0021-972X/90/7105-1396$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
RECOMBINANT HUMAN ACTIVIN-A PROMOTES PROLIFERATION OF HUMAN LUTEINIZED PREOVULATORY GRANULOSA CELLS IN VITRO JARON RABINOVICI, SUSAN J. SPENCER AND ROBERT B. JAFFE Reproductive Endocrinology Center, Department of Obstetrics, Gy necology and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143 ABSTRACT: Ovarian granulosa cells synthesize and secrete activin, a member of the transforminggrowth factorbeta (TGF-P) peptide family, during the follicular phase of the menstrual cycle. We examined the growth-promoting activity of human recombinant activin-A on human luteinized preovulatory granulosa cells obtained from women undergoing in vitro fertilization. Activin-A induced proliferation of granulosa cells on day 5 of culture in a dosedependent manner. Maximal effects were seen at concentrations £. 100 ng/mL with an ED50of 15 ng/mL. We suggest that activin-A is able to modulate growth of ovarian granulosa cells.
Activin and inhibin are structurally related dimeric glycoproteins initially characterized by their ability to alter FSH secretion from the pituitary (1). In contrast to inhibin, activin stimulates FSH release from the pituitary in vitro and in vivo (2). Both activin and inhibin modulate intragonadalgonadotropin-stimulated steroidogenesis (1). Inhibin is composed of an alpha and one of two beta (PA or Pn) subunits that form inhibin-A and inhibin-B, respectively. The three possible dimers consisting of the two (3 subunits have been designated activin-A (pA/pA), activin-AB (PA/pB) and activin-B (PB/pB) (1, 3-5). Activin-A is identical to the polypeptide dimer that induces differentiation of the mouse Friend erythroleukemia cell line named erythroid differentiation factor (EDF) (6). The P-subunit common to these proteins has DNA and amino acid sequence homology with transforming growth factor-beta (TGFP), a ubiquitous homodimeric protein with actions on many tissues and cell types (1). Ovarian TGF-P, probably of theca/interstitial cell origin, has been shown to locally regulate granulosa cell function, differentiation and proliferation (7-13). Specifically, murine granulosa cells responded in vitro to TGF-P with increased FSH-stimulated aromatase activity and DNA synthesis as assessed by thymidine incorporation (9,11,12). In other studies, activin/EDF also augmented FSH-stimulated aromatase activity and induced FSH receptor expression in murine granulosa cells (6, 10). A receptor for EDF with a Kd = 3.4 x 1010 was identified on rat granulosa cells (14). Based on the structural and functional similarity to TGF-P we hypothesized that activin-A modulates proliferation of ovarian follicular cells. Therefore, the aim of the present study was to examine the ability of reSubmittcd April 20, 1990 Address all correspondence to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, University of California San Francisco, San Francisco, CA 94143.
combinant human (rh) activin-A to regulate in vitro mitogenesis of human luteinized preovulatory granulosa cells obtained from women undergoing ovarian hyper stimulation for in vitro fertilization (IVF). We report that rh-activin-A exerts a proliferative effect on cultured ovarian granulosa cells. MATERIALS AND METHODS Materials: Medium 199 supplemented with Earle's balanced salt solution (EBSS), glutamine, gentamicin, fetal bovine serum (FBS), and saline with 0.05 % trypsin-versine were prepared in and obtained from the Cell Culture Facility, University of California, San Francisco (UCSF). Collagenase-dispase was obtained from Boehringer-Mannheim (Mannheim, West Germany) and Ficoll-Paque from Pharmacia Fine Chemicals (Piscataway, NJ). Tissue culture dishes were from Falcon Plastic (Los Angeles, CA). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were obtained from Dr. Denis Gospodarowicz (UCSF). EGF was purified as described by Savage and Cohen (15) from the submaxillary glands of adult male Swiss Webster mice. FGF was purified from bovine pituitary glands and brains (16). Rh-activin-A was generously provided by Genentech, South San Francisco, CA. Cell culture: Human luteinized granulosa cells were obtained by follicular aspiration from regularly menstruating women undergoing ovum retrieval for IVF performed because of tubal obstruction. Follicular development was stimulated by human menopausal gonadotropins(Serono, Randolph, MA) in the presence of a GnRHanalog(Lupron, TAP Pharmaceuticals, North Chicago, IL) until adequate response was achieved. The criteria for adequate response included 3 follicles ^14 mm with at least 1 follicle > 16 mm andanestradiol concentration >600 pg/mL which was inc7-easingdaily. Final maturation of the oocytes was effected with 10,000 IU human chorionic gonadotropin (hCG, Serono, Randolph, MA). Retrieval was accomplished 35 h after that injection. Granulosa cells aspirated from
