Browsing by Author "Väyrynen, Pia"

Follicle-stimulating hormone (FSH) regulates mammalian reproduction. The hormonal function of FSH is exerted through its receptor, FSHR. Hormone binding leads to signal transduction by the synthesis of the second messenger (intracellular cAMP) and the activation of other downstream signaling pathways. FSH receptors are mainly found in ovaries and testes where FSH stimulates e.g. follicular growth and spermatogenesis, respectively. It has been previously shown that FSHR mutations are linked to infertility through abnormalities in the receptor function. For example, inactivating FSHR mutation (Ala189Val) leads to arrest of follicular development in females and reduced sperm counts in males. FSH action seems to be especially critical for folliculogenesis and essential for female fertility. The development of gonads and dysfunctions affecting reproduction are the focus of our research group. Studies of recent years have demonstrated extragonadal FSHR expression, including endothelial cells of female reproductive tract and developing placenta. The physiological relevance and function of this extragonadal FSHR is still not well known, especially during embryogenesis. In addition, the expression or function of the mutated FSHR has not been studied in any cells endogenously expressing the receptor. As a consequence, the purpose of this thesis was to study FSHR expression and function in human pluripotent stem cells (hPSCs) as a model for early human development. The study was conducted in a human embryonic stem cell line (hESC H9, 46, XX) and two human induced pluripotent stem cell lines (hiPSC HEL127.6 and HEL128.5; 46, XX). iPSCs were obtained from female patients carrying the A189V mutation in the FSHR gene. Cells were differentiated for 8/12 days using two in vitro cell culturing protocols for distinct differentiation attempts. The first protocol (protocol D) was designed for embryonal cell differentiation, the other (trophoblast protocol) for extraembryonal differentiation. Cells at different stages of differentiation were studied with qRT-PCR and immunocytochemistry. At d8, cells were stimulated with FSH for qPCR studies and for cAMP assay. Control cells (H9) differentiated by protocol D endogenously expressed functional FSHR. These cells responded dose-dependently to FSH stimulation by substantially increasing cAMP production, downregulating FSHR mRNA expression and altering inhibin gene expression. Patient-derived iPSCs carrying the mutation expressed FSHR as well but the receptors were non-functional as expected. Both H9 and patient-derived cells that differentiated into trophoblast-like cells with the other protocol, also expressed FSHR at low level but did not respond to FSH stimulation. Preliminary results with protocol D indicate that the cells resemble extraembryonic cell types. The study has revealed novel information and insight about extragonadal FSHR expression and function during differentiation. However, the exact identity and the biological role of these cells is yet to be confirmed.