0013-7227/91/1294-1690$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 4 Printed in U.S.A.
Why I was Told not to Study Inhibin and What I did about it We entered the inhibin field accidentally. We were trying to account for our failure to inhibit serum FSH in the ovariectomized rat with estradiol plus progesterone. We were also puzzled by what accounted for the secondary FSH surge which follows the preovulatory gonadotropin surges in the rodent. It had been shown by others that once the primary surges occurred, the prolonged rise in serum FSH lasting to estrus was not dependent on further GnRH action. We wondered if the preovulatory LH surge turned off secretion from the ovary of an FSHsuppressing substance simultaneously with causing resumption of meiosis and ovulation (1, 2). These observations suggested that another feedback hormone from the ovary might specifically be responsible for FSH negative feedback. But that sounded like the elusive nonsteroidal substance "inhibin" that scientists working with testicular feedback had been searching for since the early 1920s (3). Because of the failure to purify such a substance after so many years, the field was seen as "questionable" by many scientists. In the mid 1970s the late Cornelia (Nina) Channing, of the Department of Physiology, University of Maryland School of Medicine, was engaged in a search for an ovarian "oocyte meiosis inhibitor" (OMI). It had been known for some time that mammalian oocytes explanted within their surrounding follicles did not exhibit germinal vesicle breakdown (GVB) or resumption of meiosis. However, if the oocytes within cumulus were explanted after removal from their surrounding follicles, they resumed meiosis quickly. These observations led some reproductive scientists, including Channing, to postulate that endogenous follicular fluid contained an OMI which was suppressed by the preovulatory LH surge, allowing the rapid GVB and meiosis resumption seen on the afternoon of proestrus in the rat. Channing and colleagues (4) had shown that porcine follicular fluid (pFF) contained a peptide which could at least partly suppress or delay GVB in explanted follicles. It occurred to me that the same OMI could be our hypothetical FSH-suppressing substance, since both activities were postulated to be reduced by the natural LH Received June 7,1991. "Remembrance" articles discuss people and events as remembered by the author. The opinoion(s) expressed are solely those of the writer and do not reflect the view of the Journal or The Endocrine Society.
surge. I called Nina and she agreed to send me some charcoal extracted pFF to test on the secondary FSH surge in the female rat. We injected it after the primary gonadotropin surges were complete, and collected blood at 0400 h of estrus, when the secondary FSH surge was maximum. To our great satisfaction, pFF caused a major
fall in serum FSH, which was dose dependent! We submitted an abstract to the FASEB 1977 meeting, and then sent an expanded paper to PNAS (5). In that paper we suggested naming the substance "folliculostatin," since it suppressed FSH secretion, but the name never caught on outside our own laboratories, and the substance continued to be called "inhibin." In the meantime DeJong and Sharpe (6) had shown that bovine FF could suppress serum FSH selectively in castrated male rats, scooping us in the demonstration that FF was a better source of inhibin than was testicular extract. After the FASEB meeting in early 1977, where I presented the paper, I received several phone calls from good friends in endocrinology, and I suppose that Nina did also, counseling me "not to get into the inhibin field." The rationale was that it seemed strange that people had searched for so long to no avail, and that while some laboratories claimed to have a partially purified inhibin other laboratories could not confirm it. Thus, many scientists had concluded it did not exist. In retrospect, the probable reasons for the lag in the field were several: testicular extract does not contain as high a concentration of inhibin as FF; the in vivo bioassays were never standardized and were insensitive; the male bioassay recipient is not as sensitive to inhibin as the female (2). Nina and I went on to show that OMI and inhibin were not the same molecule, since the FSH-suppressing activity was in the greater than 10,000 mol wt fraction, whereas OMI was in the greater than 2,000 mol wt fraction (7). As it turned out, conventional biochemical protein isolation techniques never yielded isolation of inhibin, but the techniques of molecular biology were successful in identifying the two subunits of inhibin in four laboratories simultaneously (8-11). Unfortunately, Nina died just before the existence of the inhibin molecule was confirmed. The issue of what substance in pFF is OMI is still controversial (12). 1690
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 15:25 For personal use only. No other uses without permission. . All rights reserved.
REMEMBRANCE
Why did I persist in looking for ovarian inhibin in the face of so many friendly warnings from respected colleagues and in the light of a 50-year history of unresolved research on testicular inhibin? For two reasons, I think. Had I been more junior in my career in the mid-70s I might have abandoned the search for fear of adding myself to the long list of investigators who had failed to confirm the existence of inhibin. As it was, I assumed that I could survive the possible failure if it occurred. Additionally, however, our physiological analysis of ovarian/pituitary relationships demanded another feedback hormone from the ovary! Something had to account for the relative nonsuppressibility of serum FSH after ovariectomy, and for the prolonged postsurge FSH rise in rats and hamsters. And I was stubborn enough to take the chance that follicular fluid might contain the nonsteroidal hormone. Consequently, it was a real high to show, with Kelly Mayo's laboratory, that messenger RNA for inhibin subunits declined on the afternoon of proestrus (13). Finally, using a RIA for the a-inhibin subunit, it has been possible to demonstrate that serum inhibin drops on the afternoon of proestrus, just as we had predicted (14)! Neena B. Schwartz Department of Neurobiology/Physiology Northwestern University
3. 4. 5.