1396
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RAPID COMMUNICATIONS follicles £.15 mm in diameter were used in this study. The cells were washed with medium 199 twice. Granulosa cells and red blood cells were transferred to a 12ml tube containing 3.5 mL Ficoll-Paque and separated by centrifugation at 600 x g for 5 minutes. Granulosa cells formed a thin layer between the upper phase (culture medium) and lower phase (Ficoll-Paque), and red blood cells settled to the bottom of the tube. Granulosa cells were dispersed at 37 C for 30 min with gentle shaking in 5 mL culture medium containing 0.1 % collagenase-dispase and 20 mg DNase/mL. The dispersed cells were washed in culture medium, counted and plated at a density of 10,000 cells/well. The culture medium consisted of medium M-199 containing 10%FBS, 2 mM glutamine, and 50 mg/mLgentamicin. The cells were plated on plastic dishes (24 multiwell plates) and were cultured at 37 C in 95% air, 5% CO2 with or without rh-activin-A (1-300 ng/mL). Media were changed every other day. Spent media were collected and stored at -20 C. Cell number in each well was determined with a Coulter counter (Hialeah, FL) after trypsinization on days 2-5 after initiation of culture. Each data point represents £. 3 experiments in duplicate or triplicate and each experiment was performed with cells obtained from 1 or 2 different patients. Statistical comparisons between groups were made by oneway analysis of variance (ANOVA) and Fisher PLSD or Scheffe F-test as post-tests. Results are expressed as mean ± SE. Significance was assumed at P ^ 0.05. 35000
5
30000 •
25000 •
20000 •
15000
Actlvin-A ng/ml (log)
Fig. 1: Differences in cell number on day 5 of culture after treatment with different doses of activin-A(l-300 ng/mL). (* p< 0.05 vs. control).
1397
RESULTS Stimulation of granulosa cells with activin-A resulted in a statistically significant, dose-dependent increase in granulosa cell number on days 4 and 5 of culture. On day 5, a maximal increase in cell number (mean increase of 39% over unstimulated control wells) was observed at concentrations ;>100 ng/mL (Fig. 1). The ED50for this proliferative effect was 15 ng/mL. No apparentmorphological changes were observed in wells containing activin-A compared to untreated wells. Figure 2 demonstrates the effects of activin-A in another series of experiments on granulosa cell number on day 5 in comparison to the proliferative effects of two known granulosa cell mitogens, EGF and bFGF (17). After 5 days in culture, the proliferative effect of activin at a dose of 100 ng/mL (4 x 10'9M) was equivalent to that of bFGF at a dose of 10 ng/mL (5 x 1010 M) and EGF at 50 ng/mL(7.7 x 109 M). DISCUSSION The data presented provide the first evidence that activin-A may act as a growth-promoting factor on ovarian granulosa cells. This stimulatory effect of acti vin A was comparable to responses seen with two known stimulants of granulosa cell proliferation in vitro, EGF and bFGF (Fig. 2) (17). These effects were seen at similar molar concentrations for EGF and activin-A while bFGF was about 10-fold more effective on a molar basis. Previous research in murine granulosa cells indicated that these ovarian cells are a target tissue for activin-A/EDF action and that this peptide enhances steroidogenic competence during the follicular phase in these cells (6,10,14). The ED50ofthe growth promoting effect of activin reported here correlates well with the Kd previously reported for EDF on rat granulosa cells (14). Thus, activin-A in addition to its reported differentiative effects also has the ability to act as a growth modulator of ovarian granulosa cells. In a recent study, rh-activin-A was found to influence DNA synthesis of two gonadal cell lines derived from Chinese hamster ovaries (CHO-Kl) and a rat Leydigcell testicular tumor (R2C) (18). Activin-A inhibited the uptake of a thymidineagonist(bromo-deoxyuridine)by CHO-Kl cells( 18). In a parallel study to the one reported here, we have recently found an inhibitory effect ofrh-activin-A on in uitro proliferation of human fetal adrenal cells (manuscript in preparation). Therefore, we suggest that, similar to TGF-P, activin-A can act as either a positive or a negative modulator of cell growth of different human cell types. Ample evidence exists that ovarian cells synthesize and secrete inhibin/activin. Follicular fluid contains biologically active activin-A and activin-AB (1). Im-
