Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis

Yan Yin,Liren Liu,Chenyi Yang,Congxing Lin,George Michael Veith,Caihong Wang,Peter Sutovsky,Pengbo Zhou,Liang Ma

doi:10.1074/jbc.m115.699660

Abstract

CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation ofCul4ain mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4bgerm cell-specific conditional knock-out (Cul4b(Vasa)),as well asCul4bglobal knock-out (Cul4b(Sox2)) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4bled to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore,Cul4b(Vasa)spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless,Cul4bglobal knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis.

Highlights

Protein degradation via the ubiquitin-proteasome system plays a critical role during mammalian spermatogenesis
We have previously reported that the CRL4 proteins exhibited complementary expression patterns in adult mouse testis, where CUL4A was predominantly detected in primary spermatocytes, whereas CUL4B was highly expressed in Sertoli cells, spermatogonia, and spermatids [5]
We assessed the function of the E3 ubiquitin ligase CUL4B in spermatogenesis using genetically modified mouse models

Summary

Experimental Procedures

Mice—Generation of conditional Cul4b mice was described previously [7]. The Vasa(Ddx4)-Cre (stock no. 006954), AmhCre (stock no. 007915), and Sox2-Cre (stock no. 008454) transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Flow Cytometric Analysis—Single testicular cell suspensions and epididymal spermatozoa were prepared as previously described [5]. Cell pellets were washed three times in PBS and resuspended in 0.2– 0.5 ml immunoprecipitation assay buffer After removing supernatant containing unbound proteins, affinity-purified complexes were pelleted, washed in PBS three times, and submitted to the Proteomics and Mass Spectrometry Program core facility at Washington University School of Medicine for mass spectrometry analysis (detailed method available upon request). Trypsinized cells were pelleted and washed in PBS, and 1/20 of the cell aliquots were boiled in 2ϫ SDS-PAGE buffer for Western blot analysis. Technology), and 1:4000 for anti-V5 (Abcam) 1:500 for ␤-actin (Santa Cruz)

Results

Average litter size

Discussion