Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells 已报道的全基因组选择性消除分析中发现 IGF2BP2 基因的插入/缺失变异与山羊产羔数相关 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xuyinxue@njau.edu.cn †E-mail:xuyinxue@njau.edu.cn xuyinxue@njau.edu.cn College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated ‘gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly ( P < 0.05) changed the expression of Cyclin D2 , CDK4 , Bcl-2 , and Cyp19a1 . These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs. 目的 2 验证 IGF2BP2 插入/缺失与陕北白绒山羊产羔数之间的关系，并选择有利的等位基因。 创新点 2 首次筛选、验证山羊 IGF2BP2 基因的InDel位点，确定其与山羊产羔性状的相关性及作用，为山羊经济性状改良及新品种培育提供理论依据。 方法 2 采用实时荧光定量聚合酶链式反应（qRT-PCR）方法分析 IGF2BP2 在单羔组和多羔组中的表达水平；通过PCR扩增-琼脂糖凝胶电泳在918只陕北白绒母山羊中鉴定 IGF2BP2 基因P4-Ins-13bp和P5-Del-12bp缺失/插入位点基因型，并进行测序以验证突变；利用SHEsis平台分析两个位点的连锁不平衡；采用方差分析（ANOVA）和 χ 2 检验分析山羊 IGF2BP2 基因变异与产羔数的关系；并利用在线数据库预测转录因子（TF）和突变区域序列的结合。 结论 2 本研究发现， IGF2BP2 的mRNA表达水平在单羔组明显高于多羔组。在陕北白绒母山羊中验证了 IGF2BP2 基因内的插入/缺失（indels），包括P4-Ins-13bp和P5-Del-12bp。 χ 2 检验表明，P4-Ins-13bp基因型（ χ 2 =14.479， P =0.006）在单羔和多羔组间分布差异显著；相关分析表明，P4-Ins-13bp与山羊产仔数显着相关（ P =0.022），具有缺失/缺失（DD）基因型的山羊个体产仔数高于其他山羊。因此， IGF2BP2 基因的P4-Ins-13bp突变可作为一种潜在的与繁殖性状显著相关的分子标记，以优化母羊繁殖力，提高山羊产业的经济效益。 Porcine granulosa cells Bone morphogenetic protein (BMP)/Smad Smad4 RNA interference (RNAi) Growth Steroidogenesis 山羊 IGF2BP2 产仔数 插入/缺失 分子标记辅助选择 Introduction 1 Female fertility in mammals is dependent on the growth and development of follicles. Folliculogenesis involves a series of sequential steps in which a growing follicle either develops to the ovulation stage or undergoes atresia. Significant disruption occurring at any of the stages of folliculogenesis impairs fertility (McGee and Hsueh, 2000 2000 ). Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGF-β) superfamily, and are key factors for normal folliculogenesis. Recent studies have established that BMPs play a crucial role in female fertility in mammals by regulating steroidogenesis as well as mitogenesis in granulosa cells (GCs) (Shimasaki et al., 2003 2003 ; 2004 2004 ). BMP-6 inhibits follicle-stimulating hormone (FSH)-induced progesterone synthesis through the suppression of cellular cyclic adenosine monophosphate (cAMP) synthesis (Otsuka et al., 2001a 2001a ). BMP-15 also exhibits potent suppression of FSH actions by inhibiting FSH receptor (FSHR) expression (Otsuka et al., 2001b 2001b ). In addition, growth differentiation factor-9 (GDF-9), which is highly homologous with BMP-15, also inhibits FSH-induced steroidogenesis and luteinizing hormone (LH) receptor expression in rat GCs (Vitt et al., 2000 2000 ; Miyoshi et al., 2006 2006 ). TGF-β activates a cell surface receptor complex, which consists of two different transmembrane serine/threonine kinases that phosphorylate members of the downstream receptor-regulated Smads (R-Smads) upon ligand binding (Schmierer and Hill, 2007 2007 ). The closely related R-Smads, Smad1, Smad5, and Smad8, transmit BMP and GDF signals, while Smad2 and Smad3 are phosphorylated in response to TGF-β, activin and nodal signals. The phosphorylated R-Smads in combination with the common mediators, Smad and Smad4, recruit appendant cofactors to form complexes that in turn regulate target gene expression (Massagué et al., 2005 2005 ). Smad4, originally discovered to be a tumor suppressor gene, shares structural features with the R-Smads. However, its MH2 domain lacks the C-terminal SXS motif required for receptor-mediated phosphorylation. Smad complexes regulate various kinds of biological processes, including cell proliferation, differentiation, and apoptosis during embryonic development and adult tissue homeostasis (Costello et al., 2009 2009 ). In mammals, Smad4 is the only co-mediating Smad, translocating to the nucleus with phosphorylated R-Smads, and then modulating transcription of BMP target genes. Smad4 is extensively expressed in embryonic and adult diverse tissues. Smad4 nucleocytoplasmic shuttling is not required for R-Smad phosphorylation or nuclear localization (Biondi et al., 2007 2007 ). R-Smad dephosphorylation and nuclear export are thought to be essential for optimal TGF-β signaling (Lin et al., 2006 2006 ; Dai et al., 2009 2009 ). Loss of Smad4 leads to increased steady state levels of both BMP and TGF-β phosphorylated R-Smads. Enhanced R-Smad phosphorylation levels potentially reflect reduced dephosphorylation and/or nuclear export (Lin et al., 2006 2006 ; Dai et al., 2009 2009 ; Costello et al., 2009 2009 ). Therefore, Smad4 is crucial for BMP/Smad