Expression Of LRF In Mice Testis And The Biological Functions In Testosterone Production

Luman recruiting factor?LRF?was also called CREB3 regulate factor?CREBRF?or basic leucine zipper protein transcription factor,which was detected by the Yeast two-hybrid screening and isolation from the human brain cDNA Library by Lu Ray's laboratory in 2008.LRF was expressed in multiple adult mouse tissues.Previous studies have found that abnormally high levels of GR and low concentration of PRL in LRF^-/-knockout mice,eventrally lead to maternal behavioral defect.Our laboratory previous study had found that the expression of LRF protein significantly increased in granulosa cells of atresia follicles in mice,revealing that LRF may play an pro-apoptotic effect on granulosa cells through endoplasmic reticulum stress?ERS?-mediated apoptotic pathways.Due to the existing research results,we suspect that LRF may have important physiological functions in the reproduction of male animals.In the present study,immunohistochemistry,qRT-PCR,immunofluorescence,and siRNA interference were used to examine the expression of LRF in mouse testis during sexual maturation and epididymis at sexual maturation.We analysis that the LRF had an effect on testosterone production and steroid enzymes in mouse leydig cells so that it laid the foundation for the function of LRF in the reproductive system of male mice.The main results are the following:?1?Testis tissue were collected in mouse from the postnatal to sexually mature and sexually mature epididymis.LRF staining was specific localized to the head and tail of sperm at sexually mature epididymis.After day 21,LRF immunohistochemistry staining results show that LRF was a positive staining in interstitial leydig cells in testis,and LRF mRNA related to days after brith by qRT-PCR.The expression of LRF protein was specifically located at the nucleus and cytoplasm of mouse leydig cells,while it is mainly expressed in the cytoplasm.Above all,the results suggest that LRF play an essential role in testosterone production and sperm maturation.?2?LH/hCG is an essential hormore for the production of testosterone in mouse leydig cells.The concentrations of testosterone in serm were significantly increased with the stimulation of 5 IU hCG in male mice by intraperitoneal injection for 12 h and 24 h.LRF m RNA was expressed with a significant decresed trend with treatment by 5 IU hCG for 12 h,while there is also the same trend with 5 IU hCG for 12 h and 24 h in vivo.?3?In vitro,the primary mouser leydig cells were isolated and cultured with purtuiy of over 95%.LRF mRNA was inhibited by hCG with a dose-dependent and time dependent-manner,and which negatively regulated to the producton of testosterone in the supernatant and 3?-HSD mRNA.?4?The overexpression vector pcDNA3.1?+?-LRF and the effective interference siLRF were successfully aquired.The results showed that down-regulation of LRF by si-LRF could markedly suppress the mRNA expression of StAR,P450c17,17?-HSD,3?-HSD and StAR with treatment of 1 IU hCG in primary mouse leydig cells.Conversely,up-regulation of LRF by transfection of pcDNA3.1?+?-LRF can significantly increase StAR mRNA expression.In addition,there is no significant difference in the proliferation after tranfection of si-LRF and pcDNA3.1?+?-LRF and treated by hCG in mouse leydig cells.Taken together,this study suggest that LRF is specifically expressed in mouse testicular leydig cells and may have a relevation with the production of testosterone through steroid synthase expression.