DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis

doi:10.1007/s11684-021-0851-8

Front. Med.

2021, Vol. 15

Issue (2) : 302-312 https://doi.org/10.1007/s11684-021-0851-8

RESEARCH ARTICLE

DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis

Xiuli Zhang¹, Zhizhou Xia¹, Xingyu Lv², Donghe Li¹, Mingzhu Liu¹, Ruihong Zhang¹, Tong Ji³(

), Ping Liu¹(

), Ruibao Ren¹(

)

¹. Shanghai Institute of Hematology, State Key Laboratory for Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². Key Laboratory of Laparoscopic Technology of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
³. Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China

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Abstract

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.

Keywords Dcaf8 male infertility spermatogenesis

Corresponding Author(s): Tong Ji,Ping Liu,Ruibao Ren

Just Accepted Date: 11 March 2021 Online First Date: 12 April 2021 Issue Date: 23 April 2021

Cite this article:

Xiuli Zhang,Zhizhou Xia,Xingyu Lv, et al. DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis[J]. Front. Med., 2021, 15(2): 302-312.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0851-8
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I2/302

Tab.1 List of primers used in this study

Fig.1 Dcaf8 expression among tissues and Dcaf8 KO mice generation. (A) RT-PCR showing tissue expression profile of Dcaf8 mRNA in adult mouse. (B) Expression of DCAF8 in different tissues of mice were immunoblotted. (C) The strategy for disruption of the Dcaf8 gene of mouse. (D) Agarose gel image of three different Dcaf8 genotypes. The expected size of PCR products (arrowheads) is shown on the left of the images. The band of WT product was 538 bp, while that of Dcaf8 KO was 653 bp. (E and F) Western blot analysis showing the effect of Dcaf8 KO in testis and epididymis. GAPDH was used as a loading control for total cellular proteins.

Tab.2 Mating experiment of Dcaf8^+/+ and Dcaf8^−/− mice

Fig.2 Localization of DCAF8 in testis. (A and C) Immunofluorescence and immunohistochemistry assays revealing the localization of the DCAF8 protein in the adult mice testis. (B and D) Quantification of anti-DCAF8 staining density in (A) and (C). The mean fluorescence intensity of DCAF8 staining in (A) was calculated using ImageJ. In (C), fluorescent signaling abundance was calculated by staining intensity. Colorless was set as zero points, while light yellow as one point, brown and yellow for two points, and tan for three points. (E) Western blot of nuclear and cytoplasmic proteins further confirmed the localization of DCAF8. GAPDH and H3 served as the internal controls for cytoplasmic and nuclear proteins, respectively. (Two-tailed unpaired Student’s t-test was used to compare the quantification of DCAF8 staining in Fig. 1A and 1C. **** P<0.0001.)

Fig.3 Histology of the reproductive system, sperm count, and motilities of different genotypes. (A) Total body weight of WT and Dcaf8 KO adult mice. (B) Assessing the testis and epididymis sizes of mice by calculating the ratio of testis or epididymis weight to body weight (n = 7 for WT and Dcaf8 KO mice). (C and D) Sperm count analyses of WT and Dcaf8 KO mice. (E and F) Sperm motility analyses of WT and Dcaf8 KO mice. Five fields were observed for each replicate of sperm motility analysis. We used three pairs of mice to analyze sperm counts and four pairs of mice for motility. All the above studies were conducted with 10-week-old male mice. (NS, no significance; * P<0.05, ** P<0.01.)

Fig.4 Sperm morphology of Dcaf8 KO mice. (A) Scanning electron microscopy (SEM) images of sperm from 10-week-old WT and Dcaf8 KO mice observed under a microscope at a magnification of 1000×. (B) Statistical results of sperm head reflexes in three different visual fields of SEM. At least 100 sperms were included in each field. (C and D) SEM and transmission electron microscopy (TEM) micrographs of typical head reflexes of sperm in Dcaf8 KO mice. (E,G) Confocal images of sperm from 10-week-old WT and Dcaf8 KO mice co-stained with the acrosome marker PNA (green) and with DAPI (blue) under low- and high-power microscopy, respectively. (F) Statistical analysis of (E) to detect the number of PNA(−) cells in the testis of Dcaf8 KO mice compared with that in the WT ones. (NS, no significance; *** P<0.001.)

