Why I was Told not to Study Inhibin and What I did about it
(Endocrinology 129:1690-1691)
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 FSH-suppressing substance
simultaneously with causing resumption or 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
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 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, but
the name never caught on outside our own laboratories, and the substance continued to the
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 in 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
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).
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 nonsteriodal 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-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 |
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 (ed.) Recent Progress Hormone Research.
Academic Press, New York, vol. 38:409-456
3. DeJong FH 1988 Inhibin. Physiol Rev 68:555-607
4. 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
5. 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
6. DeJong FH, Sharpe RM 1976 Evidence for inhibin-like activity in bovine follicular
fluid. Nature 263:71-72
7. 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
8. Robertson DM, Foulds LM, Leresha L, Morgan FJ, Hearn MTW, Burger HG, Wetenhall REH,
deKretser DM 1985 Isolation of inhibin from bovine follicular fluid. Biochem Biophys
Res Commum 126:220-226
9. 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 129:396-403
10. 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
11. Ling N, Ying S-Y, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial
characterization of Mr 32,000 protein with inhibin activity from porcine follicular fluid.
Proc Natl Acad Sci USA 82:7217-7221
12. 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
14. 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 43:347-35