Role of Insulin-like Growth Factors in Granulosa Cell Differentiation and Ovarian Follicle Maturation
thesisposted on 27.10.2017 by Sarah Catherine Baumgarten
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Role of Insulin-like Growth Factors in Granulosa Cell Differentiation and Ovarian Follicle Maturation Infertility affects more than 7% of couples in the United States. Approximately 40% of these infertility cases can be attributed to ovarian dysfunction, where a woman is unable to produce and/or ovulate mature oocytes for fertilization. For those who seek assisted reproductive technologies such as in vitro fertilization (IVF), anovulation is often overcome by the administration of follicle stimulating hormone (FSH) to induce the development of the oocyte-containing ovarian follicles. During follicle development to the preovulatory stage, undifferentiated granulosa cells proliferate and differentiate into two distinct populations: mural granulosa cells that line the wall of the follicle and cumulus granulosa cells that surround the oocyte. These cells work together to foster steroid hormone synthesis and oocyte development, respectively, both of which are required for normal fertility. In humans, little is understood about the process of granulosa cell differentiation because access to undifferentiated granulosa cells is extremely rare. However, cumulus and mural granulosa cells can be collected from the follicular aspirates of women undergoing IVF. Studies in rodents have revealed that the cumulus granulosa cells are protected from FSH-induced differentiation by factors secreted from the oocyte. Thus, we hypothesized that cumulus cells obtained from patients undergoing in vitro fertilization (IVF) treatment could be cultured to study human granulosa cell differentiation, once separated from the oocyte and its influence. In a novel approach, cumulus and mural granulosa cells were collected from the follicular aspirates of IVF patients and cultured in serum-free, phenol red-free media. Gene expression analysis revealed that cumulus granulosa cells expressed significantly lower levels of differentiation genes including the luteinizing hormone receptor (LHR) and steroidogenic genes such as CYP19A1 (aromatase), CYP11A1 (P450 side chain cleavage), and STAR (steroidogenic acute regulatory protein) than the mural granulosa cells. Additionally, in cumulus, but not in mural, cells, treatment with FSH stimulated the expression of key differentiation genes including CYP19A1, CYP11A1, and StAR at both the mRNA and protein level. The fact that cumulus cells are less differentiated than mural cells in culture and differentiate in response to FSH treatment indicate the cultured cumulus granulosa cells can be used as an experimental approach to study human granulosa cell differentiation. Using cultured cumulus granulosa cells, we sought to understand the role of locally produced ovarian factors, such as insulin-like growth factors (IGFs), in granulosa cell differentiation and follicle maturation. Previous studies demonstrated that IGFs enhance FSH-induced granulosa cell differentiation. We found that FSH upregulated IGF2 at both the RNA and protein level, and that this stimulation was driven specifically by the P3-promoter of IGF2. We then showed that IGF2 enhanced FSH-induced CYP19A1 expression and synergized with FSH to stimulate granulosa cell proliferation. Because IGF2 is produced by the granulosa cells and is constantly present, we inhibited IGF1 receptor (IGF1R) activity to eliminate IGF2 action, and study mechanisms by which FSH promotes granulosa cell differentiation. The FSH-induced expression of steroidogenic genes was inhibited at both the mRNA and protein level and, accordingly, the stimulation of estradiol production by FSH was eliminated. Further inspection revealed that FSH and IGF2 cooperatively enhance AKT and ERK simulation, and that IGF1R activity is required for FSH to stimulate AKT phosphorylation. Without this activation of AKT, FSH is not able to stimulate the expression of aromatase or enhance estradiol production. Additionally, FSH is not able to stimulate IGF2 expression when AKT activation is inhibited, demonstrating that by activating the IGF1R and allowing for FSH-induced AKT activation, IGF2 is an auto-regulator in human granulosa cells. Taken together, our results suggest that granulosa cells establish a niche environment within the follicle in which locally produced IGF2 stimulates AKT activation through the IGF1R to allow FSH to simulate human granulosa cell differentiation