A selective increase in circulating inhibin and inhibin
pro-
C at the time of ovulation in the mare
Kentaro
Nagaoka1,
Yasuo
Nambo2,
Natsuko
Nagamine1,4,
Shun-Ichi
Nagata3,
Yumiko
Tanaka1,
Hiromi
Shinbo1,
Nobuo
Tsunoda4,
Hiroyuki
Taniyama5,
Gen
Watanabe1,
Nigel P.
Groome6, and
Kazuyoshi
Taya1
1 Laboratory of Veterinary
Physiology, Tokyo University of Agriculture and Technology, Tokyo
183-8509; 2 Equine Research
Institute, Japan Racing Association, Tochigi 320-0856;
3 Laboratory of Racing Chemistry,
Tochigi 320-0851; 4 Shadai
Corporation, Hokkaido 059-1432; and
5 Rakuno Gakuen
University, Hokkaido 069-0836, Japan; and
6 School of Biological and
Molecular Sciences, Oxford Brookes University, Headington Oxford OX3
0BP, UK
 |
ABSTRACT |
The relationship
between a selective increase in circulating immunoreactive (ir)-inhibin
and the time of ovulation was investigated in mares. Concentrations of
plasma ir-inhibin were measured every 4 h during the periovulatory
period. Inhibin pro-
C, a precursor protein of the inhibin
-subunit, was also measured. The changes in ir-inhibin and inhibin
pro-
C in circulation were parallel. Concentrations of both
ir-inhibin and inhibin pro-
C in the plasma increased at the same
time when ovulatory follicles ruptured, and the peak levels of
circulating ir-inhibin and inhibin pro-
C were maintained for
4-8 h. There was no selective increase in plasma concentrations of
estradiol-17
during the process of ovulation. These results suggest
that the selective increase in ir-inhibin and inhibin pro-
C was
caused by the absorption of follicular fluid after the rupture of
ovulatory follicles. These results also suggest that the measuring of
plasma concentrations of ir-inhibin or inhibin pro-
C in mares might
be a useful method for detecting the time of ovulation.
luteinizing hormone; follicle-stimulating hormone; progesterone; marker of ovulation
 |
INTRODUCTION |
HORSES ARE TYPICAL SEASONAL BREEDERS. Mares repeat
ovulation from spring to summer in the northern hemisphere. There are
many unique phenomena in horse reproduction. For example, mares show estrous behavior for 1 wk, and ovulation occurs 2 days before the end
of the estrus. It is not easy to detect the most suitable time for
mating a mare with a stallion, as there are no valid methods by use of endocrine signs. Although one
possibility for detecting the time of ovulation is measuring the
preovulatory luteinizing hormone (LH) surge, it is not as valid a
method in mares as in other mammals, because the duration of the LH
surge is almost 1 wk, and the peak level of LH surge is only 4-5
times higher than the basal level, indicating that the LH surge is not a suitable sign for detecting ovulation in mares (5, 7, 20, 37). On the
other hand, our previous report indicated that circulating
immunoreactive (ir)-inhibin increases selectively at the day of
ovulation (20, 28).
Inhibins are heterodimers consisting of disulfide-linked
- and
either
A- or
B-subunits (1). Inhibins are generally produced by
granulosa cells of ovarian follicles in several mammalian species, such
as rats (17, 18), hamsters (24), guinea pigs (32), pigs (27), cows
(27), sheep (2), monkeys (31), and horses (20). Initially, these
subunits are produced as larger precursors and then processed to
produce smaller forms. Only the dimeric forms of inhibin have bioactive
effects, follicle-stimulating hormone (FSH)-suppressing effects,
although the
-subunit circulates in excess amounts as biologically
inactive monomers.
In the present study, to define the relationship between the selective
increase in circulating inhibin and ovulation in mares, peripheral
concentrations of inhibin were measured during the process of ovulation
by use of a specific radioimmunoassay (11). In addition, a precursor
protein of inhibin
-subunit, inhibin pro-
C, was also measured
using a two-site ELISA (10).
 |
MATERIALS AND METHODS |
Collection of plasma.
In the first experiment, jugular venous blood samples were collected
into heparinized tubes once daily from seven adult thoroughbred mares
between 0800 and 1000 throughout the estrous cycle. The ovulation was confirmed by transrectal palpation and ultrasound scanning every day after blood sampling.
In the second experiment, ovaries of three mares were examined by
transrectal palpation and ultrasound scanning once daily between 0800 and 1000, and blood samples were also collected. When growing follicles
larger than 3.5 cm in diameter were found in ovaries, blood samples
were collected every 4 h (at 0400, 0800, 1200, 1600, 2000, and 2400).
