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Effect of Inhibin-Like Substance Isolated from Porcine Follicular Fluid on the Follicle-Stimulating Hormone (FSH) Level in Mouse Serum and on FSH Binding to Porcine Granulosa Cells*
:0 m cld 5: III
FERTILITY AND STERILITY Copyright'" 1980 The American Fertility Society
Vol. 34, No.1, July 1980 Printed in U.8A.
EFFECT OF INHIBIN-LIKE SUBSTANCE ISOLATED FROM PORCINE FOLLICULAR FLUID ON THE FOLLICLE-STIMULATING HORMONE (FSH) LEVEL IN MOUSE SERUM AND ON FSH BINDING TO PORCINE GRANULOSA CELLS*
rrs
3, to rrg
:01
.an ~B
ive !ty, Les, Perdis'siol iter ~65,
EIMEI SATO, PH.D.t TAKEHIKO ISHIBASHI, PH.D. AKIRA IRITANI, PH.D .
Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan
An inhibin-like substance, a follicle-stimulating hormone (FSH) inhibitor, was isolated from porcine follicular fluid and purified. Serum FSH levels of unilaterally ovariectomized mice which received injections of the inhibitor were lower than those of unilaterally ovariectomized mice which received injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this protein is an FSH inhibitor and that it has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells . . Fertil Steril 34:55,1980
Iral Fed nin :tuli Rec lann rnus concomlterirats. .1:ensfungo 1970 er.In lct 6: Physciety, ,f cat.974
MATERIALS AND METHODS
An inhibitor of compensatory ovarian hypertrophy (COH) in mice has been reported in both bovine and porcine follicular fluid, 1-3 and inhibitor deri ved from both species has been purified by a selective precipitation method and gel chromatography.2, 3 Furthermore, immunohistochemical studies have indicated that this substance is located in the granulosa cells. 3 Since COH is suppressed in mice given injections of the inhibitor, it is suggested that it inhibits gonadotropin (follicle-stimulating hormone [FSH]) by suppressing either concentrations in peripheral blood or activity at the ovarian level, since FSH plays an important role in the mechanism of COH.4 The present study was undertaken to examine the effect of the inhibitor on FSH levels in serum and on FSH binding to the granulosa cells.
Preparation of the Inhibitor. The inhibitor was prepared by the method previously described. 3 Porcine ovaries were obtained at a local slaughterhouse and transported to the laboratory (within 3 hours of death) in physiologic saline at 4° C. Follicular contents were aspirated from follicles 5 to 10 mm in diameter within 5 hours of death, and centrifuged at 10,000 x g for 20 minutes at 4° C. The supernatant, i.e., the follicular fluid, was kept at - 20° C until use. A fraction of the follicular fluid was obtained by "salting out" with ammonium sulfate at saturations of 14.5% to 18.5%. Salting out was repeated twice, and the resulting fraction was loaded on a column of Sephadex G200. A column (1.5 x 100 cm) was packed (height of bed, 95 cm) with Sephadex G-200 which had been washed several times with distilled water . Elution was carried out with distilled water, and the absorbance of the eluted fractions was measured at 280 nm. Gel filtration was performed at 4° C at a flow rate of 4.5 mllhour. The volume of each fraction was 1.85 ml. Fractions comprising a peak were pooled and lyophilized, and it was confirmed that the substance eluted in the second peak suppressed COH in mice. 3
Received October 24, 1979; revised March 10, 1980; accepted March 14, 1980. *Supported in part by Grant 740-0404 from The Ford Foundation and by Grant 276164 from the Ministry of Education, Japan. tReprint requests: E. Sato, Ph.D., Department of Animal Science, College of Agriculture, Kyoto University, Kyoto 606, Japan.
55
SATO ETAL.
