Animals
All CD1 mice were purchased from the Laboratory Animal Center of the Institute of Genetics in Beijing and kept in mouse facilities that met the requirements of the China Agricultural University Institutional Animal Care and Use Committee. Mice were housed in China Agricultural University with 16/8-h light/dark cycles, at 26 °C, with free access to food and water (Rat & Mouse Maintenance Diet 1022, HFK Bio-tech, China). Female mice (6 to 8 weeks old) were mated with adult males at a ratio of 1:1 overnight. Mice with a vaginal plug in the next morning were defined at 0.5 day post-coitus (dpc). The day after partum was considered to be 1 day post partum (dpp).
Ovary culture
Neonatal females were sacrificed by cervical dislocation on the designated time. Mouse ovaries were separated by microdissection in cold phosphate buffered saline (PBS) under a stereomicroscope (ZSA302, COIC, China) in sterile conditions. The isolated ovaries were cultured on an insert (PICM0RG50, Millipore, USA) in 6-well culture dishes (NEST, China) in 1200 μL Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12) (GIBCO, Life Technologies, USA) plus ITS (1:100, Sigma, USA) and Penicillin-Streptomycin Solution at 37 °C, 5% CO2 and saturated humidity. Ovaries were cultured for 2 or 5 days to assess the role of CDC42, in either medium alone or medium supplemented with ML141 (5uM, Selleck, China) or ZCL278 (50uM, Selleck, China). ML141 is a selective reversible CDC42 inhibitor. ZCL278 is a selective CDC42 inhibitor.
Histological staining and follicle counts
Ovaries were fixed in 4% PFA overnight, embedded in paraffin, and sectioned serially at 5 μm. After stained with hematoxylin, every fifth section was analyzed for the presence of oocytes and follicles. The counting results were multiplied by five to estimate the total numbers of oocytes and follicles in each ovary. The follicles were distinguished from each other as follows: primordial follicle (a single oocyte surrounded by several flattened pre-granulosa cells) and activated follicle (an enlarged oocyte surrounded by a mixture of squamous and cuboidal somatic cells or an enlarged oocyte surrounded by one or several layers of cuboidal granulosa cells).
Ovarian follicles at different stages of development, including primordial (type 2), primary (type 3), secondary (type 4 and 5), and antral (type 6 and 7) follicles were counted in all sections of an ovary based on the well-accepted standards established by Pedersen and Peters [38].
Immunofluorescence staining
Fresh separated ovaries were fixed in cold 4% PFA overnight, embedded in paraffin, and serially sectioned at 5 μm. The sections were deparaffinized, rehydrated, and subjected to high temperature (95–98 °C) antigen retrieval with 0.01% sodium citrate buffer (pH 6.0). The sections were then blocked with ADB [3% BSA, 1% normal donkey serum in TBS (0.05 M Tris-HCl pH 7.6 and 0.15 M NaCl)] for 60 min at room temperature and incubated with primary antibodies for 12–16 h at 4 °C. The antibodies were diluted as follows: anti-CDC42 antibody (Abcam, UK) at 1:250, DDX4 (mouse, 1:200, Abcam, UK), anti-BrdU antibody (Abcam, UK) at 1:200, and anti-p-FOXO3a antibody (Cell Signaling Technologies, USA) at 1:400, anti-PTEN antibody (Cell Signaling Technologies, USA) at 1:200, anti-p110β antibody (Abcam, UK) at 1:200. Subsequently, after rinsing thoroughly with PBS, the sections were incubated with fluorophore-conjugated secondary antibody dissolved in ADB for 2 h at 37 °C. The antibodies were diluted as follows: Alexa Fluor 488- or 555-conjugated donkey secondary antibodies against mouse and rabbit IgG (1:200, Life Technologies, USA). Slides were then rinsed in PBS, stained with Hoechst 33342 (B2261, Sigma, USA) for 5 min and sealed in anti-fade fluorescence mounting medium (Applygen, China) with coverslips. Sections were examined and photographed using Nikon Eclipse 80i digital fluorescence microscope or Nikon A1 laser scanning confocal microscope. Antibodies were listed in Additional file 8: Table S3.
qRT-PCR
Eight ovaries in each group were extracted by TRIZOL Reagent (Invitrogen, Life Technologies, USA) according to the manufacturer’s protocol. cDNA was reverse transcripted using 1 μg total RNA (Promega Reverse Transcription System, Promega, USA). Quantitative RT-PCR reaction were operated and analyzed by Applied Biosystems 7300 Real-Time PCR System (Life Technologies, USA). Data were normalized by β-actin. Primers were listed in Additional file 9: Table S2.
RNAi knockdown of Cdc42 in ovaries
Ovaries at 1 dpp were injected with 0.3 μl Cdc42 esiRNAs (200 ng/μL, diluted in PBS), a cocktail of siRNAs (Sigma, USA), or scrambled siRNA by a thin glass needle with a mouthpiece. After this, ovaries were performed electric transfection (ECM2001, BTX, CA) to assist siRNA transfection. The parameters of the electric transfection were three 5-ms-long quasi-square pulses at a pulse-field strength of up to 40 V/cm. The efficiency of RNAi was detected by qRT PCR after 24-h culture and Western blot after 48-h culture.