6. 7.
8.
9.
10. 11.
12. 13.
References 1. Schwartz N, Talley W 1978 Effects of exogenous LH or FSH on endogenous FSH, progesterone and estradiol secretion. Biol Reprod 18:820-828 2. Grady R, Charlesworth MC, Schwartz NB 1982 Characterization of the FSH-suppressing activity in follicular fluid. In: Greep RO
14.
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(ed) Recent Progress Hormone Research. Academic Press, New York, vol 38:409-456 DeJong FH 1988 Inhibin. Physiol Rev 68:555-607 Tsafriri A, Pomerantz SH, Channing CP 1976 Inhibition of oocyte maturation by porcine follicular fluid: partial characterization of the inhibitor. Biol Reprod 14:511-516 Schwartz NB, Channing CP 1977 Evidence for ovarian "inhibin" suppression of the secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of porcine follicular fluid. Proc Natl Acad Sci USA 74:5721-5724 DeJong FH, Sharpe RM 1976 Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71-72 Lorenzen JR, Channing CP, Schwartz NB 1978 Partial characterization of FSH-suppressing activity (folliculostatin) in porcine follicular fluid using the metestrous rat as an in vivo model. Biol Reprod 19:635-640 Robertson DM, Foulds LM, Lerersha L, Morgan FJ, Hearn MTW, Burger HG, Wetenhall REH, deKretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 129:396403 Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 Tsafriri A 1988 Local nonsteroidal regulators of ovarian function. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 527-565 Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE 1988 Dynamic changes in inhibin mRNAs in rat ovarian follicles during the reproductive cycle. Science 239:1296-1299 Ackland JF, D'Agostino JB, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB 1990 Circulating radioimmunoassayable inhibin during periods of transient FSH rise: secondary surge and unilateral ovariectomy. Biol Reprod 43:347-352
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Vol. 129, No. 4 Printed in U.S.A.
Why I was Told not to Study Inhibin and What I did about it We entered the inhibin field accidentally. We were trying to account for our failure to inhibit serum FSH in the ovariectomized rat with estradiol plus progesterone. We were also puzzled by what accounted for the secondary FSH surge which follows the preovulatory gonadotropin surges in the rodent. It had been shown by others that once the primary surges occurred, the prolonged rise in serum FSH lasting to estrus was not dependent on further GnRH action. We wondered if the preovulatory LH surge turned off secretion from the ovary of an FSHsuppressing substance simultaneously with causing resumption of meiosis and ovulation (1, 2). These observations suggested that another feedback hormone from the ovary might specifically be responsible for FSH negative feedback. But that sounded like the elusive nonsteroidal substance "inhibin" that scientists working with testicular feedback had been searching for since the early 1920s (3). Because of the failure to purify such a substance after so many years, the field was seen as "questionable" by many scientists. In the mid 1970s the late Cornelia (Nina) Channing, of the Department of Physiology, University of Maryland School of Medicine, was engaged in a search for an ovarian "oocyte meiosis inhibitor" (OMI). It had been known for some time that mammalian oocytes explanted within their surrounding follicles did not exhibit germinal vesicle breakdown (GVB) or resumption of meiosis. However, if the oocytes within cumulus were explanted after removal from their surrounding follicles, they resumed meiosis quickly. These observations led some reproductive scientists, including Channing, to postulate that endogenous follicular fluid contained an OMI which was suppressed by the preovulatory LH surge, allowing the rapid GVB and meiosis resumption seen on the afternoon of proestrus in the rat. Channing and colleagues (4) had shown that porcine follicular fluid (pFF) contained a peptide which could at least partly suppress or delay GVB in explanted follicles. It occurred to me that the same OMI could be our hypothetical FSH-suppressing substance, since both activities were postulated to be reduced by the natural LH Received June 7,1991. "Remembrance" articles discuss people and events as remembered by the author. The opinoion(s) expressed are solely those of the writer and do not reflect the view of the Journal or The Endocrine Society.
