Abstract
FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor β (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.
Introduction
FSH is the major regulator of folliculogenesis, particularly in regulating granulosa cell (GC) proliferation and steroidogenesis. Accumulating evidence highlights the intraovarian transforming growth factor β (TGFB) superfamily signaling cascades as key regulatory pathways of fundamental female reproductive events, including folliculogenesis and ovulation (Findlay et al. 2002, Juengel & McNatty 2005, Kaivo-oja et al. 2006, Knight & Glister 2006, Pangas 2007). Recent studies have established the concept that TGFBs, particularly bone morphogenetic proteins (BMPs), play a crucial role in female fertility in mammals by regulating steroidogenesis as well as mitogenesis in GCs (Shimasaki et al. 2003, 2004). Notably, in GCs, a major regulatory process governed by BMPs involves the control of FSH signaling. For example, BMP6 inhibits FSH-induced progesterone (P4) synthesis through suppression of cellular cAMP synthesis (Otsuka et al. 2001a). BMP15 also exhibits potent suppression of FSH actions by inhibiting FSH receptor (FSHR) expression (Otsuka et al. 2001b). In addition, growth and differentiation factor-9 (GDF9), which is highly homologous with BMP15, also inhibits FSH-induced steroidogenesis and LH receptor expression in rat GCs (Vitt et al. 2000). Using a human granulosa KGN cell line, Miyoshi & Otsuka found that FSH preferentially upregulates the expression of type I receptors (BMPR1A (ALK-3) and BMPR1B (ALK-6)) as well as type II receptors (ACVR2A (ActRII) and BMPR2). In addition, FSH increased the expression levels of SMAD1/5 and decreased the expression of inhibitory SMADs, SMAD6/7. Thus, it is most likely that FSH augments the bioavailability of BMP signaling in human granulosa (Miyoshi et al. 2006).
SMADs are the downstream signaling molecules mediating the intracellular TGFB/SMAD signal transduction pathways and are divided into three functional groups: common SMAD (co-SMAD), receptor-regulated SMADs (R-SMADs), and inhibitory SMADs. Classical TGFB signaling pathway is initiated when the ligands bind their serine/threonine kinase type 2 and type 1 (activin receptor-like kinase) receptors to form an oligomeric receptor complex (Kaivo-oja et al. 2006, Knight & Glister 2006, Schmierer & Hill 2007). Subsequently, R-SMADs are phosphorylated and activated by type 1 receptor. The R-SMADs then form heteromeric complexes with co-SMAD and accumulate in the nucleus to regulate ligand-specific gene expression via recruitment of distinct transcription factors, coactivators, and corepressors (Massague 1992, 2000). Activation of SMADs can be modulated at multiple levels by numerous molecules including the ligand-induced inhibitory SMADs, SMAD6 and SMAD7, the function of which is to prevent R-SMADs from phosphorylation and signaling activation by their binding to type 1 receptors (Kawabata et al. 1998, Massague 2000, Moustakas et al. 2001, Mehra & Wrana 2002, ten Dijke & Hill 2004).
All TGFBs can transduce their signals in vitro through SMAD2 and SMAD3, which share more than 90% identity in their amino acid sequences (Brown et al. 2007), whereas BMPs activate their signaling pathways through SMAD1, SMAD5, and SMAD8 (Chang et al. 2001, Brown et al. 2007). However, in both cases, TGFB superfamily signaling cascades transmit their signals through co-mediating SMAD. In mammals, SMAD4 is the only co-mediating SMAD, which translocates to the nucleus with phosphorylated receptor-activated SMADs and then modulates transcription of TGFB target genes. SMAD4 is broadly expressed in embryonic and adult tissues. SMAD4 nucleocytoplasmic shuttling is not required for R-SMAD phosphorylation or nuclear localization (Biondi et al. 2007). R-SMAD dephosphorylation and nuclear export are thought to be required for optimal TGFB signaling (Lin et al. 2006, Dai et al. 2009). SMAD4 loss leads to increased steady-state levels of both BMP and TGFB phosphorylated R-SMADs. Enhanced R-SMAD phosphorylation levels potentially reflect decreased dephosphorylation and/or nuclear export (Lin et al. 2006, Dai et al. 2009). SMAD4 is thus crucial for TGFB/SMAD signaling.
