Effects of transforming growth factor-β1 and activin-A on in vitro porcine granulosa cell steroidogenesis

ELSEVIER

EFFECTS OF TRANSFORMING GROWTH FACTOR-P, AND ACTIVIN-A ON IN VITRO PORCINE GRANULOSA CELL STEROIDOGENESIS W.Y. Chang, F. Shidaifat, M. Uzumcu’ and Y.C. Lin* Laboratory of Reproductive and Molecular Endocrinology Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine The Ohio State University, Columbus, OH, USA Received for publication: Accepted:

August January

23, 1995 23, 1996

ABSTRACT The mammalian ovarian cycle is a strictly regulated process that is dependent on the intimate interactions among the 3 cell types in the follicle - theta, granulosa, and oocyte. The cycle has been shown to be controlled by gonadotropins as well as locally produced peptide factors. In this study, an in vitro culture system was used to study the roles of 2 locally produced ovarian peptide factors, transforming growth factor- 0, (TGF-P, ) and activin-A, on porcine granulosa cell steroidogenesis. Gonadotropinstimulated cultured porcine granulosa cells (from medium-sized follicles) were pretreated with IO0 &ml follicle-stimulating hormone (FSH) for 48 h and then treated with I ng/ml TGF-P,, 100 ng/ml activin-A, TGF-0, plus activin-A, or received no treatment (control) for 48 h. From our previous studies, the concentrations of the 2 growth factors were determined to produce maximal antisteroidogenic effects in porcine granulosa cells. Progesterone (P4) production, estradiol- l7jJ (6 ) production, and aromatase activity for gonadotropin-stimulated porcine granulosa cells treated with TGF-P,, activin-A, and TGF-0, plus activin-A were significantly (P < 0.05) reduced fromthat of the control. The same procedures were conducted on basal steroidogenesis studies in which no pretreatment with FSH was performed. Both P, and Ez production and aromatase activity for porcine granulosa cells treated with TGF-B,, activin-A and TGF-P, plus activin-A were significantly (P < 0.05) inhibited compared with the control. Our results indicate that both TGF-/3, and activin-A can inhibit FSH-stimulated and basal steroidogeneses in porcine granulosa cells and, thus, may act as local atretic factors during follicular development. When the 2 growth factors were given in combination at concentrations that would produce maximal steroidogenic inhibition, they were not able to produce a synergistic effect. These results are consistent with the current theory that TGF-0, and activin-A may act via the same messenger system, a serine-threonine kinase. Key words: pig, ovary, aromatase, progesterone,

estradiol- l7p

Acknowledgments This study was partially supported by NIH grants P30CA- 16058, DK459 16, CA-66 I 93 and the NATO Scientific Training Program/Scientific and Technical Research Council of Turkey. The FSH used in this study was supplied by the NIH National Hormone and Pituitary Program. Activin-A was generously provided by the Ajinomoto Co., Inc., Japan, through an arrangement by Dr. M. Takahashi of the Veterinary Medical Sciences, the University of Tokyo, Japan. ‘Current Address - Department of Animal Science, Washington State University, Pullman, WA 99 164. 6332. ‘To whom correspondence should be addressed - Laboratory of Reproductive and Molecular Endocrinology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210. 1092. TELE: 614-292-9706. FAX: 6 14-292-2077.

Theriogenology 45:1463-1472,

1996 0 1996 by Elsevier Science Inc.

