PURIFICATION AND ACTION SITES OF A FOLLICLE STIMULATING HORMONE INHIBITOR FROM BOVINE FOLLICULAR FLUID t

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1 PURIFICATION AND ACTION SITES OF A FOLLICLE STIMULATING HORMONE INHIBITOR FROM BOVINE FOLLICULAR FLUID t E. Sato, T. Ishibashi and A. Iritani Kyoto university 2, Kyoto 606, Japan Summary The purification and action sites of a follicle stimulating hormone (FSH) inhibitor obtained from bovine follicular fluid were examined. Bovine follicular fluid was salted out with ammonium sulfate as a concentration of 14.5 to 18.5%, and the salted out fraction was separated into two peaks by Sephadex G-200 column chromatography. The second peak, detectable as a single band by polyacrylamide gel disc electrophoresis, contained all the inhibitory activity to compensatory ovarian hypertrophy in mice. Serum FSH levels of unilaterally ovariectomized mice given injections of the inhibitor were lower than those of unilaterally ovariectomized mice given injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this inhibitor has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells. (Key Words: Follicle Stimulating Hormone Inhibitor, Inhibin-Like Substance, Follicle Stimulating Hormone Binding Inhibitor, Bovine Follicular Fluid.) I ntroduetion It has been reported that an inhibin-like substance that depresses plasma follicle stimulating hormone (FSH) levels is present in both bovine and porcine follicular fluid (De Jong and Sharpe, 1976; Hopkinson et al., 1977; Marder et al., 1977; Welschen et al., 1977), and it also appears that bovine and porcine follicular fluid contains an inhibitor of compensatory ovarian hypertrophy (COH) in mice (Sato and Ishi- t A portion of this work was supported by a grant from The Ford Foundation (No ) and a grant-in-aid from The Ministry of Education, Science and Culture, Japan (No ). 2 Dept. of Anim. Sei., College of Agriculture. 3Green Cross Co., Tokyo, Japan. bashi, 1977; Sato et al., 1978). The porcine COH inhibitor was purified by a selective precipitation method and gel chromatography (Sato et al., 1978). Furthermore, an immunohistochemical study indicated that the porcine inhibitor was located in the granulosa cells (Sato et al., 1978). Because COH is suppressed in mice given injections of the inhibitor, it was concluded that the inhibitor is a gonadotropin (FSH) inhibiting substance that suppresses either FSH levels in systemic blood or FSH activity at the ovarian level (Sato et al., 1978). The present experiments were undertaken to purify the inhibitor from bovine follicular fluid and to determine its inhibiting mechanisms. Materials and Methods Bovine ovaries were obtained at a local slaughterhouse and transported to the laboratory in physiological saline (.154 M NaCI) at 4 C within 3 h. Follicular contents were aspirated from follicles of 5 to 10 mm in diameter within 5 h of death, then centrifuged at 10,000 x g for 20 min at 4 C. The supernatant, i.e., the follicular fluid, was kept at -20 C until use. Purification of the Inhibitor. A fraction of the follicular fluid was obtained by salting out with ammonium sulfate at concentrations of 14.5 to 18.5%. Salting out was repeated twice and the resulting fraction was dialyzed for 24 h against distilled water and loaded onto a column of Sephadex G-200. A column ( cm) was packed (height of bed, 95 cm) with Sephadex G-200 that 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 ml/h. The volume of each fraction was 1.85 ml. Each fraction was assayed for progesterone using a radioimmunoassay kit (lowest detectable quantity; 7.0 pg)3 and fractions constituting a peak were pooled and 873 JOURNAL OF ANIMAL SCIENCE, Vol. 55, No. 4, 1982