signaling. The aim of this study was to determine how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs using highly potent small interfering RNA (siRNA) interference to suppress Smad4 gene expression. Materials and methods 1 Isolation and culture of GCs 2 Porcine ovaries were obtained from a local slaughterhouse, and transported to the laboratory at 37 °C in saline containing gentamicin and amphotericin. The ovaries were washed three times with pre-warmed phosphate-buffered saline (PBS) supplemented with gentamicin and amphotericin. GCs were collected from follicles (3–5 mm) by needle (20 G) aspiration, and filtered through a stainless steel filter (100 μm) to remove the cumulus-oocytes complexes (COC). The cells were centrifuged at 800× g per min and washed twice in DMEM/F12 medium (Dulbecco’s modified Eagle medium/Ham’s nutrient mixture F-12; Shanghai Invitrogen Biotechnology Co., Ltd., China). GCs were cultured as described previously (Picton et al., 1999 1999 ). GCs were counted in a hemocytometer, and the viability was determined by trypan blue dye exclusion, and then the GCs were seeded in six-well culture plates (Costar; Corning Inc., NY, USA) at a density of (3–5)×10 6 cells per well in 2 ml of DMEM/F12 medium containing 10% (v/v) fetal calf serum (FCS; Minhai Co., Lanzhou, China), 5 μl/ml of gentamicin, and 10 μl/ml amphotericin. The cells were cultured for 24 h at 37 °C in a 5% (v/v) CO 2 atmosphere and then the wells were washed with PBS to remove unattached cells. Exponentially growing cells were used for the experiments. Design and transfection of siRNA 2 The complementary DNA (cDNA) sequence of porcine Smad4 (GenBank accession No. NM_214072) was examined using Invitrogen’s web-based siRNA design software ( https://rnaidesigner.invitrogen.com/rnaiexpress/ https://rnaidesigner.invitrogen.com/rnaiexpress/ ) to select appropriate siRNA target sites. A pair of oligonucleotides corresponding to Smad4 cDNA were designed. A BLAST (basic local alignment search tool) search of these sequences confirmed their specificity to Smad4 only. In addition, a nonsense sequence with no similar match to any known sequence was used as control. The Smad4 small RNA interference (RNAi) was named Smad4-siRNA, and the nonsense sequence to Smad4 was named negative control siRNA (NC-siRNA). GC cultures were transfected with Smad4-siRNA or NC-siRNA using Lipofectamine™ RNAiMAX Reagent (Shanghai Invitrogen Biotechnology Co., Ltd.). Briefly, RNAi duplex, Lipofectamine™ RNAiMAX, and 250 μl DMEM/F12 medium were combined and incubated for 20 min at room temperature. The transfection complex was added to the cells, and incubated for 48 h at 37 °C. Measurement of Smad4 expression by real-time RT-PCR 2 Total RNA was extracted from GCs using TRIzol following the manufacturer’s instructions. RNA concentration and purity were determined by measuring optical density (OD) at wavelengths of 260 and 280 nm using a standard spectrophotometer. The OD 260 /OD 280 ratios were more than 1.8 for all samples. Aliquots (100 ng) of total RNA from each pool of cultured GCs were independently reverse-transcribed to cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega Corporation, USA) and oligonucleotide primers. Target genes and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) were quantified by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) with an ABI 7300 using a commercial kit (SYBR® Premix Ex Taq™, TaKaRa, Dalian, China). The gene-specific primers were designed on the basis of porcine mRNA sequences (Table 1 1 ). The cDNA generated was used as a template for PCR using a 25-μl reaction mixture for 40–45 cycles. Relative concentrations of mRNA were calculated using the 2 −ΔΔ C T method (Livak and Schmittgen, 2001 2001 ). A water control was included to identify possible contamination in all reactions. After amplification, each sample was applied to a 2% (w/v) agarose/ethidium bromide gel for electrophoresis. Target gene (accession number) Primer sequence Product size (bp) Annealing temperature (°C)   Smad4 (NM_214072) Forward: TTTGCGTCAGTGTCATCG; Reverse: TGCTCTGCCTTGGGTAAT 236 59.5   Cyclin D2 (NM-214088) Forward: TTACCTGGACCGCTTCTTG; Reverse: GAGGCTTGATGGAGTTGTCG 155 55.0   CDK4 (NM-001123097) Forward: GCATCCCAATGTTGTCCG; Reverse: GGGGTGCCTTGTCCAGATA 125 60.5   Bcl-2 (AB271960) Forward: TTCTTTGAGTTCGGTGGGG; Reverse: CCAGGAGAAATCAAATAGAGGC 195 62.0   Bax (AJ606301) Forward: CCGAAATGTTTGCTGACG; Reverse: AGCCGATCTCGAAGGAAGT 154 58.0   