鹅 卵泡

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 www.jgenetgenomics.org 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. www.jgenetgenomics.org 1110 Journal of Genetics and Genomics 遗传学报 Vol. 34 No. 12 2007 www.jgenetgenomics.org 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 www.jgenetgenomics.org 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