0093.691XfS6/$15.00 PII s0093-691x(96)00114-3

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INTRODUCTION During each estrous cycle, in the mammal, a cohort of quiescent primordial follicles is recruited to develop. However, only a select few, in polytocous animals, eventually mature to a stage capable of ovulation. Most of the developing follicles undergo atresia and degenerate. This selective process is at least partially regulated by gonadotropins, but many local factors are also implicated (1). The general morphology of a mammalian ovarian follicle consists of an outer layer of theta cells and inner layers of granulosa cells which surround the oocyte. The 3 cell types within the follicle (theta, granulosa, and oocyte) interact both structurally and functionally, incorporating endocrine, paracrine, and autocrine processes. In the granulosa cell, these processes regulate cellular differentiation, which contributes to the state of follicular development (14). Differentiation of the follicle beyond the small follicle is characterized by increased aromatase activity, androgen synthesis, and estrogen synthesis (14). Transforming growth factor-p (TGF-P) and activin are 2 growth factors which belong to the same family as inhibin. Both TGF-0 (IO) and activin (4,23) have been isolated in mammalian ovaries; TGF-B is an approximately 25 kDa peptide with homodimeric subunits and is produced by almost all mammalian cells investigated, normal and transformed (I 2). The TGF-B family of peptide growth factors currently includes 5 members, TGF-P, to TGF-P,. Our laboratory has previously demonstrated that TGF-P, can regulate progesterone (PJ production in gonadotropin-stimulated porcine granulosa cells in vitro (5,6). Activin is an approximately 24 kDa peptide consisting of disulfide dimers of inhibin j3-subunits. The 3 members of activin are named depending on the dimeric constituents of inhibin PA- and/or pa-subunits. Activin possesses demonstrable roles in reproductive physiology both in vivo and in vitro (9). Our laboratory has also shown that activin-A, which is formed from a homodimer of inhibin j3,-subunits, can inhibit porcine granulosa cell steroidogenesis in vitro (25). Since TGF-P and activin are protein products, they presumably act by a transmembrane receptor. Although receptors for TGF-P and activin have been identified, their mechanisms of action are indefinite (I 8.24). Both factors bind to known receptors, a class (type II) of which belongs to a new subfamily of transmembrane protein serine kinases (I 9). Expression cloning of cDNA for type II TGF-0 receptors indicates that it is closely related to the activin receptor (ActRII) (I 7). In the current study, we treated porcine granulosa cells with doses of TGF-p, (I ng/ml) and activinA ( 100 ng/ml) that were determined in previous experiments to produce maximal inhibition of steroidogenesis (525). In order to assess the effects of these factors on cultured porcine granulosa cell differentiation, we measured FSH-stimulated and basal P4 productions, estradiol (I$) productions, and aromatase activities, three indicators of differentiation beyond the small follicle stage. We also treated cells with a combination of TGF-P, and activin-A to assess any synergistic effects of these 2 growth factors on porcine granulosa cell differentiation. At doses that produce maximal inhibition, if TGF-0, and activin-A utilize the same messenger system, then their combination would not be expected to produce synergistic effects. MATERIALS

AND METHODS

Chemicals Bovine albumin, gelatin (from swine skin), Triton X- 100, progesterone

(P4), estradiol- I7p (EJ and

Theriogenology

1465

activated charcoal were obtained from Sigma Chemical Company (St. Louis, MO). monobasic (NaH2P0,), (Fair Lawn, NJ).

Sodium phosphate

sodium azide, and ScintiPrep 2 were purchased from Fisher Scientific Company

Monoclonal

antibodies to Pq and E

were obtained from Biodesign International

(Kennebunkport, ME). Purified ether and methanol were acquired from JT Baker, Incorporated (Phillipsburg, NJ). Sodium phosphate diphasic (Na,HPO,) was purchased from EM Science (Cherry Hill, NJ). Sodium chloride was obtained from Jenneile Chemical Company (Cincinnati, OH). Dextran T-70 was acquired from Pharmacia (Piscataway, NJ). Toluene was purchased from Mallinckrodt, incorporated (Paris, KY). Porcine Granulosa Cell Culture All media used contained Dulbecco’s Modified Eagle/Ham’s F- I2 medium mixture (DME/F- 12, Sigma Chemical Co.) supplemented with 250 rig/L amphotericin B, 100 It-J/ml penicillin, and 100 ug/ml streptomycin (Gibco Laboratories, Grand Island, NY). Additional supplementations are mentioned as needed. Porcine granulosa cell culture procedures were adopted from Akira et al (2).

