Journal of Genetics and Genomics
(Formerly Acta Genetica Sinica)
December 2007, 34(12): 1106-1113
Received: 2007-04-05; Accepted: 2007-05-15
This work was supported by the National Natural Science Foundation of China (No. 30300253) and Wuhan Chenguang Science and
Technology Project (No. 20065004116−25).
① Corresponding author. E-mail: xunping@gmail.com; Tel & Fax: +86-27-8728 2092
www.jgenetgenomics.org
Research Article
Effects of Downregulation of Inhibin α Gene Expression on
Apoptosis and Proliferation of Goose Granulosa Cells
Fengjian Chen1, Xunping Jiang1, ①, Xiuping Chen1, 2, Guiqiong Liu2, Jiatong Ding2
1. College of Animal Science, Huazhong Agricultural University, Wuhan 430070, China;
2. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
Abstract: Inhibin α is one of the candidate genes that control the ovulation in poultry. To study the genetic effects of inhibin α on
apoptosis and proliferation of goose granulosa cells cultured in vitro, two RNA interference (RNAi) expression vectors,
psiRNA-INHα1 and psiRNA-INHα2, were constructed to knock down inhibin α gene expression. After 48 h of transfection, the
efficiency of these two RNAi expression vectors was examined by fluorescence microscopy. Meanwhile, inhibin protein expression
levels, apoptosis indexes (AI) and proliferation indexes (PI) of granulosa cells were analyzed by flow cytometry. In addition, the
supernatants were collected to assay the concentrations of estrogen (E2) and progesterone (P) by radioimmunoassay. The results
showed that the expression level of inhibin α in the RNAi group were decreased 30%–40% than those in the control groups (P
<0.05) and the apoptosis indexes and proliferation indexes in the RNAi groups were significantly higher than those in the control
groups (P <0.05). However, the E2 concentrations in the RNAi groups were lower than those in the control groups (P <0.05). These
results indicate that inhibin α has antagonistic effect on granulosa cell apoptosis.
Keywords: inhibin α; RNAi; granulosa cell apoptosis; proliferation; goose
Inhibin is one of the gonadal glycoprotein hor-
mones and is principally produced by granulosa cells
of ovarian follicle in the female and sertoli cells of
testis in male [1]. Inhibin forms a disulphide-linked
dimer which share a common α-subunit and differs in
β-subunit (βA-subunit and βB-subunit), βA in inhibin
A (αβA) and βB in inhibin B (αβB). Both inhibin A
and inhibin B have the capacity to specifically sup-
press follicle stimulating hormone (FSH) secretion by
pituitary cells, without affecting LH secretion [2–6].
Immunizations against inhibin α-subunit result in an
increased ovulation in sheep [7], pig [8], chicken [9],
mouse [10] and cow [11]. Therefore, α-subunit is the
functional center of inhibin, and the inhibin α-subunit
may be a potential gene that can increase the ovula-
tion in poultry.
Although the mechanism involved in the nega-
tive regulation of inhibin is relatively clear [12], its di-
rect local effects on granulosa cells is uncertain and
the effects of inhibin α on apoptosis and hormone
secretion of goose granulosa cells have not yet been
reported. The RNA interference (RNAi) has emerged
as a powerful tool for selective inhibition of gene
expression for the study of gene function. In this
study [13−16], we used RNAi technique to silence the
inhibin α gene expression to study the effects of
downregulation of inhibin α gene expression on
granulosa cell apoptosis, proliferation, secretion of
Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation…… 1107
www.jgenetgenomics.org
estrogen and progesterone. Goose is a type of popular
and important poultry in China, however, its repro-
ductive ability remains poor. Most Chinese native
breeds lay less than 30 eggs per year. Thus, improving
egg production is the main focus in goose breeding
and management. Understanding the mechanism in-
volved in the regulation of inhibin α will expand our
knowledge of goose reproduction, which can in return
helps us develop new methods to increase egg pro-
duction and the efficiency of goose production.
