Isolation and

Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success in fertility clinics. In vitro culture systems enable researchers to analyse important molecular and genetic regulators of oocyte maturation and fertilisation. Here, we describe in detail a highly reproducible technique for the isolation and culture of fully grown mouse oocytes. We include the considerations and precautionary measures required for minimising the detrimental effects of in vitro culture conditions. This technique forms the starting point for a wide range of experimental approaches such as post-transcriptional gene silencing, immunocytochemistry, Western blotting, high-resolution 4D time-lapse imaging, and in vitro fertilization, which are instrumental in dissecting the molecular determinants of oocyte quality. Hence, this protocol serves as a useful, practical guide for any oocyte researcher beginning experiments aimed at investigating important oocyte molecular factors. Graphic abstract: [Image: see text] A step-by-step protocol for the isolation and in vitro culture of oocytes from mice.