56
Bioassay of FSH in Serum. Young adult mice of the JCL-ICR strain were kept in an air-conditioned room under a 13-hour light schedule. Under ether anesthesia, 160 mice were unilaterally ovariectomized (left side) 9 to 11 hours into diestrus, and the inhibitor (200 fl.g in 0.2 ml of physiologic saline/mouse) was injected subcutaneously into 80 animals. The remaining 80 mice received injections of physiologic saline alone. Twenty-four hours after surgery the animals were killed by decapitation and sera were collected from both groups of animals. All samples from each group were pooled. The pooled sera were dialyzed overnight at 4° C against three changes of physiologic saline and stored at - 20° C. Bioassay of FSH was performed by the technique described by Benson et al. 4 based on the synergistic effect ofFSH and'human chorionic gonadotropin (hCG) on ovarian weight in intact, immature female mice. Each animal received a subcutaneous injection of 1.5 ml of serum to which 20 IU ofhCG had been added. Injections of 0.3 ml each were given at 6 P.M. on the 1st day and at 9 A.M. and 6 P.M. on the 2nd and 3rd days. The animals were killed on the morning of the 4th day; the ovaries were removed and dissected free of the oviduct, fat, and connective tissue, and weighed on a torsion balance. Differences between mean ovarian weights were evaluated with Student's t-test. Assay~of FsIi and Luteinizing Hormone (LH) Binding to Granulosa Cells. Human 1251-labeled FSH and 1251-labeled LH (200 fl.Ci/fl.g, Green Cross Co., Tokyo, Japan) were used. Granulosa cells were collected, as described by Channing,5 by aspiration offollicles 3 to 5 mm in diameter from 6to 12-month-old ~igs. The granulosa cells were removed from the follicular fluid by centrifugation, washed, and resuspended in TCM-1996 as described by Nakano et al. 7 The number of cells in TABLE 1. Effect of Sera Collected from Unilaterally Ovariectomized Mice Which Received Injections with or without Inhibitor on Ovarian Weights of21-Day-Old Mice Treatment
No. of mice treated
Ovarian weights of recipients (mean ± SD)
10
8.2 ± 0.3
"'II
hCG 20 IU + 1.5 ml saline (control) hCG201U + 1.5mlserum" hCG201U + 1.5mlserumb
10 10
9.7 ± O.4c 8.4 ± 0.3
"Serum collected from unilaterally ovariectomized mice which received injections of saline. bSerum collected from unilaterally ovariectomized mice which received injections of inhibitor. CSignificantly different from the control value (P < 0.05).
July 1980 TABLE 2. Effect of Inhibitor on the Binding of Iodinated hFSH and hLH to Porcine Granulosa Cells Recovered from Follicles 3 to 5 mm in Diameter Doee of inhibitor
No. of experiments
% of iodinated hormones bound (mean'±
8m
fJlflml
125I_Iabeled hFSH
0 10 50 100
3 3 3 3
21.8 15.6 13.8 12.6
125I-Iabeled hLH
0 50 100
3 3 3
10.7 ± 0.7 9.6 ± 0.3 9.2 ± 0.3
± ± ± ±
0.6 0.4" 0.4" 0.3"
"Significantly different from the control value (P < 0.05).
the suspension was adjusted with TCM-199 containing 0.1 % egg albumin to give a final concentration of5 x 106 cells/ml. Aliquots (500 fl.!) of the cell suspension were pipetted into 10 x 75 mm test tubes in which 100 fl.l of 1251-labeled hFSH or 1251_ labeled hLH solution (14,000 cpm) and different concentrations of the inhibitor in TCM-1996 containing 0.1% egg albumin were contained. Nonspecific binding of 1251-labeled hFSH and 1251_ labeled hLH to granulosa cells was determined in the presence of100 fl.g of unlabeled hormones. The test tubes were incubated for 60 minutes at 37° C in an incubator, th'en centrifuged at 2,500 x g for 10 minutes. The supernatant was removed and the cells were resuspended and washed three times in 1 ml of TCM-1996 containing 0.1% egg albumin. Radioactivity of the cell suspension was counted in an automatic 'Y counter, and values were expressed as a percentage of the original amount of hormone given, corrected for nonspecific binding of iodinated hormones to the surface of the glass tube and granulosa cells. RESULTS
Effect of the Inhibitor on FSH Levels in Serum. The average ovarian weight of recipients of serum from animals which had been unilaterally ovariectomized was significantly higher than that of animals which received physiologic saline (P < 0.05), but the average ovarian weight of recipients of serum from animals which had been unilaterally ovariectomized and which received injections of the inhibitor was not increased as compared with that of the control group (Table 1). This suggests that the FSH level in the serum of unilaterally ovariectomized mice which received injections of the inhibitor was lower than that of unilaterally ovariectomized mice. Effect of the Inhibitor on Binding Of 125I -Labeled
