No abstract

Germline specification underlies human reproduction and evolution, but it has proven difficult to study in humans since it occurs shortly after blastocyst implantation. This process can be modeled with human induced pluripotent stem cells (hiPSCs) by differentiating them into primordial germ cell-like cells (hPGCLCs) through an incipient mesoderm-like cell (iMeLC) state. Here, we elucidate the key transcription factors and their interactions with important signaling pathways in driving hPGCLC differentiation from iPSCs. Germline competence of iMeLCs is dictated by the duration and dosage of WNT signaling, which induces expression of EOMES to activate SOX17, a key driver of hPGCLC specification. Upon hPGCLC induction, BMP signaling activates TFAP2C in a SOX17-independent manner. SOX17 and TFAP2C then cooperatively instate an hPGCLC transcriptional program, including BLIMP1 expression. This specification program diverges from its mouse counterpart regarding key transcription factors and their hierarchies, and it provides a foundation for further study of human germ cell development.

Summary Segregation of homologous chromosomes at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that allows them to make a monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC) [1]. Although this appears to be the case in the male [2–5], the presence of one or several univalents does not cause cell cycle delay or arrest in the mammalian oocyte [6, 7]. The spindle assembly portion of the SAC appears to function normally in the oocyte [8–10], but two hypotheses have been proposed to explain the surprising lack of response to univalent chromosomes: 1) reduced stringency of the oocyte SAC to aberrant chromosome behavior [7], and 2) the ability of univalents to form bipolar attachments that satisfy SAC requirements [6]. Results of the present study of Mlh1 mutant mice demonstrate that metaphase alignment is not a prerequisite for anaphase onset and provide strong evidence that MI spindle stabilization and anaphase onset requires stable bipolar attachment of a critical mass - but, importantly, not all - chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error-prone and provide a biological explanation for the high rate of aneuploidy in humans.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.