The Expression And Location Of A Novel Protein Prolactin-like Protein I In Mouse Testis

Objective: Prolactin-like protein I(PLP-I, also named PLP-J, Prlpj,Prlpi, prl3c1) is a novel prolactin-like gene in rodents. In our previousstudy, we found that PLP-I signal was present in mouse testis. In this study,we aimed to investigate the expression pattern of PLP-I in mouse testis andprepare the anti-PLP-I polyclonal antibody for further study on the roles ofPLP-I in testis.Methods: In situ hybridization, immunohistochemistry andImmunofluorescence method were employed to identify the expressionsignal of PLP-I in mouse testis.The expression pattern of PLP-I in differentage mouse was detected by Real-time PCR and WB.To prepare the anti-PLP-I polyclonal antibody, the purified antigenwas used to immunize rabbit. The specificity and titer of antibody wereidentified via ELISA and western blotting.To investigate the role of PLP-I in TM3Leydig cells, PLP-I wascloned into the lentivirus expression vector and transfected into293T cells.After packaging in293T cells, the lentivirus were transfected into TM3cells. The PLP-J-positive TM3cells were screened by resistance to puromycin. The cell cycle and cell apoptosis were analyzed by FACS.Results:1. The results of immunocytochemical and in situhybridization indicated that PLP-I is located at the mouse testis Leydig cells.2. RT-PCR and western blotting results showed that PLP-I mRNA andprotein level is low in8-day-old mouse, reaching the peak in adult mouse.3. The anti-PLP-I polyclonal antibody was proved to recognize PLP-Iprotein specifically, and its titers reached1:102400.4. Lentivirus-mediatedup-expression of PLP-I in TM3Leydig cells showed no alteration in cellcycle, which indicated that PLP-I have no roles in TM3cell proliferation.Conclusions: We report for the first time that PLP-I is expressed in theLeydig cells of male mouse testis. In addition, The anti-PLP-I polyclonalantibody have been successfully prepared.