2 874 SATO ET AL. lyophilized. Polyacrylamide gel disc electrophoresis was performed according to the method of Davis (1964). Examination of COIl in Mice. Young adult mice of the JCL-ICR strain were kept in an air-conditioned room on a 13-h light schedule with free access to food and water. The mice were ovariectomized unilaterally under ether anesthesia between 0900 and 1100 h on the day of diestrus; the left ovaries were removed and weighed immediately to the nearest.1 mg on a torsion balance. The fractionated samples, or fractionated samples treated with charcoal were injected sc into the mice of the experimental groups. The control group received physiological saline. Each experiment was replicated once. On the third postoperative day, the animals were sacrificed and the right ovaries were removed and weighed. The amount of COH for each animal was calculated as the percentage increase in the weight of the right ovary (ovarian weight at death) compared with that of the left (ovarian weight at hemispaying). The mean percentage of COH increase was calculated for each group and the significance of differences between the means was determined by the use of Student's t test. Bioassay of FSH in Serum. Young adult mice of the JCL-ICR strain were kept in an air-conditioned room on a 13-h light schedule. Under ether anesthesia, 160 mice were unilaterally ovariectomized (left side) between 0900 and 1100 h on the day of diestrus and the inhibitor (200/ag/mouse) was injected ip into 80 mice. The remaining 80 mice were given injections of physiological saline. Twelve hours after surgery, the animals were killed by decapitation and the sera collected from the two groups of animals. All samples in a group were pooled. The pooled sera were dialyzed overnight at 4 C against three changes of physiological saline and stored at -20 C. Bioassay of FSH was performed by the technique described by Benson et al. (1969), based on the synergistic effect of FSH and hcg on ovarian weight in intact, immature female mice. Each animal was given a sc injection of 1.5 ml of serum to which 20 IU of hcg has been added. Injections of. 3 ml each were given at 1800 h on the first day and at 0900 and 1800 h on the second and third days. The 4 Green Cross Co., Tokyo, Japan. animals were killed on the morning of the fourth 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 by Student's t-test. Assay of FSH Binding to Granulosa Cells. Human 12SI-FSH 4 (200 /aci//lg) were used. Granulosa cells were collected, as described by Channing (1970), by aspiration of follicles 3 to 5 mm in diameter. The granulosa cells were removed from the follicular fluid by centrifugation, washed and resuspended in tissue culture medium (TCM 199) as described by Nakano et al. (1977). The number of cells in the suspension was adjusted with TCM 199 containing.1% egg albumin to give a final concentration of 5 x 10 ~ ceus/ml. Aliquots (500/~1) of the cell suspension were pipetted into 10 x 75 mm test tubes containing 100 ~tl of t2si-hfsh (14,000 cpm), and different concentrations of the inhibitor in TCM 199 and.1% egg albumin. Nonspecific binding of 12Sl-hFSH to granulosa cells was determined in the presence of 100/ag of unlabeled hormones. The test tubes were incubated for 60 min at 37 C in an incubator, then centrifuged at 2,500 x g for 10 rain. The supernatant was removed and the cells were resuspended and washed three times in 1 ml of TCM 199 containing. 1% egg albumin. Radioactivity of the cell suspension was counted in an automatic gamma 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 ganulosa cells, Results Purification of the Inbibitor. As depicted in figure 1, the precipitate salted out at 14.5 to 18.5% ammonium sulfate was separated into two peaks by gel chromatography, and no protein was detected in the fractions eluted earlier than peak 1. The lyophilized material of the second peak (fraction 84 to 90) suppressed the COH in the mice (table 1); injection of 100 /~g of the materials from the second peak suppressed COH by about 25% and 200 /ag suppressed it by about 32%, while the material from peak 1 (fraction 70 to 77) had no suppressive effect. The material in the second peak, treated with charcoal, also had a suppressive effect (table 1). Progesterone was not detected (<7.0 pg) in the second peak.