In oocytes, GCs, and theca interna cells (TIC) of both immature and cycling rats, SMAD4 (Guéripel et al. 2004) has been detected. However, the regulation of SMAD4, as the only co-mediating SMAD, in FSH stimulation of porcine ovarian GCs remains poorly understood. In this study, we applied siRNA approach to knockdown SMAD4 expression and investigated the role of SMAD4 in FSH-induced proliferation and steroidogenesis of porcine GCs.
Results
Inhibition of SMAD4 expression by RNA interference
We knocked down SMAD4 expression in porcine GCs by RNA interference (RNAi). Cells transfected with negative control siRNA (NC-siRNA) and SMAD4-siRNA were analyzed for the expression of SMAD4 mRNA and protein 48 h post-transfection by real-time PCR, immunofluorescence, and western blotting, as shown in Fig. 1. The results as in Fig. 1A showed that SMAD4 mRNA expression was inhibited after transfection with SMAD4-siRNA, the SMAD4 transcript level being reduced by ∼89.0% compared with that in the control cultures (blank). The expression of SMAD4 protein clearly revealed the suppressive effect of SMAD4-siRNA in the GCs by immunofluorescence staining and western blotting (Fig. 1B and C), with the level of SMAD4 protein being decreased by ∼56.6% 48 h post-transfection with the SMAD4-siRNA (Fig. 1D).
(A) Expression of SMAD4 mRNA after transfection for 48 h. (B) Silencing effect of expression SMAD4 protein measured by immunofluorescence staining. (C) Silencing effect of expression SMAD4 protein measured by western blotting. (D) Expression of SMAD4 protein after transfection for 48 h. Statistically significant differences from the blank group are indicated by *(P<0.05).
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Effect of SMAD4 knockdown on SMAD1, 3, and 5 expressions
To test whether other SMADs were affected by SMAD4-siRNA in GCs, cells were collected after treatment, and SMAD1, 3, and 5 mRNA levels were determined by quantitative real-time PCR. The results showed that the SMAD1, 3, and 5 mRNA levels were not affected by SMAD4 silencing in porcine GCs (Fig. 2A–C).
(A) Expression of SMAD1 mRNA after transfection for 48 h. (B) Expression of SMAD3 mRNA after transfection for 48 h. (C) Expression of SMAD5 mRNA after transfection for 48 h.
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Effect of SMAD4 knockdown on FSH-induced FSHR expression
To better understand the effects of SMAD4 knockdown on the role of FSH in GCs, we verified the expression of FSHRs. GCs were collected after treatment, and mRNA levels were determined by quantitative real-time PCR. The results in Fig. 3 showed that FSHR is significantly downregulated in SMAD4-siRNA cells without FSH, and in FSH+SMAD4-siRNA treatment, the expression of FSHR mRNA significantly decreased as compared with that of FSH.
Effect of knockdown of endogenous SMAD4 on FSH-induced FSHR mRNA levels. Significant differences among the groups are indicated by different letters (P<0.05).
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Effect of SMAD4 knockdown on the number of living cells in FSH-induced GCs
MTT (methyl thiazolyl tetrazolium) analysis was performed to test the effect of SMAD4 knockdown on the number of living cells in FSH-induced GCs. As expected, FSH induced an increase in the number of living cells in untransfected cells, compared with blank group (Fig. 4). In contrast, the increase of living cells was downregulated for cells transfected with SMAD4-siRNA, comparing FSH+SMAD4-siRNA group with FSH group.
Effect of knockdown of endogenous SMAD4 expression on the number of living cells in FSH-induced GCs. Significant differences among the groups are indicated by different letters (P<0.05).
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Effect of SMAD4 knockdown on FSH-induced cell cycle
To further determine the effect of knockdown of SMAD4 expression on FSH-induced cell growth, flow cytometry was used to analyze cells for the changes of cell cycle. Cells were labeled with propidium iodide (PI) after treatment. The effects of FSH on the cell cycle were investigated in untransfected cells as well as cells transfected with the SMAD4-siRNA.
The results in Table 1 show that knockdown of SMAD4 expression significantly increases the proportion of cells in G0/G1 phase. And with FSH+SMAD4-siRNA treatment, the number of G0/G1 phase cells significantly increased compared with FSH. Silencing SMAD4 expression can significantly decrease the cells of S phase in both the presence and the absence of FSH. No significant differences were observed in the proportion of cells in G2/M phase between groups.