Cyp19a1 (SSU92246) Forward: GCTGCTCATTGGCTTAC; Reverse: TCCACCTATCCAGACCC 187 60.5   Cyp11a1 (Bx674071) Forward: AGACACTGAGACTCCACCCCA; Reverse: GACGGCCACTTGTACCAATGT 110 69.5   GAPDH (AF017079) Forward: GGACTCATGACCACGGTCCAT; Reverse: TCAGATCCACAACCGACACGT 220 57.2 Measurement of Smad4 expression by immunofluorescence 2 GCs were grown in glass chamber slides. After treatments were applied, cells were washed with PBS three times, fixed with 4% (w/v) paraformaldehyde, washed in PBS, permeabilized with 0.3% (v/v) Triton X-100 in PBS, and blocked in 0.5% (w/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X-100 in PBS for 30 min at room temperature. Slides were then incubated with primary antibodies as follows: rabbit monoclonal antibody against Smad4 (1:200 dilution in PBS; Abcam; ab40759) for 2 h at room temperature. Slides were washed three times with cold PBS and incubated with biotinylated anti-rabbit immunoglobulin G (IgG; 1:200 dilution in PBS; Abcam; ab6939) for 1 h at room temperature, and then mounted with Vectashield mountant containing the nuclear counter-stain 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA). Fluorescent images were captured with a Leica DMLB2 fluorescent microscope. Morphometric analysis was carried out using the ImageJ program. Measurement of Smad4 expression by Western blotting 2 Total proteins from GCs were extracted in a cell lysis buffer for Western and immunol precipitation (Beyotime, China) containing 10 mmol/L phenylmethyl sulfonylfluoride (PMSF; Beyotime, China), and the resultant cell suspension was centrifuged at 12 000 r/min for 5 min. The supernatant was removed and the amount of protein was quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime, China). The homogenized samples (15 μg crude proteins per lane) were subjected to electrophoresis on 12% (w/v) poly-acrylamide-sodium dodecyl sulfate (SDS) gels under standard reducing conditions with precision protein molecular weight marked (MBI Fermentas, China), and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) at 2.5 mA/cm 2 for 45 min. Then they were blocked with 5% (w/v) BSA in PBS (blocking buffer) for 1 h at room temperature. Immunoblotting was carried out by incubating the membranes overnight at 4 °C with antibodies against Smad4 (Abcam; 1:2000 dilution) or GAPDH (Sigma, 1:2000 dilution). The membranes were washed three times with 1% (v/v) Tween-20 in PBS, then hybridized with horse radish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., California, USA) diluted at 1:5000 for 2 h. After three washes with 1% (v/v) Tween-20 in PBS, the signals were detected as chemical luminescene by X-ray film with BeyoECL Plus kit (Beyotime, China). Measurement of cell growth by MTT assay 2 GCs were counted in a hemocytometer, and the viability was determined by trypan blue dye exclusion, and then the GCs were seeded in 96-well culture plates (Costar; Corning Inc., NY, USA) at a density of (3–5)×10 4 cells per well. GCs were treated with or without siRNA. The number of live cells at the end of the process of culture was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) kit (KeyGEN Biotechnology Co., Ltd., Nanjing, China), as recommended by the manufacturer. The optical density at 550 nm (OD 550 ) was determined using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-TEK Instruments, USA). Measurement of cell cycle and apoptosis by flow cytometry 2 After culture, GCs were digested and prepared by washing twice with PBS, and then fixed in 70% (v/v) ethanol and stained with 100 μg/ml propidium iodide (PI) at 4 °C for 1 h. DNA content was monitored by FACScan (COULTESR, Elite, USA). Also after culture, GCs were digested and prepared by washing twice with PBS, and then 5 μl annexin V-rhodamine isothiocyanate (RITC) and PI were added to the cells, combined, and incubated for 15 min at room temperature. The rate of apoptosis was monitored by FACScan (COULTESR, Elite, USA). Measurement of estradiol and progesterone 2 GCs (2×10 5 viable cells) were cultured in 24-well plates. After treatment, the culture medium was collected and stored at −80 °C until assay. The levels of estradiol (E2) and progesterone (P4) in the medium were measured by chemiluminescent immunoassay (CLIA; ACCESS, Beckman Coulter Co., USA). Steroid contents were negligible (P4 < 0.1 ng/ml and E2 < 10 pg/ml). The intra- and inter-assay coefficients of variation were 15% and 20% for E2, and 5% and 10% for P4, respectively. Statistical analysis 