Porcine ovaries

obtained from a local slaughterhouse were transported on ice within 30 min of slaughter to the laboratory. The ovaries were then soaked in 70% ethanol for approximately I min and washed IO times with doubledistilled water. Granulosa cells were obtained by aspirating medium-sized follicles (2 to 5 mm in diameter) with a 20-gauge needle and a IO-cc syringe. Follicular fluids were pooled and centrifuged for 5 min at approximately 200 x g. The supernatant was removed, and cells were resuspended by gentle mixing in DME/F- I2 and recentrifuged. Removal of supernatant, resuspension, and centrifugation was repeated. After removing the remaining supernatant, the cells were suspended in DME/F- 12 supplemented (DME/F12/S) with I pg/ml insulin (Sigma), I II-J/ml thrombin (Sigma), and IO pg/ml low density lipoprotein (Sigma). Cells were then seeded into 24-well Corning culture plates (#X820, Corning Glass Works, Corning, NY) at a density of 2.0 x IO5 live cells/well. The number of live cells was determined by trypan exclusion method. The wells had been previously coated with IO% fetal calf serum (FCS, Hyclone, Logan, UT) by adding 1.0ml DME/F-I 2/S plus 10% FCS to each well 18 to 24 h prior to seeding. The 10% FCS media was completely removed approximately I h prior to seeding, and FCS coating was used to enhance cell attachment. Each well contained a total volume of I .O ml DME/F- 12/S. The cells were cultured ~OI 48 h (5% CO,, 95% air, 37” C) at which time porcine granulosa cells reached a monolayer of close to IOO% confluency. For gonadotropin stimulation, the wells were then washed 3 times with DME/F- 12, and I .O ml DME/F-12/S containing 100 ng/ml follicle- stimulating hormone (FSH, ovine NIH-FSH-SY, National Institutes of Health) was added to each well. The cells were allowed to incubate (5% CO,, 95% air, 37” C) for another 48 h. In the basal steroidogenesis study, the cells were washed 3 times and incubated (5% CO?, 95% air, 37” C) with DME/F-12/S for 48 h. Media were then removed and the cells were treated with control media (DMEIF-12/S only), I ng/ml TGF-0, (from porcine platelets, R&D Systems, Minneapolis, MN), 100 ng/ml activin-A, or TGF-P, plus activin-A in DME/F-12/S for 48 hours. Media and cells were collected at the end of treatment for P, and E? radioimmunoassay (RIA) and protein assay, respectively. Radioimmunoassay (RlA) The RLA procedures for Pd and E, measurements follow the methods described by Bartke et al (3). Before steroids were extracted from media samples, [I ,2,6,7-ZH]-P, (0.0 I8 yCi: DuPont NEN, Boston, MA) or ]2,4,6,7-‘H(N)]estradiol (0.018 pCi; DuPont NEN, Boston, MA) was added to each sample for measurement of extraction efficiency. Aliquots of media were mixed with tritiated steroid (0. I8 uCi) and monoclonal anti-steroid antibody (Sigma) and then incubated at room temperature and at 4°C

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Theriogenology

Mean f SEM $

2500

n=6

-3 z 2000 0 ‘G 2 1500 0

$B

1000

b _, !l& 500 0

FE

0 Control

TGF-6 1

Activin-A

TGF-O1 & Activin-A

Figure I.

Progesteroneproductionsfor FSH-stimulated porcine granulosa cells treated with transforming growth factor-o, (TGF-0,) and activin-A. Progesteroneproductions were significantly inhibited by treatments with TGF-P, (I ng/ml), activin-A (100 ng/ml), and TGF-0, (I @ml) plus activin-A (100 ng/ml). Bars with different letters are significantly different (P < 0.05).

for 6 h and I h, respectively.

Free and bound steroids were separated with dextran-coated activated

charcoal (Sigma), and the radioactivity of the bound fraction was measured on a liquid scintillation counter. Both the P., and Er concentrations were determined from a standard curve ranging from 12.5 to IO0 pg/ml. Steroid concentrations were expressed as pg/pg cell protein. Aromatase Activity Assay Immediately

after media were collected for P, and E2 RIA, cells in each well were cultured with

