Ovarian Sample Extraction during Estrous Cycle

To retrieve ovarian samples from adult mice (8 weeks old) during each of the four stages of the estrous cycle, we performed the vaginal smear tests to identify the specific estrous cycle stages. Briefly, we placed the tip of a plastic pipette filled with 40 μl of phosphate buffered saline after (PBS; Hyclone, USA) into the mouse vagina, and gently flushed the vagina 3–5 times. Vaginal contents (cells and vaginal fluids) from each mouse were collected into a PCR tube and then smeared onto a glass slide. The evaluation of the vaginal smear images was performed under a microscope IX71 system (Olympus, Japan). Proestrus was characterized by the mostly nucleated and some cornified epithelial cells in the smear; estrus by cornified squamous epithelial cells without visible nuclei; metestrus by the predominance of leucocytes and a few nucleated epithelial and/or cornified squamous epithelial cells; and diestrus was characterized by a predominance of leukocytes [24 (link), 25 ]. The reader is directed to further citations for a full description [24 (link), 25 ]. Vaginal smear tests were performed at 08:00 and 20:00 each day. When the stages were identified, the mice were sacrificed via cervical dislocation and their ovaries were collected.