Secretion of Inhibin in Female Japanese Quails (Coturnix japonica) from Hatch to Sexual Maturity
To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.
Journal of Reproduction and Development, Vol. 54, No. 1, 2008, 19112
Secretion of Inhibin in Female Japanese Quails (Coturnix japonica) from
Hatch to Sexual Maturity
, Qiang WENG
, Gen WATANABE
, Mohamed M.M. KANDIEL
, Akira K SUZUKI
, Shinji TANEDA
and Kazuyoshi TAYA
Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University
of Agriculture and Technology, Tokyo 183-8509, Japan,
College of Biological Science and Technology, Beijing Forestry
University, Beijing 100083, P.R. China,
Department of Theriogenology, Faculty of Veterinary Medicine, Benha University,
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project and
Environmental Nanotoxicology Section, Research Center for Environmental Risk, National Institute for Environmental
Studies, Ibaraki 305-8506, Japan
Abstract. To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular
development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after
hatching. Localization of the inhibin/activin α,
subunts was investigated by immunohistochemistry. To
monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone
concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly
increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6
weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased
significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin α,
subunts were localized in the
granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin α,
subunts were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that
the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The
immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in
female Japanese quails.
Key words: Development, Female quail, Inhibin, Luteinizing hormone (LH), Progesterone
(J. Reprod. Dev. 54: 52–57, 2008)
nhibins and activins are structurally related dimeric gonadal
proteins with the ability to regulate follicle-stimulating hor-
mone (FSH) secretion from pituitary glands in mammals [1, 2].
Inhibin consists of an α subunit linked by a disulfide bridge to one
of the 2 highly homologous
) to form inhibin
A (α and
) or inhibin B (α and
) . Apart from their action
on FSH secretion, inhibins and activins have been shown to exert
paracrine/autocrine effects within the gonads [4, 5] and other tis-
sues . In mammalian females, the roles played by inhibin/
activin in modulating FSH secretion by the pituitary gland in vivo
and in vitro have been well documented. Inhibins selectively sup-
press [7–12] while activins stimulate [11–15] the release of FSH.
Previous studies have shown that the avian ovary also produces
inhibins, which plays important roles in the regulation of pituitary
FSH [16–21]. However, the roles of inhibins have received little
attention in quail. As a laboratory animal, the female Japanese
quail has been extensively used in reproductive research, because
of its adaptability to battery breeding cages, small body size, early
sexual maturation, short generation interval, regular egg laying and
high egg production. However, studies on the reproductive endo-
crinology of quails are scarce. Specifically, no research results
have been published concerning the secretion of inhibins in female
quails. In order to clarify the possible endocrine interaction of the
plasma concentrations of immunoreactive (ir) inhibins, the local-
ization of inhibin subunits was investigated in female Japanese
quails from hatching to sexual maturity. The aim of the present
study was to elucidate the relationship between inhibin secretion
and ovarian function during sexual maturation of the Japanese quail
from hatching to sexual maturity.
Materials and Methods
Female Japanese quails (Coturnix japonica) were selected from
low antibody response (L) lines for use in this study from hatching
to sexual maturity [22, 23]. Fertilized eggs were incubated using a
Showa Incubator Laboratory (Trade Mark Showa Furanki, Urawa,
Japan) under regular conditions, such as temperature (38.7 C),
humidity (55 ± 10%) and turning (once every hour), that were con-
trolled daily. One day before hatching (at day 16 of incubation),
candling was performed. The birds were provided with food
(Kanematsu quail diet; Kanematsu Agri-tech, Ibaraki, Japan) and
water and were allowed to feed ad libitum. They were housed in
metal cages in a controlled environment (lights on 0500–1900 h;
Accepted for publication: October 3, 2007
Published online: November 13, 2007
Correspondence: G. Watanabe (e-mail: Gen@cc.tuat.ac.jp)
53INHIBIN IN FEMALE JAPANESE QUAILS
temperature, 23 ± 2 C; humidity 55 ± 10%; air exchanged 20 times
hourly). This study was conducted in accordance with the Guiding
Principles in the Use of Animals in Toxicology and was approved
by the Animal Care and Use Committee of the Japanese National
Institute for Environmental Studies.