2 pCi [ Ip-‘HI-androstenedione (Amersham, Amersham, UK) in I .O ml DMEW12/S for 3 h. As previously described (3), aromatase activity was determined by measuring the amount of ‘H-H,0 released into the media after the 3-h exposure time. Aromatase activities were expressed as pmol ‘H-HI0 released/pg protein. Protein Assay Protein contents of the cells were determined by Bio-Rad microassay techniques (Bio-Rad Laboratories, Richmond, CA). Briefly, 0.8 ml of 0. I N NaOH (Sigma) in double distilled water was added to each well after removal of media. The NaOH solution was then removed and mixed with 0.2 ml BioRad protein assay dye reagent concentrate (Bio-Rad Laboratories, Richmond, CA) and absorbance

1467

Theriogenology

1000

Mean f SEM -g a 8 ‘5

n=6

40

30

b

b

T

3 g z ‘fi ii w”

I

i

t

800 600

20

400

10

200

0

0

TGF-6 1

Control

Activin-A

TGF-I+

0

& Activin-A Figure 2.

Estradiol-17B productionsand aromatase activities for FISH-stimulated porcine granulosa cells cells treated with transforming growth factor-p, (TGF-P, ) and activin-A. Estradiol- I7/3 productions (solid bars) and aromatase activities (cross-hatched bars) were significantly inhibited by treatments with TGF-B, (I q/ml), activin-A (100 ng/ml), and TGF-B, (I @ml) plus activin-A (100 ng/ml). Bars with different letters are significantly different (P < 0.05).

measured with a Beckman DU-70

Spectrophotometer (Beckman Instruments Inc., Fullerton, CA) using

S95-nm visible light. Statistical Analyses For all experiments, the results are presented as mean * SEM for 6 replicate cultures of each treatment. Each experiment utilized cells from different pools of granulosa cells, and each was repeated at least twice. The absolute steroid production values are often very different among batches of granulosa cells. Therefore, data shown for all assays performed on FSH-stimulated steroidogenesis were performed on the same pool of granulosa cells. Data shown for basal steroidogenesis were performed on a different pool of granulosa cells. Additionally, only representative experiments are reported rather than pooled data

from all replicate experiments. Data were evaluated with one-way analysis of variance. Post-tests were A P value of less than 0.05 was performed using the Bonferroni multiple comparisons procedure. considered statistically significant. RESULTS In FSH-stimulated

porcine granulosa cells, treatment with TGF-B, and activin-A were able to Fj productions (pg/pg total cell protein; mean f

significantly inhibit P4 and L$ accumulation in media.

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Theriogenology

Mean * SEM n=6

Control

TGF-8 1

A&k-A

TGF-I3z &

Activin-A Figure 3.

Basal progesterone productions for porcine granulosa cells cells treated with transforming growth factor-p, (TGF-P,) and activin-A. Progesterone productions were significantly inhibited by treatments with TGF-P, (I r&ml), activin-A (100 r&ml), and TGF-fi (1 nglml) plus activin-A (100 nglml). Bars with different letters are significantly different (P < 0.05).

SEM) for gonadotropin-stimulated porcine granulosa cells treated with control, I ng/ml TGF-B,, 100 ng/ml activin-A, and TGF-P, plus activin-A were 2554 + 297, 1164 + 102, 692 f 73, and 673 f 67, respectively (Figure 1). Ez steroidogenesis was assessed by 2 parameters, total E, detected secreted into media and aromatase activity. The E, concentrations in media (pglpg total cell protein; mean f SEM) from gonadotropin-stimulated porcine granulosa cells treated with control, 1 @ml TGF-B,, 100 nglml activin-A, and TGF-P, plus activin-A were 40.6 + 3.7,23.5 k I .9, 20.1 f 1.5, and 24.0 f 3.3, respectively (Figure 2). Aromatase assays were also performed in conjunction to assess whether altered Ez production may be due to altered aromatase activity, the enzyme that converts testosterone to Ez,. Aromatase activities (pmol/ug total cell protein/3 h; mean f SEM) for gonadotropin-stimulated porcine granulosa cells treated with control, 1 ng/ml TGF-P,, 100 ng/ml activin-A, and TGF-P, plus activin-A were 614.1 k 81.5, 27 1.8 f I 18.1,203.1 r 28.8, and 136.7 + 44.2, respectively (Figure 2). All 3 treatments were able to suppress PAand E2 production and aromatase activity significantly compared with that of the control (P c 0.05). Treatment with TGF-B, and activin-A were also able to significantly inhibit basd P and E steroidogenesis. Production of PJ (pg/pg total cell protein; mean + SEM) for porcine granulosa cells treated with control, 1 ng/ml TGF-B,, 100 @ml activin-A, and TGF-P, plus activin-A were 580.6 + 156.0, 67.7 -1- 1 I .7, 133.9 f 35.5 and 7 I .2 f 17.3, respectively (Figure 3). Production of E, (pglug total cell protein; mean -e SEM) from porcine granulosa cells treated with control, 1 nglml TGF-P,, 100 ng/ml nctivin-A and TGF-B, plus activin-A were 5.97 + 0.38, 3.20 r 0.34, 3. I5 +- 0.5 1 and 2.62 + 0.24, respectively (Figure 4). Aromatase activities (fmollug total cell protein/3 h; mean rt SEM) for porcine