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RAPID COMMUNICATIONS
1398
JCE & M • 1990 Vol 71 • No 5
factor regulating human follicle/corpus luteum growth, development and differentiation. REFERENCES
Control ActiviniOOng EGF 50 ng
bFGF 1ng bFGFIOng
Fig. 2: Differences in cell number on day 5 of culture1 with activin 100 ng/mL, EGF 50 ng/mL and bFGF at V and 10 ng/mL (* p< 0.05 vs. control). munocytochemical and in situ hybridization studies have localized the subunits of inhibin/activin to granulosa cells of follicles in murine, porcine and primate ovaries (2,19-21). The expression of the mRNA of these subunits was shown to undergorapid changes throughout the stages of the menstrual cycle and to depend on the degree of development of the ovarian follicle (5, 19, 20). In monkey ovaries, Schwall et al. detected only f3Bsubunit mRNA expression in granulosa cells of small antral follicles, while granulosa cells of dominant follicles contained both the a- and (3A-, but not (3D-subunit mRNA (5). These investigators indicated that in another study cells transfected with the (3B-subunit cDNA secreted the (3B-subunit dimer activin-B, which exhibited similar biological activities to activin-A(5). The authors concluded that granulosa cells of small antral follicles secrete activin-B that in turn influences follicular development through autocrine/paracrine mechanisms (5). The secretion patterns of inhibin/activin by the larger follicles are difficult to interpret based on the presence of both a and |3A-subunits and their mRNAs. The presence of detectable activin in follicular fluid of larger follicles indicates that it is also produced at later stages of follicular development (1). Taken together, these data indicate that activin is produced and/or present in the ovarian follicle throughout the follicular phase. The development of specific antibodies directed against the intact activin molecule should further elucidate the secretion patterns and levels of ovarian activin throughout the menstrual cycle. Until recently, most studies have focused on activin's stimulatory role on pituitary FSH secretion. The data obtained in this study, coupled with previous studies on the action of activin on ovarian granulosa cells, suggest that this dimer, similar to the structurally related peptide TGF-P, may be an important paracrine/autocrine
1. Vale W, Rivier C, Hsueh A, ct al. Chemical and biological characterization of the inhibin family of protein hormones. Rec Prog Horm Res. 1988;44:1. 2. Schwall R, SchmelzerCH.Matsuyama E,Ma6on AJ. Multiple actions of recombinant activin-A in vivo. Endocrinology. 1989; 125:1420-3. 3. Vale W, Rivier J, Vaughan J, ct al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986,321:776-9. 4. LingN, YingSY, UcnoN, ctal. A homodimer of the bcta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem Biophys Res Commun. 1986;138:1129-37. 5. Schwall R, Mason AJ, Wilcox J, BasscttS, Zcleznick A. Localization ofinhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol. 1990;4:75-9. 6. Hasegawa Y, Miyamoto K, Abe Y, etal. Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cells. Biochem Biophys Res Commun. 1988;156:668-74. 7. Ying SY, Becker A, Haird A, et al. Type beta transforming growth factor (TGF-bcta) is a potent stimulator of the basal secretion of follicle stimulating hormone (FSH) in a pituitary monolaycr system. Biochem Biophys Res Commun. 1986; 135:950-6. 8. YingS, Becker A, LingN, UcnoN, Guillcmin R. Inhibin and beta type transforming growth factor (TGFP) have opposite modulating effects on the follicle stimulating hormone (FSH)-induccd aromatasc activity of cultured rat granulosa cells. Biochem Biophys Res Commun. 1986;136:969. 9. Skinner M, Kcski-Oja J, Ostcen K, MOBCH H. Ovarian thecal cells produce transforming growth factor-beta which can regulate granulosa cell growth. Endocrinology. 1987;121:786-92. 10. Hutchinson LA, Findlay JK, de Vos F, Robertson DM. Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation. Biochem Biophys Res Commun. 1987;146:1405-12. 11. Bcndcll J, Dorrington J. Rat thccal/intcrstitial cells secrete a transforming growth factor-beta-like factor that promotes growth and differentiation in rat granulosa cells. Endocrinology. 1988;123:941-8. 