Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) Sep. 2007, 34(12): 1106-1113

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 hormones 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 suppress 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 ovulation in poultry. Although the mechanism involved in the negative regulation of inhibin is relatively clear [12], its direct 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

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: [email protected]; Tel & Fax: +86-27-8728 2092 www.jgenetgenomics.org

Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation……

estrogen and progesterone. Goose is a type of popular and important poultry in China, however, its reproductive 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 involved 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 production and the efficiency of goose production.

1 1.1

Materials and Methods 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 monitored daily by the timing of oviposition. The stage of the cycle was verified by digital palpation of the reproductive tract, and all geese were sacrificed approximately 16 h prior to a midsequence ovulation by cervical dislocation according to the management regulations for experimental animals. The restriction enzymes (BamHⅠ, EcoRⅠ, HindⅢ), Mini-BEST Plasmid Purification Kit and DNA Ligation Kit were purchased from TaKaRa (Dalian, China). Rabbit anti-human inhibin α antibody Table 1

and goat anti-rabbit IgG-FITC antibody were obtained from Boster (Wuhan, China). Estrogen (E2) and progesterone (P) radioimmunoassay kit were purchased from Beijing Chemclin Biotech (Beijing, China). 1.2

Construction of recombinant pSIREN expressing 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 criteria and BLAST searching showed no significant homology with other genes. Two RNAi expression vectors, psiRNA-INHα1 and psiRNA-INHα2, were constructed to interfere inhibin α mRNA expression. In order to accredit these vectors, HindⅢ site was inserted into the vector. The sequences of the oligonucleotides 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-RetroQZsGreen 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′ .

The sequences of the oligonucleotides of siRNAs

Name psiRNA- INHα1

psiRNA- INHα2

Scrambled control

Sense and antisense strand sequences S:

5′-gatccgaaggcatcttcacttaccttcaagagaggtaagtgaagatgccttcttttttaagcttg -3′

A:

3′-gcttccgtagaagtgaatggaagttctctccattcacttctacggaagaaaaaattcgaacttaa-5′

S:

5′-gatccgtacgagacggtgcccaacttcaagagagttgggcaccgtctcgtacttttttaagcttg-3′

A:

3′-gcatgctctgccacgggttgaagttctctcaacccgtggcagagcatgaaaaaattcgaacttaa-5′

S:

5′-gatccgcttcataaggcgcatagcttcaagagagctatgcgccttatgaagcttttttaagcttg-3′

A:

3′-gcgaagtattccgcgtatcgaagttctctcgatacgcggaatacttcgaaaaaattcgaacttaa-5′

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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 follicle were dissected from each ovary and then placed in sterile Hank’s balanced salt solution (HBSS). Early

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30 min. After washing twice with PBS, the cells were incubated with goat anti-rabbit IgG FITC-conjugated antibody for 30 min at RT. The fluorescence intensity was examined by flow cytometry (FACS Arial, Beckton Dickinson). 1.5

atretic follicles were identified on the basis of the

The analyses of apoptosis and proliferation of granulosa cells

presence of follicle haemorrhagia, collapsed mor-

The fixed cells were washed in PBS twice and

phology, and opaque appearance [19]. The granulosa

then incubated with 200 µL RNase A (100 µg/mL) at

cells of F1 and atretic follicle were separated using the

37 ℃ for 30 min. The cells were stained with

[20]

Propidium Iodide (SIGMA, CA) at 4℃ for 30 min

method described earlier

. The harvested granulosa

cells were cultured at 38.5℃ in 6-well culture plates 5

(2.5–5×10 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 reconstructed RNAi plasmids (4 µg/well) using Lipofectamine TM 2000 (Invitrogen, CA) according to the manufacturer’s protocol. To the interference groups were added 4 µg plasmids and 10 µL transfection reagents, while to the mixed groups (psiRNA-INHα1 and psiRNA-INHα2) were added 2 µg for each plas-

and immediately analyzed by flow cytometry. 1.6

The determination of concentrations of estrogen 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 supernatants were collected to assay the concentrations of estrogen (E2) and progesterone (P) by radioimmunoassay.

mid and 10 µL transfection reagents. Each group had

1.7

three replicates, and the same experiments were repeated three times.

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.

1.4

FACS analysis of interfere inhibin α protein expression

Forty-eight hours after transfection, the transfection efficiency was examined by fluorescence microscopy (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

2 2.1

Data analysis

Results 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 HindⅢ, 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 the ones

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Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation……

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 example, the inhibin level in the psiRNA-α2 group was significantly lower than those the control groups (337.3 vs

designed in this study.