2 All results are shown as mean±standard error of the mean (SEM) from at least three separate experiments, each performed with triplicate samples. Data were analyzed by one-way analysis of variance (ANOVA), followed by the Fisher’s least significant difference (LSD) test for multiple comparisons using SPSS 16.0 software. Differences were considered to be statistically significant at the 95% confidence level ( P < 0.05). Results 1 Effect of siRNA on Smad4 expression 2 We knocked down Smad4 in porcine GCs by siRNA. The expression of Smad4 mRNA and protein was analyzed by real-time PCR, immunofluorescence, and Western blotting at post-transfection, as described in Fig. 1 1 . The results in Fig. 1a 1a show that the expression of Smad4 mRNA was inhibited after transfection with Smad4-siRNA, the Smad4 transcript level being reduced by approximately 89% compared with that in the control cultures (blank and NC-siRNA). Western blotting and immunofluorescence staining analyses of Smad4 expression clearly revealed the suppressive effect of Smad4-siRNA on the expression of the Smad4 protein in the GCs (Figs. 1c and 1d 1c and 1d ). The results of the analyses demonstrate that the level of Smad4 protein also markedly decreased 48 h post-transfection with the Smad4-siRNA, by approximately 56.6% (Fig. 1b 1b ). (a) Expression of Smad4 mRNA after transfection for 48 h; (b) Expression of Smad4 protein after transfection for 48 h; (c) Silencing effect of Smad4 protein expression measured by Western blotting; (d) Silencing effect of Smad4 protein expression measured by immunofluorescence staining. After treatment, cells were fixed and stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; blue). The data are presented as mean±SEM of three independent experiments. * Statistically significant differences from the blank group ( P < 0.05) Effect of interrupted BMP/Smad signaling on growth of GCs 2 MTT analysis was performed to test the effect of interrupted BMP/Smad signaling on the growth of GCs. As expected, interrupted BMP/Smad signaling significantly inhibited the growth of cells, as compared with the control cultures (blank and NC-siRNA) cells (Table 2 2 ). Values are presented as mean±SEM. Statistically significant differences ( P< 0.05) among the groups are indicated by different letters Group OD 550 * *  Blank 0.2360±0.01 a  NC-siRNA 0.2616±0.02 a  Smad4-siRNA 0.1510±0.01 b Effect of interrupted BMP/Smad signaling on cell cycle of GCs 2 To further determine the effect of interrupted BMP/Smad signaling on the growth of GCs, flow cytometry was used to analyze the cell cycle changes of GCs. The results in Table 3 3 show that interrupted BMP/Smad signaling can significantly increase the proportion of cells in G1-phase, and decrease the cells in S-phase, and there are no significantly differences in the proportion of cells in G2-phase. Values are presented as mean±SEM. Statistically significant differences ( P < 0.05) among the groups are indicated by different letters 2 Group 3 Cell cycle G0/G1 S G2/M Blank 86.52±0.53 a 5.65±0.32 a 6.50±0.16 NC-siRNA 87.31±0.61 a 5.95±0.56 a 6.74±0.06 Smad4-siRNA 92.44±0.16 b 1.66±0.08 b 5.89±0.16 Effect of interrupted BMP/Smad signaling on cell cycle marker genes 2 To investigate the mechanism by which interrupted BMP/Smad signaling regulates the cell cycle of GCs, we measured the expression of Cyclin D2 and CDK4 , functional markers of cell proliferation (Fig. 2 2 ). In Smad4 knockdown cells, Cyclin D2 and CDK4 mRNA levels were lower than those in the controls. Data are presented as mean±SEM of three independent experiments. * Statistically significant differences ( P < 0.05) among the groups Effect of interrupted BMP/Smad signaling on apoptosis of GCs 2 Flow cytometry analysis was performed to test the effect of interrupted BMP/Smad signaling on the apoptosis of GCs. Table 4 4 shows that interrupted BMP/Smad signaling increased the rate of apoptotic cells, as compared with the control cultures (blank and NC-siRNA). Values are presented as mean±SEM. Statistically significant differences ( P < 0.05) among the groups are indicated by different letters 2 Group 3 Rate (%) Living cells Apoptotic cells Dead cells Blank 83.12±0.73 15.98±0.62 a 0.90±0.06 NC-siRNA 81.89±0.95 17.26±0.46 a 0.85±0.10 Smad4-siRNA 75.16±0.56 23.95±0.88 b 0.89±0.08 Effect of interrupted BMP/Smad signaling on cell apoptosis related genes 2 To investigate the mechanism by which interrupted BMP/Smad signaling encourages the