0013-7227/90/1261-0216$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Insulin-Like Growth Factor-I (IGF-I) and IGF-II Hormonal Action in Cultured Rat Granulosa Cells: Mediation via Type I but not Type II IGF Receptors* ELI Y. ADASHI, CAROL E. RESNICK, AND RON G. ROSENFELD Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and Physiology, University of Maryland School of Medicine (E. Y.A., C.E.R.), Baltimore, Maryland 21201; and the Division of Endocrinology and Metabolism, Department of Pediatrics, Stanford University (R.G.R.), Stanford, California 94143
ABSTRACT. Both insulin-like growth factor-I (IGF-I) and IGF-II have been shown to promote granulosa cell differentiation and proliferation. While both type I and type II IGF receptors have been observed in rat granulosa cells, the identity of the IGF receptor type(s) mediating IGF hormonal action remains uncertain. Whereas the role of the rat type I IGF receptor cannot be completely evaluated at this time due to the lack of specific reagents, the availability of antibodies specific for the rat type II IGF receptor (R-II-PAB1) has made studies of this receptor type possible. To validate the utility of the RII-PABl antiserum at the level of the rat granulosa cell, its ability to immunoneutralize the granulosa cell type II IGF receptor was examined. Significantly, R-II-PAB1 (10-100 Mg/ml) proved a potent inhibitor of [125I]IGF-II (but not [125I]IGF-I) binding to granulosa cell membrane preparations. Substantial, albeit finite, R-II-PABl-mediated inhibition of the cross-linking of [128I] IGF-II was also observed. Moreover, R-II-PABl proved highly potent in immunoprecipitating the rat granulosa cell type II IGF receptor. In light of these observations, we have proceeded to use R-II-PABl to assess the functional role of the rat granu-
B
losa cell type II IGF receptor in IGF-I and IGF-II hormonal action. To this end, FSH (20 ng/ml)-primed granulosa cells were cultured for 72 h in the absence or presence of IGF-I or IGF-I I (50 ng/ml) with or without increasing (receptor-active) concentrations of R-II-PABl (10-100 jig/ml). Control incubations were carried out with an ammonium sulfate precipitate of nonimmune rabbit serum dialyzed against PBS. Significantly, both R-IIPABl and nonimmune rabbit serum were without effect on the cytodifferentiative action of either IGF-I or IGF-II. Subject to limitations inherent to the immunoneutralizing potency of RII-PAB1, these findings are in keeping with the notion that (inasmuch as the conventional cytodifferentiative process is concerned) the granulosa cell type II IGF receptor does not appear to participate in transmembrane IGF signalling. By inference, these findings also suggest that IGF-I and IGF-II hormonal action at the level of the granulosa cell may be exerted largely, if not exclusively, via the type I IGF receptor. Thus, the potential relevance and the functional role(s), if any, of the granulosa cell type II IGF receptor remain to be determined. {Endocrinology 126: 216-222,1989)
rat type II IGF receptor (6-10) has made studies of this receptor type possible for the first time. The importance of such studies derives from the possibility of highlighting the potential biological role of the type II IGF receptor, the physiological significance of which remains uncertain. Indeed, a significant body of evidence now suggets that the type II IGF receptor may not participate in transmembrane signaling and that it may, in fact, function primarily as a transporter protein the intracellular pool of which can be readily mobilized to the plasma membrane (11, 12). This perplexing situation has recently been further complicated by the discovery of apparent identity between the type II IGF receptor and the cation-independent mannose-6-phosphate receptor; the exact significance of this finding remains unknown at this time (13-15). Moreover, this polyfunctional binding protein has been reported to recognize and interact with a high degree of avidity with the PRL-related principle proliferin (16). It is the objective of this investigation to
OTH insulin-like growth factor-I (IGF-I) and IGFII have previously been shown to promote granulosa cell differentiation (1) and proliferation (2). Although both type I and type II IGF receptors have been documented in the rat granulosa cell (2-5), the identity of the IGF receptor type(s) mediating IGF hormonal action remains uncertain. Whereas the role of the rat type I IGF receptor cannot be completely evaluated at this time due to the lack of antibodies to the rat type I IGF receptor, the recent availability of antisera to the Received February 10, 1989. Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and Physiology, University of Maryland School of Medicine, Room 11-009, Bressler Research Building, 655 East Baltimore Street, Baltimore, Maryland 21201. * This work was supported in part by NIH Research Grant HD19998 and Research Career Development Award HD-00697 (to E.Y.A.) and NIH Research Grants DK-28229, DK-36054, CA-42106, and Research Career Development Award DK-01275 (to R.G.R.).
