Investigation of the differential regulation of endoplasmic reticulum signal proteins in cumulus cells with normal, hyperstimulated, hypostimulated and polycystic ovary syndromes

There is a direct relationship between the maturation of the nucleus and cytoplasmic maturation and biological activation in oocytes. Cumulus cells contribute to this process by stimulating protein synthesis in oocytes and, therefore, the maturation of oocytes. Moreover, when we consider the fact that only a mature oocyte (in MII [metaphase II]) has the capability of fertilization, it is clear to see responsibility cumulus cells have for the maturation and activation of oocytes. In addition, the fact that oocytes found in PI (prophase I) and MI (metaphase I), which are not mature enough, do not have the capability of fertilization shows another aspect of the importance of cumulus cells. Endoplasmic reticulum is an essential organelle in maintaining cells vital function. ER regulates intracellular calcium concentration,protein synthesis and homeostasis and it also enables the biosynthesis of lipids.Ca dependent molecules like 78kDA GRP78 , GRP94 and calreticulin are chaperon rich molecules due to their role in protein synthesis and its intracellular transport. These chaperons stabilize intermediary proteins which enable protein folding.Several factors may be the basis for the aggregation of unfolding proteins in ER lumen. This aggregation of unfolding proteins in ER lumen induces a reaction called Unfolding Protein Response (UPR), an ER dependent cellular reaction which is evolutionally quite well conserved. The effects of UPR induced by ER stress are categorized into 3 major groups; adaptation, stimulation and apoptosis. Hypoxia and damage to the redox pathway caused by oxidative agents bring about termination of the disulfide bonds within ER lumen and the unfolding or misfolding of the proteins.Of the patients applying for IVF techniques, 3 subtypes of patient groups are distinguished according to their response to follicle stimulations; the first group being composed of those demonstrating normal stimulation, the second group composed of patients demonstrating hypostimulation, and the third group demonstrating hyperstimulation. Normostimulation is considered as retrieval of 10-20 oocytes in IVF patients aged less than 35 years as a response to follicle stimulation. In contrast, retrieval of 30 or more oocytes as a response to the same stimulus is considered to be ovarian hyperstimulation syndrome, which includes polycystic ovary syndrome. Retrieval of 6 or less oocytes is considered to be hypostimulation. There are significant differences among these 3 groups in terms of both follicle development and the number of mature oocytes (MII) or immature (PI [GV] or M I) oocytes retrieved.In this study we examined the regulation of UPR due to ER stress which may occur in patients responding differently to the induction of ovulation.The protein level of GRP78, which is the main regulator protein of UPR pathway, is analysed by the Western Blot method. No significant change is observed between the GRP78 protein levels groups. On the other hand, we have determined that GRP78 protein levels in the cumulus cells of the hyporesponsive patients are two times higher than the other groups.In order to evaluate the ER stress at mRNA level; XBP1 and sXBP1 mRNA levels are examined by the RT?PCR method. In our study, when the quantity of XBP1s mRNA in the whole form XBP1 mRNA is compared to the other groups, it has only shown an increase in the hyporesponsive patients. This result is similar to the result which we have found for GRP78. Also the increased ER stress or the impaired protein doubling mechanism which we have determined in the hyporesponsive patients, support our hypothesis about folliculogenesis and oocyte ripening in these patients in a significant way.Cumulus cells are reproduced with the cell culture and the tunicamycine which is the TUDCA and ER stress stimulus of the UPR pathway inhibitor is processed with tunicamicyne and proliferation and apoptosis indexes are determined with MTT ad TUNEL methods.After the cumulus has performed the cell culture, we have processed the cumulus cells for 48 hours with tunicamicyne which is the TUDCA and ER stress stimulus of the UPR pathway inhibitor.MTT analysis is performed in order to determine the cell activity. According to our findings of MTT proliferation analysis; cumulus cell proliferation is pressurized as a result of the activation of UPR by ER stress which is induced with tunicamycine. When we have pressurized ER stress by applying TUDCA and tunicamycine on the cumulus cells together; we have observed a little increase in the cell proliferation when compared to the tunicamycine treated group.As a result of the MTT analysis, the ER stress in the cumulus cells are induced with tunicamycine and their apoptosis indexes are determined and then they are compared to the control group in order to determine whether the significantly decreased cell proliferation in the cumulus cells stimulated with ER stress and tunicamycine is related to the decrease in proliferation or the increased apoptosis.As a result of the TUNEL method; it is observed that apoptosis in the cumulus cells is increased by the tunicamycine when compared to the control group. This finding shows us that the cell proliferation pressurized by the ER stress in MTT analysis has probably risen from the increased apoptosis, not by the decreased proliferation.Based on the response to the IVF treatment and the clinical findings, the patients are inspected in 4 different groups related to the oocyte quantity as normostimulation, polycystic over syndrome, hyperstimuli and hypostimuli. When the patient groups are analysed statistically according to the ages, no significant difference is observed between the groups.When the patient groups are evaluated according to the collected average oocyte quantities, PKOS and hyperstimuli patient groups have statistically significant more oocyte quantity than the normal and hypostimuli patient groups. Also the normal patient groups have significantly more oocyte quantity than the hypostimuli patient groups.In this study, we have shown in the literature for the first time that ER signal pathway and its molecules GRP78, XBP1 and sXBP1 are present in the cumulus cells of the human ovarian follicle. We have shown that when the cumulus cells are exposed to the signal pathway related to the ER stress; a decrease in the cell proliferation and/or increase in apoptosis can occur. We have demonstrated that an increased ER stress in the cumulus cells of the ovarian follicle of the patients forming the hyporesponsive (hyperstimuli) group from the patients subject to IVF treatment is present and we have shown this stress by sXBP1 at the mRNA level.Finally we recommend that a new treatment approach can be foreseen by keeping the ER stress and UPR signal pathway in the physiological limits in order to obtain a health folliculogenesis and a qualified oocyte and also by using the agents which will decrease the pathological ER stress levels in the hyporesponsive patient groups.