Knockdown of endogenous expression of SMAD4 alters FSH-induced cell cycle progression.
Group | G0/G1 (%) | S (%) | G2/M (%) |
---|---|---|---|
Blank | 92.08±0.66bc | 3.25±0.21a | 4.67±0.87a |
FSH | 90.64±0.27c | 4.12±0.18a | 5.24±0.09a |
SMAD4-siRNA | 94.41±0.35a | 0.95±0.69c | 4.65±0.34a |
FSH+SMAD4-siRNA | 92.38±0.36b | 2.15±1.25b | 5.48±0.90a |
Significant differences among the groups are indicated by different letters (P<0.05).
Effect of knockdown of SMAD4 on the expression of cell cycle marker genes of FSH-induced GCs
To investigate the mechanism by which SMAD4 knockdown represses FSH-induced growth of GCs, we measured the expression of cyclin B, cyclin D2, cyclin-dependent kinase 2 (CDK2), and CDK4, the functional markers of cell cycle (Fig. 5). FSH increased levels of mRNA encoding cyclin B and cyclin D2 in untransfected cells. In SMAD4 knockdown cells, cyclin D2 mRNA levels were lower than that in the blank group. In FSH+SMAD4-siRNA group, FSH-induced increase in the expression of cyclin D2 was significantly inhibited, as compared with that of FSH. However, the expression of cyclin B did not significantly change in the FSH and FSH+SMAD4-siRNA groups. The CDK2 and CDK4 mRNA levels were decreased by SMAD4 silencing based on whether cells were stimulated by FSH or not.
Knockdown of endogenous expression of SMAD4 effect FSH-induced cell cyclin B, cyclin D2, CDK2, and CDK4 mRNA levels. Significant differences among the groups are indicated by different letters (P<0.05).
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Effect of SMAD4 knockdown on FSH-induced E2 and P4 production
We examined the effects of SMAD4 on steroidogenesis regulated by FSH. As shown in Table 2, FSH increased the levels of E2, and SMAD4-siRNA decreased the levels of E2, and SMAD4 knockdown attenuated FSH-induced augmentation of E2. FSH raised the production of P4 but SMAD4-siRNA did not, and SMAD4 knockdown did not affect FSH-induced secretion of P4. SMAD4 interference decreased FSH-induced E2 production and had no significant effect on FSH-induced P4, comparing FSH group with FSH+SMAD4-siRNA group.
Effects of knockdown of endogenous expression of SMAD4 on FSH-induced estradiol (E2) and progesterone (P4) production.
E2 (pmol/l) | P4 (nmol/l) | |
---|---|---|
Blank | 117.25±2.75b | 3.075±0.19b |
FSH | 154.75±8.75a | 4.688±0.04a |
si-SMAD4 | 104.25±5.25c | 2.703±0.02b |
FSH+siSMAD4 | 124.00±6.50b | 4.145±0.07a |
Significant differences among the groups are indicated by different letters (P<0.05).
Effect of SMAD4 knockdown on FSH-induced steroidogenic enzyme genes
To confirm whether the effects of SMAD4 were involved in the expression changes of steroidogenic enzyme genes, levels of mRNA encoding CYP19a1, CYP11a1, STAR, and HSD 3B, the key enzymes regulating E2 and P4 production in GCs, were examined by quantitative real-time PCR. Knockdown of SMAD4 decreased CYP19a1 and CYP11a1 mRNA expression induced by FSH as compared between the FSH and FSH+SMAD4-siRNA groups. In contrast, the STAR and HSD 3B mRNA levels induced by FSH were not affected by SMAD4 silencing (Fig. 6).
Effects of knockdown of endogenous SMAD4 expression on FSH-induced steroidogenic enzyme genes. Significant differences among the groups are indicated by different letters (P<0.05).
Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0098
Discussion
The TGFB superfamily has been implicated in multiple GC processes, including proliferation, differentiation, and regulation of steroidogenesis. SMADs are downstream signaling molecules mediating the intracellular TGFB/SMAD signal transduction pathways. SMAD4 is the only co-SMAD in mammals. This study aimed to determine the role of SMAD4 in growth and steroidogenesis of porcine GCs in vitro. We have uncovered a cellular mechanism of regulation of GC growth and steroidogenesis involved in the interaction between FSH and SMAD4 in porcine GCs.
