0013-7227/90/1275-2372102.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 5 Printed in U.S.A.

The Production of Transforming Growth Factor Porcine Ovary and its Secretion in Vitro*

in the

BHUSHAN K. GANGRADE AND JEFFREY V. MAY Department of Obstetrics and Gynecology, The University of Kansas School of Medicine- Wichita, and The Women's Research Institute, Wichita, Kansas 67214

ABSTRACT. Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-/3 (TGF/3) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF/3 to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF/3 in the theca cell conditioned medium was quantitatively estimated by generating a TGF/3-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF/3-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF/3-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF/3 which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF/3-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF/3-neutralizing antibody (which recognizes TGF/3-1 and 2) but not nonimmune serum attenuated the TGF/3like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF/3. Since many cell types

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RANSFORMING growth factor type-/? (TGF/3), a 25,000 mol wt homodimeric protein, was first isolated from media conditioned by transformed cells and identified as the protein responsible for the phenotypic transformation of murine fibroblasts (1). TGF/3 is a multifunctional polypeptide since it can induce or inhibit proliferation of different cell types at least in vitro (2). Since its discovery a number of reports have appeared suggesting the occurrence of this growth factor in a wide variety of both normal and neoplastic tissues. The ubiq-

ReceivedJune 1, 1990. Address all correspondence and reprint requests to: Jeffrey V. May, Ph.D., The Women's Research Institute, Department of Obstetrics and Gynecology, The University of Kansas School of Medicine-Wichita, 2903 E. Central, Wichita, KS, 67214. * Presented in part at the 72nd Annual Meeting of The Endocrine Society, June 20-23, 1990, Atlanta, GA (Abstract 676). This work was supported in part by The Women's Research Institute, The Wesley Foundation, and a grant (J.V.M.) from The Wesley Medical Research Institutes, Wichita, KS.

secrete latent TGF/3 in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF/3 was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF/3-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acidethanol extracted protein fraction was mixed with trace amounts of 125I-TGF/3 for detection and chromatographed on Bio-Gel P60 column under acidic conditions. Elution of TGF/3 bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF/3. Preincubation of TGF/3-like activitycontaining fractions with TGF/3-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF/3 activity was also observed in fractions extracted from porcine corpora lutea. However, porcine follicular fluid collected from large size (>7 mm in diameter) follicles did not exhibit TGF/3 activity. The occurrence of TGF/3 in ovarian theca and corpora lutea, TGF/3 secretion in vitro by thecal cells, and regulation of granulosa cell proliferation by TGF/3 in vitro suggest the possibility of an autocrine/paracrine role of this growth factor in folliculogenesis and overall ovarian function. The lack of detectable levels of TGF/3 in the follicular fluid despite theca cell secretion of this growth factor suggests that TGF/3 actions are mediated locally. {Endocrinology 127: 2372-2380, 1990)

uitous distribution of TGF/3 along with its diverse effects on various cells prompted the search for this growth factor in the reproductive system. It has been shown that rat granulosa cells not only express TGF/3-mRNA (3) but also secrete high levels of TGF/3 when cultured in vitro (4). TGF/3 is also secreted by murine (5, 6) and bovine (6) thecal-interstitial cells. In addition to its effect on the proliferation of murine (7) and porcine (8) granulosa cells in culture, TGF/3 has also been reported to affect several ovarian phenomena such as FSH-dependent LH receptor induction in rat granulosa cells (9, 10), FSH-stimulated estrogen and progesterone production by granulosa cells (11-13), progesterone and androgen production by rat thecal-interstitial cells (14), and LHinduced maturation of follicle-enclosed rat oocytes (15). These findings suggest that TGF0 may be an important locally acting hormone involved in the regulation of follicle growth, differentiation of granulosa and theca

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TGF/3 PRODUCTION IN THE PORCINE OVARY

cells, oocyte maturation, and ovarian steroidogenesis. Despite the fact that the porcine ovary is used extensively in folliculogenic research, little information relating to the occurrence and/or secretion of TGF/3 by this species is available. Therefore, in the present study we have examined the occurrence of TGF/3 activity in various ovarian compartments in the pig (viz. the theca, follicular fluid and the corpus luteum). In addition we have also investigated the secretion of this growth factor by porcine theca cells in culture.

