Study Of The Function And Regulatory Mechanism Of SUMO-1 In Mouse Ovarian Granulosa Cells

Most of the cellular activities including regulation of protein functions and cell signaling networks are dependent on Post translational modifications. These modifications doesnot change the concentration of protein synthesis but it does alter the protein function. Therefore, the function of protein was complicated. There are many types of post-translational modifications in eukaryotes. SUMO (small ubiquitin-related modifier) is a kind of small molecular ubiquitin modifications (Ubls) and similarity with ubiquitin in structures. At present, there were four kinds of SUMO in mammal:SUMO-1, SUMO-2, SUMO-3 and SUMO-4. SUMO-1 is about 12 KDa and widespread in eukaryotes. It acts to combine with the target protein under the catalytic action of El, E2 and E3 enzymes. Thespecificity of combination process is called the SUMOylation. SUMOylation is one of Post-translational modification and dynamic reversible; It could be reversed by SUMO specific protease (SENP). The dynamic balance involved in a variety of biological processes, such as cell cycle, apoptosis, cell proliferation and differentiation, interaction between two or more proteins, signal transduction, regulation of the target protein molecule, the cellular localization/transport, stability, transcriptional activity, biological activity and so on. At present, the role and the function of SUMO-1 in granulosa cells are not clear. So, the aim of this study is the function and regulatory mechanism of SUMO-1 in mouse ovary granulosa cells. At the same time, identification of unknown SUMO-1 target protein and their regulatory role on cell.The aim of this study is research the positioning and expression patterns of SUMO-1 and UBC9 in mouse ovary, and investigated their function in ovary granulosa cells. Reveal the regulation function of SUMO-1 and UBC9 on cell cycle, proliferation and apoptosis of ovarian granular cells. At the same time, identification of unknown SUMO-1 target protein and their regulatory role on cell. This it also provides a strong theoretical base for understand a variety of physiological and biochemical adjustment mechanism of ovaries.This project of this study was used mouse as experimental animal. PMSG/hCG injected mouse. Gain the ovaries of mouse after different times. Using western blotting and immunohistochemistry to detected the expression and location of SUMO-1 and UBC9 in follicles at different developmental stages. In addition, we constructed eight vectors of pCMV-N-HA/Flag-SUMO-1/UBC9,pCMV-N-Flag-SUMO-1G96-97A, pEGFP-C1-SUMO-1/UBC9, pCMV-N-HA-PTX3 and pCMV-N-HA-PTX3K203R by PCR. In vitro cultured of granulosa cells, and carry out the expression vector transected into cells. Using immunofluorescence to tested the subcellular location and distribution of SUMO-1, UBC9 and PTX3 in cells. Unveil SUMO-1 and UBC9 activities in cell cycle, cell apoptosis, cell proliferation in the process of regulating function and regulation mechanism in ovarian granulosa cells by flow cytometry and ELIASA. Focusing on its participation in the specific cellular process, screening and identification with target protein, whether PTX3 can be modified by SUMO-1 and to know which one is the main SUMOylation locus by western blotting and co-immunoprecipitation. These researches established firm foundation to study SUMO-1 modification.The results as follows:(1) pCMV-N-HA/Flag-SUMO1/UBC9, pCMV-N-Flag-SUMO1G96-97A, pEGFP-C1-SUMO-1/UBC9, pCMV-N-HA-PTX3 and pCMV-N-HA-PTX3K203R vectors were constructed;(2) Western blotting showed that SUMO-1 and UBC9 have different expression patterns in different development period of ovarian tissue in mice. There was different SUMO-1 conjugates at 34,43, and 72 KDa were highly expressed in mice ovaries and the expression pattern was different. The amount of SUMO-1 conjugates at ～72 KDa and global proteins significantly increased at 1h, and decreased at 12,24, and 36 h, then increased at 48 h after PMSG injection (P<0.001), at 34-43 KDa, SUMO-1 conjugates significantly increased at 1h, and decreased at 12 and 24 h, then increased at 36 h (P< 0.001). Furthermore, the amount of SUMO-1 conjugates at ～34-43 KDa significantly