During this period, ovaries were examined by transrectal palpation and
ultrasound scanning every 8 h (at 0800, 1600, and 2400) to detect the
ovulation. After ovulation occurred, the 4-h sampling was continued for
the next 24 h. Blood samples were centrifuged at 1,700 g and 4°C for 10 min, and plasma was collected and stored at
30°C until assayed for
ir-inhibin, FSH, LH, estradiol-17
, progesterone, and inhibin
pro-
C concentrations.
Radioimmunoassay of ir-inhibin, FSH, LH,
estradiol-17
, and progesterone.
Concentrations of ir-inhibin in plasma were measured using a rabbit
antiserum against bovine 32-kDa inhibin (TNDH-1) and
125I-labeled 32-kDa bovine
inhibin, as described previously (11). The results were expressed in
terms of 32-kDa bovine inhibin. The sensitivity of the assay was 7.8 pg/tube (78 pg/ml). The intra- and interassay coefficients of variation
were 7.4 and 10.5%, respectively. The anti-inhibin serum (TNDH-1) used
in the present study does not cross-react with equine chorionic
gonadotropin, activin, and human transforming growth factor-
(11).
Concentrations of FSH in plasma were measured using a rabbit antiserum
against human FSH (no. 6, provided by the National Institute of
Diabetes and Digestive and Kidney Diseases, Bethesda, MD).
Highly purified equine FSH (provided by Dr. J. Roser, Department of
Animal Science, University of California, Davis, CA and Dr. H. Papkoff,
Hormone Research Laboratory, University of California, San Francisco,
CA) was used as a standard and for iodination. Plasma LH was measured
using a rabbit antiserum against ovine LH (YM no. 18, provided by Dr.
Y. Mori, Laboratory of Veterinary Ethology, University of Tokyo, Tokyo,
Japan). Highly purified equine LH was provided by Dr. J. Roser and Dr.
H. Papkoff. The sensitivity of LH and FSH assays was 31.2 pg/tube (312 pg/ml) and 312.5 pg/tube (1,560 pg/ml), respectively. The intra- and interassay coefficients of variation were 6.9 and 9.7% for the FSH
assay and 8.8 and 13% for the LH assay, respectively.
Concentrations of estradiol-17
and progesterone in plasma were
measured by the double-antibody RIA systems by use of
125I-labeled radioligands, as
described previously (21, 35). Antisera against estradiol-17
(GDN
244) (14) and progesterone (GDN 337) (6) were kindly provided by Dr. G. D. Niswender (Animal Reproduction and Biotechnology Laboratory,
Colorado State University, Fort Collins, CO). The sensitivity of
estradiol-17
and progesterone assays was 0.31 pg/tube (0.62 pg/ml)
and 1.25 pg/tube (12.5 pg/ml), respectively. The intra- and interassay
coefficients of variation were 4.8 and 5.8% for estradiol-17
assay
and 3.5 and 13.4% for the progesterone assay, respectively.
Two-site ELISA for inhibin pro-
C.
Concentrations of inhibin pro-
C in plasma were measured using the
ELISA kits (Serotec, Oxford, UK) for the measurement of human
inhibin pro-
C without modifications. In the assay, two monoclonal antibodies against the pro and
C regions were
used (10). Serial dilutions of pooled equine follicular fluid (eFF) and
plasma of mares were assayed to test for parallelism.
Statistical analyses of data.
All data were presented as means ± SE. The significance of daily
changes in concentrations of each hormone during the estrous cycle was
analyzed by a two-way ANOVA, with the animal and the time from
ovulation as the two factors. Significance of difference between means
was compared by Duncan's multiple range test (33). The linear
coefficients of correlation (r) were
calculated between the following pairs of plasma concentrations:
ir-inhibin and FSH, and estradiol-17
and progesterone. All
differences with a value of
P <0.05 were considered significant.
 |
RESULTS |
Characterization of the inhibin pro-
C ELISA system.
Serial dilutions of eFF (0.03-3.9 nl) and peripheral plasma
(1.56-50 µl) of mares produced excellent dose-response curves. These curves were parallel with the human inhibin pro-
C standard curve, indicating that it is possible to measure the concentration of
inhibin pro-
C in plasma and eFF of mares using the present ELISA
method (Fig. 1).

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Fig. 1.
Dose-response curves of human inhibin pro- C standard ( ), mare's
plasma ( ), and equine follicular fluid ( ) in ELISA assay for
inhibin pro- C.
|
|
Hormonal changes during the estrous cycle.