Materials and Methods Materials and reagents Fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (Logan, UT). Ham's F-12, Dulbecco's modified Eagle's medium, penicillin, streptomycin, fungizone and trypsin-EDTA were purchased from Gibco (Grand Island, NY). Purified preparations of human platelet-derived TGF/3 and TGF/3-neutralizing antibody were obtained from R & D Systems (Minneapolis, MN). 125I-TGF/3 was obtained from Biomedical Technologies, Inc. (Stoughton, MA). Mouse EGF was obtained from Collaborative Research (Bedford, MA). Collagenase, DNase, and globulin-free BSA prepared from fraction V bovine albumin were purchased from Sigma Chemical Company (St. Louis, MO). Falcon tissue culture flasks and Costar 48-well culture plates were obtained from Fisher Scientific Company (Springfield, NJ). Bio-Gel P-60 series of polyacrylamide gel beads were purchased from Bio-Rad Laboratories (Richmond, CA). All other reagents were analytical grade and obtained from either Fisher Scientific Company (Springfield, NJ) or Sigma Chemical Co. (St. Louis, MO). Cell culture Ovarian granulosa cells were isolated essentially as described by May and Schomberg (16). Briefly, porcine ovaries from adult cycling females were obtained from a local slaughterhouse. Ovaries were collected asceptically within 30 min of killing and carried to the laboratory in ice-cold normal saline supplemented with penicillin (20 U/ml), streptomycin (20 Mg/ml), and fungizone (0.5 Mg/ml). Hemifollicle linings from small (1-3 mm diameter) and large (>7 mm) follicles were dissected and the granulosa cells were removed by mechanical agitation. The isolated cells were suspended in serum-free nutrient medium (FD; Ham's F-12 + Dulbecco's modified Eagle's medium, 1:1) supplemented with penicillin (100 U/ml) and streptomycin (100 Mg/ml). Theca cells were prepared by collagenase (1 mg/ml) and DNase (5 Mg/ml) digestion of granulosa cell-free follicular linings at 37 C (60-90 min) followed by filtration through a 177 MM diameter nylon mesh screen to remove undigested tissue. Theca cell suspension was extensively washed with media (FD) to remove digestion enzymes. Cell viability, routinely 50-60%, was determined by trypan blue dye exclusion. Preparation and activation of conditioned media Granulosa and theca cells from small and large follicles were seeded in tissue culture flasks (Falcon; 25 cm2; 5 X 106 cells per

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flask) in FD supplemented with 5% fetal bovine serum (vol/ vol) and allowed to attach for 2 days. The unattached cells and media were removed by aspiration and the monolayer was washed ( 3 x 5 ml) with serum-free medium. The monolayer was then incubated at 37 C in FD (8 ml/flask) for 3 days after which the media were collected, centrifuged to remove debris, and stored at 4 C. Conditioned media were used within 14 days of collection. For heat activation of TGF/3-like activity in the conditioned media, the media were heated at 95 C for 5 min, cooled to the room temperature, supplemented with L-glutamine (final concentration 2 mM) and then used in culture. The untreated conditioned media were likewise supplemented with glutamine and used to compare the effect of heat-treatment of conditioned medium on TGF/?-like activity. Acid-activation of conditioned media involved dialysis against 1 M acetic acid using Spectropor dialysis membrane (mol wt cut off, 1000) followed by lyophilization of the dialysate. The dialysate was then dissolved in an appropriate volume of 4 mM HCl/0.1% BSA and tested in the bioassay for TGF/3-like activity. Isolation of TGFfi activity Hemifollicle linings after removal of the granulosa cells, consisting of mostly theca, were isolated from small (7 mm diameter) follicles by needle aspiration, centrifuged to remove cells, and stored frozen at —20 C. Tissues and follicular fluid were collected and pooled until enough material accumulated for extraction. Granulosa cell-free hemifollicle linings (33 g), corpora lutea (50 g), and follicular fluid (170 ml) were used for extraction. For the extraction and purification of TGF/3, the procedure of Assoian (17) was followed. Tissues (hemifollicle linings and corpora lutea) were washed twice in a solution containing 9 vol Tris buffer (17 mM Tris base, 150 mM NaCl, 0.1% glucose brought to pH 7.5 with HC1) and 1 vol citrate buffer (38 mM citric acid, 88 mM sodium citrate, 2.2% glucose). Washed tissues were added to a solution (4 ml/g tissue) of acid-ethanol (375 ml 95% ethanol, 7.5 ml concentrated HC1, 33 mg phenylmethylsulfonyl fluoride and 1.9 mg Pepstatin) and extracted in a homogenizer until homogeneous. The follicular fluid was mixed with acidethanol (1:4). The acid-ethanol extracts were incubated overnight at 4 C and precipitated proteins were removed by centrifugation. The resulting supernatant was adjusted to pH 3.0 by the addition of concentrated ammonium hydroxide. Proteins were precipitated by addition of freshly opened ethyl ether to the pH 3.0 solution and incubated overnight at 4 C. The precipitate was collected, either by centrifugation or filtration through Whatman no. 1 filter paper, and dissolved in 1 M acetic acid by incubating at 4 C overnight with stirring. The solution was clarified by centrifugation and lyophilized. Tissue and follicular fluid extracts were dissolved in (5-6 ml) 1 M acetic acid and gel-filtered at a flow rate of 9 ml/h on a Bio-Gel P-60 column (2.2 X 85 cm; bed volume, 422 ml; void volume, 120 ml) equilibrated in 1 M acetic acid. The column was characterized using BSA (mol wt 67,000), ovalbumin (mol wt 43,000) chymotrypsinogen A (mol wt 25,000) and cytochrome c (mol wt

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TGF/3 PRODUCTION IN THE PORCINE OVARY

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12,400) as mol wt markers. The sample load was mixed with approximately 2.0 X 105 cpm 125I-TGF/3 (