12. Adashi E, Resnick C, Hernandez E, May J, Purchio A, Twardzik D. Ovarian transforming growth factor-beta (TGF beta): cellular sitc(s), and mechanism(s) of action. Mol Cell Endocrinol. 1989;61:247-56. 13. Adashi E, Resnick C. Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation. Endocrinology. 1986; 119:1879. 14. SuginoH,NakamuraT, Hasegawa Y, etal. Identification of a specific receptor for erythroid differentiation factor on folliculargranulosa cells. J Biol Chcm. 1988;263:15249-52. 15. Savage C, Cohen S. Epidermal growth factor and a new derivative. J Biol Chcm. 1972;247:7609. 16. Gospodarowicz D, Bialecki H, Grecnburg G. Purification of the fibroblast growth factor activity from bovine brain. .1 Biol Chem. 1978;253:3736. 17. Tapancinen J, Lcinoncn P, Tapancincn P, Yamamoto M, JafTc R. Regulation of human granulosa-lutcal cell progesterone production and proliferation by gonadotropins and growth factors. Fertil Steril. 1987;48:576. 18. Gonzalez-Manchon C. Activin-A, inhibin and transforming growth factor-P modulate proliferation of twogonadal cell lines. Endocrinology. 1989;125:1666-72. 19. Meunier H, Cajander S, Roberts V, et al. Rapid changes in the expression ofinhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat cstrous cycle. Mol Endocrinol. 1988;2:1352-63. 20. Torney A, Hodgson Y, Forage R, de Krctscr D. Cellular localization ofinhibin mRNA in the bovine ovary by in-situ hybridization. J Rcprod Fertil. 1989;86:391-9. 21. Woodruff TK, D'Agostino J, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. Science. 1988;239:1296-9. J. R. is a recipient of a Fogarty scholarship.
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Vol. 71, No. 5 Printed in U.S.A.
RECOMBINANT HUMAN ACTIVIN-A PROMOTES PROLIFERATION OF HUMAN LUTEINIZED PREOVULATORY GRANULOSA CELLS IN VITRO JARON RABINOVICI, SUSAN J. SPENCER AND ROBERT B. JAFFE Reproductive Endocrinology Center, Department of Obstetrics, Gy necology and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143 ABSTRACT: Ovarian granulosa cells synthesize and secrete activin, a member of the transforminggrowth factorbeta (TGF-P) peptide family, during the follicular phase of the menstrual cycle. We examined the growth-promoting activity of human recombinant activin-A on human luteinized preovulatory granulosa cells obtained from women undergoing in vitro fertilization. Activin-A induced proliferation of granulosa cells on day 5 of culture in a dosedependent manner. Maximal effects were seen at concentrations £. 100 ng/mL with an ED50of 15 ng/mL. We suggest that activin-A is able to modulate growth of ovarian granulosa cells.
Activin and inhibin are structurally related dimeric glycoproteins initially characterized by their ability to alter FSH secretion from the pituitary (1). In contrast to inhibin, activin stimulates FSH release from the pituitary in vitro and in vivo (2). Both activin and inhibin modulate intragonadalgonadotropin-stimulated steroidogenesis (1). Inhibin is composed of an alpha and one of two beta (PA or Pn) subunits that form inhibin-A and inhibin-B, respectively. The three possible dimers consisting of the two (3 subunits have been designated activin-A (pA/pA), activin-AB (PA/pB) and activin-B (PB/pB) (1, 3-5). Activin-A is identical to the polypeptide dimer that induces differentiation of the mouse Friend erythroleukemia cell line named erythroid differentiation factor (EDF) (6). The P-subunit common to these proteins has DNA and amino acid sequence homology with transforming growth factor-beta (TGFP), a ubiquitous homodimeric protein with actions on many tissues and cell types (1). Ovarian TGF-P, probably of theca/interstitial cell origin, has been shown to locally regulate granulosa cell function, differentiation and proliferation (7-13). Specifically, murine granulosa cells responded in vitro to TGF-P with increased FSH-stimulated aromatase activity and DNA synthesis as assessed by thymidine incorporation (9,11,12). In other studies, activin/EDF also augmented FSH-stimulated aromatase activity and induced FSH receptor expression in murine granulosa cells (6, 10). A receptor for EDF with a Kd = 3.4 x 1010 was identified on rat granulosa cells (14). Based on the structural and functional similarity to TGF-P we hypothesized that activin-A modulates proliferation of ovarian follicular cells. Therefore, the aim of the present study was to examine the ability of reSubmittcd April 20, 1990 Address all correspondence to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, University of California San Francisco, San Francisco, CA 94143.