Fig.5 Inability of Dcaf8 KO sperm to fertilize eggs in vitro. (A–C) The eggs obtained from super-ovulation female mice were incubated with capacitated WT or Dcaf8 KO sperm in vitro. Dcaf8 KO sperm showed severely deficient oocyte binding. The IVF rate was dramatically lower in samples from Dcaf8 KO male mice than in those from WT mice. The average of total cells and division cells counted was shown in graphics. Graphics A, B, and C represent the ratio of total oocytes developed to a 2-cell, 4-cell, and 8-cell stage, respectively. T, total cells counted; D, division cells counted at different stages. (*P<0.05, ***P<0.001.)

1	FT Neto, PV Bach, BB Najari, PS Li, M Goldstein. Genetics of male infertility. Curr Urol Rep 2016; 17(10): 70 https://doi.org/10.1007/s11934-016-0627-x pmid: 27502429
2	PN Schlegel. Evaluation of male infertility. Minerva Ginecol 2009; 61(4): 261–283 pmid: 19745794
3	KL O’Flynn O’Brien, AC Varghese, A Agarwal. The genetic causes of male factor infertility: a review. Fertil Steril 2010; 93(1): 1–12 https://doi.org/10.1016/j.fertnstert.2009.10.045 pmid: 20103481
4	C Krausz. Male infertility: pathogenesis and clinical diagnosis. Best Pract Res Clin Endocrinol Metab 2011; 25(2): 271–285 https://doi.org/10.1016/j.beem.2010.08.006 pmid: 21397198
5	EM Eddy. Male germ cell gene expression. Recent Prog Horm Res 2002; 57(1): 103–128 https://doi.org/10.1210/rp.57.1.103 pmid: 12017539
6	DM de Kretser, KL Loveland, A Meinhardt, D Simorangkir, N Wreford. Spermatogenesis. Hum Reprod 1998; 13(Suppl 1): 1–8 https://doi.org/10.1093/humrep/13.suppl_1.1 pmid: 9663765
7	N Schultz, FK Hamra, DL Garbers. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl Acad Sci USA 2003; 100(21): 12201–12206 https://doi.org/10.1073/pnas.1635054100 pmid: 14526100
8	CC Hou, WX Yang. New insights to the ubiquitin-proteasome pathway (UPP) mechanism during spermatogenesis. Mol Biol Rep 2013; 40(4): 3213–3230 https://doi.org/10.1007/s11033-012-2397-y pmid: 23268313
9	WM Baarends, R van der Laan, JA Grootegoed. Specific aspects of the ubiquitin system in spermatogenesis. J Endocrinol Invest 2000; 23(9): 597–604 https://doi.org/10.1007/BF03343782 pmid: 11079455
10	D Kopanja, N Roy, T Stoyanova, RA Hess, S Bagchi, P Raychaudhuri. Cul4A is essential for spermatogenesis and male fertility. Dev Biol 2011; 352(2): 278–287 https://doi.org/10.1016/j.ydbio.2011.01.028 pmid: 21291880
11	T Ma, JA Keller, X Yu. RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis. Acta Biochim Biophys Sin (Shanghai) 2011; 43(5): 339–345 https://doi.org/10.1093/abbs/gmr016 pmid: 21444325
12	S Wang, H Zheng, Y Esaki, F Kelly, W Yan. Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis. Biol Reprod 2006; 74(1): 102–108 https://doi.org/10.1095/biolreprod.105.045484 pmid: 16162871
13	LY Lu, J Wu, L Ye, GB Gavrilina, TL Saunders, X Yu. RNF8-dependent histone modifications regulate nucleosome removal during spermatogenesis. Dev Cell 2010; 18(3): 371–384 https://doi.org/10.1016/j.devcel.2010.01.010 pmid: 20153262
14	MD Petroski, RJ Deshaies. Function and regulation of Cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 2005; 6(1): 9–20 https://doi.org/10.1038/nrm1547 pmid: 15688063
15	S Jackson, Y Xiong. CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem Sci 2009; 34(11): 562–570 https://doi.org/10.1016/j.tibs.2009.07.002 pmid: 19818632