surge. I called Nina and she agreed to send me some charcoal extracted pFF to test on the secondary FSH surge in the female rat. We injected it after the primary gonadotropin surges were complete, and collected blood at 0400 h of estrus, when the secondary FSH surge was maximum. To our great satisfaction, pFF caused a major
fall in serum FSH, which was dose dependent! We submitted an abstract to the FASEB 1977 meeting, and then sent an expanded paper to PNAS (5). In that paper we suggested naming the substance "folliculostatin," since it suppressed FSH secretion, but the name never caught on outside our own laboratories, and the substance continued to be called "inhibin." In the meantime DeJong and Sharpe (6) had shown that bovine FF could suppress serum FSH selectively in castrated male rats, scooping us in the demonstration that FF was a better source of inhibin than was testicular extract. After the FASEB meeting in early 1977, where I presented the paper, I received several phone calls from good friends in endocrinology, and I suppose that Nina did also, counseling me "not to get into the inhibin field." The rationale was that it seemed strange that people had searched for so long to no avail, and that while some laboratories claimed to have a partially purified inhibin other laboratories could not confirm it. Thus, many scientists had concluded it did not exist. In retrospect, the probable reasons for the lag in the field were several: testicular extract does not contain as high a concentration of inhibin as FF; the in vivo bioassays were never standardized and were insensitive; the male bioassay recipient is not as sensitive to inhibin as the female (2). Nina and I went on to show that OMI and inhibin were not the same molecule, since the FSH-suppressing activity was in the greater than 10,000 mol wt fraction, whereas OMI was in the greater than 2,000 mol wt fraction (7). As it turned out, conventional biochemical protein isolation techniques never yielded isolation of inhibin, but the techniques of molecular biology were successful in identifying the two subunits of inhibin in four laboratories simultaneously (8-11). Unfortunately, Nina died just before the existence of the inhibin molecule was confirmed. The issue of what substance in pFF is OMI is still controversial (12). 1690
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 15:25 For personal use only. No other uses without permission. . All rights reserved.
REMEMBRANCE
Why did I persist in looking for ovarian inhibin in the face of so many friendly warnings from respected colleagues and in the light of a 50-year history of unresolved research on testicular inhibin? For two reasons, I think. Had I been more junior in my career in the mid-70s I might have abandoned the search for fear of adding myself to the long list of investigators who had failed to confirm the existence of inhibin. As it was, I assumed that I could survive the possible failure if it occurred. Additionally, however, our physiological analysis of ovarian/pituitary relationships demanded another feedback hormone from the ovary! Something had to account for the relative nonsuppressibility of serum FSH after ovariectomy, and for the prolonged postsurge FSH rise in rats and hamsters. And I was stubborn enough to take the chance that follicular fluid might contain the nonsteroidal hormone. Consequently, it was a real high to show, with Kelly Mayo's laboratory, that messenger RNA for inhibin subunits declined on the afternoon of proestrus (13). Finally, using a RIA for the a-inhibin subunit, it has been possible to demonstrate that serum inhibin drops on the afternoon of proestrus, just as we had predicted (14)! Neena B. Schwartz Department of Neurobiology/Physiology Northwestern University
3. 4. 5.
6. 7.
8.
9.
10. 11.
12. 13.
References 1. Schwartz N, Talley W 1978 Effects of exogenous LH or FSH on endogenous FSH, progesterone and estradiol secretion. Biol Reprod 18:820-828 2. Grady R, Charlesworth MC, Schwartz NB 1982 Characterization of the FSH-suppressing activity in follicular fluid. In: Greep RO
14.
1691
(ed) Recent Progress Hormone Research. Academic Press, New York, vol 38:409-456 DeJong FH 1988 Inhibin. Physiol Rev 68:555-607 Tsafriri A, Pomerantz SH, Channing CP 1976 Inhibition of oocyte maturation by porcine follicular fluid: partial characterization of the inhibitor. Biol Reprod 14:511-516 Schwartz NB, Channing CP 1977 Evidence for ovarian "inhibin" suppression of the secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of porcine follicular fluid. Proc Natl Acad Sci USA 74:5721-5724 DeJong FH, Sharpe RM 1976 Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71-72 Lorenzen JR, Channing CP, Schwartz NB 1978 Partial characterization of FSH-suppressing activity (folliculostatin) in porcine follicular fluid using the metestrous rat as an in vivo model. Biol Reprod 19:635-640 Robertson DM, Foulds LM, Lerersha L, Morgan FJ, Hearn MTW, Burger HG, Wetenhall REH, deKretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun 126:220-226 Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M, Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin of 32K daltons. Biochem Biophys Res Commun 129:396403 Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun 133:120-127 Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:7217-7221 Tsafriri A 1988 Local nonsteroidal regulators of ovarian function. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 527-565 Woodruff TK, D'Agostino JB, Schwartz NB, Mayo KE 1988 Dynamic changes in inhibin mRNAs in rat ovarian follicles during the reproductive cycle. Science 239:1296-1299 Ackland JF, D'Agostino JB, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB 1990 Circulating radioimmunoassayable inhibin during periods of transient FSH rise: secondary surge and unilateral ovariectomy. Biol Reprod 43:347-352
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