increased at 9 and 11 h and relatively decreased at 7 and 15 h after hCG injection (P<0.001). Whereas, there was no significantly different at 72 KDa and global proteins were increased at 11 and 24 h after PMSG/hCG injection. At the same time, the relative level of UBC9 at ～34-43 KDa was significantly increased at 24,36 and 48 h, but the global protein was significantly increased at 48 h (P<0.001),11 h decreased after hCG (P<0.001). This implied that whose expression is regulated by PMSG and hCG, the changes of SUMO-1 conjugates were related to the process of folliculogenesis in ovary. And participate in the regulation of follicular development, oocytes maturation and granular cell development. In vitro cultured GCs also showed different expression pattern of SUMO-1 and UBC9 conjugates, it was increased at 48 h (P<0.05);(3) Immunohistochemical analysis demonstrated the positive signals of SUMO-1 and UBC9 in the granulosa cells, oocytes and corpus luteum during all stages of follicular development. On day 8 and 10, we found very strong immunostaining signals of SUMO-1 and UBC9 in the primary and secondary follicles, the localization was mainly dominated in the nucleus of oocytes and GCs, treatment with PMSG had no significant effect on immunostaining signals of SUMO-1 and UBC9 in the oocytes and GCs of all stages of follicles, Whereas, strong immunostaining signal was observed in the follicles at 12 h after PMSG injection. After hCG treatment, there were mainly accumulated in the GCs of antral follicles and pre-ovulatory follicles, Furthermore, the immunostaining signal of SUMO-land UBC9 was also observed in the corpus luteum after hCG treatment;(4) SUMO-1 and UBC9 vectors transfected in granulosa cells. Indirect immunofluorescence showed that endogenous and over expressed SUMO-1, UBC9 were predominantly localized in the nucleus of GCs, However, mutant SUMO-1G96-97A was localized both in the nucleus and cytoplasm, These results suggested that mutation of SUMO-1 at G96-97A affected the normal localization of SUMO-1 in GCs, implying that SUMO-1 has important functions in the cytoplasm m granular cells;(5) Over-expression SUMO-1, UBC9 and mutant of SUMO-1 could affect the cell cycle, apoptosis and proliferation of granulosa cells, the results showed that, SUMO-1 and UBC9 have some influence on the cell cycle, but it is not significant (P>0.05), SUMO-1 and UBC9 can promote the proliferation of granule cells (P< 0.001), over-expression SUMO-1 and UBC9 significantly induce the apoptosis of granulosa cells. However, mutant SUMO-1 at G96-97 could more significantly induce the apoptosis of granulosa cells (P< 0.001), suggesting the important function of SUMO-1 for apoptosis;(6) Immunoblotting and immunoprecipitation analysis indicated that PTX3 was modified by SUMO-1 and the binding site was lys203. Mutation of PTX3 at K203R significantly reduced the modification of PTX3 by SUMO-1, suggesting that Lysine 203 is a key amino acid for SUMO-1’s modification of PTX3. Further study indicated that endogenous PTX3 was mainly located in the cytoplasm of GCs and the overexpressed PTX3 was also detected the localization in both cytoplasm and nucleus in GCs, while mutation of PTX3 at K203R was only localized in the nucleus. We propose that SUMOylation of PTX3 at Lysine 203 is crucial for its cytoplasm localization and might be involved in its secretion from cytoplasmic contents into extracellular matrix, which are important for the formation of cumulus extracellular matrix.Our research confirmed that SUMO-1 and UBC9 have specificity of time and space expression patterns in different development period of ovarian tissue, the expression levels were regulated by hormones, and showed time dependence; SUMO-1 and UBC9 were localized in follicles at different developmental stages, granular cell and corpus luteum. SUMO-1 and UBC9 were predominantly localized in thenucleus of GCs, However, mutant SUMO-1G96-97A was changed its localization. Over-expression SUMO-1, UBC9 and mutant of SUMO-1 could affect the cell cycle, and promoted the proliferation and apoptosis of granulosa cells. PTX3 could be modified by SUMO-1 and lysine 203 was the site of SUMO, while mutation of PTX3 could be changed its localization.