Vol. 34, No.1
INHIBIN-LIKE SUBSTANCE AND FSH
hFSH and 125[ -Labeled hLH to Porcine Granulosa Cells. The inhibitor interfered with the binding of 125I-Iabeled hFSH to granulosa cells (Table 2). In the absence of inhibitor, 21.8% of the incorporated 125I-Iabeled hFSH was bound specifically to the granulosa cells 1 hour after incubation at 37° C. However, in the presence of100 JJ.g of the inhibitor, specific binding dropped to 12.6%, indicating that approximately 42.2% ofthe original binding found in the control group was inhibited by the addition of inhibitor. The inhibitor did not significantly inhibit the binding of 125I-Iabeled hLH to granulosa cells. DISCUSSION
The level ofFSH in serum of unilaterally ovariectomized mice which received injections of the inhibitor was lower than that in unilaterally ovariectomized mice, suggesting that the inhibitor may depress the FSH level in serum. An inhibinlike substance -which depresses serum FSH levels has been reported in bovine and porcine follicular fluid. 8-11 The results of the present experiments suggest that the inhibin-like activity of follicular fluid reported previously8-11 may be identical with the protein isolated in the present study. The present report also suggests that the inhibitor is a substance which inhibits FSH action at the ovarian level, i.e., that it is localized to the granulosa cells3 and inhibits FSH binding to its receptor on these cells. It was previously reported that follicular fluid inhibits the binding ofFSH to granulosa cells. 12 The results of the present experiments suggest that this inhibition of binding may be ascribed to the isolated follicular protein. Thus this inhibitor from porcine follicular fluid may act in the following two ways: suppression of FSH levels in serum and inhibition of FSH binding to its receptor. Richards and Midgley13 have pointed out that ovarian follicles are unequally responsive to the same cyclic hormonal condition and that there
57
are local factors inductive of follicular growth in individual follicles. The present results suggest that the follicles possessing this inhibitor in their fluid may be prevented from progressing further in development, and that this protein may be one of the local regulators of follicular growth. REFERENCES 1. Sato E, Ishibashi T: Inhibition of compensatory ovarian hypertrophy in the mouse by the administration of the non-dialyzable fraction of bovine follicular fluid. J ap J Zootech Sci 48:782, 1977 2. Sato E, Ishibashi T: Partial purification of the gonadotropin inhibiting substance found in bovine follicular fluid. Jap J Zootech Sci 49:313, 1978 3. Sato E, Miyamoto H, Ishibashi T, Iritani A: Identification, purification and immunohistochemical detection of the inhibitor from porcine ovarian follicular fluid to compensatory ovarian hypertrophy in mice. J Reprod Fertil 54:263, 1978 4. Benson B, Sorrentino S, Evans JS: Increase in serum FSH following unilateral ovariectomy in the rat. Endocrinology 84:369, 1969 5. Channing CP: Effect of stage of the estrous cycle and gonadotropin upon luteinization of porcine granulosa cells in culture. Endocrinology 87:156, 1970 6. Morgan JF, Morton HJ, Parker RC: Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proc Soc Exp BioI Med 73:1, 1950 7. Nakano R, Akahori T, Katayama K, Tojo S: Binding ofLH and FSH to porcine granulosa cells during follicular maturation. J Reprod Fertil 51:23, 1977 8. De Jong FH, Sharpe RM: Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71, 1976 9. Hopkinson CRN, Daume E, Strum G, Fritze E, Kaiser S, Hirschhauser C: Inhibin-like activity of bovine ovarian extracts in male and female rats. J Reprod Fertil 50:93, 1977 10. Marder ML, Channing CP, Schwartz NB: Suppression of serum follicle-stimulating hormone in intact and acutely ovariectomized rats by porcine follicular fluid. Endocrinology 101:1639, 1977 11. Welschen R, Hermans WP, Dullaart J, De Jong FH: Effect of an inhibin-like factor present in bovine and porcine follicular fluid on gonadotropin levels in ovariectomized rats. J Reprod Fertil 50:129,1977 12. Dorga NC, Reichert LE: Some properties of the interaction of follicle-stimulating hormone with bovine granulosa cells and its inhibition by follicular fluid. BioI Reprod 19:235, 1978 13. Richards JS, Midgley AR: Protein hormone action: a key to understanding ovarian follicular and luteal cell development. BioI Reprod 14:82, 1976
FERTILITY AND STERILITY Copyright'" 1980 The American Fertility Society
Vol. 34, No.1, July 1980 Printed in U.8A.
