Researchers from the Karolinska Institute in Sweden have proven that hypothesized egg stem cells do not exist.
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The study, published in Nature Communications, reported single-cell transcriptomes and cell surface antigen profiles of over 24,000 Egg cells to reveal transcriptional profiles of several main cells. This data has revealed the absence of germline Sam cells, supporting the existing dogma of limited ovarian reserves in adult human ovaries.
This work builds on the understanding that although ovaries ceased to function at the age of menopause, there may be stem cells capable of differentiating into various cell lineages past this point, enabling continuous regeneration of egg cells throughout the adult female lifetime.
A brief history of the egg stem cell hypothesis
Oogonial stem cells (OSCs) are a hypothesized cell type thought to be present in the ovarian cortex, the outer lining of the ovary. They are thought to give rise to new oocytes and originate from fetal germ cells. Fluorescence-activated cell sorting (FACS) has been used to isolate OSCs using an antibody targeted to the DDX4 protein.
Existing work that characterized somatic cells derived from the inner part of human ovaries provided the basis for Wagner et al.’s work, extensively characterizing the germ and somatic cells in the human ovary. Enzymic digestion liberated 24,000 individual cells for scRNA-seq and cell surface marker profiling.
Tissue samples were derived from 21 healthy patients’ follicles, and single-cell RNA-sequencing (scRNA-seq) was performed alongside surface marker screening. The latter was conducted to determine the surface proteome of the cells present in the ovarian cortex.
The cells derived were taken from healthy individuals, proven fertile with no known conditions affecting ovarian function.
Both DDX4 antibody marking and validation of this surface marker by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (qPCR) was performed alongside scRNA-seq.
Six main cell types populate the human ovarian cortex
A total of 12,160 cells were used for cell characterization, revealing six cell clusters common to all donors. The identities of these cell types were determined via gene expression analysis and enriched gene expression profiles.
These included endothelial, granulosa, oocyte, immune, perivascular and stromal cells. This data was integrated with recently published single-cell profiling of a subtype of cells (antral cells and tissue fragments taken from the ovarian medulla) to determine a complete map of all egg cell types present in the adult ovary.
Wagner et al. found that most somatic cell types overlapped with cortex cell types they identified (immune cells, perivascular (or smooth muscle) cells, and endothelial cells). This merged map revealed that both vasculature and clusters of immune cells are found throughout the ovaries.
Using DDX4 to identify perivascular cells
The presence of OSCs was investigated using OSC-specific markers, DDX4. Expressing cells were manually pooled and their transcriptomes analyzed. Plots of gene expression revealed that DDX4 is mainly localized to oocytes. An additional 15 somatic cells expressing ddx4 were also analyzed.
Further investigation revealed that DDX4 Ab+ cells made up the cluster identified as perivascular cells. Validation of this finding was achieved by staining of ovarian tissue; DDX4 similarly identifies perivascular cells.
A comparison of the 24,000 cells to existing transcriptome data from both human fetal ovaries and the ovarian medulla, the inner region of the ovary, also failed to reveal any oogonial stem cells.
This revealed that the DDX4 isolation technique is not indicative of the presence of egg stem cells. Moreover, DDX4-Ab+ cells that have been previously used to treat infertility in women, have in fact been perivascular cells.
The theca and stromal cells of the ovary
A notable finding was that stromal and theca cells closely resemble one another. This was determined as a result of the close cluster expression of the genes STAR and CYP7A1.
This similarity is further reflected by the inability to separates both subtypes. To understand how the theca cell type relates to stromal cells requires the identification of factors that triggered its recruitment to the cortex and medulla.
Thecal and stromal cells have implications - thecal cells are endocrine cells whose principal function is to produce steroid hormones. These mediate signal transduction between egg cells and granulosa cells. They additionally provide nutrients and a structural basis along with stromal cells. An understanding of their function informs fertility.
The implications of stem cell inexistence for infertility treatment
Previously, the lack of knowledge about the constituents of the ovary has limited the developments made for the treatment of infertility. Through revealing several cell types that exist in the ovary, this study lays the foundation for new methodologies that focus on cells of proven existence.
Wagner M. et al. (2020) Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. Nat Commun. DOI:10.1038/s41467-020-14936-3.
Fan X. et al. (2019) Single-cell reconstruction of follicular remodeling in the human adult ovary. Nat Commun. DOI:10.1038/s41467-019-11036-9.