1 Materials and Methods
1.1 Animals and reagents
Yangzhou geese (Yangzhou, China), 8−10
months old and laying regular sequences of at least
2–3 eggs, were used in this study. On the basis of
Chen’s report [17], geese were individually caged in
laying batteries, provided with free access to feed and
water, and exposed to a photoperiod of 15L: 9D (light
on at midnight). Individual laying cycles were moni-
tored daily by the timing of oviposition. The stage of
the cycle was verified by digital palpation of the re-
productive tract, and all geese were sacrificed ap-
proximately 16 h prior to a midsequence ovulation by
cervical dislocation according to the management
regulations for experimental animals.
The restriction enzymes (BamH Ⅰ, EcoR Ⅰ,
Hind Ⅲ), Mini-BEST Plasmid Purification Kit and
DNA Ligation Kit were purchased from TaKaRa (Da-
lian, China). Rabbit anti-human inhibin α antibody
and goat anti-rabbit IgG-FITC antibody were obtained
from Boster (Wuhan, China). Estrogen (E2) and pro-
gesterone (P) radioimmunoassay kit were purchased
from Beijing Chemclin Biotech (Beijing, China).
1.2 Construction of recombinant pSIREN ex-
pressing siRNA
RNAi-Ready pSIREN-RetroQ-ZsGreen (BD
Bioscience, CA) was used for DNA vector-based
siRNA synthesis under the control of U6 promoter in
vivo. Inhibin α (GenBank, NM-001031257) siRNAs
were designed according to Ambion web-based crite-
ria and BLAST searching showed no significant ho-
mology with other genes. Two RNAi expression vec-
tors, psiRNA-INHα1 and psiRNA-INHα2, were con-
structed to interfere inhibin α mRNA expression. In
order to accredit these vectors, Hind Ⅲ site was in-
serted into the vector. The sequences of the oligonu-
cleotides are shown in Table 1.
The oligonucleotides (5 μL 20 pmol/μL of sense
strand and 5 μL 20 pmol/μL of antisense strand in 40
μL ultrapure water) were annealed by incubating at
95℃ for 5 min followed by slow cooling at room
temperature. The double-stranded hairpin siRNA
templates were inserted into the pSIREN-RetroQ-
ZsGreen RNAi plasmid and transfected to E. coli
DH5α competent cells, as previously reported [18].
Plasmid DNA isolated from the positive clones was
digested with Hind Ⅲ to confirm the presence of the
insert fragment and sequenced using a primer: 5′-
ATGGACTATCATATGCTTACCGTA-3′.
Table 1 The sequences of the oligonucleotides of siRNAs
Name Sequences
S: 5′-gatccgaaggcatcttcacttaccttcaagagaggtaagtgaagatgccttcttttttaagcttg-3′
psiRNA- INHα1
A: 3′-gcttccgtagaagtgaatggaagttctctccattcacttctacggaagaaaaaattcgaacttaa-5′
S: 5′-gatccgtacgagacggtgcccaacttcaagagagttgggcaccgtctcgtacttttttaagcttg-3′
psiRNA- INHα2
A: 3′-gcatgctctgccacgggttgaagttctctcaacccgtggcagagcatgaaaaaattcgaacttaa-5′
S: 5′-gatccgcttcataaggcgcatagcttcaagagagctatgcgccttatgaagcttttttaagcttg-3′
Scrambled control
A: 3′-gcgaagtattccgcgtatcgaagttctctcgatacgcggaatacttcgaaaaaattcgaacttaa-5′
S means sense strand; A means antisense strand.
1108 Journal of Genetics and Genomics 遗传学报 Vol. 34 No. 12 2007
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1.3 Granulosa cells culture and transfection
Ovaries from geese were harvested after cervical
dislocation and immediately placed in ice-cold saline.
The largest preovulatary follicle (F1) and atresic folli-
cle were dissected from each ovary and then placed in
sterile Hank’s balanced salt solution (HBSS). Early
atretic follicles were identified on the basis of the
presence of follicle haemorrhagia, collapsed mor-
phology, and opaque appearance [19]. The granulosa
cells of F1 and atretic follicle were separated using the
method described earlier [20]. The harvested granulosa
cells were cultured at 38.5℃ in 6-well culture plates
(2.5–5×105 viable cells/well) for 18 h to allow the
cells to reach a confluence in Dulbecco’s modified
eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum at 5% CO2 and 95% humidity.