The mean concentrations of ir-inhibin, LH, FSH, estradiol-17
, and
progesterone throughout the estrous cycle are shown in Fig.
2. The follicular phase and luteal phase
were characterized by the concentrations of estradiol-17
and
progesterone (Fig. 2C). Plasma
concentrations of ir-inhibin increased slowly in the follicular phase
and then decreased until just before ovulation. Thereafter, there was a
selective increase in plasma concentrations of ir-inhibin on the day of
ovulation (Fig. 2A). Concentrations of FSH in the plasma were high during the luteal phase compared with
the follicular phase. Change in plasma concentrations of FSH was
inversely correlated to that of ir-inhibin
(r =
0.297, n = 196, P < 0.05). Concentrations of LH in
the plasma began to rise 3 days before the ovulation and reached a peak
on the day after ovulation. The LH surge started to increase slowly,
and the peak value of the LH surge was ~4-5 times higher than
the basal levels (Fig. 2B). Changes
in circulating estradiol-17
and progesterone showed a clear inverse
relationship throughout the estrous cycle
(r =
0.406,
n = 196, P < 0.05). Plasma concentrations of
estradiol-17
increased abruptly during the late follicular phase and
reached the peak at 2 or 3 days before ovulation, followed by a
gradual decline when the LH surge started (Fig.
2C).

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Fig. 2.
Changes in plasma concentrations of immunoreactive (ir)-inhibin ( ,
A), luteinizing hormone (LH, )
and follicle-stimulating hormone (FSH, ), both
B, estradiol-17 ( ) and
progesterone ( ), both C, during the
estrous cycle in mares. Values are aligned relative to the day of
ovulation. Each value represents mean ± SE of 7 mares. Dotted
vertical lines represent the day of ovulation (day
0 of estrous cycle).
|
|
The periovulatory changes in plasma concentrations of ir-inhibin and
inhibin pro-
C are shown in Fig. 3.
Changes in circulating inhibin pro-
C were parallel with those of
ir-inhibin. Both circulating ir-inhibin and inhibin pro-
C showed a
selective increase at the day of ovulation, indicating that there is a
relationship between ovulation and a selective increase in circulating
ir-inhibin and inhibin pro-
C.

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Fig. 3.
Changes in plasma concentrations of ir-inhibin ( ,
A) and inhibin pro- C ( ,
B) around the day of ovulation in
mares. Values are aligned relative to the day of ovulation during the
estrous cycle in mares. Each value represents mean ± SE of 14 observations. Dotted vertical lines represent day of ovulation
(day 0 of estrous cycle).
|
|
Hormonal changes around the ovulation.
Individual changes in the concentrations of LH, FSH, ir-inhibin,
inhibin pro-
C, estradiol-17
, and progesterone in the plasma around the time of ovulation are shown in three mares
(mares A, B, and
C) in Figs.
4, 5, and
6. The selective increase in plasma concentrations of ir-inhibin and inhibin pro-
C was observed when ovulatory follicles were ruptured, whereas concentrations of
estradiol-17
in plasma showed an abrupt decline but not a selective
increase. Changes in circulating inhibin pro-
C were completely
parallel with those of ir-inhibin during the periovulatory period in
these mares. Plasma concentrations of FSH increased, parallel with LH, before ovulation and declined when the postovulatory increase in
ir-inhibin and inhibin pro-
C occurred. The peak of ir-inhibin and
inhibin pro-
C was observed at the same time when the ovulation was
confirmed by transrectal palpation and ultrasound scanning, and the
increase of circulating ir-inhibin and inhibin pro-
C was observed
for 8-12 h, followed by an abrupt decline. Plasma concentrations
of progesterone began to increase at 3 days after ovulation.

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Fig. 4.
Changes in plasma concentrations of LH ( ) and FSH ( ),
A, ir-inhibin ( ) and inhibin
pro- C ( ), B, and estradiol-17
( ) and progesterone ( ), C, around ovulation during
estrous cycle in mare A. Shadow area
represents sampling of 4-h interval, and open area represents sampling
of 24-h interval. Ovulation occurred in the hatched
area.
|
|

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Fig. 5.
Changes in plasma concentrations of LH ( ) and FSH ( ),
A, ir-inhibin ( ) and inhibin
pro- C ( ), B, and estradiol-17
( ) and progesterone ( ), C, around ovulation during
estrous cycle in mare B. Shadow area
represents sampling of 4-h interval, and open area represents sampling
of 24-h interval. Ovulation occurred in the hatched area.
|
|

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Fig. 6.