Theriogenology

1469

200 Mean f SEM n=6 150 b

I

b

0

0 Control

TGF4 1

Activin-A

,, ,,y; + I!&

TGF-I3 1 & Activin-A

Figures 4.

Basal estradiol- I7p productions and aromatase activities for porcine granulosa cells treated with TGF-P, and activin-A. EstradioL17j3 productions (solid bars) and aromatase activities (crosshatched bats) were significantly inhibited by treatments with TGF-0, (1 ng/ml), activin-A (100 ng/ml), and TGF-P, (1 ng/ml) plus activin-A (100 ng/ml). Bars with different letters are significantly different (P < 0.05).

granulosa cells treated with control, 1 ng/ml TGF-P,, 100 ng/ml activin-A and TGF-P, plus activin-A were I3 I .8 2 3 I .6, 58.4 + 10.2,53.7 f 9. I, and 57.8 f 4.2, respectively (Figure 4). All 3 treatments were able to suppress P4 production, E, production, and aromatase activity significantly compared with the control (P < 0.05). DISCUSSION It has become apparent through recent research that growth factors produced by theta and granulosa cells may locally modulate ovarian cell proliferation, differentiation, steroidogenesis and angiogenesis (I 5). This study demonstrated that 2 growth factors, TGF-P, and activin-A, can inhibit gonadotropin-stimulated and basal steroidogeneses in cultured porcine granulosa cells from medium-sized follicles. Both of these growth factors have been isolated within the mammalian ovary (4,10,23) and have been previously shown in our laboratory to possess steroidogenic regulatory capabilities in cultured porcine granulosa cells (5,6,25). More specifically, this study examined the ability of TGF-P, and activin-A to regulate the biosynthetic pathways for androgen and estrogen, parameters for differentiation beyond the small follicle stage (14). Both TGF-P, (I @ml) and activin-A (I 00 r&ml) were able to attenuate P4 and E, production and aromatase activity, as assessed by 3H-Hz0 release from [I p’ HI-androstenedione. Our data show that