216
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 17:42 For personal use only. No other uses without permission. . All rights reserved.
IGF-I AND IGF-II ACTION IN RAT GRANULOSA CELLS
assess the functional role of the rat granulosa cell type II IGF receptor in mediating the cytodifferentiative actions of IGF-I and IGF-II.
Materials and Methods Animals Immature (23-25 days old) Sprague-Dawley female rats from Johnson Laboratories, Inc. (Bridgeview, IL), were implanted sc with Silastic capsules containing diethylstilbestrol (DES) and killed between 3-4 days after surgery. Reagents and hormones McCoy's 5a medium (modified, without serum), penicillinstreptomycin solution, L-glutamine, and trypan blue stain (0.4%, wt/vol) were obtained from Gibco (Grand Island, NY). [N-3H]Progesterone (20 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Antiserum R-II-PABl was raised in rabbits against purified type II IGF receptors from 18,54-SF cells (7). This antibody is capable of immunoprecipitating the rat type II receptor and of specifically inhibiting IGF-II binding to a variety of rat tissues (7, 17). Monoclonal antibody A2-1 to the rat type II receptor was employed for immunoprecipitation studies (8). Highly purified IGF-I prepared from Cohn fraction IV of human plasma was generously provided by Dr. Judson J. Van Wyk, University of North Carolina (Chapel Hill, NC). Highly purified [Thr59] IGF-I prepared from an E. coli host by recombinant DNA procedures was obtained from Amgen Biologicals (Thousand Oaks, CA). Highly purified synthetic IGF-II was generously provided by Dr. C. H. Li, University of California (San Francisco, CA). Ovine FSH (oFSH; NIH FSH S14; FSH potency equal to 9 NIH FSH Si units/mg; LH activity, 0.02 NIH LH Si units/mg; PRL activity,
Vol. 126, No. 1 Printed in U.S.A.
Insulin-Like Growth Factor-I (IGF-I) and IGF-II Hormonal Action in Cultured Rat Granulosa Cells: Mediation via Type I but not Type II IGF Receptors* ELI Y. ADASHI, CAROL E. RESNICK, AND RON G. ROSENFELD Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and Physiology, University of Maryland School of Medicine (E. Y.A., C.E.R.), Baltimore, Maryland 21201; and the Division of Endocrinology and Metabolism, Department of Pediatrics, Stanford University (R.G.R.), Stanford, California 94143
ABSTRACT. Both insulin-like growth factor-I (IGF-I) and IGF-II have been shown to promote granulosa cell differentiation and proliferation. While both type I and type II IGF receptors have been observed in rat granulosa cells, the identity of the IGF receptor type(s) mediating IGF hormonal action remains uncertain. Whereas the role of the rat type I IGF receptor cannot be completely evaluated at this time due to the lack of specific reagents, the availability of antibodies specific for the rat type II IGF receptor (R-II-PAB1) has made studies of this receptor type possible. To validate the utility of the RII-PABl antiserum at the level of the rat granulosa cell, its ability to immunoneutralize the granulosa cell type II IGF receptor was examined. Significantly, R-II-PAB1 (10-100 Mg/ml) proved a potent inhibitor of [125I]IGF-II (but not [125I]IGF-I) binding to granulosa cell membrane preparations. Substantial, albeit finite, R-II-PABl-mediated inhibition of the cross-linking of [128I] IGF-II was also observed. Moreover, R-II-PABl proved highly potent in immunoprecipitating the rat granulosa cell type II IGF receptor. In light of these observations, we have proceeded to use R-II-PABl to assess the functional role of the rat granu-
B
losa cell type II IGF receptor in IGF-I and IGF-II hormonal action. To this end, FSH (20 ng/ml)-primed granulosa cells were cultured for 72 h in the absence or presence of IGF-I or IGF-I I (50 ng/ml) with or without increasing (receptor-active) concentrations of R-II-PABl (10-100 jig/ml). Control incubations were carried out with an ammonium sulfate precipitate of nonimmune rabbit serum dialyzed against PBS. Significantly, both R-IIPABl and nonimmune rabbit serum were without effect on the cytodifferentiative action of either IGF-I or IGF-II. Subject to limitations inherent to the immunoneutralizing potency of RII-PAB1, these findings are in keeping with the notion that (inasmuch as the conventional cytodifferentiative process is concerned) the granulosa cell type II IGF receptor does not appear to participate in transmembrane IGF signalling. By inference, these findings also suggest that IGF-I and IGF-II hormonal action at the level of the granulosa cell may be exerted largely, if not exclusively, via the type I IGF receptor. Thus, the potential relevance and the functional role(s), if any, of the granulosa cell type II IGF receptor remain to be determined. {Endocrinology 126: 216-222,1989)
