Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries
Trascrizione
In a mammalian ovary, fully grown oocytes derived from the antral follicles only need a short period of maturation in culture, a crucial factor for in vitro embryogenesis. These oocytes are surrounded by a cluster of somatic cells, which must be removed through denudation to facilitate sperm injection.
To isolate denuded mouse antral oocytes, begin with an anesthetized female mouse pre-injected with pregnant mare serum gonadotropin hormone to stimulate follicular growth. Prep the mouse and incise the lower abdomen.
Exteriorize the intestine to reveal ovaries at the top of V-shaped uterine tubes. Dissect an ovary. Transfer it to a pre-equilibrated culture medium to maintain the viability of oocytes. Under a stereomicroscope, identify the antral follicles by their distinctive fluid-filled cavity.
Using a sterile needle, puncture the antral follicles of the ovary to release oocytes. Using a pipette of an optimum diameter, carefully aspirate the oocytes to protect them from mechanical damage. Avoid aspirating follicular cells and other tissue debris.
Subsequently, transfer the oocytes into multiple media drops covered with mineral oil. This step helps remove the surrounding somatic cells and generate denuded oocytes. Lastly, transfer the denuded oocytes into fresh media drops with mineral oil and preserve them for further experiments.