Lentiviral production and ovary infection
Lentiviruses were produced in 293 T cells by co-transfecting 5 μg pMD2.G, 15 μg psPAX2, and 20 μg of the transfer vector (pSicoR or pLVX-IRES-ZsGreen1). CDC42 overexpressing lentiviruses were constructed by cloning open reading frame of Cdc42 into the pLVX-IRES-ZsGreen1 vector. This lentivirus expressing Cdc42 followed a GFP tag. Therefore, cells expressing CDC42 were also labeled with GFP. The transfection was performed by Lipofectamine 3000 (Invitrogen, USA), and the transfection media were replaced 6 h post transfection. The viral supernatants were harvested at 24 and 48 h with 0.45-μm membrane filtration and centrifuged at 40,000 rpm at 4 °C for 2 h [39]. The lentiviruses were injected into the ovary using a thin glass needle with a mouthpiece. For each injection, the optimal volume was 0.3 μL per ovary. The lentiviral construct pMD2.G and psPAX2 were obtained from Dr. Sheng Cui (China Agricultural University). The lentiviral construct pSicoR and pLVX-IREX-ZsGreen1 were obtained from Dr. Haibin Wang (Xiamen University).
Immunoblotting and CDC42 activity detection
Mouse ovaries were homogenized in WIP Tissue and Cell lysis solution containing 1 mM phenylmethylsulfonyl fluoride (Cell Signaling Technologies, USA). Next, proteins were prepared according to the manufacturer’s instructions. The concentration of proteins was measured by a BCA assay (Beyotime, China). Samples containing 50 μg protein were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, USA). The membranes were then incubated overnight at 4 °C with the appropriate primary antibody listed below: CDC42 (21 kDa, 1:300, Abcam, UK), p-AKT (56 kDa, Thr308, 1:500, Beyotime, China), AKT (56 kDa, 1:500, Beyotime, China), p-FOXO3a (90 kDa, 1:300, Santa Cruz Biotechnology, USA), FOXO3a (90 kDa, 1:200, Santa Cruz Biotechnology, USA), PTEN (54 kDa, 1:200, Cell Signaling Technologies, USA), and p110β (123 kDa, 1:200, Abcam, UK). After rinsing thoroughly with TBST, the membranes were incubated with secondary antibody (1:5000, ZSGB-BIO, China). Membranes were visualized using SuperSignal West Pico chemiluminescent detection system (Prod 34080, Thermo, USA). β-actin (42 kDa, Sigma, USA) was used as an intrinsic control. An Alpha Imager 2200 was used to quantify the relative amount of protein. Antibodies were listed in Additional file 8: Table S3.
CDC42 activity (CDC42-GTP) was determined using RAC1/CDC42 Activation Magnetic Beads Pull-down Assay Kits from Merck Millipore following the instructions. In brief, 100 μg of clarified total ovary lysates were incubated with PAK-1 PBD magnetic beads in microfuge tube at 4 °C for 45 min in a spin column. The beads were pelleted by setting the tubes on a magnetic tube stand for 10 s. Removed and discarded the supernatant. The magnetic beads were washed for three times with MLB buffer. The magnetic beads were resuspended in 40 μL of 2X Laemmli reducing sample buffer and boil for 5 min. Addition of 2 μL of 1 M dithiothreitol prior to boiling. The samples were run on a 10% SDS-PAGE separating gel and subjected to Western blot analyses using an antibody against CDC42. CDC42 activity was indicated by the amount of PAK1-PBD-bound CDC42 (GTP-CDC42). Total ovary lysates were also directly immunoblotted for total CDC42 levels for normalization.
Immunoprecipitation
Magnet beads (Life Technologies, USA) were washed and resuspended in 200 μL Ab Binding and Washing Buffer containing 5 μg Ab. The mix were incubated 30 min with rotation at RT. Supernatant were removed and magnet beads-Ab complex were washed by 200 μL Ab Binding and Washing Buffer. The same amount of ovaries (35 each group or 70 total) were lysed in 200 μL IP buffer, and lysates were cleared by centrifugation, 10 μL of the lysates were kept as input. Supernatant were removed from magnet beads, and ovary lysates were added to beads-Ab complex, gently resuspended by pipetting and incubated 30 min at RT with rotation. Supernatant were transferred to a clean tube, and beads-Ab-Ag complex was washed three times, using 200 μL washing buffer. The washing buffer was removed, and the beads-Ab-Ag complex was gently resuspended in 20 μL elution buffer. Incubate 5 min at RT. 5x SDS sample buffer were added to the mix and the tubes were placed at 100 °C for 10 min. Then, the samples were separated by SDS-PAGE and detected by relevant antibodies (Additional file 8: Table S3).
In vitro transient treatment of mouse ovaries and ovarian kidney capsules transplantation
Paired ovaries from mice at 6 dpp were collected, and one ovary served as control and the other was incubated with CDC42 activator II (1 IU, Cytoskeleton, USA) for 30 min on an insert (PICM0RG50, Millipore, USA) in six-well culture dishes. Ovaries from mice at 35 dpp were cut into 4 pieces before treatment. The culture media was DMEM/F12 (GIBCO, Life Technologies, USA) supplemented with ITS (1:100, Sigma, USA) and penicillin-streptomycin solution. After the treatment, paired ovaries from the same donor were randomly inserted under each kidney capsule of the same ovariectomized host. Mice were sacrificed at 14 days after transplantation to assess follicular development. For Western blot assay, ovaries were incubated with or without CDC42 activator II on an insert in six-well culture dishes in culture media described above for 30 min, respectively. Ovaries were transferred to drug-free medium and cultured for 12 h and then harvested to Western blot analyses.
Statistical analysis
All experiments were repeated at least three times, and the values are presented as the means ± SEM. Data were analyzed by t test and considered statistically significant at P < 0.05.