SMADs are a class of very important factors regulating ovarian function in mammals, but they are also mediated by TGFB/SMAD signal transduction pathways. The SMAD2 cKO mouse model supplied evidence that SMAD2 is not requisite in transducing TGFB signals in GCs (Li et al. 2008). The results from SMAD3 cKO mice support the concept that SMAD3 is capable of activating essential target genes downstream of TGFB-related ligands (Gong & McGee 2009). Furthermore, results from SMAD2 and SMAD3 double cKO mice showed that the loss of SMAD2/3 signaling in GCs led to disrupted follicular development and reduced ovulation efficacy (Li et al. 2008). Recently, SMAD1/5/8 cKO mice have been generated by Pangas' group, and these mice developed metastatic tumors with increased signaling of SMAD2/3 in GCs (Pangas et al. 2008). Moreover, ablation of all TGFB signaling in the ovarian GCs by conditionally inactivating SMAD4 resulted in premature luteinization of GCs, leading to the predisposition of premature ovarian failure (Pangas et al. 2006). The R-SMADs form heteromeric complexes with SMAD4, which translocates to the nucleus and then modulates transcription of TGFB target genes. We chose to knock down endogenous SMAD4, as an alternative, in porcine GCs to characterize a potential interaction between FSH and SMAD4. We adopted an RNAi technique in part because the sequence-specific knockdown of target mRNA is more efficient than antisense oligonucleotides or ribozymes (Dorsett & Tuschl 2004, Mittal 2004). Our results showed that our SMAD4-siRNA significantly inhibits the mRNA and protein expression of SMAD4, as demonstrated by real-time RT-PCR, immunofluorescence, and western blotting. The role of SMAD4 as a co-SMAD is not required for R-SMAD phosphorylation or nuclear localization (Biondi et al. 2007) with which our results are consistent. Therefore, we detected the expression of SMAD1, 3, and 5 mRNA, with their mRNA levels being not affected by SMAD4 loss in porcine GCs.
We demonstrated that SMAD4, the only co-mediating SMAD, plays a key role in FSHR expression in vitro. Our findings showed that FSHR is significantly down-regulated in SMAD4-siRNA-treated cells, which is in agreement with a previous study in which FSHR was slightly downregulated in GCs from pregnant mare's serum gonadotrophin (PMSG)-stimulated SMAD4 cKO ovaries (Pangas et al. 2006). Knockdown of SMAD4 also diminished the ability of FSH to upregulate its own receptor. Genes regulating FSHR activity may be of great functional importance. SMAD4 deficiency does not turn off the FSHR, but it does reduce the ability of the GCs to respond to gonadotropins. Further studies are necessary to clarify the levels of regulation and the role of signaling cross talk in determining FSHR regulation and downstream function.
FSH stimulates porcine GC proliferation, but SMAD4 loss inhibits cell proliferation in this study. It has been well documented that cell proliferation depends on progression through the cell cycle, and in this study, we showed that SMAD4 knockdown resulted in G0/G1 arrest and a decrease in the proportion of cells in S phase. This study also investigated the mechanism(s) whereby SMAD4-siRNA suppresses FSH-induced cell proliferation in GCs. FSH potently increases cell cycle progression at the G0/G1 phase (Paterson et al. 2002, Matsuura et al. 2004). The G0/G1 checkpoint can be viewed as a master checkpoint of the mammalian cell cycle (Paterson et al. 2002, Matsuura et al. 2004). Regulation of the G0/G1 phase of the cell cycle involves many different families of cyclins: cyclin B, cyclin D, and CDKs. Inhibition of FSH-induced proliferation of GCs by SMAD4-siRNA was associated with decrease in cyclin D2 expression, instead of cyclin B. Cyclin D2 acts as a downstream gene of SMAD4, participating in the regulation of FSH in porcine GCs, and it may be involved in FSH-induced cell growth in a SMAD4-dependent manner. So we postulate that at least part of the SMAD4-siRNA effect on FSH-induced GC proliferation is mediated by a decrease in cyclin D2 expression. This hypothesis needs further studies to be verified.