apoptosis of GCs, we measured the mRNA expression of Bcl-2 and Bax , related markers of apoptosis cell (Fig. 3 3 ). In Smad4 knockdown cells, Bcl-2 mRNA levels were significantly lower than those in controls. In contrast, the Bax mRNA levels were not affected by interrupted BMP/Smad signaling. Data are presented as mean±SEM of three independent experiments. * Statistically significant differences ( P < 0.05) among the groups Effect of interrupted BMP/Smad signaling on estradiol and progesterone production 2 We examined interrupted BMP/Smad signaling effects on steroidogenesis. As shown in Table 5 5 , interrupted BMP/Smad signaling decreased E2 production, and had no significant effect on P4 production. Values are presented as mean±SEM. Statistically significant differences ( P < 0.05) among the groups are indicated by different letters Group E2 (pmol/L) P4 (nmol/L)  Blank 117.25±2.75 a 3.075±0.19  NC-siRNA 120.86±6.15 a 3.642±0.14  Smad4-siRNA 102.65±3.25 b 2.943±0.08 Effect of interrupted BMP/Smad signaling on steroidogenic enzyme genes 2 To confirm whether the effects of interrupted BMP/Smad signaling were involved in the expression changes of steroidogenic enzyme genes, levels of mRNA encoding Cyp19a1 and Cyp11a1 , key enzymes regulating E2 and P4 production in GCs (Lee et al., 2001 2001 ), were examined by quantitative real-time PCR. Interrupted BMP/Smad signaling decreased Cyp19a1 mRNA expression (Fig. 4 4 ). Data are presented as mean±SEM of three independent experiments. * Statistically significant differences ( P < 0.05) among the groups Discussion 1 The BMP family controls a variety of intraovarian reproductive processes (Shimasaki et al., 2004 2004 ). To further understand the effect of endogenous BMP/Smad signaling on porcine GCs, in the present study, RNAi was employed to silence the Smad4 gene, the key mediator of BMP/Smad signaling. It was observed that the knockdown of endogenous Smad4 gene blocked BMP/Smad signaling, inhibited the growth of GCs, induced cell apoptosis, and decreased E2 production, suggesting that the endogenous BMP/Smad signaling is important to porcine GCs. Pangas et al. ( 2006 2006 )’s study on the Smad4 conditional knockout (cKO) ovary suggested that one of the primary defects of Smad4 loss is premature luteinization of GCs. We silenced endogenous Smad4, as an alternative to block BMP/Smad signaling, in porcine GCs to characterize a potential role of BMP/Smad signaling. We selected RNAi technique in part because the sequence-specific knockdown of target mRNA is more efficient than that of antisense oligonucleotides or ribozymes (Dorsett and Tuschl, 2004 2004 ; Mittal, 2004 2004 ). Our results show that Smad4-siRNA can significantly inhibit the mRNA and protein expression of Smad4, as demonstrated by real-time RT-PCR, Western blotting, and immunofluorescence staining. We established a strategy for identifying efficient siRNA sequence for gene knockdown by transfecting porcine GCs. It has been well documented that cell proliferation depends on the cell cycle. Therefore, we investigated the effects of blocked BMP/Smad signaling on cell cycle of porcine GCs. Blocked BMP/Smad signaling significantly increased and decreased the cells of G1 and S phase stages, respectively. We also investigated the mechanism by which blocked BMP/Smad signaling suppresses cell growth in GCs. The G1 checkpoint can be viewed as a master checkpoint of the mammalian cell cycle (Paterson et al., 2002 2002 ; Matsuura et al., 2004 2004 ). Regulation of the G1 phase of the cell cycle involves many different families of cyclins, Cyclin D and cyclin-dependent kinases (CDKs). Inhibition of growth of GCs by blocked BMP/Smad signaling was associated with a decrease in Cyclin D2 and CDK4 expression. Cyclin D2 and CDK4 act as a downstream gene of Smad4 , participating in the regulation of BMPs in porcine GCs, and they may be involved in BMPs-induced cell growth in a Smad4-dependent manner. Thus, we postulate that at least part of blocked BMP/Smad signaling effect on GCs growth is mediated by the decrease in Cyclin D2 and CDK4 expression. RNAi-mediated Smad4 silencing resulted in decreased proliferation of the cells and G1 cell cycle arrest, indicating that BMP/Smad signaling plays a key role in the normal growth of porcine GCs. Follicular development in mammals is dependent on many factors, such as gonadotropin hormones, FSH and LH, and the steroid hormones, E2, and P4, as well as ovarian local factors including