter incresed from 4. mm t h to 5. ±.2 mm t 48 h postwening (p <.5). Similrly, estrdiol in folliculr fluid incresed from 2.9 to ng/ml t nd 48 h postwening, respectively (p <.5). DNA content, which did not chnge with dosge of des (-3) or IGF-, ws 3. jkg/well for tightly bound nd.2.5 rg/well for wekly ssocited grnulos cells (p <.). Wekly ssocited cells collected t h postwening hd greter progesterone secretion during 96 h of culture thn tightly bound grnulos cells (p <.5), wheres the two subpopultions hd similr profiles of progesterone production when collected t 48 h postwening. Regrdless of grnulos cell subpopultion or time postwening, progesterone production ws.6-fold greter in des (-3)-treted cells thn in IGF--treted cells (p <.5); nd t both postwening times, secretion 3

5 - 2 HOWARD AND FORD J TB - IGF TB -des(l-3) WA - IGF- '"" WA- des(-3) hr PW X ' 8 --O-- WA - IGF- -'t*- WA -des(l-3) -- C-- TB- IGF- - TB - des(l3) 4-44 kd IGFBP w 2 'I w,llllllil~ll. &. C m 4 no I...-" i m t hr PW o, eo X C O c w C m m Dosge of IGF- or Anlog (ng/ml) I,SOf FIG. 5. Concentrtion (48-96 h of culture) of estrdiol in culture medium of tightly bound (TB) nd wekly ssocited (WA) porcine grnulos cells from follicles of sows t (top pnel) or 48 (bottom pnel) h postwening (PW). Cells were treted with dosges of - ng/ml of IGF- or the IGF- nlog, des (-3). SM:.4 ng/ml. Probbility: interction of h post-wening with cell type with dosge, p <.5. of progesterone incresed with dosge of des (-3) or IGF- (p <.5). From 48 to 96 h of culture, estrdiol production by tightly bound grnulos cells ws greter t incresing dosges of des (-3) or IGF- for cells obtined t 48 h postwening thn for those obtined t h postwening (Fig. 5, p <.5). No significnt differences were detected between des (-3) or IGF- tretments, but mgnitude of the response by tightly bound grnulos cells to incresing dosges of either compound ws greter thn tht observed in wekly ssocited cells (p <.5). For cells from sows t 48 h fter wening, the dose response for des (-3) vs. IGF- tretments ws shifted left by nerly one log dosge, but the difference in estrdiol production did not chieve sttisticl significnce. No differences in IGFBP production were detected for grnulos cells obtined t nd 48 h postwening, so dt were pooled cross these times. Strong trends in the dt were not evident; the IGFBP doublet of 4-44 kd ws similr between grnulos cell subpopultions. The 4-44-kD IGFBP incresed with dosge of IGF-, but decresed with nlog tretment reltive to the ng/ml dosge (p <.5, Dosge of IGF- or Anlog (ng/ml) FIG. 6. Chnges in IGFBP (4-44 kd, top pnel; 28-3 kd, bottom pnel) from 48 to 96 h in culture for wekly ssocited (WA) nd tightly bound (TB) grnulos cells collected from follicles of sows t nd 48 h postwening (pooled cross postwening times). Cells were treted with to ng/ml IGF- or the IGF- nlog, des (-3). SM: 4-44 kd,.2; 28-3 kd,.3. Probbilities: 4-44 kd: interction of IGF tretment with dosge, p <.5; 28-3 kd: interction of cell type with dosge, p <.; interction of IGF tretment with dosge, p <.. Fig. 6). The 28-3-kD IGFBP incresed with dosge of IGF- to lrger extent thn with des (-3) for both subpopultions of grnulos cells (p <.). This IGFBP lso showed lrger increses with dosge of IGF- or nlog in wekly ssocited compred to tightly bound grnulos cells (p <.). DISCUSSION In the present studies we determined tht IGF- nd two of its nlogs, des (-3) nd Long R3, will mintin estrdiol production by porcine grnulos cells from mediumsized follicles (4-6 mm); but the response ws dependent upon origin of grnulos cells within the follicles, stge of folliculr development, nd durtion of culture. Tightly bound cells (from the murl lyers of the follicle) produced greter quntities of estrdiol when stimulted by IGF- or its nlogs thn wekly ssocited cells (from the ntrl lyers). Tightly bound grnulos cells produced more estrdiol in response to IGF- or des (-3) if collected from sows t 48 h thn t h postwening, reflecting folliculr D

