多项使用单细胞RNA测序（scRNAseq）的研究未能检测到成年卵巢中的干细胞。我们坚信，在经过苏木精和伊红染色的卵巢表面上皮（OSE）细胞涂片中，可以轻松地检测到两个卵巢干细胞群体，包括多能的，非常小胚胎样干细胞（VSELs）和组织专用的“前体细胞”，被称为卵巢干细胞（OSCs），这些样本可以从小鼠和人类卵巢中制备。可能是因为干细胞只通过将细胞悬液离心1000 g来沉淀，而用于scRNAseq的细胞则始终通过将细胞悬液离心200-400 g来制备。最近一篇文章进一步解释了scRNAseq未能检测到卵巢干细胞的原因。使用先进的软件对(scRNAseq)产生的数据进行了广泛的重新分析，并成功检测到了OSCs和有支持成年人卵巢新生的减数分裂标记物。但这篇文章仍然对VSELs的生物学相关性及其与OSCs之间的关系保持批评态度。通过仔细研究通过部分胰蛋白酶消化完整小鼠卵巢在不同的发情周期阶段制备的OSE细胞涂片（其中包含VSELs，OSCs和生殖细胞巢GCNs），我们成功地描绘了VSELs / OSCs在生理条件下的新功能。 VSELs经历不对称的分裂进行自我更新，并产生稍微大一些的OSC，而这些OSC再进行对称分裂和克隆扩增，形成GCNs，被认为是新生卵和卵泡组装的正常过程。在胎儿卵巢和FSH处理下的成年卵巢OSE细胞培养中，GCNs已经被早期描述过。新生儿时期暴露于内分泌干扰物的毒害会导致VSELs / OSCs（这些细胞表达ERα，ERβ和FSHR）功能失调，从而导致卵巢功能不足和多囊卵巢。VSELs也与卵巢癌有关。年龄相关的卵巢老化/更年期也与VSELs / OSCs的功能失调和阻塞分化有关。这些新的真实发现以及存在于已发表文献中的丰富的体外和线索追踪研究数据为进一步的研究提供了巨大的空间，提供了管理卵巢病理的新途径，并呼吁重新编写教科书。 ©2023年作者。

Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in adult ovaries. We have maintained that two populations of ovarian stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed 'progenitors' termed ovarian stem cells (OSCs) can easily be detected in Hematoxylin and Eosin-stained ovary surface epithelial (OSE) cells smears prepared from both mice and human ovaries. Most likely the stem cells never get subjected to scRNAseq since they pellet down only by centrifuging cells suspension at 1000 g while cells for scRNAseq were invariably prepared by centrifuging at 200-400 g. A recent article provided further explanation for the failure of scRNAseq to detect ovarian stem cells. Extensive reanalysis of data (generated by scRNAseq) using an advanced software successfully detected OSCs and meiotic markers supporting neo-oogenesis in adult human ovaries. But this article remained critical on the biological relevance of VSELs and their relationship with OSCs. By carefully studying the OSE cells smears (which hold VSELs, OSCs and germ cell nests GCNs), prepared by partial trypsin digestion of intact mice ovaries during different stages of estrus cycle, we have successfully delineated novel functions of VSELs/OSCs in vivo under physiological conditions. VSELs undergo asymmetrical divisions to self-renew and give rise to slightly bigger OSCs which in turn undergo symmetrical divisions and clonal expansion to form GCNs, regular neo-oogenesis and follicle assembly. GCNs have been earlier described in fetal ovaries and during OSE cells culture (from adult ovaries) in response to FSH treatment. Dysfunction of VSELs/OSCs (which express ERα, ERβ, FSHR) due to neonatal exposure to endocrine disruption results in ovarian insufficiency and polycystic ovaries. VSELs have also been implicated in ovarian cancer. Age-related ovarian senescence/menopause is also due to dysfunction and blocked differentiation of VSELs/OSCs. These novel findings in vivo along with abundant in vitro and lineage tracing studies data in published literature provides huge scope for further research, offers novel avenues to manage ovarian pathologies and calls for re-writing of textbooks.© 2023. The Author(s).