Birds were decapitated weekly and blood samples were col-
lected into heparinized tubes between 1000 h and 1200 h. The
blood samples were centrifuged at 4 C for 15 min at 1700 g. The
plasma was separated and stored at –20 C until it was assayed for
ir-inhibin, LH and progesterone by specific radioimmunoassay
(RIA). After dissection, the chicks were sexed, and their ovaries
were removed, weighed and fixed for 24 h in 4% paraformaldehyde
(Sigma-Aldrich Chemical, St. Louis, MO, USA) in 0.05 M PBS
(pH 7.4) for immunohistochemical examination.
RIA of LH, ir-inhibin and progesterone
Luteinizing hormone (LH) concentrations were measured with a
USDA-ARS RIA kit (Beltsville, MD, USA) for chicken LH. The
antiserum used was anti-avian LH (HAC-CH27-01 RBP75). The
hormone for iodination was chicken USDA-cLH-I-3. The results
were expressed in terms of USDA-cLH-K-3. The sensitivity of the
assay for LH was 15.6 pg/tube, and the intra- and interassay coeffi-
cients of variation were 5.2 and 11.2%, respectively. USDA-cLH-
I-3 and USDA-cLH-K-3 were kindly provided by Dr. John A.
Proudman, Biotechnology and Germplasm Laboratory, Animal and
Natural Resources Institute, Beltsville, MD, USA . The antise-
rum against avian LH was kindly provided by the Biosignal
Research Center, Institute for Molecular and Cellular Regulation,
Gunma University, Gunma, Japan . Plasma concentrations of
ir-inhibin were measured as described previously . The iodi-
nated preparation used was 32 kDa bovine inhibin, and the
antiserum used was rabbit antiserum against bovine inhibin
(TNDH-1). The results were expressed in terms of 32 kDa bovine
inhibin. Serial dilutions of plasma from female and male quails
were compared with the standard curve for 32 kDa bovine inhibin.
All curves obtained from the serially diluted samples were parallel
to the standard curve, indicating that it was possible to measure the
concentration of ir-inhibin in the peripheral plasma of the quails
using the present RIA system. The sensitivity of the assay for ir-
inhibin was 19.5 pg/tube of purified bovine 32 kDa inhibin, and the
intra- and interassay coefficients of variation were 8.8 and 14.4%,
respectively. The concentrations of progesterone were determined
by a double-antibody RIA system with
I-labeled radioligands as
described previously . The antiserum against progesterone
(GDN 337) was kindly provided by Dr. G.D. Niswender (Colorado
State University, Fort Collins, CO, USA). The sensitivity of the
assay for progesterone was 2.5 pg/tube, and the intra- and interas-
say coefficients of variation were 6.3 and 7.2%, respectively.
The ovaries were dehydrated through a series of graded concen-
trations of ethanol and xylene, embedded in paraffin, sectioned
serially at 4
m, mounted on glass slides coated with 3-aminopro-
pyltriethoxysilane (APS; Sigma Diagnostics, St. Louis, MO, USA)
and dried overnight at 37 C.