6 2 AND IGFBP FROM SUBSTS OF PORCIN GRANULOSA CLLS 3 fluid estrdiol concentrtions t these times (experiment 2). Regrdless of this difference in quntity of estrdiol produced, the mgnitude of the increse ws fold for cells collected on both dys. Tightly bound cells produced greter quntities of estrdiol in response to IGF- or nlog tretment from 48 to 72 h of culture thn from 72 to 2 h (experiment ); but the mgnitude of the difference between stimultion by IGF- nd its nlogs incresed with time in culture. These findings indicte tht reltive to estrdiol production, the proportion of IGF--responsive cells increses with time fter wening nd tht these cells re present in medium-sized follicles during much of the estrous cycle. The stimultory effects of IGF- nd insulin on in vitro steroidogenesis nd function of grnulos cells hve been well documented [, 2, 24], but this is the first report of differentil responses by subpopultions of these cells within the sme follicles. Observtion of the potentil for tightly bound grnulos cells from medium-sized follicles (4-6 mm) to mintin estrdiol secretion in culture extends previous studies with this subpopultion of cells from smll (-3 mm) porcine follicles [6,7]. Tightly bound grnulos cells originte from the murl lyers tht border the bsement membrne of the follicle, wheres wekly ssocited cells re from the ntrl lyers [7]. Murl grnulos cells hve greter concentrtions of LH receptors [25, 26] nd greter mounts of cytochrome P45 cholesterol side-chin clevge enzyme thn do ntrl grnulos cells [27]. In contrst, IGF- mrna concentrtions nd prolifertion, s ssessed by tritited thymidine incorportion, re greter in ntrl thn in murl lyers of the follicle [28, 29]. The present results emphsize the impct of conducting studies with follicles t defined stges of development nd heterogeneous responses by cells from different loctions within follicle. We suggest tht IGFBPs hve role in modulting IGF- 's stimultory effects on estrdiol production by tightly bound grnulos cells becuse both des (-3) nd Long R3, compred to IGF-, hve reduced ffinity to IGFBPs nd greter potency for estrdiol stimultion. Tightly bound cells produced less 28-3-kD IGFBP in vitro thn wekly ssocited cells, nd nlogs to IGF- were less potent thn IGF- for production of this IGFBP. This ltter observtion indictes tht IGF- intercts with IGFBPs to further enhnce secretion of 28-3-kD IGFBP, but this conclusion ssumes tht differences in ffinity to IGF- receptors re not the cuse for the observed differences between IGF- nd the nlogs in stimultion of IGFBP secretion. These results illustrte tht subpopultions of grnulos cells tht differed in steroidogenic cpbilities lso hd different IGFBP profiles. The observtion tht IGF- nlogs filed to enhnce estrdiol production by wekly ssocited cells indictes tht IGFBPs probbly do not contribute to diminished estrdiol production by this subpopultion of cells. In contrst, IG.FBPs re implicted s inhibitory for progesterone production in both cell types, becuse IGF- nlogs incresed progesterone production over tht induced by IGF-. In previous studies with porcine grnulos cells, decresed secretion of IGFBP-2 nd -3 with time in culture hs been reported [9], nd mximl secretion of these IGFBP occurred t lower dosges of des (-3) or Long R3 thn of IGF- [3]. In contrst, we detected very little IGFBP in medium from cells during the first 24 to 48 h in culture (unpublished observtions); IGF- provided greter stimultion for secretion of 28-3-kD IGFBP (IGFBP-2) thn the nlogs; nd IGF- or its two nlogs hd no mjor effect on the concentrtion of 4-44-kD IGFBP (IGFBP-3). In the erlier studies, grnulos cells from prepubertl nimls were evluted, nd the cells were plted in collgen-coted wells. Adhesion fctors modulte cellulr processes [3, 32], but how these fctors influence IGFBP secretion hs not