FSH plays a central role in regulating GC steroidogenesis. In particular, FSH enhances the expression of steroidogenic enzymes such as CYP11a1 and CYP19a1. In vitro, FSH-stimulated steroidogenesis is modulated by growth factors, including members of the BMP family. Recombinant BMP4, BMP6, BMP7, BMP15, and GDF9 inhibited FSH-induced P4 secretion by rat GCs in culture (Shimasaki et al. 1999, Otsuka et al. 2000, Vitt et al. 2000, Fraser et al. 2001, Lee et al. 2001), whereas BMP4 and BMP7 enhanced FSH-dependent E2 production, suggesting that BMP factors may delay luteinization (Shimasaki et al. 1999). In this study, SMAD4 knockdown inhibited FSH-induced E2 secretion but did not affect FSH-induced P4 secretion. These data led us to analyze the expression of CYP19a1, CYP11a1, STAR, and HSD 3B genes, the four of the key regulators in the steroidogenesis implicated in the E2 and P4 synthesis pathway. Our results showed that SMAD4-siRNA significantly inhibits FSH-induced CYP19a1 and CYP11a1 expression. There is a conflicting report regarding the effect of SMAD4 on steroidogenesis in other species. Studies by Pangas et al. (2006) showed that SMAD4 loss did not alter CYP19a1 mRNA levels in mouse GCs, but upregulated CYP11a1 mRNA levels. These results demonstrate the difficulty in extrapolating data between species and also between different conditions (in vivo and vitro). The mRNA expression of STAR and HSD 3B induced by FSH were not affected by SMAD4 silencing. Our results are consistent with the studies by Pangas et al. Their results showed that STAR was significantly upregulated by PMSG, even in SMAD4 cKO mice. FSH signaling pathway is a complex regulatory network, in which related gene expression is affected by many factors. Our results showed that the STAR and HSD3B mRNA levels induced by FSH were not affected by SMAD4 silencing. However, the results are inconsistent with our expected ones. We expected that FSH-responsive genes would be regulated by FSH in the presence of SMAD4 silencing. This needs further investigation.
In summary, we used siRNA to knock down SMAD4 expression in porcine GCs and provided evidence that SMAD4 plays an important and specific role in mediating FSH-induced GCs proliferation and steroidogenesis.
Materials and Methods
Isolation and culture of GCs
Porcine ovaries were obtained at 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 PBS supplemented with gentamicin and amphotericin. GCs were collected from follicles (3–5 mm) by aspiration using a syringe with needle (20 G) and filtered through a stainless steel filter (100 μm) to remove the cumulus–oocytes complexes. The cells were centrifuged at 800 g/min and washed twice in DMEM/F12 medium (Shanghai Invitrogen Biotechnology Co., Ltd). GCs were cultured as described previously (Picton et al. 1999). GCs were counted in a hemocytometer, and the viability was determined by trypan blue dye exclusion, then seeded in six-well culture plates (Costar, Corning, Inc., New York, NY, USA) at a density of (3–5)×106 cells per well in 2 ml of DMEM/F12 containing 10% FCS (MinHai Co., LanZhou, People's Republic of China), gentamicin (5 μl/ml) and amphotericin (10 μl/ml). The cells were cultured for 24 h at 37 °C in a 5% CO2 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
The cDNA sequence of SMAD4 (GenBank accession number, NM_214072) was examined using Invitrogen's web-based siRNA design software (https://rnaidesigner.invitrogen.com/rnaiexpress/) to select appropriate siRNA target sites. A pair of oligonucleotides corresponding to SMAD4 cDNA at the start position 925 bp was designed. The siRNA sequence of SMAD4 is as follows: sense, CAC CAG GAA UUG AUC UCU CAG GAU U; antisense, AAU CCU GAG AGA UCA AUU CCU GGU G. A Blast search of these sequences confirmed their specificity to SMAD4 only. In addition, a nonsense sequence with no similar match to any known sequence was designed as control. The siRNA sequence of nonsense sequence is as follows: sense, CAC AAG GAG UUC UCU GAC UGA CAU U; antisense, AAU GUC AGU CAG AGA ACU CCU UGU G. The SMAD4 small RNAi was named SMAD4-siRNA, the nonsense sequence to SMAD4 was named NC-siRNA.