combinant human (rh) activin-A to regulate in vitro mitogenesis of human luteinized preovulatory granulosa cells obtained from women undergoing ovarian hyper stimulation for in vitro fertilization (IVF). We report that rh-activin-A exerts a proliferative effect on cultured ovarian granulosa cells. MATERIALS AND METHODS Materials: Medium 199 supplemented with Earle's balanced salt solution (EBSS), glutamine, gentamicin, fetal bovine serum (FBS), and saline with 0.05 % trypsin-versine were prepared in and obtained from the Cell Culture Facility, University of California, San Francisco (UCSF). Collagenase-dispase was obtained from Boehringer-Mannheim (Mannheim, West Germany) and Ficoll-Paque from Pharmacia Fine Chemicals (Piscataway, NJ). Tissue culture dishes were from Falcon Plastic (Los Angeles, CA). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were obtained from Dr. Denis Gospodarowicz (UCSF). EGF was purified as described by Savage and Cohen (15) from the submaxillary glands of adult male Swiss Webster mice. FGF was purified from bovine pituitary glands and brains (16). Rh-activin-A was generously provided by Genentech, South San Francisco, CA. Cell culture: Human luteinized granulosa cells were obtained by follicular aspiration from regularly menstruating women undergoing ovum retrieval for IVF performed because of tubal obstruction. Follicular development was stimulated by human menopausal gonadotropins(Serono, Randolph, MA) in the presence of a GnRHanalog(Lupron, TAP Pharmaceuticals, North Chicago, IL) until adequate response was achieved. The criteria for adequate response included 3 follicles ^14 mm with at least 1 follicle > 16 mm andanestradiol concentration >600 pg/mL which was inc7-easingdaily. Final maturation of the oocytes was effected with 10,000 IU human chorionic gonadotropin (hCG, Serono, Randolph, MA). Retrieval was accomplished 35 h after that injection. Granulosa cells aspirated from
1396
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RAPID COMMUNICATIONS follicles £.15 mm in diameter were used in this study. The cells were washed with medium 199 twice. Granulosa cells and red blood cells were transferred to a 12ml tube containing 3.5 mL Ficoll-Paque and separated by centrifugation at 600 x g for 5 minutes. Granulosa cells formed a thin layer between the upper phase (culture medium) and lower phase (Ficoll-Paque), and red blood cells settled to the bottom of the tube. Granulosa cells were dispersed at 37 C for 30 min with gentle shaking in 5 mL culture medium containing 0.1 % collagenase-dispase and 20 mg DNase/mL. The dispersed cells were washed in culture medium, counted and plated at a density of 10,000 cells/well. The culture medium consisted of medium M-199 containing 10%FBS, 2 mM glutamine, and 50 mg/mLgentamicin. The cells were plated on plastic dishes (24 multiwell plates) and were cultured at 37 C in 95% air, 5% CO2 with or without rh-activin-A (1-300 ng/mL). Media were changed every other day. Spent media were collected and stored at -20 C. Cell number in each well was determined with a Coulter counter (Hialeah, FL) after trypsinization on days 2-5 after initiation of culture. Each data point represents £. 3 experiments in duplicate or triplicate and each experiment was performed with cells obtained from 1 or 2 different patients. Statistical comparisons between groups were made by oneway analysis of variance (ANOVA) and Fisher PLSD or Scheffe F-test as post-tests. Results are expressed as mean ± SE. Significance was assumed at P ^ 0.05. 35000
5
30000 •
25000 •
20000 •
15000
Actlvin-A ng/ml (log)
Fig. 1: Differences in cell number on day 5 of culture after treatment with different doses of activin-A(l-300 ng/mL). (* p< 0.05 vs. control).