EFFECT OF INHIBIN-LIKE SUBSTANCE ISOLATED FROM PORCINE FOLLICULAR FLUID ON THE FOLLICLE-STIMULATING HORMONE (FSH) LEVEL IN MOUSE SERUM AND ON FSH BINDING TO PORCINE GRANULOSA CELLS*
rrs
3, to rrg
:01
.an ~B
ive !ty, Les, Perdis'siol iter ~65,
EIMEI SATO, PH.D.t TAKEHIKO ISHIBASHI, PH.D. AKIRA IRITANI, PH.D .
Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan
An inhibin-like substance, a follicle-stimulating hormone (FSH) inhibitor, was isolated from porcine follicular fluid and purified. Serum FSH levels of unilaterally ovariectomized mice which received injections of the inhibitor were lower than those of unilaterally ovariectomized mice which received injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this protein is an FSH inhibitor and that it has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells . . Fertil Steril 34:55,1980
Iral Fed nin :tuli Rec lann rnus concomlterirats. .1:ensfungo 1970 er.In lct 6: Physciety, ,f cat.974
MATERIALS AND METHODS
An inhibitor of compensatory ovarian hypertrophy (COH) in mice has been reported in both bovine and porcine follicular fluid, 1-3 and inhibitor deri ved from both species has been purified by a selective precipitation method and gel chromatography.2, 3 Furthermore, immunohistochemical studies have indicated that this substance is located in the granulosa cells. 3 Since COH is suppressed in mice given injections of the inhibitor, it is suggested that it inhibits gonadotropin (follicle-stimulating hormone [FSH]) by suppressing either concentrations in peripheral blood or activity at the ovarian level, since FSH plays an important role in the mechanism of COH.4 The present study was undertaken to examine the effect of the inhibitor on FSH levels in serum and on FSH binding to the granulosa cells.
Preparation of the Inhibitor. The inhibitor was prepared by the method previously described. 3 Porcine ovaries were obtained at a local slaughterhouse and transported to the laboratory (within 3 hours of death) in physiologic saline at 4° C. Follicular contents were aspirated from follicles 5 to 10 mm in diameter within 5 hours of death, and centrifuged at 10,000 x g for 20 minutes at 4° C. The supernatant, i.e., the follicular fluid, was kept at - 20° C until use. A fraction of the follicular fluid was obtained by "salting out" with ammonium sulfate at saturations of 14.5% to 18.5%. Salting out was repeated twice, and the resulting fraction was loaded on a column of Sephadex G200. A column (1.5 x 100 cm) was packed (height of bed, 95 cm) with Sephadex G-200 which had been washed several times with distilled water . Elution was carried out with distilled water, and the absorbance of the eluted fractions was measured at 280 nm. Gel filtration was performed at 4° C at a flow rate of 4.5 mllhour. The volume of each fraction was 1.85 ml. Fractions comprising a peak were pooled and lyophilized, and it was confirmed that the substance eluted in the second peak suppressed COH in mice. 3
Received October 24, 1979; revised March 10, 1980; accepted March 14, 1980. *Supported in part by Grant 740-0404 from The Ford Foundation and by Grant 276164 from the Ministry of Education, Japan. tReprint requests: E. Sato, Ph.D., Department of Animal Science, College of Agriculture, Kyoto University, Kyoto 606, Japan.
55
SATO ETAL.
