Whole Mount Immunostaining of Fetal and Neonatal Mouse Ovaries

The ovarian reserve consists of the oocytes available to a female for reproduction and are found in primordial follicles, each containing one oocyte surrounded by several granulosa cells. The oocytes start out as primordial germ cells that form outside the gonad and migrate to the developing ovary during embryonic development. Once they reach the ovary, they become oogonia and divide by mitosis, with incomplete cytokinesis, forming clusters of interconnected cells called germ cell cysts. While in cysts, the cells enter meiosis and become oocytes. In the mouse, two days before birth, the cysts begin to break apart, and each oocyte is packaged into a primordial follicle. Only some of the oocytes survive during this process, the rest undergo apoptosis. The regulation of cyst breakdown and primordial follicle formation is only partially understood, and identifying the molecules involved is crucial to understanding this process. Whole mount immunostaining of developing mouse ovaries can be used to determine the localization of potential regulatory molecules and to assess effects on primordial follicle formation due to mutation or other molecular changes using oocyte and somatic cell markers. Ovaries are harvested from fetal or neonatal mice, fixed in formaldehyde, and blocked. The ovaries are then exposed to antibodies against a protein of interest, followed by secondary antibodies conjugated to a fluorescent tag that detect the primary antibodies. Finally, the ovaries are mounted and examined by confocal microscopy. This procedure can detect multiple proteins of interest and can be applied to other small tissues, such as fetal mouse testes.