503.0, 337.3 vs 514.5, P <0.05) of F1 follicle granuFig. 1 Agrose gel electrophoresis of recombinant plasmids digested with HindⅢ 1: psiRNA- INHα1; 2: psiRNA- INHα2; 3: scrambled control; M: λ -EcoT14Ⅰdigested Marker; 750: pSIREN-RetroQZsGreen

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 microscope after 48 h of transfection, more than 85% of them emitted green fluorescence. Table 2

AF GC*

2.3 Effects of inhibin α gene silencing on the apoptosis and proliferation As the inhibin levels in granulosa cells were reduced, the apoptosis indexes and proliferation indexes of granulosa cells in the RNAi groups were significantly 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

Effects of inhibin α gene silencing on apoptosis, proliferation and E2 and P secretion of cultured granulosa cells Group

F1 GC*

lose cells. This result suggests that the interference vectors can specifically and efficiently knock down the inhibin mRNA expression in granulosa cells (Table 2).

Inhibin level**

Apoptosis index, AI%

Proliferation index, PI%

E2 level (pg/mL)

P level (ng/mL)

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

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

–

b

b

b

b

–

psiRNA-α1

188.7±21.6

33.223±5.677

18.678±2.757

0.624±0.071

psiRNA-α2

173.2±11.5b

35.123±3.165b

21.003±3.276b

0.645±0.042b

–

0.607±0.081b

–

psiRNA-α1and 34.692±3.011b 21.550±3.178b 172.7±19.5b psiRNA-α2 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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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 inhibin 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 proliferation 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 Table 3

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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 secretion of estrogen and progesterone

After inhibin levels in granulosa cells were reduced, the E2 concentrations decreased and P concentrations 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).

Effects of inhibin α gene silencing on cell cycle stages of cultured granulosa cells Group

F1 GC*

AF GC*

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

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

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. 1110 www.jgenetgenomics.org

Fengjian Chen et al.: Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation……

3

Discussions

During ovarian follicle growth and development, the follicular atresia is a negatively selective degenerative 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 crucial for the growth, development and differentiation of ovarian follicles both before birth and during the reproductive life [23]. Inhibin is a member of the transforming growth factor β (TGFβ) 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 expression 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 expression 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 decreased, the apoptosis indexes were increased. The pro-apoptotic action induced by inhibin gene silencing 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 accumulation was reduced [28]. In this study, the concentrations of E2 secreted by granulosa cells were reduced and the P levels were increased with inhibin gene silencing. These results indicate that inhibin has a direct effect on E2 and P secretion by granulosa cells. In the present study, inhibin levels in F1 follicle granulosa cells were significantly higher (514.5) than atretic follicular granulosa cells (295.7). High levels of inhibin have been observed in the dominant follicle, and low or undetectable levels of inhibin have been found in atretic follicles. This finding is also in agreement with the results in rat [29] and duck [30]. When inhibin α gene expression was silenced, the inhibin protein expression was significantly decreased and the apoptosis indexes were increased. Inhibin A expression is increased during the preovulatory follicle development, and the endogenous inhibin works to reduce the apoptosis of granulosa cells to prevent follicular atresia. Acknowledgements: We thank Dr. Zhengrong (Huazhong Agricultural University ) for providing us the RNAi plasmid, and also thank the technicians in Testing Centre of Yangzhou University who helped us during sampling, flow cytometry material preparation and scanning. References 1

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502−509 (in Chinese with an English Abstract).

抑制素 α 基因表达下调对鹅卵泡颗粒细胞凋亡和增殖的影响 陈锋剑1, 姜勋平1, 陈秀萍1,2, 刘桂琼2, 丁家桐2 1. 华中农业大学动物科技学院, 武汉 430070; 2. 扬州大学动物科学与技术学院, 扬州 225009 摘 要: 抑制素 α 基因是控制家禽排卵率的一个重要候选基因。为研究抑制素α基因对体外培养的鹅卵泡颗粒细胞增殖凋亡 的遗传作用, 构建 psiRNA-INHα1 和 psiRNA-INHα2 两个抑制素 α 基因干扰载体, 以降低抑制素 α 基因表达。体外培养鹅 F1 和闭锁卵泡的颗粒细胞, 经抑制素 α 基因干扰 48h 后, 荧光显微镜检测转染效率, 同时用流式细胞术检测抑制素 α 基因表 达水平, 以及细胞生长周期、细胞凋亡指数和增殖指数的变化, 用放免法测定细胞培养上清中雌激素和孕酮水平。结果表明, 抑制素 α 基因干扰后卵泡颗粒细胞抑制素表达水平比对照组降低 30–40％(P <0.05); 细胞凋亡指数和增值指数显著高于对照 组(P <0.05); 细胞分泌雌激素水平显著下降(P <0.05)。由此推断, 抑制素α基因对颗粒细胞具有显著的抗凋亡效应。 关键词: 抑制素 α 基因; RNA 干扰; 颗粒细胞凋亡; 细胞增殖; 鹅 作者简介: 陈锋剑(1982−), 男, 硕士研究生, 研究方向: 动物生殖生物学。E-mail: [email protected]

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