1397
RESULTS Stimulation of granulosa cells with activin-A resulted in a statistically significant, dose-dependent increase in granulosa cell number on days 4 and 5 of culture. On day 5, a maximal increase in cell number (mean increase of 39% over unstimulated control wells) was observed at concentrations ;>100 ng/mL (Fig. 1). The ED50for this proliferative effect was 15 ng/mL. No apparentmorphological changes were observed in wells containing activin-A compared to untreated wells. Figure 2 demonstrates the effects of activin-A in another series of experiments on granulosa cell number on day 5 in comparison to the proliferative effects of two known granulosa cell mitogens, EGF and bFGF (17). After 5 days in culture, the proliferative effect of activin at a dose of 100 ng/mL (4 x 10'9M) was equivalent to that of bFGF at a dose of 10 ng/mL (5 x 1010 M) and EGF at 50 ng/mL(7.7 x 109 M). DISCUSSION The data presented provide the first evidence that activin-A may act as a growth-promoting factor on ovarian granulosa cells. This stimulatory effect of acti vin A was comparable to responses seen with two known stimulants of granulosa cell proliferation in vitro, EGF and bFGF (Fig. 2) (17). These effects were seen at similar molar concentrations for EGF and activin-A while bFGF was about 10-fold more effective on a molar basis. Previous research in murine granulosa cells indicated that these ovarian cells are a target tissue for activin-A/EDF action and that this peptide enhances steroidogenic competence during the follicular phase in these cells (6,10,14). The ED50ofthe growth promoting effect of activin reported here correlates well with the Kd previously reported for EDF on rat granulosa cells (14). Thus, activin-A in addition to its reported differentiative effects also has the ability to act as a growth modulator of ovarian granulosa cells. In a recent study, rh-activin-A was found to influence DNA synthesis of two gonadal cell lines derived from Chinese hamster ovaries (CHO-Kl) and a rat Leydigcell testicular tumor (R2C) (18). Activin-A inhibited the uptake of a thymidineagonist(bromo-deoxyuridine)by CHO-Kl cells( 18). In a parallel study to the one reported here, we have recently found an inhibitory effect ofrh-activin-A on in uitro proliferation of human fetal adrenal cells (manuscript in preparation). Therefore, we suggest that, similar to TGF-P, activin-A can act as either a positive or a negative modulator of cell growth of different human cell types. Ample evidence exists that ovarian cells synthesize and secrete inhibin/activin. Follicular fluid contains biologically active activin-A and activin-AB (1). Im-
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RAPID COMMUNICATIONS
1398
JCE & M • 1990 Vol 71 • No 5
factor regulating human follicle/corpus luteum growth, development and differentiation. REFERENCES
Control ActiviniOOng EGF 50 ng
bFGF 1ng bFGFIOng
Fig. 2: Differences in cell number on day 5 of culture1 with activin 100 ng/mL, EGF 50 ng/mL and bFGF at V and 10 ng/mL (* p< 0.05 vs. control). munocytochemical and in situ hybridization studies have localized the subunits of inhibin/activin to granulosa cells of follicles in murine, porcine and primate ovaries (2,19-21). The expression of the mRNA of these subunits was shown to undergorapid changes throughout the stages of the menstrual cycle and to depend on the degree of development of the ovarian follicle (5, 19, 20). In monkey ovaries, Schwall et al. detected only f3Bsubunit mRNA expression in granulosa cells of small antral follicles, while granulosa cells of dominant follicles contained both the a- and (3A-, but not (3D-subunit mRNA (5). These investigators indicated that in another study cells transfected with the (3B-subunit cDNA secreted the (3B-subunit dimer activin-B, which exhibited similar biological activities to activin-A(5). The authors concluded that granulosa cells of small antral follicles secrete activin-B that in turn influences follicular development through autocrine/paracrine mechanisms (5). The secretion patterns of inhibin/activin by the larger follicles are difficult to interpret based on the presence of both a and |3A-subunits and their mRNAs. The presence of detectable activin in follicular fluid of larger follicles indicates that