Effects Of DNALI1 On Spermatogenesis And Fertility In Male Mice

Objective:To establish the DNALI1 conditional knock out mouse model and investigate the role of DNALI1 in the spermatogenesis and reproductive ability in the male mice.Methods:At in vitro experiments,Dnali1 and PACRG plasmids were co-transfected into HEK293T cells and CHO cells,then Gaussia Luciferase system was selected to detect the interaction between the DNALI1 and PACRG.The gel filtration test was also chosen to examine the DNALI1 and PACRG interaction in the bacterial and mammalians.The Cre/Loxp system was used to generate the conditional knockout Dnali1 mice model in germ cells,then these Dnali1 ^flox/flox;Stra8-i Cre⁺knockout（Dnali1 KO）male mice were used in the experimental group,with Dnali1 ^flox/+male mice as the control group.PCR technology was used to analyze the mouse genotypes to verify whether Dnali1 KO transgenic model was successfully constructed.Western blot was used to detect the expression of DNALI1 protein in the testis tissues from Dnali1 KO male mice and Dnali1 ^flox/+male mice.Male mice aged2-3 months from these two groups were mated with wild-type female mice to assess the fertility rate of the male mice.Sperm from the epididymis in these two groups were collected and performed the number count and the morphology analysis,then HE staining was used to identify the morphological changes during spermatogenesis in the testis and epididymis tissues.Immunofluorescence staining was used to detect the location of DNALI1 and PACRG in spermatogenic cells,in order to explore whether there is a direct interaction between DNALI1 and PACRG.Results:It was found that DNALI1 and PACRG strongly interact in cells.DNALI1can recruit and co-localize with PACRG and increase the expression of PACRG protein,and DNALI1 can stabilize PACRG in bacteria.We could confirmed Dnali1KO mice model have obtained according genotyping analysis,and furtherly found the expression of DNALI1 protein in the testis was significantly reduced in Dnali1 KO mice.Although the Dnali1 KO male mice maintained normal mating behavior,they had nearly infertility.Compared to control mice(Dnali1 ^flox/+),the sperm count and sperm motility in the epididymis of Dnali1 KO male mice were abundantly reduced,and the sperm showed abnormal morphology（P<0.05）.HE staining showed that the number of elongated sperms in the testis tissue of Dnali1 KO mice was significantly reduced,following that a small amount of them was released into the lumen.And there were many cytoplasmic bodies appeared in the epididymis with the state of cavities.Under the light microscope,abnormal heads were observed in the sperm of Dnali1 KO mice,and the sperm tails were uneven and broken.Immunofluorescence staining showed that DNALI1 and PACRG were co-localized on the manchette,while the localization of DNALI1was independent of expression of PACRG.Conclusions:The expression of DNALI1 plays a critical role in the spermatogenesis and affect reproductive ability in the male mice,which could be potentially through the interaction with the manchette.