56
Bioassay of FSH in Serum. Young adult mice of the JCL-ICR strain were kept in an air-conditioned room under a 13-hour light schedule. Under ether anesthesia, 160 mice were unilaterally ovariectomized (left side) 9 to 11 hours into diestrus, and the inhibitor (200 fl.g in 0.2 ml of physiologic saline/mouse) was injected subcutaneously into 80 animals. The remaining 80 mice received injections of physiologic saline alone. Twenty-four hours after surgery the animals were killed by decapitation and sera were collected from both groups of animals. All samples from each group were pooled. The pooled sera were dialyzed overnight at 4° C against three changes of physiologic saline and stored at - 20° C. Bioassay of FSH was performed by the technique described by Benson et al. 4 based on the synergistic effect ofFSH and'human chorionic gonadotropin (hCG) on ovarian weight in intact, immature female mice. Each animal received a subcutaneous injection of 1.5 ml of serum to which 20 IU ofhCG had been added. Injections of 0.3 ml each were given at 6 P.M. on the 1st day and at 9 A.M. and 6 P.M. on the 2nd and 3rd days. The animals were killed on the morning of the 4th day; the ovaries were removed and dissected free of the oviduct, fat, and connective tissue, and weighed on a torsion balance. Differences between mean ovarian weights were evaluated with Student's t-test. Assay~of FsIi and Luteinizing Hormone (LH) Binding to Granulosa Cells. Human 1251-labeled FSH and 1251-labeled LH (200 fl.Ci/fl.g, Green Cross Co., Tokyo, Japan) were used. Granulosa cells were collected, as described by Channing,5 by aspiration offollicles 3 to 5 mm in diameter from 6to 12-month-old ~igs. The granulosa cells were removed from the follicular fluid by centrifugation, washed, and resuspended in TCM-1996 as described by Nakano et al. 7 The number of cells in TABLE 1. Effect of Sera Collected from Unilaterally Ovariectomized Mice Which Received Injections with or without Inhibitor on Ovarian Weights of21-Day-Old Mice Treatment
No. of mice treated
Ovarian weights of recipients (mean ± SD)
10
8.2 ± 0.3
"'II
hCG 20 IU + 1.5 ml saline (control) hCG201U + 1.5mlserum" hCG201U + 1.5mlserumb
10 10
9.7 ± O.4c 8.4 ± 0.3
"Serum collected from unilaterally ovariectomized mice which received injections of saline. bSerum collected from unilaterally ovariectomized mice which received injections of inhibitor. CSignificantly different from the control value (P < 0.05).
July 1980 TABLE 2. Effect of Inhibitor on the Binding of Iodinated hFSH and hLH to Porcine Granulosa Cells Recovered from Follicles 3 to 5 mm in Diameter Doee of inhibitor
No. of experiments
% of iodinated hormones bound (mean'±
8m
fJlflml
125I_Iabeled hFSH
0 10 50 100
3 3 3 3
21.8 15.6 13.8 12.6
125I-Iabeled hLH
0 50 100
3 3 3
10.7 ± 0.7 9.6 ± 0.3 9.2 ± 0.3
± ± ± ±
0.6 0.4" 0.4" 0.3"
"Significantly different from the control value (P < 0.05).
the suspension was adjusted with TCM-199 containing 0.1 % egg albumin to give a final concentration of5 x 106 cells/ml. Aliquots (500 fl.!) of the cell suspension were pipetted into 10 x 75 mm test tubes in which 100 fl.l of 1251-labeled hFSH or 1251_ labeled hLH solution (14,000 cpm) and different concentrations of the inhibitor in TCM-1996 containing 0.1% egg albumin were contained. Nonspecific binding of 1251-labeled hFSH and 1251_ labeled hLH to granulosa cells was determined in the presence of100 fl.g of unlabeled hormones. The test tubes were incubated for 60 minutes at 37° C in an incubator, th'en centrifuged at 2,500 x g for 10 minutes. The supernatant was removed and the cells were resuspended and washed three times in 1 ml of TCM-1996 containing 0.1% egg albumin. Radioactivity of the cell suspension was counted in an automatic 'Y counter, and values were expressed as a percentage of the original amount of hormone given, corrected for nonspecific binding of iodinated hormones to the surface of the glass tube and granulosa cells. RESULTS
Effect of the Inhibitor on FSH Levels in Serum. The average ovarian weight of recipients of serum from animals which had been unilaterally ovariectomized was significantly higher than that of animals which received physiologic saline (P < 0.05), but the average ovarian weight of recipients of serum from animals which had been unilaterally ovariectomized and which received injections of the inhibitor was not increased as compared with that of the control group (Table 1). This suggests that the FSH level in the serum of unilaterally ovariectomized mice which received injections of the inhibitor was lower than that of unilaterally ovariectomized mice. Effect of the Inhibitor on Binding Of 125I -Labeled