Cells were transfected with purified recon-
structed RNAi plasmids (4 μg/well) using Lipofec-
tamine TM 2000 (Invitrogen, CA, USA) according to
the manufacturer’s protocol. To the interference
groups were added 4 μg plasmids and 10 μL transfec-
tion reagents, while to the mixed groups (psiRNA-
INHα1 and psiRNA-INHα2) were added 2 μg for
each plasmid and 10 μL transfection reagents. Each
group had three replicates, and the same experiments
were repeated three times.
1.4 FACS analysis of interfere inhibin α protein
expression
Forty-eight hours after transfection, the transfec-
tion efficiency was examined by fluorescence mi-
croscopy (BH2-RFT-T3, Olympus).
Granulosa cells were fixed in 75% alcohol for
16 h, and then washed in phosphate buffered solution
(PBS) (0.1 mol/L, PH 7.2, 0.1% Triton X-100) twice
and permeabilized on ice for 5 min. The cells were
blocked in PBS with 1% bovine serum albumin (BSA)
at room temperature (RT) for 1 h, and then incubated
with rabbit anti-human inhibin α antibody at 37℃ for
30 min. After washing twice with PBS, the cells were
incubated with goat anti-rabbit IgG FITC-conjugated
antibody for 30 min at room temperature. The fluo-
rescence intensity was examined by flow cytometry
(FACS Arial, Beckton Dickinson).
1.5 The analyses of apoptosis and proliferation
of granulosa cells
The fixed cells were washed in PBS twice and
then incubated with 200 μL RNase A (100 μg/mL) at
37℃ for 30 min. The cells were stained with Propid-
ium Iodide (SIGMA, CA, USA) at 4℃ for 30 min
and immediately analyzed by flow cytometry.
1.6 The determination of concentrations of es-
trogen and progesterone
In order to reduce the effect of extrinsic blood
serum on hormone secretion by granulosa cells, the
cells were cultured for 6 h after interference at 38.5℃
in 6-well culture plates in DMEM (GIBCO, CA)
without serum. Forth-eight hours later, the super-
natants were collected to assay the concentrations of
estrogen (E2) and progesterone (P) by radioimmuno-
assay.
1.7 Data analysis
Data were presented as mean ± SD. Statistical
comparisons were performed using a one-way
ANOVA followed by the Tukey test for multiple
comparisons, and the probability values of <0.05 were
considered to represent significant differences.
2 Results
2.1 The construction of RNAi expression vector
As shown in Fig. 1, recombinant plasmids
yielded two bands, 4.1 kb and 2.5 kb after digestion
with Hind Ⅲ , which showed that inhibin α gene
fragments were successfully reconstructed into the
RNAi expression vectors. The results of sequencing
showed that all the inserted fragments were similar to
Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation…… 1109
the ones designed in this study.
Fig. 1 Agrose gel electrophoresis of recombinant plasmids
digested with Hind Ⅲ
1: psiRNA- INHα1; 2: psiRNA- INHα2; 3: scrambled control;
M: λ-EcoT14Ⅰdigested Marker; 750: pSIREN-RetroQ-
ZsGreen
2.2 The efficiency of RNAi expression vectors
Transfection efficiency of RNAi expression
vectors encoding siRNA for inhibin α mRNA in
granulosa cells was assayed by co-transfection with
pEGFP-1 that expresses green fluorescence protein.
When cells were examined under a fluorescence mi-
croscope after 48 h of transfection, more than 85% of
them emitted green fluorescence.
The inhibin levels of atretic follicular granulosa
cells were significantly lower than those of F1 follicle
granulosa cells. Forty-eight hours after transfection,
the inhibin levels in interference groups of F1 and
atretic follicular granulosa cells were decreased,
compared with those in the control groups. For exam-
ple, the inhibin level in the psiRNA-α2 group was sig-
nificantly lower than those the control groups (337.3 vs
503.0, 337.3 vs 514.5, P <0.05) of F1 follicle granulose
cells. This result suggests that the interference vectors
can specifically and efficiently knock down the inhibin
mRNA expression in granulosa cells (Table 2).
