Study On Function Of LRF In Female Mouse Reproduction And Granulosa Cell Apoptos1s

Luman recruitment factor （LRF） is novel identified transcription factor, but thebiological function of LRF remains unknown. Previous studies with LRF knockout mouse hasbeen suggested that LRF has relationship with the mouse reproduction, the litter size and thematernity of homozygote LRF knockout female mouse （LRF-/-） reduced when mated withwild type （LRF+/+） and heterozygote （LRF+/-） male mouse, but sterility arised when thehomozygote LRF knockout female mouse （LRF-/-） mated with homozygote LRF knockoutmale mouse （LRF-/-）. The patterns of prolactin （PRL）, glucocorticosteroid, ocytocin in thepregnant female mice were remarkably changed. These results suggested that LRF might playimportant roles in the mouse reproduction, but the mechanism and the accurate function ofLRF in reproduction were unknown. The expression patterns of LRF in the mouse werechecked by Real time PCR, immunohistochemistry and Western blot during different time andgerminal tissue, including ovary, oviduct and uterus during estrous cycle; Ovarian follicle andpreimplantation embryo; Peri-implantation mouse uteri, the uteri of pseudopregnant mouse,the uteri of delayed implantation and activiated implantation, artificial induced decidual uteri,the uteri administrated with steroid hormone. The biological functions of LRF were revealedthrough these studies. In addition, the biological function of LRF in the ERS during ovariangranulosa cell apoptosis was studied.1. Constructed GST-LRF-N terminal recombination vector and chosen the optimalexpression conditions, the GST-LRF-N fusion protein was purified by affinity chromatographand LRF antiserum was gained from the mouse and rabbit immuned with the GST-LRF-Nfusion protein. The LRF antiserum was identified by Western blot and the results suggestedthat the antiserum might be used for the next studies.2. The expression patterns of LRF in the ovary, oviduct and uterus during estrous cyclewere checked by Real time PCR, immunohistochemistry and Western blot. The expression ofLRF remarkably changed at different stages during estrous cycle, LRF was lower atpreoestrus and estrous than others, but higher at dioestrus. LRF protein mainly localized in theampulla and isthmus of oviduct, in the glandular epithelium and luminal epithelium of uterusat different stages. The results suggested that LRF might play important roles in the female mouse reproduction and the biological functions of LRF might be regulated by steroidhormone.3. The expression patterns of LRF in the preimplantation embryo were checked by Realtime PCR and immunofluorescence. The LRF mRNA and protein were all checked in zygote,2-cell,4-cell,8-cell, morula and blastula. The LRF mRNA and protein were remarkablyincreased in the4-cell and blastula, LRF protein were detected in the trophectoderm （TE） andinner cell mass （ICM）. The results suggested that LRF might involve in embryonicdevelopment and differentiation, as well as embryo implantation.The localization of LRF protein in the ovarian follicle were checked, the LRF proteinexisted in primordial follicle, primary follicle, secondary follicle, tertiary follicle and vesiculagraafianae, LRF protein mainly localized in the oocyte and ovarian granulosa cell. The resultssuggested that LRF might involve in the development, growth, mature and ovulation ofoocyte. In addition, the LRF might play important role in proliferation and differentiation ofgranulosa cell.4. The expression of LRF in the peri-implantation pregnant mouse uterus,pseudopregnant mouse uterus, delayed implantation uterus and activated uterus, artificalinduced decidual uterus were checked by Real time PCR, immunohistochemistry and relatedmouse model.Compared with pregnant uterus during days1-4, LRF mRNA remarkably increased inthe uterus on day5when embryo implantation happened, LRF mainly localized in theimplantation site, stromal cell and glandular cell. The result suggested that LRF might involvein embryo implantation. The expression patterns of pseudopregnant uteri and implantationactivated uterus suggested that increased expression of LRF and the receptivity of uterus onday5was activated by the embryo.LRF mRNA remarkably increased in the uterus at day6,7when decidualizationhappened, the LRF protein localized in the primary decidual zone （PDZ） on day6and in thesecondary decidual zone （SDZ） on day7. LRF mRNA significantly higher in artifical induceddecidual uterus than control, little protein detected in the luminal epithelial cell in control, butmass of decidual cell were detected and stained positive in the artifical induced decidualuterus. The results suggested that LRF might involve in decidualization.Compared with the control, the LRF mRNA was changed in the uteri administrated withE₂, P₄, E₂+P₄. Little protein detected in the luminal epithelial cell in the control, but LRFprotein were detected in the luminal epithelial cell, glandular epithelial cell of uteri treatedonly with E₂, in the luminal epithelial cell, glandular epithelial cell and stroma-cell of uteritreated with P₄, E₂+P₄.The results suggested that the function of LRF regulated by E₂and P₄. 5. Mouse granulosa cell were cultured in vitro and induced by thapsigargin （Tg） andtunicamycin （Tm） for ERS model, the biological functions of LRF was studied by Real timePCR, immunohistochemistry and flow cytometry. The LRF protein was checked in theapoptotic ovarian granulosa cell, the LRF protein and pro-apoptotic CHOP, Caspase-12wereco-localized in the apoptotic ovarian granulosa cell. ERS related genes were remarkablychanged companied with the increased apoptotic ovarian granulosa cell. The results suggestedthat LRF might involve in the mouse granulosa cell apoptosis.