Abstract
Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, “muscle cell development”, “muscle cell differentiation”, “muscle contraction”, “muscle organ development”, “muscle organ morphogenesis”, “muscle structure development”, “muscle system process” and “muscle tissue development”. The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.
Highlights
Together with oocytes, theca cells and ovarian surface epithelial cells, granulosa cells (GCs) are found in the follicular fluid and are fundamental for folliculogenesis and oogenesis [1–4]
GO families that we focused on were ranked at positions between 153 and 387 according to number of represented genes and statistical significance, except for one GO term (“muscle organ morphogenesis”) which ranked at the 886th position
Stem cells sustain the formation of oocyte and follicle in the postnatal mammalian ovary with some reports even suggesting that the consensus stating a defined number of primordial follicles formed in the embryonic period may be wrong [27]
Summary
Theca cells and ovarian surface epithelial cells, granulosa cells (GCs) are found in the follicular fluid and are fundamental for folliculogenesis and oogenesis [1–4]. Mural GCs surround the oocyte, forming the follicular wall around the antrum and communicate with the female gamete through the cumulus cells, which remain in direct contact with the oocyte and, through gap junctions, allow bi-directional stimulation as well as reciprocal exchange of nutrients and metabolites promoting folliculogenesis and oogenesis [7]. GCs stimulate the maturation of oocytes which, in turn, promote GCs proliferation and differentiation required for proper follicular growth. They display a structural and functional role in the active preovulatory and ovulatory follicle. GCs can differentiate towards multiple different cell lineages showing great potential for diverse clinical therapies and applications. Further studies revealed the ability of GCs to differentiate into muscle cells and cardiac cells [10,11]
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