Steroidogenesis-inducing protein (SIP) molecular characterization and mechanism of action

The major function of the female gonad is the differentiation and release of the mature oocyte for fertilization, and the sex steroids production. Although it is well established that the pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play key regulatory roles in follicle development, oocyte maturation and luteinization, it has become evident that their effects in ovary are modulated or “fine-tuned” by paracrine and autocrine actions of steroid hormones, growth factors, cytokines, neuropeptides and other putative locally produced intraovarian regulators. In the previous studies a protein has been identified in human ovarian follicular fluid, which was named steroidogenesis-inducing protein (SIP) due to its significant stimulatory effects on steroid production in ovarian, testicular and adrenal cells. Later studies identified SIP as a potent growth factor that stimulated proliferation of rat testicular Leydig cells, rat ovarian granulosa cells and human ovarian cancer cells. Its effects on target cells were mediated via a tyrosine-kinase signaling pathway, although the identity of SIP receptor and other signaling components remained unknown. Recently the bioactive SIP has been purified to homogeneity. The N-terminal sequence analysis revealed that it is a novel human protein. Our overall hypothesis is that SIP is a novel gonadal protein which is secreted by granulosa cells in response to stimulation with pituitary gonadotropins (FSH and/or LH) and mediates the effects of tropic hormones on proliferation, differentiation and function of granulosa cells and may be involved in luteinization of granulosa cells, and in steroidogenic function of luteal cells. In this dissertation, I report molecular characterization of SIP and study on the mechanism of its action. I purified SIP from human follicular fluid collected from women undergoing in vitro fertilization (IVF) by FPLC. Three peptides were obtained by protein sequencing analysis. Peptide 2 revealed the uniqueness of SIP in human protein databases and Pepetide1 and 3 showed homology with human immunoglobulin heavy chain sequences. A number of human cDNAs have been reported from different human tissues which contain peptide 1 and 3 sequences but do not contain peptide 2 sequences. The peptide 2 sequences have been found in recently characterized DING family of proteins. None of the eukaryotic DING proteins have been cloned and a recent study suggests that DING proteins may be from Pseudomonas. Antibodies raised against SIP peptide 2 recognized the existence of SIP in human follicular fluid, granulosa cells from different species, human ovarian epithelial cancer cells and other human cancer cell lines. Based on SIP peptide sequences, I tried to clone SIP cDNA from human granulosa cells by RT-PCR and by screening a human ovarian cDNA library for SIP coding sequences. Studies on the mechanism of SIP action provided us with important information about signal transduction pathways involved in SIP effects on both cell proliferation and steroidogenesis in target cells. Studies on immature rat Leydig cells revealed that stimulatory effects of SIP on DNA synthesis and androgen production were mediated though tyrosine kinase-coupled signaling pathways, which can be blocked by pre-treating cells with genistein, a general tyrosine kinase inhibitor. Investigation on two major tyrosine kinase based signaling pathways (MAPK/ERK and PI3K/Akt) revealed that SIP stimulated activation of both pathways and both pathways were involved in SIP effects on DNA synthesis but not in its effects on steroidogenesis. Finally, studies on SIP peptides revealed that synthetic SIP peptide 2 has similar stimulatory effect as its parent protein SIP on DNA synthesis in different cell types and might be responsible for SIP mitogenic effects.