Single-cell analysis on specification of mammalian germline and its role in health and diseases

Abstract

Germ cell specification is the first step for developing reproductive cells. The specification requires formative pluripotent stem cells as precursors. Previous attempts to capture formative pluripotency used opposite manipulation of Wnt signaling, activating or inhibiting, and achieved two distinctive states that indicated two ends of the formative pluripotency spectrum. Study I explored the role of Wnt signaling in the formative pluripotency spectrum. We produced a new form of formative pluripotency in epiblast-like stem cells (EpiLSCs) with activation of Wnt. We developed a computational single-cell method for transcriptionally aligning various cell lines within the formative pluripotency spectrum. Our analysis highlighted that EpiLSCs filled the gap in the pluripotency spectrum between previously published cell lines. Additionally, we revealed context-dependent roles of Wnt signaling in sustaining pluripotency and facilitating differentiation at the two ends of the formative pluripotency spectrum.

Female germ cell specification in humans generally exhibits lower efficiencies than males in vitro. Human X chromosome inactivation has a large extent of incompleteness, resulting in escapees. Whether the female lower efficiencies are related to X-linked escapees is unknown. Study II investigated the influence of Xlinked escapees on germ cell specification in females and individuals with Klinefelter syndrome (KS), a condition typically characterized by an extra copy of the X chromosome and infertility. Through RNA sequencing and functional assays, we identified critical X-linked escapees, CHRDL1, IGSF1, and USP9X, inhibiting germ cell specification in females and KS. We found that USP9X elevated SOX2 to repress oxidative phosphorylation, promote mitochondria fusion and clustering, and perturb SOX17's regulation, exerting a profound reduction in germ cell specification.

Germ cells carry both genetic and epigenetic information. Sperm's small RNA composition is shaped by the soma-to-germline communication pathway and, therefore, is responsive to environmental exposure. Polycystic ovary syndrome (PCOS) is an epigenetically heritable disorder affecting female offspring. The possibility of PCOS equivalent in males has prompted questions about the potential impact on male offspring and whether sperm small RNA plays a role in it. In study III, using a Swedish registered cohort, a Chile longitudinal cohort, and a mouse model, we found that women with PCOS transgenerationally transmitted reproductive and metabolic dysfunction into their male offspring. Using small RNA sequencing, we identified transgenerationally altered sperm small RNA in the mice, which overlapped with changes in the sons of women with PCOS, suggesting potential mechanistic parallels of inheritance between mice and humans.

In this thesis, we uncover critical insights into the Wnt's context-dependent roles in the formative pluripotency spectrum, the repression of XCI escapees on germ cell development, and sperm small RNAs' transgenerational transmission. Additionally, this research paves the way for further exploration into germ cell development for individuals with KS and the inheritance of PCOS in male offspring, offering potential avenues for therapeutic interventions.