3 FSH INHIBITOR 87.5 I.4 A i 0! FRACTION NUMBER Figure 1. Chromatogram of the fraction salted out at 14.5 to 18.5% ammonium sulfate on Sephadex G- 200 ( cm). Each fraction contains 1.85 ml of the eluate. Patterns of polyacrylamide gel disc electrophoresis of the follicular fluid and the material from peak 2 are shown in figure 2. A concentrated sample from peak 2 was detected as a single band, being at the head of the albumin fractions of the follicular fluid. As figure 1 shows, the second peak was eluted between the positions of ribonuclease A (MW, 13,700) and chymotrypsinogen A (MW, 25,000), so the molecular weight of the inhibitor was estimated at 17,000. Effect of the Inhibitor on FStt Levels in Serum. As shown in table 2, the average ovarian weight of mice given serum from animals that had been unilaterally ovariectomized was higher than that of animals given physiological saline (P<.O.5), but the average ovarian weight of mice given serum from animals that had been unilaterally ovariectomized and given injections of the inhibitor was not increased over that of the control group. This suggests that the FSH level in the serum of unilaterally ovariectomized mice given injections of the inhibitor was lower than that of unilaterally ovariectomized mice. Effect of the Inhibitor on Binding of 12Sl bfsh to Bovine Granulosa Cells. As shown in table 3, the inhibitor interfered with the binding of 12Sl-hFSH to granulosa cells. In the absence of the inhibitor, 21% of the incorporated *2Sl-hFSH was bound specifically to the granulosa cells 1 h after incubation at 37 C. However, in the presence of 100 #g of the inhibitor, specific binding dropped to 9.4% indicating that approximately 57% of the original binding observed in the control was inhibited by the addition of inhibitor Discussion It had been previously reported that progesterone suppressed COH of the unilaterally ovariectomized rat (Benson et al., 1969). On the other hand, in the present study, an inhibitor separated from bovine follicular fluid was nondialyzable and a proteinaceous substance, indicating that this separated inhibitor is not progesterone. We had previously obtained an inhibitor of COH from porcine follicular fluid by dialysis, salting out and gel chromatography. It was detected as a single band on polyacrylamide gel disc electrophoresis (Sato et al., 1978). Although purification of the inhibitor from TABLE 1. EFFECT OF THE FRACTIONS SEPARATED BY COLUMN CHROMATOGRAPHY ON COMPENSATORY OVARIAN HYPERTROPHY Mean SE Dose a, No. of Weights of % hypertrophy Fractions jag/mouse mice treated left ovary, mg of right ovary Saline (control) Peak Peak b b Peak 2 treated with charcoal * b i aeach sample was dissolved in.2 ml saline and injected subcutaneously. bdifferent (P<.05) from the control value.

4 876 SATO ET AL. Figure 2. Patterns of polyacrylamide gel disc electrophoresis in 7.5% gel at ph 8.3. Right:follicular fluid; left:peak 2 (fraction no. 84 to 90). bovine follicular fluid was more difficult than that of the porcine inhibitor, we could separate the bovine inhibitor by salting out twice and column chromatography using Sephadex G-200. When the porcine and bovine inhibitors are compared, their patterns of elution by Sephadex G-200 column chromatography and by disc electrophoresis and their modes of action are very similar, inidicating similarity of molecular weights and physico-chemical properties (Sato et al., 1978, 1980). However, the suppression of COH in mice by the porcine inhibitor is almost double that produced by the bovine inhibitor. Previous investigators have suggested that an inhibin-like substance that decreases serum FSH levels is present in both bovine and porcine follicular fluid (De Jong and Sharpe, 1976; Hopkinson et al., 1977; Marder et M., 1977; Welschen et al., 1977). As shown in table 2, the separated substance from bovine follicular fluid appeared to decrease serum FSH levels. This suggests that the inhibin-like substance may be identical to the protein separated in the present study. An inhibin-like substance from human follicular fluid has been purified (Chariet al., 1979). The molecular weight of this human inhibinqike substance however, was slightly larger than that of the porcine and bovine inhibitors. The present report also suggests that the inhibitor is a substance that inhibits FSH action at the ovarian level; that is, it is present in follicular fluid and inhibits FSH binding to its receptor on the granulosa cells. It was previously reported that follicular fluid inhibits the binding of FSH to the granulosa cells (Dorga and Reichert, 1978). The results of the present experiments suggest that this inhibition of binding may be ascribed to the protein separated in the present study. Richards and Midgley (1976) have pointed out that the follicles in the ovary are not equally responsive to the same cyclic hormonal TABLE 2. EFFECT OF SERA COLLECTED FROM UNILATERALLY OVARIECTOMIZED MICE GIVEN INJECTIONS WITH OR WITHOUT INHIBITOR ON OVARIAN WEIGHTS OF 21-D-OLD MICE Treatment No. of mice treated Mean (-+ SE) ovarian weights of recipients, mg hcg 20 IU ml saline (control) hcg 20 IU ml serum a hcg 20 IU ml serum b c aserum collected from unilaterally ovariectomized mice given injections of saline. bserum collected from unilaterally ovariectomized mice given injections of inhibitor. CDifferent (P<.05) frem the control value.

