Inherent mitochondrial activity influences specification of the germ line in pluripotent stem cells.

Herein we investigated whether inherent differences in mitochondrial activity in mouse pluripotent cells could be used to identify populations with an intrinsic ability to differentiate into primordial germ cells (PGCs). Notably, we determined that stem cells sorted based on differences in mitochondrial membrane activity exhibited altered germline differentiation capacity, with low-mitochondrial membrane potential associated with an increase in PGC-like cells. This specification was not further enhanced by hypoxia. We additionally noted differences between these populations in metabolism, transcriptome, and cell-cycle. These data contribute to a growing body of work demonstrating that pluripotent cells exhibit a large range of mitochondrial activity, which impacts cellular function and differentiation potential. Furthermore, pluripotent cells possess a subpopulation of cells with an improved ability to differentiate into the germ lineage that can be identified based on differences in mitochondrial membrane potential.

A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics.

Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi-parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.

Dynamics of WNT signaling components in the human ovary from development to adulthood.

WNT signaling has been shown to play a pivotal role in mammalian gonad development and sex differentiation; however, its role in the developing human ovary has not been investigated. We analyzed a quantitative mass spectrometry dataset to determine the expression of WNT signaling components between 47 and 137 days of development and in adult ovarian cortex tissue. WNT signaling was identified within the top ten canonical pathways of proteins detected at every developmental stage examined. We further examined the specific localization of WNT signaling components glycogen synthase kinase 3 (GSK3B), frizzled 2 (FZD2), and ß-catenin (CTNNB1) within ovarian tissue. GSK3B was nearly ubiquitously expressed during fetal development, while FZD2 was specific to germ cell nests during early development. ß-catenin exhibited translocation from primarily membrane bound during early ovarian development to cytoplasmic and nuclear staining specifically in early primordial follicles in the fetal ovary. This cytoplasmic and nuclear ß-catenin persisted in primordial follicles in adult ovarian tissue, but returned to membrane-bound localization in secondary follicles. We conclude that WNT signaling components are expressed in the human ovary from early to mid-gestation and remain in the adult ovary, and observed evidence for canonical WNT signaling only in the oocytes of primordial follicles. Together, these data are indicative of a role for canonical WNT signaling via ß-catenin nuclear translocation during human follicle formation and follicle maintenance.

Quantitative Proteomic Profiling of the Human Ovary from Early to Mid-Gestation Reveals Protein Expression Dynamics of Oogenesis and Folliculogenesis.

The in vivo gene networks involved in coordinating human fetal ovarian development remain obscure. In this study, quantitative mass spectrometry was conducted on ovarian tissue collected at key stages during the first two trimesters of human gestational development, confirming the expression profiling data using immunofluorescence, as well as in vitro modeling with human oogonial stem cells (OSCs) and human embryonic stem cells (ESCs). A total of 3,837 proteins were identified in samples spanning developmental days 47-137. Bioinformatics clustering and Ingenuity Pathway Analysis identified DNA mismatch repair and base excision repair as major pathways upregulated during this time. In addition, MAEL and TEX11, two key meiosis-related proteins, were identified as highly expressed during the developmental window associated with fetal oogenesis. These findings were confirmed and extended using in vitro differentiation of OSCs into in vitro derived oocytes and of ESCs into primordial germ cell-like cells and oocyte-like cells, as models. In conclusion, the global protein expression profiling data generated by this study have provided novel insights into human fetal ovarian development in vivo and will serve as a valuable new resource for future studies of the signaling pathways used to orchestrate human oogenesis and folliculogenesis.