Changes in plasma concentrations of LH ( ) and FSH ( ),
A, ir-inhibin ( ) and inhibin
pro- C ( ), B, and estradiol-17
( ) and progesterone ( ), C,
around ovulation during estrous cycle in mare
C. Shadow area represents sampling of 4-h interval, and
open area represents sampling of 24-h interval. Ovulation occurred in
the hatched area.
|
|
 |
DISCUSSION |
The present study clearly demonstrates that circulating levels of
ir-inhibin and inhibin pro-
C increase selectively when ovulatory
follicles rupture. The peak levels of both ir-inhibin and inhibin
pro-
C were detected at the same time, and the peak levels of both
inhibins were maintained for 8-12 h after ovulation. The selective
increase in circulating ir-inhibin and inhibin pro-
C demonstrated in
the present study is a specific phenomenon in mares. Previous reports
in other mammals, such as rats (36), guinea pigs (32), cows (13), goats
(19), pigs (12), monkeys (22), and humans (16), have shown that plasma
levels of ir-inhibin increase during the follicular phase and abruptly
decline when the LH surge is initiated, followed by low levels during
the process of ovulation. Although exact mechanisms responsible for the
selective increase in circulating ir-inhibin and inhibin pro-
C in
mares are not clear at the present time, they may be closely correlated with ovulation. One of the reasons for the selective increase in
circulating inhibin and inhibin pro-
C during the process of ovulation in mares may be the size of ovulatory follicles. The size of
mature follicles is extremely large compared with the other animals.
The size of fully mature ovulatory follicles in mares reaches >6 cm
in diameter. In comparison, the size of mature ovulatory follicles in
cows is ~1.5-2.0 cm in diameter (13). This
indicates that the volume of follicular fluid in mature follicles of
mares is much larger than that in mature follicles of cows. In
addition, large preovulatory follicles of mares contain a large amount
of ir-inhibin, inhibin pro-
C, and estradiol-17
in follicular fluid, and the amount of hormones was significantly greater than in
follicular fluid from small and medium-sized follicles (34). These
observations indicate that the preovulatory follicle contains the large
volume and high concentrations of ir-inhibin, inhibin pro-
C, and
estradiol-17
. It was supposed that large preovulatory follicles
rupture and release follicular fluids into the peritoneal cavity.
Ir-inhibin and inhibin pro-
C may be absorbed immediately into the
circulation, which would induce a temporary increase in circulating
levels of ir-inhibin and inhibin pro-
C. On the other hand, another
follicular hormone, estradiol-17
, which is a fat-soluble steroid
hormone, may be absorbed into the fat of the peritoneal cavity rather
than into the circulation. This may be one of the reasons that the
temporary increase in circulating estradiol-17
was not observed at
the time of ovulation.
In the present study, we used the human inhibin pro-
C ELISA assay
kit to measure the plasma concentrations of inhibin pro-
C in mares.
It was already reported that this assay kit was useful for measuring
inhibin pro-
C in hamsters (23). Inhibin A and Inhibin B ELISA assay
kits were also developed and used to measure inhibin A and inhibin B in
humans (8, 9, 25), rats (3), and hamsters (23). However, such kits were
not able to measure circulating concentrations of inhibin A and inhibin
B in mares, whereas concentrations of these hormones in follicular
fluid were detectable, probably due to low cross-reaction (34). It was reported that free forms of the
-subunit, including particularly inhibin pro-
C, were also found abundantly in serum and follicular fluids in humans (15, 26, 29). Previous papers indicated that free
forms of the
-subunit might have some role in the endocrine, autocrine, or paracrine systems. Furthermore, other evidence exists that immunization of sheep with inhibin
N region impaired fertility (4) and that pro-
N-
C modulates the binding of FSH to its receptor
(30).
In summary, the present study is the first to demonstrate
that a selective increase in circulating inhibin and inhibin pro-
C occurs during the process of ovulation in mares. This unique
phenomenon, an ovulatory surge of inhibin, may be a useful method for
detecting the time of ovulation in mares.
 |
ACKNOWLEDGEMENTS |
We are grateful to National Institute of Diabetes and Digestive and
Kidney Diseases and Drs. Roser, Papkoff, Mori, and Niswender, all
identified in MATERIALS AND METHODS,
for their generous gifts.
 |
FOOTNOTES |
This work was supported by a grant-in-aid from the Equine Research
Institute of the Japan Racing Association.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: K. Taya, Laboratory of
Veterinary Physiology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
(E-mail: taya{at}cc.tuat.ac.jp).
Received 31 March 1999; accepted in final form 16 June 1999.
 |
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