Effect Of LncRNA Hand2os1 During Embryo Implantation In Mice

Embryo implantation play a crucial role in pregnancy,which is an ideal link for reproductive regulation.Although artificial insemination and embryo transfer technology have been widely used in the animal husbandry production practice,the rate of pregnancy is still below expectancy.The failure of endometrial receptivity is an important reason,and it has become a major problem in the application of assisted reproductive technology.Endometrial receptivity and embryo-maternal interaction are the determinants of successful implantation.In addition to the dominance of steroid hormones,the endometrium during the implantation window has specific gene cluster expression and cross-linking of a series of signal transduction pathways,which in turn promotes the implantation of embryos and the subsequent decidualization process,but the exact molecular mechanism is still unclear.The role of long non-coding RNA（Lnc RNA）in reproduction has been found clearly in recent years,such as spermatogenesis,follicular development,ovulation and placental formation.In the previous study,we conducted a RNA-sequencing analysis to compare gene expressions between decidual of days six and eight,and normal pregnant endometrium（day four）.And the result showed that the expression of Lnc RNA Hand2os1/Uph was significantly increased in the decidual tissue on the 6th and 8th day of pregnancy.Hand2os1is a lncRNA which is located in the antisense strand of Hand2（heart and neural crest derivatives-expressed transcript 2）,and Hand2 plays a key role in embryo implantation as an important transcription factor downstream of P₄.Therefore,on the basis of above research,our study using RNAscope and RT-q PCR technology explores the expression,regulation and biological functions of Lnc RNA Hand2os1 during mouse endometrium decidualization by constructing early pregnancy,pseudopregnancy and artificial induced decidualization models;using the model of hormone treatment in vitro to clarify the regulatory effects of E₂and P₄ on Hand2os1;using technology of overexpression and si RNA to analyze the effect of Hand2os1on decidualization in vitro;establishing the model of PMSG/h CG-induced corpus luteum Formation and degeneration,normal pregnancy,estrus and cyclic luteal to analysis the Hand2os1 expression in the process of mouse corpus luteum formation and degradation.The results clarifying the biological function of Hand2os1 in the process of decidualization and revealing the regulation of Hand2os1 in mouse.Besides,these results laid the foundation for revealing the molecular mechanism of Hand2os1 in regulating embryo implantation of mouse.The main findings obtained are as follows:（1）Hand2os1 was specifically expressed in endometrial stromal cells on the 2nd to 5th day of pregnancy in mouse,but no positive signal was detected in the luminal epithelium or glandular epithelial cells;the expression level of Hand2os1 significantly increased alongside the process of decidualization,and Hand2os1 was mainly located in the nucleus of decidual cells on the 6th to 8th day of pregnancy;（2）The results of artificial induced decidualization and pseudopregnancy models showed that Hand2os1 was highly expressed in decidualized cells,and the expression of Hand2os1 in stromal cells and decidualized cells was not dependent on the presence of embryos;（3）Hormone treatment model and experiments in vitro confirmed that P₄ can significantly promote the expression level of Hand2os1 in stromal cells,and the effect was most significant at three hour after subcutaneous injection of P₄,and the up-regulation can be impeded by RU486;E₂ appeared to have no regulating effect on Hand2os1 expression and has no antagonistic effect on the regulation of Hand2os1 by P₄;（4）Hand2os1 interference or overexpression dramatically increased decidualization markers Prl8c2 and Prl3c1 in vitro,but no regulating effect was found on Hand2 expression;（5）In the model of luteal formation and degeneration induced by PMSG/h CG of 21day mice,there has no positive signal of Hand2os1 was detected;in the ovaries of the estrous cycle,Hand2os1 was specifically localized in proestrum and estrous luteal cells,and the expression level of Hand2os1 in the estrus was higher than proestrum;in luteal cells of pregnancy,Hand2os1 was significantly expressed on the 4th and 18th of pregrance;the expression level of Hand2os1 was significantly reduced as the corpus luteum degenerates after delivery.In summary,Hand2os1 is specifically localizes in mouse endometrial stromal cells and decidualized cells.Hand2os1 plays an important role in the decidualization process by regulating the expression of decidualization marker genes Prl8c2 and Prl3c1;the expression of Hand2os1 is mainly affected by P₄,and the regulation showed PR pathway dependence.In addition,Hand2os1 is expressed in the corpus luteum of estrus and pregnancy,indicating that Hand2os1 plays an important role in the establishment and maintenance of mouse pregnancy.