The serial sections of ovaries were incubated with 10% normal
goat serum to reduce background staining caused by the second
antibody. The sections were then incubated with primary antibod-
ies raised against chicken inhibin α subunit (1-25)-NIe-Tyr, cyclic
(#305-24-D) and cyclic inhibin
(#305-25-D) for 12 h at room temperature. The antibody
against chicken inhibin α subunit was kindly provided by Dr. Pat
Johnson (Animal Physiology, Cornell University, NY, USA). The
antibodies for inhibin/activin (
) were kindly provided by
Dr. W. Vale (Salk Institute for Biological Studies, La Jolla, CA,
USA). The sections were then incubated with a second antibody,
goat anti-rabbit IgG conjugated with biotin and peroxidase with
avidin, using a rabbit ExtrAvidin staining kit (Sigma). This was
followed by visualizing with 30 mg 3,3-diaminobenzidine (Wako,
Osaka, Japan) solution in 150 ml of 0.05mol Tris-HCL 1-1 buffer
(pH 7.6) and 30
. Finally, the reacted sections were coun-
terstained with hematoxylin solution (Merck, Tokyo, Japan). The
control sections were treated with normal rabbit serum (Sigma)
instead of the primary antisera. The specificity of antibodies
against the inhibin α,
subunits was not examined using
neutralized antibodies instead of primary antibodies in the present
Mean values (± SEM) were calculated and analyzed using one-
way ANOVA. Duncan’s multiple-range test was used for detection
of significant differences using the SAS computer software pack-
age. A value of P<0.05 was considered to be statistically
Body and ovarian weight
The body and ovarian weights of the female Japanese quails
from hatching to sexual maturity are shown in Fig. 1. Body weight
increased gradually and reached a maximum at 7 weeks of age (Fig.
1A). On the other hand, ovarian weight did not show any signifi-
cant changes by 6 weeks of age, but there was an abrupt increase at
7 weeks of age (Fig. 1B).
Plasma concentrations of LH, ir-inhibin and progesterone
The plasma concentrations of LH did not change until 5 weeks
of age. They then increased significantly at 6 weeks of age and
declined at 7 weeks of age (Fig. 2A).The plasma concentrations of
ir-inhibin remained at a low level until 6 weeks of age and then
abruptly increased at 7 weeks of age (Fig. 2B). There were non-
significant fluctuations in the plasma concentrations of progester-
one until 6 weeks of age. A significant increase in the plasma
concentration of progesterone was observed at 7 weeks of age (Fig.
2C). The pituitary contents of LH began to increase at 6 weeks of
age and showed a significant increase at 7 weeks of age (Fig. 3).
SEDQYAR et al.54
Immunolocalization of inhibin/activin subunits
Immunolocalization of the inhibin α,
subunits in the
Japanese quail ovaries is shown in Fig. 4. Positive staining for
inhibin α-subunit was clearly seen in the granulosa cells of ovarian
follicles from 1–7 weeks post-hatching. However, immunostaining
of theca interna cells was weak or negative in all follicular catego-
ries. Immunostaining for inhibin
was found in the same
cells as inhibin α subunit. Although the theca cell layer showed
lower affinity to the inhibin α,
subunits, some cells
showed positive staining for the inhibin
subunit. The intensity
of immunostaining for inhibin α and
was usually stronger than
in all stages of follicles. Positive staining of the number
of interstitial cells for all the inhibin subunits increased and tended
to surround the health large follicle. No immunostaining was
detected in control sections in which normal rabbit serum was sub-
stituted in place of the primary antibody (data not shown).
The present study demonstrated that developmental changes in
plasma ir-inhibin, LH and progesterone occurred in the female
quails from hatching to sexual maturity and that the plasma concen-
trations of ir-inhibin and progesterone were significantly increased
at 7 weeks of age. These results showed that ovarian activity are
accompanied by developmental changes in circulating ir-inhibin
Fig. 1. Changes in the body (A) and ovarian (B) weights of the
female Japanese quails from 1 to 7 weeks after hatching.
Values are means ± SEM (n=5–12). Within the same panel,
values without common superscripts are significantly
Fig. 2. Changes in the concentrations of LH (A), immunoreactive
(ir) inhibin (B) and progesterone (C) in the female Japanese
quails from 1 to 7 weeks after hatching. Values are means ±
SEM (n=5–12). Within the same panel, values without
common superscripts are significantly different (P<0.05).