been investigted. A relevnt difference between the erlier studies nd ours is the increse in IGFBP-3 we obtined fter tretment of grnulos cells with IGF- nd its nlogs. The mrna for this protein is not detected in freshly collected porcine follicles but is bundnt in CL [33, 34]. Thus, the presence of this protein should be regrded s n indictor of cellulr differentition in cultured grnulos cells; however, steroid production ws not reported in the erlier studies. Additionlly, chnges in IGFBP-3 concentrtion cn result from chnges in its rte of degrdtion [35]. At present, these divergent results hve not been resolved, but culture conditions or source of grnulos cells my modify direct effects of IGFBPs reltive to effects medited through binding of IGF- [36]. Depending on culture conditions, the effect of IGF- cn be mnifested by IGF- itself [37] but my require interction with FSH [38]. It is unknown t this time why the response to FSH ws not significnt in the present studies, given tht it is generlly ccepted tht gondotropin tretment is necessry if the full effects of IGF- responsiveness re to be observed [, 39]. In the present studies, IGF- nhogs were more potent thn IGF- t stimultion of estrdiol production by tightly bound grnulos cells (experiment nd 48 h postwening, experiment 2) nd in the stimultion of progesterone production by tightly bound nd wekly ssocited cells (both experiments). Dose-response comprisons were compromised becuse the potencies of IGF- reltive to IGF- nlogs were compred t vrible concentrtions of IGFBPs. In cultures of rt grnulos cells, ddition of IGFBP-2 or -3 inhibited romtse ctivity [4,4]. Furthermore, Adshi et l. [42] observed tht the potency of des (-3) ws greter thn tht of IGF- in the stimultion of cell prolifertion nd progesterone secretion. This ws probbly due to reduced ffinity of des (-3) for grnulos-derived IGFBPs, but which IGFBPs were involved ws not known. One vrible in both experiments ws 2- to 3-fold greter cellulr DNA t the end of the culture period for wekly ssocited grnulos cells thn for tightly bound cells, even

7 4 HOWARD AND FORD though the number of vible cells plted for ech subpopultion ws similr. In contrst, when these subpopultions from smll porcine follicles were cultured in medium supplemented with fetl bovine serum (% t plting, 2.5% t subsequent medium chnges), cellulr DNA ws similr for tightly bound nd wekly ssocited grnulos cells [7]. Initil vibility ws > 9% for tightly bound grnulos cells nd 4-6% for wekly ssocited cells. In greement with the current studies, cellulr DNA ws higher for wekly ssocited thn for tightly bound grnulos cells cultured s described in the present studies but with insulin supplementtion [43]. Thus, some component or combintion of components of the serum-free system (Albumx, x-cyte, Peptite 2) pprently enhnced ttchment, survivl, or repir of some of the wekly ssocited cells tht initilly took up trypn blue. In conclusion, we demonstrted differences in estrdiol nd IGFBP biosynthetic cpbilities between subpopultions of grnulos cells from medium-sized porcine follicles. Most importntly, this is the first demonstrtion of enhnced romtse ctivity in tightly bound porcine grnulos cells in the context of compring the dose responsiveness of des (-3) or Long R3-nlogs with reduced ffinity to IGFBPs-with the dose responsiveness of IGF-. Furthermore, this subpopultion of grnulos cells, which hs the bility to mintin estrdiol production in long-term culture, lso produced less 28-3-kD IGFBP. Chnges in the 28-3-kD IGFBP re relevnt s modultor of estrdiol production in vivo becuse concentrtion of this IGFBP in folliculr fluid decreses s estrdiol concentrtions increse [2]. The reltive potencies of the IGF- nlogs nd IGF- on romtse ctivity in tightly bound grnulos cells indicte tht IGFBPs hve n importnt negtive modultory role on IGF- ctions in vitro. 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