Cultures of GCs were transfected with SMAD4-siRNA or NC-siRNA using Lipofectamine RNAiMAX Reagent (Shanghai Invitrogen Biotechnology Co., Ltd). Briefly, 8 pmol RNAi duplex, 4 μl Lipofectamine RNAiMAX, and 250 μl DMEM/F12 were combined and incubated for 20 min at room temperature. The transfection complex was added to the cells and incubated for 8 h at 37 °C. Then the medium was drawn, and the new DMEM/F12 containing 1% FCS (MinHai Co.), gentamicin (5 μl/ml), and amphotericin (10 μl/ml) was added. At the same time, the cells in FSH group and FSH+SMAD4-siTNA group were treated with FSH (NingBo Hormone Products Co., Ltd, NingBo, People's Republic of China) at the concentration of 1 IU/ml for 48 h.
Real-time quantitative RT-PCR
Total RNA was extracted from GCs using TRIzol following the manufacturer's instructions. RNA concentration and purity was determined by measuring optical density (OD) at wavelengths of 260 and 280 nm using a standard spectrophotometer. The OD260/280 ratios were ≥1.8 for all samples. Aliquots (100 ng) of total RNA from each pool of cultured GCs were independently reverse transcribed to cDNA using M-MLV reverse transcriptase (Promega Corporation) and oligonucleotide primers.
Target genes and the housekeeping gene GAPDH were quantified by real-time PCR with an ABI 7300 using a commercial kit (SYBR Premix Ex Taq, TaKaRa, Dalian, People's Republic of China). The gene-specific primers were designed on the basis of porcine mRNA sequences (Table 3). 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
Details of primers for target gene amplification.
Target gene (accession number) | Primer sequence | Product size (bp) | Annealing temperature (°C) |
---|---|---|---|
SMAD4 (NM_214072) | Forward: TTTGCGTCAGTGTCATCG | 236 | 59.5 |
Reverse: TGCTCTGCCTTGGGTAAT | |||
SMAD1 (NM_213965) | Forward: CTGCGAGTTTCCTTTTGGT | 133 | 63.5 |
Reverse: GAGGCTGTGCTGAGGGTTA | |||
SMAD3 (NM_214137) | Forward: GACTACAGCCATTCCATCCC | 134 | 55.5 |
Reverse: CTGTGGTTCATCTGGTGGTC | |||
SMAD5 (NM_001001419.1) | Forward: TGTTGCCTATGAAGAGCCTAA | 215 | 57.0 |
Reverse: CACCTTTTCCAATATGTCGC | |||
FSHR (NM_214386) | Forward: TCCCTCGGTTCCTTATGT | 269 | 53.5 |
Reverse: ATGGCGGACTTGCACTTT | |||
Cyclin B (NM_001170768) | Forward: TGGCTAGTGCAGGTTCAG | 199 | 54.5 |
Reverse: CAGTCACAAAGGCAAAGT | |||
Cyclin D2 (NM-214088) | Forward: TTACCTGGACCGCTTCTTG | 155 | 55.0 |
Reverse: GAGGCTTGATGGAGTTGTCG | |||
CDK2 (XM-001928965) | Forward: AAACAAGTTGACGGGAGA | 298 | 56.0 |
Reverse: GTGAGAATGGCAGAAAGC | |||
CDK4 (NM-001123097) | Forward: GCATCCCAATGTTGTCCG | 125 | 60.5 |
Reverse: GGGGTGCCTTGTCCAGATA | |||
Cyp19a1 (SSU92246) | Forward: GCTGCTCATTGGCTTAC | 187 | 60.5 |
Reverse: TCCACCTATCCAGACCC | |||
Cyp11a1 (Bx674071) | Forward: AGACACTGAGACTCCACCCCA | 110 | 69.5 |
Reverse: GACGGCCACTTGTACCAATGT | |||
STAR (NM_213755) | Forward: GGAGAGCCGGCAGGAGAATG | 191 | 54.5 |
Reverse: CTTCTGCAGGATCTTGATCTTCTTG | |||
HSD 3B (NM_001004049) | Forward: AGGGTTTCTGGGTCAGAGGATC | 107 | 56.5 |
Reverse: CGTTGACCACGTCGATGATAGAG | |||
GAPDH (AF017079) | Forward: GGACT CATGACCACG GTCCA T | 220 | 57.2 |
Reverse: TCAGATCCAC AACCG ACACG T |
Immunofluorescence
GCs were grown in glass chamber slides. After treatments, cells were washed with PBS three times, fixed in 4% paraformaldehyde for 15 min, washed three times with PBS, and then pre-incubated with PBS supplemented with 0.5% BSA for 30 min at room temperature, to permeabilize the cells in order to allow antibody penetration for the detection of intracellular as well as cell surface antigens. Cells were incubated with rabbit MAB against SMAD4 (1:200 dilution in PBS; Abcam, Shanghai, People's Republic of China; ab40759) for 2 h at room temperature. Slides were washed three times with cold PBS and incubated with biotinylated anti-rabbit IgG (1:200 in PBS; Abcam; ab6939) for 1 h at room temperature. Then, the slides were washed three times with cold PBS and were mounted with Vectashield mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI, Sigma). Cells were counterstained with DAPI. The immunofluorescent signals were examined with a Leica DMLB2 fluorescence microscope. All images were captured using the same settings and saved in the same format. The gray-scale signal of 20 fields was analyzed for each replicate using the ImageJ Software (http://rsbweb.nih.gov/ij/).