Fig. 3. Changes in the pituitary content of LH in the female Japanese
quails from 1 to 7 weeks after hatching. Values are means ±
SEM (n=5–12). Within the same panel, values without
common superscripts are significantly different (P<0.05).
55INHIBIN IN FEMALE JAPANESE QUAILS
and progesterone. In addition, our immunohistochemical results
demonstrated that positive staining for the inhibin α,
subunits is observed in granulosa and interstitial cells, whereas it is
not clear in the theca cells of female quails from hatching to sexual
maturity. These results suggest that the granulosa and interstitial
cells of female quails may secrete bioactive inhibins from hatching
to sexual maturity and that inhibins may play an important role in
the follicular development of Japanese quails. The developmental
changes in inhibins in female Japanese quails were observed for the
first time in the present study, and these observations showed that
Fig. 4. Immunohistochemical localization of the inhibin/activin subunits in the Japanese quail ovary from 1 to 7 weeks after hatching.
Immunostaining of the inhibin α,
subunits was observed in the granulosa and interstitial cells of all follicles at all ages from 1 to 7
weeks of age. Results at 1 week (Ai-A iii), 2 weeks (Bi-B iii), 3 weeks (C i-C iii), 4 weeks (Di-D iii), 5 weeks (Ei-E iii), 6 weeks (F i-F iii) and
7 weeks (G i-G iii) of age are shown. F1 follicles are shown from 5 to 7 weeks of age. The scale bar represents 100
m. G: granulosa cells.
I: interstitial cells.
SEDQYAR et al.56
plasma ir-inhibins were significantly increased at 7 weeks of age.
In other avian species, such as chickens [18–21, 28, 29] and ducks
[30, 31], similar developmental changes in circulating ir-inhibin
concentrations have been observed in accordance with the ovarian
activity. Previous studies in female chickens have shown that
inhibins are involved in regulation of their reproductive function
[18, 19]. Ir-inhibins in plasma have been shown to increase during
sexual maturation , and it has been reported that the ir-inhibin
content of granulosa cells and secretion into culture medium is cor-
related with the plasma levels . Yang et al.  reported that
the rise in ir-inhibin is correlated with age at sexual maturity in
female ducks, while a progressive increase in steroid hormones
may be consistent with a progressive increase in the steroidogenic
processes of the ovary. In the present study, the developmental
changes in circulating ir-inhibins parallelled progesterone and
exhibited higher values at 7 weeks of age. Supporting the present
study, previous studies have shown similar developmental profiles
for ir-inhibin and progesterone that reportedly correspond to Japa-
nese quails. In hens, circulating progesterone concentrations are
positively correlated with those of plasma inhibin A and ir-inhibin,
and the concentrations of inhibin A, ir-inhibin and progesterone
increase progressively prior to the onset of laying . Therefore,
our results are in agreement with the proposals that the maturing
ovary and, in particular, the large preovulatory follicles that are
more steroidogenically active  are primary sources of ir-inhibin
and progesterone .