TGF-β superfamily. Yamashita et al. ( 2007 2007 ) and Kawashima et al. ( 2008 2008 ) believed that the pituitary gonadotropin FSH is essential for GCs’ E2 production and increases estrogen concentrations in follicular fluid during the development of mammalian ovarian follicles. Changes in the expression of mRNA encoding steroidogenic genes exhibit patterns of expression that coordinated with steroid production. In vivo, the growth and differentiation of GCs are tightly regulated by gonadotropins and ovarian local factors such as estrogen, GDF-9/BMP-15, and insulin-like growth factor 1 (IGF-1). However, in vitro, people cannot mimic these conditions. We investigated the effects of interrupted endogenous BMP/Smad signaling on steroidogenesis of porcine GCs. After transfection, the E2 level of Smad4-siRNA group significantly decreased, while transfection had no significant effect on P4. A possible reason is that we measured the basal levels of E2 and P4 in cultured GCs; thus, unknown factors in the serum may have affected GC steroidogenesis. These require further elucidation. A widely accepted opinion is that the Ras-MAPK (mitogen-activated protein kinase) pathway (activated after LH surge) plays a major role in regulating GC steroidogenesis (Fan et al., 2008 2008 ; 2009 2009 ). The TGF-β superfamily members are critical in maintaining cell growth and differentiation in the ovary (Pangas et al., 2008 2008 ). BMPs play a key role in regulating GC steroidogenesis. In particular, they change the expression of steroidogenic enzymes such as Cyp19a1 (aromatase) and Cyp11a1 (p450scc) (Winters et al., 1998 1998 ), and these actions are important, and are modulated by various growth factors, acting in a paracrine or autocrine way (Khamsi and Roberge, 2001 2001 ). In vitro, BMP-4 and BMP-7 enhanced FSH-dependent E2 production (Shimasaki et al., 1999 1999 ), whereas BMP-4, BMP-6, BMP-7, BMP-15, and GDF-9 inhibited FSH-induced P4 secretion by rat GCs in culture (Shimasaki et al., 1999 1999 ; Otsuka et al., 2000 2000 ; Vitt et al., 2000 2000 ; Fraser et al., 2001 2001 ; Lee et al., 2001 2001 ), suggesting that BMP factors regulate GC differentiation, particularly in delaying luteinization. In the present study, blocked BMP/Smad signaling inhibited E2 secretion, but did not affect P4 secretion. In order to evaluate the regulation of E2 and P4 production, we examined the steady-state level of mRNA for Cyp19a1 and Cyp11a1 genes, two of the key regulators in the steroidogenesis implicated in the E2 and P4 synthesis pathways. Our results show that blocked BMP/Smad signaling significantly inhibited Cyp19a1 expression. In conclusion, the interruption of endogenous BMP/Smad signaling by RNAi-mediated Smad4 gene silencing not only inhibits the growth but also induces the apoptosis of porcine GCs. In addition, interrupted BMP/Smad signaling changes steroidogenesis. Our findings provide a new insight that interrupted BMP/Smad signaling plays an important and specific role in mediating porcine GC growth and steroidogenesis. References CA Biondi , D Das , M Howell , A Islam , EK Bikoff , CS Hill , EJ Robertson . Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling . Biochem J , 2007 . 404 ( 2 ): 235 - 245 . 17300215 DOI: 10.1042/BJ20061830 http://doi.org/10.1042/BJ20061830 . Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling I Costello , CA Biondi , JM Taylor , EJ Bikoff , EJ Robertson . Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development . BMC Devel Biol , 2009 . 9 ( 1 ): 54 DOI: 10.1186/1471-213X-9-54 http://doi.org/10.1186/1471-213X-9-54 . Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development F Dai , X Lin , C Chang , XH Feng . Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-β signaling . Dev Cell , 2009 . 16 ( 3 ): 345 - 357 . 19289081 DOI: 10.1016/j.devcel.2009.01.022 http://doi.org/10.1016/j.devcel.2009.01.022 . Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-β signaling Y Dorsett , T Tuschl . siRNAs: applications in functional genomics and potential as therapeutics . Nat Rev Drug Discov , 2004 . 3 ( 4 ): 318 - 329 . 15060527 DOI: 10.1038/nrd1345 http://doi.org/10.1038/nrd1345 . siRNAs: applications in functional genomics and potential as therapeutics HY Fan , M Shimada , ZL Liu , N Cahill , N Noma , Y Wu , J Gossen , JS Richards . Selective expression of Kras G12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation . Development , 2008 . 135 ( 12 ): 2127 - 2137 . DOI: 10.1242/dev.020560 http://doi.org/10.1242/dev.020560 . Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation HY Fan , ZL Liu , M Shimada , E Sterneck , PF Johnson , SM Hedric , JS Richards . MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility . Science , 2009 . 324 ( 5929 ): 938 - 941 . 19443782 DOI: 10.1126/science.1171396 http://doi.org/10.1126/science.1171396 . MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility M Fraser , GA Braems , JR Challis . Developmental regulation of corticotrophin receptor gene expression in the adrenal gland of the ovine fetus and newborn lamb: effects of hypoxia during late pregnancy . J Endocrinol , 2001 . 169 ( 1 ): 1 - 10 . 11250641 DOI: 10.1677/joe.0.1690001 http://doi.org/10.1677/joe.0.1690001 . Developmental regulation of corticotrophin receptor gene expression in the adrenal gland of the ovine fetus and newborn lamb: effects of hypoxia during late pregnancy I Kawashima , T Okazaki , N Noma , M Nishibori , Y Yamashita , M Shimada . Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and vitro . Reproduction , 2008 . 136 ( 1 ): 9 - 21 . 18456902 DOI: 10.1530/REP-08-0074 http://doi.org/10.1530/REP-08-0074 . Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and vitro F Khamsi , S Roberge . Granulosa cells of the cumulus oophorus are different from mural granulosa cells in their response to gonadotrophins and IGF-1 . J Endocrinol , 2001 . 170 ( 3 ): 565 - 573 . 11524236 DOI: 10.1677/joe.0.1700565 http://doi.org/10.1677/joe.0.1700565 . Granulosa cells of the cumulus oophorus are different from mural granulosa cells in their response to gonadotrophins and IGF-1 WS Lee , F Otsuka , RK Moore , S Shimasaki . Effect of bone morphogenetic protein-7 on folliculo-genesis and ovulation in the rat . Biol Reprod , 2001 . 65 ( 4 ): 994 - 999 . 11566718 DOI: 10.1095/biolreprod65.4.994 http://doi.org/10.1095/biolreprod65.4.994 . Effect of bone morphogenetic protein-7 on folliculo-genesis and ovulation in the rat X Lin , XY Duan , YY Liang , Y Su , KH Wrighton , JY Long , M Hu , CM Davis , JR Wang , FC Brunicardi , 等 . PPM1A functions as a Smad phosphatase to terminate TGFβ signaling . Cell , 2006 . 125 ( 5 ): 915 - 928 . 16751101 DOI: 10.1016/j.cell.2006.03.044 http://doi.org/10.1016/j.cell.2006.03.044 . PPM1A functions as a Smad phosphatase to terminate TGFβ signaling KJ Livak , TD Schmittgen . Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔ C T method . Methods , 2001 . 25 ( 4 ): 402 - 408 . 11846609 DOI: 10.1006/meth.2001.1262 http://doi.org/10.1006/meth.2001.1262 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method J Massagué , J Seoane , D Wotton . Smad transcription factors . Genes Dev , 2005 . 19 ( 23 ): 2783 - 2810 . DOI: 10.1101/gad.1350705 http://doi.org/10.1101/gad.1350705 . Smad transcription factors I Matsuura , NG Denissova , G Wang , DM He , JY Long , F Liu . Cyclin-dependent kinases regulate the antiproliferative function of Smads . Nature , 2004 . 430 ( 6996 ): 226 - 231 . 15241418 DOI: 10.1038/nature02650 http://doi.org/10.1038/nature02650 . Cyclin-dependent kinases regulate the antiproliferative function of Smads EA McGee , AJW Hsueh . Initial and cyclic recruitment of ovarian follicles . Endocr Rev , 2000 . 21 ( 2 ): 200 - 214 . 10782364 DOI: 10.1210/er.21.2.200 http://doi.org/10.1210/er.21.2.200 . Initial and cyclic recruitment of ovarian follicles V Mittal . Improving the efficiency of RNA interference in mammals . Nat Rev Genet , 2004 . 5 ( 5 ): 355 - 365 . 15143318 DOI: 10.1038/nrg1323 http://doi.org/10.1038/nrg1323 . Improving the efficiency of RNA interference in mammals T Miyoshi , F Otsuka , J Suzuki , M Takeda , K Inagaki , Y Kano , H Otani , Y Mimura , T Ogura , H Makino . Mutual regulation of follicle-stimulating hormone signaling and bone morphogenetic protein system in human granulosa cells . Biol Reprod , 2006 . 74 ( 6 ): 1073 - 1082 . 16436528 DOI: 10.1095/biolreprod.105.047969 http://doi.org/10.1095/biolreprod.105.047969 . Mutual regulation of follicle-stimulating hormone signaling and bone morphogenetic protein system in human granulosa cells F Otsuka , ZX Yao , TH Lee , S Yamamot , GF Erickson , S Shimasaki . Bone morphogenetic protein-15. Identification of target cells and biological functions . J Biol Chem , 2000 . 275 ( 50 ): 39523 - 39528 . 10998422 DOI: 10.1074/jbc.M007428200 http://doi.org/10.1074/jbc.M007428200 . Bone morphogenetic protein-15. Identification of target cells and biological functions F Otsuka , RK Moore , S Shimasaki . Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary . J Biol Chem , 2001 . 276 ( 35 ): 32889 - 32895 . 11447221 DOI: 10.1074/jbc.M103212200 http://doi.org/10.1074/jbc.M103212200 . Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary F Otsuka , S Yamamoto , GF Erickson , S Shimasaki . Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression . J Biol Chem , 2001 . 276 ( 14 ): 11387 - 11392 . 11154695 DOI: 10.1074/jbc.M010043200 http://doi.org/10.1074/jbc.M010043200 . Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression SA Pangas , XH Li , EJ Robertson , MM Matzuk . Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice . Mol Endocrinol , 2006 . 20 ( 6 ): 1406 - 1422 . 16513794 DOI: 10.1210/me.2005-0462 http://doi.org/10.1210/me.2005-0462 . Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice SA Pangas , XH Li , L Umans , A Zwijsen , D Huylebroeck , C Gutierrez , DG Wang , JF Martin , SP Jamin , RR Behringer , 等 . Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice . Mol Cell Biol , 2008 . 28 ( 1 ): 248 - 257 . 17967875 DOI: 10.1128/MCB.01404-07 http://doi.org/10.1128/MCB.01404-07 . Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice IC Paterson , M Davies , A Stone , S Huntley , E Smith , M Pring , JW Eveson , CM Robinson , EK Parkinson , SS Prime . TGF-β1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad4 expression and ligand-induced G(1) arrest . Oncogene , 2002 . 21 ( 10 ): 1616 - 1624 . 11896591 DOI: 10.1038/sj.onc.1205217 http://doi.org/10.1038/sj.onc.1205217 . TGF-β1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad4 expression and ligand-induced G(1) arrest HM Picton , BK Campbell , MG Hunter . Maintenance of oestradiol production and exression of cytochrome P450 aromatase enzyme mRNA in long-term serum-free cultures of pig granulosa cells . J Reprod Fertil , 1999 . 115 ( 1 ): 67 - 77 . 10341724 DOI: 10.1530/jrf.0.1150067 http://doi.org/10.1530/jrf.0.1150067 . Maintenance of oestradiol production and exression of cytochrome P450 aromatase enzyme mRNA in long-term serum-free cultures of pig granulosa cells B Schmierer , CS Hill . TGFβ-SMAD signal transduction: molecular specificity and functional flexibility . Nat Rev Mol Cell Biol , 2007 . 8 ( 12 ): 970 - 982 . 18000526 DOI: 10.1038/nrm2297 http://doi.org/10.1038/nrm2297 . TGFβ-SMAD signal transduction: molecular specificity and functional flexibility S Shimasaki , RJ Zachow , DM Li , H Kim , SI Iemura , N Ueno , K Sampath , RJ Chang , GF Erickson . A functional bone morphogenetic protein system in the ovary . PNAS , 1999 . 96 ( 13 ): 7282 - 7287 . 10377406 DOI: 10.1073/pnas.96.13.7282 http://doi.org/10.1073/pnas.96.13.7282 . A functional bone morphogenetic protein system in the ovary S Shimasaki , RK Moore , GF Erickson , F Otsuka . The role of bone morphogenetic proteins in ovarian function . Reprod Suppl , 2003 . 61 323 - 337 . 14635945 . The role of bone morphogenetic proteins in ovarian function S Shimasaki , RK Moore , F Otsuka , GF Erickson . The bone morphogenetic protein system in mammalian reproduction . Endocr Rev , 2004 . 25 ( 1 ): 72 - 101 . 14769828 DOI: 10.1210/er.2003-0007 http://doi.org/10.1210/er.2003-0007 . The bone morphogenetic protein system in mammalian reproduction UA Vitt , M Hayashi , C Klein , AJ Hsueh . Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles . Biol Reprod , 2000 . 62 ( 2 ): 370 - 377 . 10642575 DOI: 10.1095/biolreprod62.2.370 http://doi.org/10.1095/biolreprod62.2.370 . Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles TA Winters , JA Hanten , JD Veldhuis . In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert . Endocrine , 1998 . 9 ( 1 ): 57 - 63 . 9798731 DOI: 10.1385/ENDO:9:1:57 http://doi.org/10.1385/ENDO:9:1:57 . In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert Y Yamashita , I Kawashima , Y Yanai , M Nishibori , JS Tichards , M Shimada . Hormone-induced expression of tumor necrosis factor α-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor . Endocrinology , 2007 . 148 ( 12 ): 6164 - 6175 . 17901238 DOI: 10.1210/en.2007-0195 http://doi.org/10.1210/en.2007-0195 . Hormone-induced expression of tumor necrosis factor α-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor We thank Prof. Christopher A. PRICE for his kind help in reviewing the manuscript. 10.1631/jzus.B1000079 Project (No. 2006AA10Z136) supported by National High-Tech R & D Program (863) of China 2010-02-22 2010-08-09 2010-05-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 11