Objective: The mutation of cytosolic carboxypeptidase (CCP1) causes male infertility. However, the underlying mechanism has not been elucidated yet.
Methods: The interacting proteins of CCP1 in mouse testis were identified by Co-IP and LC-MS/MS. The expression of specific interacting proteins in mouse testis was examined by real-time PCR, western blot and immunohistochemical analyses. The regulation between CCP1 and TUBB4B was explored by real-time PCR and western blot. shRNA-mediated gene knockdown in GC1 and GC2 cells was used to analyze the function of CCP1. RNA-seq and proteomic analyses were applied to identify the pathways involved and the results were confirmed by western blot method.
Results: The mutation or knockdown of CCP1 can lead to down-regulation of TUBB4B. Co-IP assay and immunofluorescence detection showed that CCP1 interacted with TUBB4B and co-localized in the cytoplasm. Both were expressed in the middle piece of the mouse sperm tail. The loss of CCP1 function inhibited the proliferation of GC1 spermatogonia cells, and the expressions of p-ERK1/2 and p-p65 were reduced. After inhibition of CCP1 expression, GC1 cells were arrested in G1/S phase, while GC-2 cells were arrested in G2/M phase, and the expression levels of cyclins CCND1 and CCNB1 were also decreased.
Conclusion: Our study reveals downregulation of CCP1 can regulate the cell proliferation of GC-1 and GC-2 through the MAPK/ERK-NF-κB pathway, provides the potential cellular and molecular mechanisms for CCP1 function in spermatogenesis and may provide fundamental clues for future treatment of male sterility.
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