Gonadal inhibin has been implicated as a negative regulator of
FSH secretion in the anterior pituitary gland with little or no effect
on LH [35, 36]. In this study, the plasma LH levels significantly
increased at 6 weeks of age, but declined at 7 weeks age, indicating
that the increased levels of LH stimulate maturation of large folli-
cles. Similar observations have also been reported in the chicken;
the plasma LH levels start to rise steeply between 16 and 19 weeks
of age, and then decrease at sexual maturity . The present
results suggest that the suppression of plasma LH in female quails
at 7 weeks of age maybe due to a high level of plasma progesterone
through the negative feedback regulation of gonadotropin releasing
hormone (GnRH). These results also agree with opinions
expressed in a review concerning birds, that gonadal steroids have
the ability to feedback at the hypothalamus and pituitary level to
control chicken GnRH receptor gene expression . Identifica-
tion of the sites of expression and production of inhibin subunit
messenger RNA and protein is critical to understanding the biology
of inhibins. In the present study, the inhibin α and inhibin/activin
subunits were expressed in the granulosa cells of all fol-
licles during different stages of development from 1 to 7 weeks
after hatching, indicating that granulosa cells may secrete dimeric
and bioactive inhibins during the follicular development of Japa-
nese quails. Immunolocalization of the inhibin/activin subunits in
the granulosa cells of the quail ovary is in agreement with previous
studies showing that the granulosa cells of chicken [18, 19] and
duck [30, 31] ovaries are the major source of inhibin. In hens, the
greatest amount of mRNA expression for the inhibin/activin
subunit is found in the F
follicle, while the inhibin α subunit is
expressed more abundantly than the inhibin
subunit in large pre-
ovulatory follicles. Furthermore, the greatest amount of mRNA
expression for the inhibin
subunit is found in the small yellow
developmental follicles. This suggests that the
subunit, either as
inhibin A or as activin A, may be critical for ovulatory events,
while the inhibin/activin
subunit may play an important role
during early follicular development . In the present study, the
expressions of the inhibin α,
subunits were similar in the
quail ovarian follicles during the period from hatching to sexual
maturity. This may explain the species difference in ovarian secre-
tion of inhibin during the prepubertal period. These differences
may even be found between breeds, such as in some mammals.
The expression levels of inhibin α and
mRNA have been shown
to be correlated with the status of follicular development, whereas
mRNA is predominanrly expressed in large antral folli-
cles [39–41]. The levels of inhibin α subunit mRNA have been
shown to further increase during developmental maturation of folli-
cles and to dramatically decrease in preovulatory follicles
following the proestrous gonadotropin surges . This type of
difference may extend to the period after maturity in hens ,
whereas there is a differential level of inhibin expression between
broiler breeders and layers. The present study demonstrated that
granulosa cells are primarily the source of secretion of inhibin in
the quail ovary. Moreover, interstitial cells are an additional source
of inhibin and may share in control of the final stages of follicular
development. These results provide new physiological evidence
concerning follicular development of Japanese quails, particulary
that ovarian interstitial cells have the ability to synthesize inhibins.
The presence of inhibin subunits in ovarian interstitial cells could
imply a potential function for inhibin or activin in the quail ovary.
Supporting the present study, previous studies in the golden ham-
ster clearly demonstrated that both the proteins and mRNA of the
inhibin α and inhibin/activin
subunits were found in ovarian
interstitial cells and that its expression was induced by LH surge
[44–46]. In summary, the present results demonstrated that hor-
monal changes depend on age, the rise in ir-inhibin is correlated
with ovarian activity and age at sexual maturity and ovarian granu-
losa cells and interstitial cells are the main sources of inhibin
secretion in the female Japanese quail from hatching to sexual
We thank Dr. G D Niswender, Animal Reproduction and Bio-
technology Laboratory, Colorado State University (Fort Collins,
CO, USA), for providing antiserum to progesterone (GDN 337);
Dr. J A Proudman, USDA-ARS, Biotechnology and Germplasm
Laboratory (Beltsville, MD, USA), for LH radioimmunoassay
materials; Dr. P Johnson (Animal Physiology, Cornell University,
NY, USA) for providing antiserum to chicken inhibin α subunit;
Dr. W Vale (Salk Institute for Biological Studies, La Jolla, CA,
USA) for providing anti-cyclic inhibin
D) and cyclic inhibin
(#305-25-D); and the Bio-
signal Research Center, Institute for Molecular and Cellular
Regulation, Gunma university, Gunma, Japan, for providing antise-
rum against chicken LH. This study was supported in part by
Grants-in-Aid for Scientific Research (Basic research B-18310044,
P06445) and Japan-Thailand joint research from the Japan Society
57INHIBIN IN FEMALE JAPANESE QUAILS
for the Promotion of Science and a Grant-in-Aid from the National
Natural Science Foundation of China (NSFC) (No. 30670261).
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