Unless otherwise specified, all chemicals and reagents were purchased from Sigma-Aldrich (USA).
Specific pathogen-free (SPF) ICR female mice (9–11 g in weight) were used for all the experiments. These mice were purchased either from Nanjing Medical University Animal Core Facility or Beijing Vital River Laboratory Animal Technology Co., Ltd. All experimental and animal procedures were approved by the Ethical Committee of Laboratory Animals and the Animal Care and Use Committee of Nanjing Medical University, and performed in accordance with the institutional guidelines.
Female mice were primed with 5 U of pregnant mare serum gonadotropin (PMSG) (Ningbo Second Hormone Factory, China) to stimulate the antral follicle development in the ovaries. Ovaries were picked up 46 –48 hours after the initial priming, and placed into a 3 cm dish containing MEM-alpha (Gibco, USA) medium. Large antral follicles were then punctured with a pair of syringes connected with 25 gauge needles to release the mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs). COCs were collected, and oocytes and cumulus cells were separated by repeatedly pipetting the COCs using a mouth controlled glass pipette with the inner diameter slightly smaller than the oocyte. Similar amount of MGCs, cumulus cells (CCs), and germinal vesicle (GV) stage fully-grown oocytes (FGOs) were then collected. These procedures were carried out in MEM-alpha supplemented with 75 μg/mL penicillin G, 50 μg/mL streptomycin sulfate, 25 μg/mL pyruvate, 3 mg/mL of bovine serum albumin (BSA) and 5 μmol/L milrinone (Calbiochem, Germany), a specific inhibitor of PDE3A to maintain oocyte meiotic arrest.
For oocyte culture, GV-stage oocytes were incubated in MEM (Gibco) supplemented with 75 μg/mL penicillin G, 50 μg/mL streptomycin sulfate, 25 μg/mL pyruvate, 38 μg/mL EDTA and 3 mg/mL BSA. Culture was conducted at 37 °C in an incubator infused with 5% O2, 5% CO2 and 90% N2. Oocytes at GV, Pro-metaphase Ⅰ (Pro-MⅠ), metaphase (MⅠ), anaphase Ⅰ (AⅠ)/telophase Ⅰ (TⅠ), and MⅡ stages were collected at 0, 4, 6–7, 8–10, and 12–14 hours after the culture was set up.
Total RNA was extracted from granulosa cells and oocytes using RNeasy micro kit (Qiagen, Germany), and in vitro transcription and real-time PCR analyses were then carried out using QuantiTect® Reverse Transcription Kit (Qiagen) and QuantiTec® SYBR Green PCR Kits (Qiagen), respectively, on the ABI 7500 Real-time PCR System (Applied Biosystems, USA). The relative fold changes in mRNA levels were calculated via the method of 2−ΔΔCt using Rpl19 as an internal control. The primer sequences for the tested genes are listed in Table 1.
Primers Sequence (5′→ 3′) EML1-F GCACATCTAAGGATGGAAAGCAA EML1-R CGGTCAAAAAAGCCTATTCCAA EML2-F CCCCGTCACCTGTAAGCAAA EML2-R CCAAATCCCAAACACTCCAAA EML3-F ATTGGTTCCCATGACAACATGA EML3-R TGATAAAACTGGAGTGCCCCATA EML4-F GACGCCAGTGTGACCAAAAC EML4-R GCCCATCCTGCTTTCCTCTT EML5-F CAGATGGCGCTTACCTTGCT EML5-R AAGGGAGCCGACACATTCAC EML6-F AGAAAGACCACCCGTTAGCC EML6-R ATGATGTCGGCACCATCGTT
Table 1. Primers used for real-time RT-PCR analysis
Isolated mouse ovaries initially primed with PMSG for 46 hours were fixed for 4 hours in 4% paraformaldehyde, and then embedded in paraffin and sectioned at 5-μm thickness for immunohistochemistry and immunofluorescence. Immunostaining of EML6 was then carried out using rabbit anti-EML6 primary antibody (1:100), and the VECTASTAIN ABC-AP KIT (Rabbit IgG, catalog No. AK-5001, Vector, USA) and VECTOR Red Alkaline Phosphatase (AP) Substrate Kit (catalog no. AK-5100) purchased from Vector Laboratories. Alexa Fluor-488 donkey anti-rabbit IgG was used as the secondary antibody for immunofluorescence.
Oocytes were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and then washed and blocked in PBS containing 10% fetal bovine serum (FBS) and 1% Triton X-100 for 1 hour at 37 °C or at 4 °C overnight. After blocking, oocytes were incubated overnight at 4 °C with EML6 antibody (1:100) and FITC-α-tubulin antibody (1:500, Proteintech, Wuhan, China). After three washes with PBS-1% FBS, oocytes were incubated for 2 hours in Alexa Fluor 594-conjugated secondary antibody solution (1:200), and finally counterstained with DAPI (1:100) for 10 minutes, and mounted on glass slides for imaging using a laser scanning confocal microscope (LSM 700, Carl Zeiss, Germany).
After 8 hours in culture, oocytes at MⅠ stage were transferred to pre-warmed culture medium containing 20 μg/mL nocodazole or 10 μmol/L Taxol, and incubated for 10 or 45 minutes, respectively. Control oocytes were incubated with the same concentration of dimethyl sulfoxide (DMSO) under the same culture conditions. After treatment, the oocytes were washed thoroughly and fixed for immunofluorescence.
Zona pellucida of MⅡ-stage oocytes was removed by exposure to acid Tyrode's solution (pH 2.5) for 30 seconds at 37 °C. The oocytes were put on a glass slide in a small drop of fixative containing 1% PFA and 0.15% Triton X-100, and then air dried. The fixed oocytes were incubated with the Human anti-Centromere (1:500, Antibodies Incorporated, USA) at 4 °C overnight after being blocked with 1% BSA for an hour, followed by incubation with Alexa Fluor594-conjugated donkey anti-Human IgG (1:750, Invitrogen, Shanghai, China). The chromosomes were counterstained with Hoechst 33342 for 15 minutes. The specimens were examined under a LSM 700 laser scanning confocal microscope (Carl Zeiss).
All experiments were repeated at least three times independently, with the data presented as mean ± SEM. Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, USA). Differences between the groups were analyzed using Student's t-test. P<0.05 was considered to be significantly different.
Participation of EML6 in the regulation of oocyte meiotic progression in mice
- Received Date: 2019-01-26
- Accepted Date: 2019-02-28
- Rev Recd Date: 2019-01-31
- Available Online: 2019-04-30
- Publish Date: 2020-01-01
- echinoderm microtubule-associated protein like 6 (EML6) /
- meiosis /
- spindle /
- chromosome alignment /
- oocyte aneuploidy /
- female fertility
Abstract: The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.
|Citation:||Hong Yin, Xuan Hou, Teng Zhang, Lanying Shi, You-Qiang Su. Participation of EML6 in the regulation of oocyte meiotic progression in mice[J]. The Journal of Biomedical Research, 2020, 34(1): 44-53. doi: 10.7555/JBR.33.20190014|