Measurement of SMAD4 expression by western blotting
Protein from GCs was extracted in a cell lysis buffer for western and IP (Beyotime, Shanghai, People's Republic of China) containing 10 mM phenylmethylsulphonyl fluoride (Beyotime), and the resultant cell suspension was centrifuged at 13 800 g (12 000 r.p.m.) for 5 min. The supernatant was removed and the amount of protein was quantified using the BCA Protein Assay kit (Beyotime). The homogenized samples (15 μg crude proteins per lane) were subjected to electrophoresis on 12% polyacrylamide-SDS gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA) at 2.5 mA/cm2 for 45 min. Then they were blocked with 5% 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) or GAPDH (Sigma; 1:2000). The membranes were washed three times with 1% Tween-20 in PBS and then hybridized with HRP-conjugated secondary goat anti-rabbit IgG antibody (Santa Cruz Biotechnology Shanghai, People's Republic of China) diluted at 1:5000 for 2 h. After three washes with 1% Tween-20 in PBS, the signals were detected as chemical luminescence by X-ray film with BeyoECL Plus kit (Beyotime).
Cell proliferation assay
GCs were counted in a hemocytometer, and the viability was determined by trypan blue dye exclusion and then seeded in 96-well culture plates (Costar, Corning, Inc.) at a density of (3–5)×104 cells per well, and treated with or without siRNA and FSH. The number of living cells at the end of culture was determined with the MTT kit (KeyGEN Biotechnology Co., Ltd, NanJing, JiangSu, People's Republic of China) as recommended by the manufacturer. After treatment, MTT (50 μl) was added into each well. After 4 h, the liquid in the wells was drawn out, 150 μl DMSO was added into each well, and then incubated for 10 min at 37 °C. The OD at 550 nm was determined using an ELISA reader (Bio-TEK Instruments, Winooski, VT, USA).
Flow cytometry
In order to do flow cytometry, we seeded GCs in 25 cm2 culture flask at a density of (5–8)×106 cells per flask. After treatment, GCs were digested using 0.25% pancreatic enzyme without EDTA, washed twice with PBS, fixed in 70% ethanol, and stained with 100 μg/ml propidium iodide at 4 °C for 1 h. The DNA content was monitored by FACSscan FACS Calibur (Becton Dickinson, Franklin, NJ, USA).
Measurement of P4 and E2
GCs (2×105 viable cells) were cultured in 24-well plates. After treatment, the culture media was collected and stored at −80 °C. The levels or P4 and E2 in the media were measured by chemiluminescent immunoassay (ACCESS, Beckmancoulter Co., Pasadena, CA, USA). Steroid contents were negligible (P4<0.1 ng/ml and E2<10 pg/ml). The intra- and interassay coefficients of variation were 15 and 20% for E2 respectively and were 5 and 10% for P4 respectively.
Statistical analysis
All results are shown as mean±s.e.m. of data from at least three separate experiments, each performed with triplicate samples. Data were analyzed by one-way ANOVA, followed by Fisher's least significant difference test for multiple comparisons using the SPSS 16.0 software (SPSS Inc., Shanghai, People's Republic of China). Statistical significance was accepted at P<0.05.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by Hi-Tech Research and Development Program of China (2006AA10Z136).
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