it is also produced at later stages of follicular development (1). Taken together, these data indicate that activin is produced and/or present in the ovarian follicle throughout the follicular phase. The development of specific antibodies directed against the intact activin molecule should further elucidate the secretion patterns and levels of ovarian activin throughout the menstrual cycle. Until recently, most studies have focused on activin's stimulatory role on pituitary FSH secretion. The data obtained in this study, coupled with previous studies on the action of activin on ovarian granulosa cells, suggest that this dimer, similar to the structurally related peptide TGF-P, may be an important paracrine/autocrine
1. Vale W, Rivier C, Hsueh A, ct al. Chemical and biological characterization of the inhibin family of protein hormones. Rec Prog Horm Res. 1988;44:1. 2. Schwall R, SchmelzerCH.Matsuyama E,Ma6on AJ. Multiple actions of recombinant activin-A in vivo. Endocrinology. 1989; 125:1420-3. 3. Vale W, Rivier J, Vaughan J, ct al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986,321:776-9. 4. LingN, YingSY, UcnoN, ctal. A homodimer of the bcta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem Biophys Res Commun. 1986;138:1129-37. 5. Schwall R, Mason AJ, Wilcox J, BasscttS, Zcleznick A. Localization ofinhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol. 1990;4:75-9. 6. Hasegawa Y, Miyamoto K, Abe Y, etal. Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cells. Biochem Biophys Res Commun. 1988;156:668-74. 7. Ying SY, Becker A, Haird A, et al. Type beta transforming growth factor (TGF-bcta) is a potent stimulator of the basal secretion of follicle stimulating hormone (FSH) in a pituitary monolaycr system. Biochem Biophys Res Commun. 1986; 135:950-6. 8. YingS, Becker A, LingN, UcnoN, Guillcmin R. Inhibin and beta type transforming growth factor (TGFP) have opposite modulating effects on the follicle stimulating hormone (FSH)-induccd aromatasc activity of cultured rat granulosa cells. Biochem Biophys Res Commun. 1986;136:969. 9. Skinner M, Kcski-Oja J, Ostcen K, MOBCH H. Ovarian thecal cells produce transforming growth factor-beta which can regulate granulosa cell growth. Endocrinology. 1987;121:786-92. 10. Hutchinson LA, Findlay JK, de Vos F, Robertson DM. Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation. Biochem Biophys Res Commun. 1987;146:1405-12. 11. Bcndcll J, Dorrington J. Rat thccal/intcrstitial cells secrete a transforming growth factor-beta-like factor that promotes growth and differentiation in rat granulosa cells. Endocrinology. 1988;123:941-8. 12. Adashi E, Resnick C, Hernandez E, May J, Purchio A, Twardzik D. Ovarian transforming growth factor-beta (TGF beta): cellular sitc(s), and mechanism(s) of action. Mol Cell Endocrinol. 1989;61:247-56. 13. Adashi E, Resnick C. Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation. Endocrinology. 1986; 119:1879. 14. SuginoH,NakamuraT, Hasegawa Y, etal. Identification of a specific receptor for erythroid differentiation factor on folliculargranulosa cells. J Biol Chcm. 1988;263:15249-52. 15. Savage C, Cohen S. Epidermal growth factor and a new derivative. J Biol Chcm. 1972;247:7609. 16. Gospodarowicz D, Bialecki H, Grecnburg G. Purification of the fibroblast growth factor activity from bovine brain. .1 Biol Chem. 1978;253:3736. 17. Tapancinen J, Lcinoncn P, Tapancincn P, Yamamoto M, JafTc R. Regulation of human granulosa-lutcal cell progesterone production and proliferation by gonadotropins and growth factors. Fertil Steril. 1987;48:576. 18. Gonzalez-Manchon C. Activin-A, inhibin and transforming growth factor-P modulate proliferation of twogonadal cell lines. Endocrinology. 1989;125:1666-72. 19. Meunier H, Cajander S, Roberts V, et al. Rapid changes in the expression ofinhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat cstrous cycle. Mol Endocrinol. 1988;2:1352-63. 20. Torney A, Hodgson Y, Forage R, de Krctscr D. Cellular localization ofinhibin mRNA in the bovine ovary by in-situ hybridization. J Rcprod Fertil. 1989;86:391-9. 21. Woodruff TK, D'Agostino J, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. Science. 1988;239:1296-9. J. R. is a recipient of a Fogarty scholarship.
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