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Theriogenology

suppression of E, production by both growth factors is at least in part due to suppression of aromatase activities. The actions of TGF-P, are not surprising since it has been demonstrated to also inhibit luteinizing hormone (LH) and FSH receptor formations in porcine granulosa cells, actions that suggest an, atretic role in follicular development (I 5). This study supports the atretic role theory. One mechanism through which TGF-0, can inhibit FSH-stimulated steroidogenesis is through the aforementioned inhibition on FSH receptor formation. TGF-0, can also inhibit basal steroidogenesis, which suggests that this growth factor can more directly regulate steroid enzyme activities such as aromatase. If TGF-0, can truly suppress FSH receptor formation and, therefore, inhibit FSH-stimulated differentiation and steroidogenesis in porcine granulosa cells as well as suppress steroidogenic enzyme activities directly, then TGF-P, is potentially a major regulator of follicular atresia. However, this simplified and appealing theory is confounded by our previous finding that TGF-II,, in specific conditions, can also stimulate steroidogenesis in porcine granulosa cells (6). The exact role of TGF-0, in porcine granulosa cells may not be clarified until we understand the role of steroidogenic and non-steroidogenic factors in the regulation of TGF-9, expression in the ovary. Transforming growth factor-p has demonstrated vast abilities in many different tissues (I I). The actions of TGF-P often depend on the presence of specific environmental conditions, varying with cell type, presence of growth factors, and presence of other constituents; TGF-P stimulates proliferation in rat granulosa cells (l3), but inhibits growth in pig granulosa cells (7). Our laboratory has shown that TGF-P, can stimulate P4 synthesis in granulosa cells isolated from PMSG-stimulated mature rats (8) and inhibit P, production in cultured porcine granulosa cells (5.6). Within the same species and cell type, manipulations of environmental conditions have also produced opposing action (6,22). In this study, conditions permissive for the antisteroidogenic effects of TGF-0, were established to allow comparison with the antisteroidogenic activities of activin-A. Activin belongs in the same family of proteins as TGF-P. As with its familial counterpart, TGF-0, activin-A exhibits species-specific modulation of gonadotropin-induced differentiation of granulosa cells. Activin-A stimulated FSH-induced steroidogenesis and aromatase activity in rat granulosa cells (16.21). In contrast, this study demonstrated that activin-A inhibits FSHinduced and basal steroidogenesis and aromatase activity in porcine granulosa cells. It is unknown whether activin-A has any effect on gonadotropin receptor synthesis in granulosa cells. More information is needed on the effect of activin and its regulation in the ovary, with specific emphasis on changes in activin’s role as associated with changes in stage of follicular development. Like TGF-0, activin’s differential role in the follicle may depend on cell type, presence of other growth factors, and specific intra- and extracellular environments. It has been suggested that activin may function to establish and maintain the dominant follicle(s) during the preovulatory period, and to suppress the nondominant follicles (9). Our study indicates that activin-A may act as an atretic factor on medium-sized follicles, Although receptors for TGF-0, and activin-A have been identified, a consensus second messenger system has largely been unidentified until recently (18,24). Transforming growth factor-o, binds predominantly to the type II receptor. Expression cloning of the cDNA for the type II TGF-fi and activin (A&II) receptors disclose that they are closely related and belong to a new subfamily of transmembrane protein serine/threonine kinases (I 7,I9). This study evaluated the synergistic actions of maximal doses of TGF-P, and activin-A on porcine granulosa cell steroidogenesis. Since the 2 factors did not act synergistically in either gonadotropin-stimulated or basal studies, we speculate that they may act via the same or related second messenger systems. Our data are consistent with the hypothesis that activin-A and TGF-P, share the same second messenger system, a serinelthreonine kinase. It must also be noted that our data does not exclude the possibility that TGF-P, and activin-A may act via different second messenger systems that, perhaps, converge downstream at a common pathway or that may act in a competitive fashion

1471

Theriogenology

to inhibit steroidogenesis

in porcine granulosa cells.

This study has presented data which suggest that TGF-l3

and activin-A

are potential

atresia in porcine follicles. By their abilities to inhibit both basal and FSH-stimulated growth

factors present as major regulators of follicular

differentiation.

inducers of

steroidogeneses,

As mentioned

these

previously.

both

factors also possess differential functions that are dependent on microenvironmental conditions, The abilities to respond in a differential manner to the specific environment may confer an adaptive function to these growth factors. The functions of the growth factors adapt to the environment, allowing porcine granulosa cells to respond appropriately. Since TGF-P, can stimulate and suppress porcine granulosa cell steroidogenesis, it may be the local factor that senses environmental conditions and determines whethei the follicle

will develop or undergo atresia during the estrous cycle REFERENCES

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Ackland JF, Schwartz NB, Mayo KE, Dodson RE. Nonsteroidal Physiol Rev 1992;72:73 l-787.

2. Akira S, Ohmura H, Araki T, Lin YC. granulosa cells Theriogenology I994;4

Gossypol

inhibits aromatase activity

in cultured

porcine

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4. Braw-Tal R. Expression of mRNA for follistatin and inhibin/activin J Mol Endocrinol

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3. Bartke A, Steele RE, Musto N, Caldwell BU. Fluctuation rats and mice. Endocrinology 1973;92: l223- 1228. and atresia.

signals originating

subunits during follicular- growth

1994; 13:253-264.