5 FSH INHIBITOR 877 TABLE 3. EFFECT OF THE INHIBITOR ON THE BINDING OF IODINATED hfsh TO BOVINE GRANULOSA CELLS RECOVERED FROM FOLLICLES 3 TO 5 MM IN DIAMETER lodinated hormone % of iodinated Dose of No. of hormone bound inhibitor, #g/ml experiments (mean + SE) 12Sl-labeled hfsh O a a a adifferent (P<.05) from the control value. condition and that there are local inductive factors for follicular growth in individual follicles. The present results suggest that the follicles possessing this inhibitor in their fouicular fluid may be prevented from progressing further in development, and that this protein may be one of the local regulators of follicular growth. Literature Cited Benson, B., S. Sorrentino and J. S. Evans Increase in serum FSH following unilateral ovariectomy in the rat. Endocrinology 84:369. Channing, C. P Effect of stage of the estrus cycle and gonadotropin upon luteinization of porcine granulosa cells in culture. Endocrinology 87:156. Chari, S., C.R.N. Hopkinson, E. Daume and G. Sturm Purification of "inhibin" from human ovarian follicular fluid. Acta Endocrinol. 90:157. Davis, B. J Disc electrophoresis-ll, method and application to human serum proteins. Ann. New York Acad. Sci. 121:404. De Jong, F. H. and R. M. Sharpe Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71. Dorga, N. C. and L. E. Reichert Some properties of the interaction of follicle stimulating hormone with bovine granulosa cells and its inhibition by follicular fluid. Biol. Repro& 19:235. Hopkinson, C.R.N., E. Daume, G. Strum, E. Fritze, S. Kaiser and C. Hirschhguser Inhibin-like activity of bovine ovarian extracts in male and female rats. J. Reprod. Fertil. 50:93. Marder, M. L., C. P. Channing and N. B. Schwartz Suppression of serum follicle stimulating hormone in intact and acutely ovariectomized rats by porcine follicular fluid. Endocrinology 101:1639. Nakano, R., T. Akahori, K. Katayama and S. Tojo Binding of LH and FSH to porcine granulosa cells during follicular maturation. J. Reprod. Fertil. 51:23. Richards, J. S. and A. R. Midgley Protein hormone action: A key to understanding ovarian follicular and luteal cell development. Biol. Reprod. 14:82. Sato, E. and T. Ishibashi Inhibition of compensatory ovarian hypertrophy in the mouse by the administration of the non-dialyzable fraction of bovine follicular fluid. Japanese J. Zootech. Sci. 48:782. Sato, E., T. lshibashi and A. lritani Effect of inhibin-like substance isolated from porcine follicular fluid on the folficle-stimulatinghormone (FSH) level in mouse serum and on FSH binding to porcine granulosa cells. Fert. Steril. 34:55. Sato, E., H. Miyamoto, T. Ishibashi and A. Iritani Identification, purification and immunohistochemical detection of the inhibitor from porcine ovarian follicular fluid to compensatory ovarian hypertrophy in mice. J. Reprod. Fertil. 54:263. Welschen, R., W. P. Hermans, J. Dullaart and F. H. De Jong Effect of inhibin-like factor present in bovine and porcine follicular fluid on gonadotropin levels in ovariectomized rat. J. Reprod. Fertil. 50:129.