Vol. 34, No.1
INHIBIN-LIKE SUBSTANCE AND FSH
hFSH and 125[ -Labeled hLH to Porcine Granulosa Cells. The inhibitor interfered with the binding of 125I-Iabeled hFSH to granulosa cells (Table 2). In the absence of inhibitor, 21.8% of the incorporated 125I-Iabeled hFSH was bound specifically to the granulosa cells 1 hour after incubation at 37° C. However, in the presence of100 JJ.g of the inhibitor, specific binding dropped to 12.6%, indicating that approximately 42.2% ofthe original binding found in the control group was inhibited by the addition of inhibitor. The inhibitor did not significantly inhibit the binding of 125I-Iabeled hLH to granulosa cells. DISCUSSION
The level ofFSH in serum of unilaterally ovariectomized mice which received injections of the inhibitor was lower than that in unilaterally ovariectomized mice, suggesting that the inhibitor may depress the FSH level in serum. An inhibinlike substance -which depresses serum FSH levels has been reported in bovine and porcine follicular fluid. 8-11 The results of the present experiments suggest that the inhibin-like activity of follicular fluid reported previously8-11 may be identical with the protein isolated in the present study. The present report also suggests that the inhibitor is a substance which inhibits FSH action at the ovarian level, i.e., that it is localized to the granulosa cells3 and inhibits FSH binding to its receptor on these cells. It was previously reported that follicular fluid inhibits the binding ofFSH to granulosa cells. 12 The results of the present experiments suggest that this inhibition of binding may be ascribed to the isolated follicular protein. Thus this inhibitor from porcine follicular fluid may act in the following two ways: suppression of FSH levels in serum and inhibition of FSH binding to its receptor. Richards and Midgley13 have pointed out that ovarian follicles are unequally responsive to the same cyclic hormonal condition and that there
57
are local factors inductive of follicular growth in individual follicles. The present results suggest that the follicles possessing this inhibitor in their fluid may be prevented from progressing further in development, and that this protein may be one of the local regulators of follicular growth. REFERENCES 1. Sato E, Ishibashi T: Inhibition of compensatory ovarian hypertrophy in the mouse by the administration of the non-dialyzable fraction of bovine follicular fluid. J ap J Zootech Sci 48:782, 1977 2. Sato E, Ishibashi T: Partial purification of the gonadotropin inhibiting substance found in bovine follicular fluid. Jap J Zootech Sci 49:313, 1978 3. Sato E, Miyamoto H, Ishibashi T, Iritani A: Identification, purification and immunohistochemical detection of the inhibitor from porcine ovarian follicular fluid to compensatory ovarian hypertrophy in mice. J Reprod Fertil 54:263, 1978 4. Benson B, Sorrentino S, Evans JS: Increase in serum FSH following unilateral ovariectomy in the rat. Endocrinology 84:369, 1969 5. Channing CP: Effect of stage of the estrous cycle and gonadotropin upon luteinization of porcine granulosa cells in culture. Endocrinology 87:156, 1970 6. Morgan JF, Morton HJ, Parker RC: Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proc Soc Exp BioI Med 73:1, 1950 7. Nakano R, Akahori T, Katayama K, Tojo S: Binding ofLH and FSH to porcine granulosa cells during follicular maturation. J Reprod Fertil 51:23, 1977 8. De Jong FH, Sharpe RM: Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71, 1976 9. Hopkinson CRN, Daume E, Strum G, Fritze E, Kaiser S, Hirschhauser C: Inhibin-like activity of bovine ovarian extracts in male and female rats. J Reprod Fertil 50:93, 1977 10. Marder ML, Channing CP, Schwartz NB: Suppression of serum follicle-stimulating hormone in intact and acutely ovariectomized rats by porcine follicular fluid. Endocrinology 101:1639, 1977 11. Welschen R, Hermans WP, Dullaart J, De Jong FH: Effect of an inhibin-like factor present in bovine and porcine follicular fluid on gonadotropin levels in ovariectomized rats. J Reprod Fertil 50:129,1977 12. Dorga NC, Reichert LE: Some properties of the interaction of follicle-stimulating hormone with bovine granulosa cells and its inhibition by follicular fluid. BioI Reprod 19:235, 1978 13. Richards JS, Midgley AR: Protein hormone action: a key to understanding ovarian follicular and luteal cell development. BioI Reprod 14:82, 1976