5. Chang WY, Ohmura H, Coskun S, Lin YC. Transforming growth factor-p, (TGF-@ ) inhibits progesterone secretion in cultured porcine granulosa cells. In: Leung PCK, Hsueh AJW, Friesen HG (eds), Molecular Basis of Reproductive Endocrinology. New York: Springer-Verlag, I993;234-242. 6. Chang WY, Ohmura H, Kulp SK, Lin YC. Transforming growth factor-o, regulates differentiation of porcine granulosa cells in vitro. Theriogenology 1993;40:699-7 12. 7. Dorrington J, Chuma AV, Bendell JJ. Transforming growth factor B and follicle-stimulating promote rat granulosa cell proliferation, Endocrinology 1988; I23:353-359. 8. Dye RB, Rabinovici I992;47: I73- 185.

J, Jaffe RB.

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9. Findlay JK. An update on the roles of inhibin, folliculogenesis. Biol Reprod 1993;48:15-23.

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IO. Gangrade BK, May JV. The production of transforming growth factor-beta in the porcine ovary and its secretion in vitro. Endocrinology 1990; 127:2372-2380. I I. Gitay-Goren H, Kim IC, Miggans ST, Schomberg DW. Transforming growth factor p modulates gonadotropin receptor expression in porcine and rat granulosa cells differently. Biol Reprotl I993;48: 1284.1289. 12. Hammond JM. Peptide regulators in the ovarian follicle. Australian J Biol Sci I98 13. Hsuan JJ. Transforming growth factors beta. Brit Med Bull I989;45:425-437. 14. Hsueh AJ, Adashi EY, Jones PBC, Welsh TH. Hormonal ovarian granulosa cells. Endocr Rev I984;5:76- 127. IS.

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Hunter MG, Biggs C, Faillace LS, Picton HM. Current concepts of folliculogenesis polyovular farm species. J Reprod Fertil 1992;45 (SuppI): I-38.

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16. Hutchinson LA, Findlay JK, de Vos FL, Robertson DM. Effects of bovine inhibin, transforming growth factor-r), and bovine activin-A on granulosa cell differentiation. Biochem Biophys Res Commun 1987;146:140.5-1412. 17. Lin HY, Wang X-F, Ng-Eaton E, Weinberg RA, Lodish HF. Expression cloning of the TGF-H type II receptor, a functional transmembrane serine/threonine kinase. Cell I992;68:775-785.

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18. Massague J, Andres J, Attisano L, Cheifetz S, Lopez-Cassillas F, Ohtsuki M, Wrana JL. TGF-8 receptors. Mol Reprod Dev 1992;32:99- 104. 19. Mathews LS, Vale WW. Characterization of type II activin receptors. Binding, processing, and phosphorylation. J Biol Chemistry I993;268: I90 I3- 190 18. 20. Miro F, Smyth CD, Hillier SG. Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells. Endocrinology 199 I ; 129:3388-3394. 21. Mondschein JS, Canning SF, Hammond JM. Effects of transforming growth factor-p on the production of immunoreactive insulin-like growth factor I and progesterone and on [jH]thymidine incorporation in porcine granulosa cell cultures. Endocrinology 1988; 123: 197% 1976. 22. Ohmura H, Chang WY, Uzumcu M, Coskun S, Akira S, Araki T, Lin YC. Transforming growth factor-@1 stimulates progesterone production in cultured granulosa cells from gonadotropin-primed adult rats. In: Leung PCK, Hsueh AJW, Friesen HG (eds), Molecular Basis of Reproductive Endocrinology. New York: Springer-Verlag, 1993;215-22 I. 23. Roberts VJ, Barth S, el-Roeiy A, Yen SS. Expression of inhibinlactivin system messenger ribonucleic acid and proteins in avarian follicles from women with polycystic ovarian syndrome. J Clin Endocrinol Metab I994;79: I434- 1439. 24. Segarini PR. TGF-8 receptors. In: Bock CR, Marsh J (eds), Clinical Applications of TGF-B. New York: Wiley, 1991;29-50. 25. Shidaifat F, Chang WY, Uzumcu M, Coskun S, Kulp S, Lin YC. Activin-A inhibits steroidogenesis in porcine granulosa cells. Submitted for publication to Theriogenology.