2.3 Effects of inhibin α gene silencing on the
apoptosis and proliferation
As the inhibin levels in granulosa cells were re-
duced, the apoptosis indexes and proliferation indexes
of granulosa cells in the RNAi groups were signifi-
cantly higher than those in the control groups. The
percentage of apoptotic cells in the psiRNA-α2 group
was higher than those of the control groups (11.457%
vs 7.135%, 11.457% vs 8.362%, P <0.05) of F1 follicle
granulosa cells. The proliferation in the psiRNA-α2
Table 2 Effects of inhibin α gene silencing on apoptosis, proliferation and E2 and P secretion of cultured granulosa cells
Group Inhibin level** Apoptosis index, AI%
Proliferation
index, PI%
E2 level
(pg/mL)
P level
(ng/mL)
F1GC* Blank control 514.5±38.3a 7.135±1.466a 5.061±2.373a 2.200±0.417a 8.902±0.838a
Scrambled control 503.0±33.6a 8.362±2.036a 5.546±0.885a 1.818±0.353b 11.420±4.409b
psiRNA-α1 371.3±33.6b 10.893±1.477b 6.778±1.433a 1.624±0.218c 12.870±2.001b
psiRNA-α2 337.3±26.7b 11.457±2.029b 6.918±0.715b 1.607±0.104c 13.331±1.617b
psiRNA-α1and
psiRNA-α2
359.7±29.9b 12.160±1.299b 7.673±1.278c 1.626±0.124c 13.117±2.206b
AFGC* Blank control 295.7±24.9a 25.193±5.922a 10.610±4.254a 0.785±0.076a –
Scrambled control 305.5±28.1a 25.633±1.723a 11.597±2.329a 0.775±0.082a –
psiRNA-α1 188.7±21.6b 33.223±5.677b 18.678±2.757b 0.624±0.071b –
psiRNA-α2 173.2±11.5b 35.123±3.165b 21.003±3.276b 0.645±0.042b –
psiRNA-α1and
psiRNA-α2
172.7±19.5b 34.692±3.011b 21.550±3.178b 0.607±0.081b –
Values with different superscript in the same column indicates significant difference (P <0.05).
* F1GC: F1 follicle granulosa cells; AFGC: atretic follicular granulosa cells.
** The level of inhibin protein represented by the fluorescence intensity.
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1110 Journal of Genetics and Genomics 遗传学报 Vol. 34 No. 12 2007
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group was higher than those of the control groups
(6.918% vs 5.061%, 6.918% vs 5.546%, P <0.05).
The percentage of G1 phase in the psiRNA-α2 group
was lower than those of the control groups (93.068%
vs 94.940%, 93.068% vs 94.450%, P <0.05), whereas
the percentage of S phase in the psiRNA-α2 group was
higher than those of the control groups (5.506% vs
3.120%, 5.506% vs 4.408%, P <0.05). When the in-
hibin levels were reduced, the percentage of G1 phase
were decreased with corresponding increase in S
phase (Table 3).
The apoptosis indexes and proliferation indexes
of atretic follicular granulosa cells in the RNAi
groups were higher than those of the control groups.
The apoptosis of the psiRNA-α2 group was higher
than those of the control groups (35.123% vs
25.193%, 35.123% vs 25.633%, P <0.05). The prolif-
eration of the psiRNA-α2 group was higher than those
of the control groups (21.003% vs 10.610%, 21.003%
vs 11.597%, P <0.05). The percentage of G1 phase in
the psiRNA-α2 group was lower than those of the
control groups (78.998% vs 89.390%, 78.998% vs
88.403%, P <0.05), whereas the percentage of S
phase in psiRNA-α2 group was higher than those of
control groups (14.012% vs 9.382%, 14.012% vs
7.533%, P <0.05 ) (Table 2).
The correlation coefficient between inhibin level
and apoptosis index was −0.966 (P <0.05), and the
correlation coefficient between the inhibin level and
proliferation index was −0.924 (P <0.05).
