Establishment Of A Transgenic Mouse With Testicular Leydig Cell Overexpression Of Prl3c1

Objective:Prolactin family 3,subfamily c,member 1(Prl3c1)is a memeber of Prolactin(PRL)family.Prl3c1 is mainly expressed in decidual cells during pregnancy and plays an important role in the maintenance of pregnancy.Our previous study revealed that Prl3c1 is expressed in Leydig cells of the testis.However,its roles in testicular function need to be further investigated.In the present study,we aimed to generate Prl3c1 overexpressing mice(C57BL/6)and investigate its function during male reproduction.Methods:1.The transgenic construct(Insl3-Flag-Prl3c1-Insl3PA)was generated by in-fusion PCR.2.The transgenic constructs were microinjected into the pronuclei of fertilized oocytes from C57BL/6 mice.The founder(F0)transgenic mice were screened by PCR.3.Western blotting and immunofluorescence assay were performed to investigate the expression of transgenic construct in testis.4.HE staining was used to observe the morphological changes of testis in Prl3c1 transgenic mice.5.Serum testosterone levels of the wildtype and Prl3c1 overexpressing mice were measured using enzyme-linked immunosorbent assay(6 animals in each group).Results:Three males that were positive for the mouse Prl3c1 transgene were selected for further breeding.Western blot and immunofluorescence staining showed that the Prl3c1 transgenic construct was expressed in Leydig cells.The testes of Prl3c1 overexpressing mice appeared morphologically normal.No difference in serum testosterone levels was observed between Prl3c1 overexpressing mice and wildtype mice [(2.61 ± 0.34)ng/mL vs(2.51 ± 0.35)ng/mL,P>0.05].However,HCG stimulated testosterone production was lowered in Prl3c1 overexpressing mice compared with wildtype mice [(5.35 ± 0.83)ng/mL vs(7.56 ± 1.17)ng/mL,P<0.01].Further,Prl3c1 overexpression caused a decrease in HCG-stimulated STAR expression(P<0.01).Conclusion:We have successfully established a Prl3c1 overexpressing mice,which will be beneficial to further explore the role of Prl3c1 in male reproductive function.Additionally,Prl3c1 may be involved in testosterone production by regulating the expression of STAR.