rat type II IGF receptor (6-10) has made studies of this receptor type possible for the first time. The importance of such studies derives from the possibility of highlighting the potential biological role of the type II IGF receptor, the physiological significance of which remains uncertain. Indeed, a significant body of evidence now suggets that the type II IGF receptor may not participate in transmembrane signaling and that it may, in fact, function primarily as a transporter protein the intracellular pool of which can be readily mobilized to the plasma membrane (11, 12). This perplexing situation has recently been further complicated by the discovery of apparent identity between the type II IGF receptor and the cation-independent mannose-6-phosphate receptor; the exact significance of this finding remains unknown at this time (13-15). Moreover, this polyfunctional binding protein has been reported to recognize and interact with a high degree of avidity with the PRL-related principle proliferin (16). It is the objective of this investigation to
OTH insulin-like growth factor-I (IGF-I) and IGFII have previously been shown to promote granulosa cell differentiation (1) and proliferation (2). Although both type I and type II IGF receptors have been documented in the rat granulosa cell (2-5), the identity of the IGF receptor type(s) mediating IGF hormonal action remains uncertain. Whereas the role of the rat type I IGF receptor cannot be completely evaluated at this time due to the lack of antibodies to the rat type I IGF receptor, the recent availability of antisera to the Received February 10, 1989. Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and Physiology, University of Maryland School of Medicine, Room 11-009, Bressler Research Building, 655 East Baltimore Street, Baltimore, Maryland 21201. * This work was supported in part by NIH Research Grant HD19998 and Research Career Development Award HD-00697 (to E.Y.A.) and NIH Research Grants DK-28229, DK-36054, CA-42106, and Research Career Development Award DK-01275 (to R.G.R.).
216
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 17:42 For personal use only. No other uses without permission. . All rights reserved.
IGF-I AND IGF-II ACTION IN RAT GRANULOSA CELLS
assess the functional role of the rat granulosa cell type II IGF receptor in mediating the cytodifferentiative actions of IGF-I and IGF-II.
Materials and Methods Animals Immature (23-25 days old) Sprague-Dawley female rats from Johnson Laboratories, Inc. (Bridgeview, IL), were implanted sc with Silastic capsules containing diethylstilbestrol (DES) and killed between 3-4 days after surgery. Reagents and hormones McCoy's 5a medium (modified, without serum), penicillinstreptomycin solution, L-glutamine, and trypan blue stain (0.4%, wt/vol) were obtained from Gibco (Grand Island, NY). [N-3H]Progesterone (20 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Antiserum R-II-PABl was raised in rabbits against purified type II IGF receptors from 18,54-SF cells (7). This antibody is capable of immunoprecipitating the rat type II receptor and of specifically inhibiting IGF-II binding to a variety of rat tissues (7, 17). Monoclonal antibody A2-1 to the rat type II receptor was employed for immunoprecipitation studies (8). Highly purified IGF-I prepared from Cohn fraction IV of human plasma was generously provided by Dr. Judson J. Van Wyk, University of North Carolina (Chapel Hill, NC). Highly purified [Thr59] IGF-I prepared from an E. coli host by recombinant DNA procedures was obtained from Amgen Biologicals (Thousand Oaks, CA). Highly purified synthetic IGF-II was generously provided by Dr. C. H. Li, University of California (San Francisco, CA). Ovine FSH (oFSH; NIH FSH S14; FSH potency equal to 9 NIH FSH Si units/mg; LH activity, 0.02 NIH LH Si units/mg; PRL activity,
