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Alliance"],"accepted":{"date-parts":[[2025,11,7]]},"published-print":{"date-parts":[[2026,2]]},"abstract":"<jats:p>Understanding the molecular basis of oocyte identity and function is essential not only for basic biology but also for clinical applications, as it is closely linked to female infertility. However, technical challenges remain in advancing this understanding, mainly because of the difficulty in obtaining a sufficient number of oocytes. In this study, through refining previously reported three-dimensional protocols, we established a two-dimensional culture method that efficiently generates oocyte-like cells, referred to as mini-oocytes, from mouse embryonic stem cells. This method requires minimal labor, does not rely on supporting somatic cells, and leverages a transcription factor\u2013mediated approach for oocyte-like cell generation. Our transcriptome and proteome analyses revealed significant similarities between in vitro\u2013derived mini-oocytes and in vivo oocytes, despite their relatively smaller size. Furthermore, we demonstrated the utility of mini-oocytes for investigating oocyte-specific molecular features through a small-scale knockout screen targeting the subcortical maternal complex. 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