2.4 Effects of inhibin α gene silencing on secre-
tion of estrogen and progesterone
After inhibin levels in granulosa cells were re-
duced, the E2 concentrations decreased and P concen-
trations increased in F1 follicular granulosa cells. E2
concentrations in the psiRNA-α2 group was lower
than those of the control groups (1.607 pg/mL vs
2.200 pg/mL, 1.607 pg/mL vs 1.818 pg/mL, P < 0.05),
and the P concentrations was higher than the blank
control group (13.331 ng/mL vs 8.902 ng/mL, P <
0.05). E2 concentrations in the psiRNA-α2 group of
atretic follicular was lower than those of the control
groups (0.645 pg/mL vs 0.785 pg/mL, 0.645 pg/mL vs
0.775 pg/mL, P <0.05). The P concentrations were
beyond the limit of detection (Table 2).
Table 3 Effects of inhibin α gene silencing on cell cycle stages of cultured granulosa cells
Group G1 phase (%) S phase (%) G2 phase (%)
Blank control 94.940±2.373a 3.120±1.766a 1.940±2.203c
Scrambled control 94.450±0.885a 4.408±1.183b 1.138±1.512a
psiRNA-α1 93.223±1.433a 5.790±1.489c 0.987±0.941a
psiRNA-α2 93.068±0.624b 5.506±1.262c 1.413±0.844b
F1GC*
psiRNA-α1and psiRNA-α2 92.327±1.278b 5.711±2.327c 1.962±2.687c
Blank control 89.390±4.254a 9.382±3.618a 1.228±0.819a
Scrambled control 88.403±2.329a 7.533±4.108a 4.063±3.771b
psiRNA-α1 81.322±2.757b 13.847±4.595b 4.832±3.697b
psiRNA-α2 78.998±3.276b 14.012±5.247b 6.990±5.225c
AFGC*
psiRNA-α1and psiRNA-α2 78.450±3.178b 12.787±5.182b 8.763±4.595d
Values with different superscript in the same column indicates significant difference (P <0.05).
* F1GC: F1 follicle granulosa cells; AFGC: atretic follicular granulosa cells.
Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation…… 1111
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3 Discussion
During ovarian follicle growth and development,
the follicular atresia is a negatively selective degen-
erative process which involves granulosa cells death
via apoptosis. Apoptosis is a distinct physiological
form of cell death with characteristic morphological
and biochemical changes [21, 22]. The balance between
proliferation and apoptosis of granulose cells is cru-
cial for the growth, development and differentiation
of ovarian follicles both before birth and during the
reproductive life [23]. Inhibin is a member of the trans-
forming growth factor β (TGF-β) superfamily, and has
been proposed as an autocrine/paracrine factor that
modulates follicular growth, atresia, gonadotropin
responsiveness and steroidogenesis [3, 24, 25]. In this
study, we used RNAi technique to silence inhibin α
gene expression to study the effects of inhibin α gene
silencing on granulosa cells apoptosis. When inhibin
α gene expression was knocked down, the apoptosis
indexes were significantly increased (Table 2). The
correlation coefficient between inhibin α gene expres-
sion and apoptosis index was −0.966 (P <0.05), which
suggested that inhibin α gene has a direct effect on
apoptosis of granulosa cells. It has been reported that
adding exogenous inhibin could increase the amount
of ovarian follicle [26] and inhibit apoptosis [23], which
supports our conclusion that inhibin α gene has
antagonistic effect on granulosa cells apoptosis.
However, the mechanism of increased proliferation
remains unknown.
Adding inhibin A (10 ng/mL) to the cultured
granulosa cells resulted in an enhanced expression of
prooncogenes (Bcl-2, Bcl-xl) and a reduced expres-
sion of caspase-3 and pro-apoptotic protein Bak [23].
Apoptosis in human granulosa cells is regulated
mainly by caspases and Bcl-2 family members [27].
When inhibin gene expression was significantly de-
creased, the apoptosis indexes were increased. The
pro-apoptotic action induced by inhibin gene silenc-
ing may be because of a result of the imbalance be-
tween anti-apoptotic and pro-apoptotic proteins, and it
may interfere with follicular development by a
mechanism yet unknown.
Immunoneutralization of endogenous inhibin
resulted in a significant decrease in estrogen secretion
and an increase in progesterone accumulation. When
antiserum-treated follicles were supplemented with
exogenous inhibin, estrogen secretion was restored
and progesterone accumulat