16	BC O’Connell, JW Harper. Ubiquitin proteasome system (UPS): what can chromatin do for you? Curr Opin Cell Biol 2007; 19(2): 206–214 https://doi.org/10.1016/j.ceb.2007.02.014 pmid: 17314036
17	W Zhong, H Feng, FE Santiago, ET Kipreos. CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing. Nature 2003; 423(6942): 885–889 https://doi.org/10.1038/nature01747 pmid: 12815436
18	K Sugasawa. The CUL4 enigma: culling DNA repair factors. Mol Cell 2009; 34(4): 403–404 https://doi.org/10.1016/j.molcel.2009.05.009 pmid: 19481520
19	J Cheng, J Guo, BJ North, K Tao, P Zhou, W Wei. The emerging role for Cullin 4 family of E3 ligases in tumorigenesis. Biochim Biophys Acta Rev Cancer 2019; 1871(1): 138–159 https://doi.org/10.1016/j.bbcan.2018.11.007 pmid: 30602127
20	H Wang, L Zhai, J Xu, HY Joo, S Jackson, H Erdjument-Bromage, P Tempst, Y Xiong, Y Zhang. Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage. Mol Cell 2006; 22(3): 383–394 https://doi.org/10.1016/j.molcel.2006.03.035 pmid: 16678110
21	J Lee, P Zhou. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase. Mol Cell 2007; 26(6): 775–780 https://doi.org/10.1016/j.molcel.2007.06.001 pmid: 17588513
22	S Angers, T Li, X Yi, MJ MacCoss, RT Moon, N Zheng. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 2006; 443(7111): 590–593 https://doi.org/10.1038/nature05175 pmid: 16964240
23	Y Yin, L Liu, C Yang, C Lin, GM Veith, C Wang, P Sutovsky, P Zhou, L Ma. Cell autonomous and nonautonomous function of CUL4B in mouse spermatogenesis. J Biol Chem 2016; 291(13): 6923–6935 https://doi.org/10.1074/jbc.M115.699660 pmid: 26846852
24	CY Lin, CY Chen, CH Yu, IS Yu, SR Lin, JT Wu, YH Lin, PL Kuo, JC Wu, SW Lin. Human X-linked intellectual disability factor CUL4B is required for post-meiotic sperm development and male fertility. Sci Rep 2016; 6(1): 20227 https://doi.org/10.1038/srep20227 pmid: 26832838
25	Y Yin, C Lin, ST Kim, I Roig, H Chen, L Liu, GM Veith, RU Jin, S Keeney, M Jasin, K Moley, P Zhou, L Ma. The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis. Dev Biol 2011; 356(1): 51–62 https://doi.org/10.1016/j.ydbio.2011.05.661 pmid: 21624359
26	A Ali, BV Mistry, HA Ahmed, R Abdulla, HA Amer, A Prince, AM Alazami, FS Alkuraya, A Assiri. Deletion of DDB1- and CUL4-associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice. Sci Rep 2018; 8(1): 9202 https://doi.org/10.1038/s41598-018-27379-0 pmid: 29907856
27	Y Wu, L Zhou, X Wang, J Lu, R Zhang, X Liang, L Wang, W Deng, YX Zeng, H Huang, T Kang. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A. Cell Discov 2016; 2(1): 16014 https://doi.org/10.1038/celldisc.2016.14 pmid: 27462461
28	M Nowak, B Suenkel, P Porras, R Migotti, F Schmidt, M Kny, X Zhu, EE Wanker, G Dittmar, J Fielitz, T Sommer. DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy. J Cell Sci 2019; 132(17): jcs233395 https://doi.org/10.1242/jcs.233395 pmid: 31391242
29	G Li, T Ji, J Chen, Y Fu, L Hou, Y Feng, T Zhang, T Song, J Zhao, Y Endo, H Lin, X Cai, Y Cang. CRL4DCAF8 ubiquitin ligase targets histone H3K79 and promotes H3K9 methylation in the liver. Cell Rep 2017; 18(6): 1499–1511 https://doi.org/10.1016/j.celrep.2017.01.039 pmid: 28178526
30	D Huang, C Liu, X Sun, X Sun, Y Qu, Y Tang, G Li, T Tong. CRL4DCAF8 and USP11 oppositely regulate the stability of myeloid leukemia factors (MLFs). Biochem Biophys Res Commun 2020; 529(2): 127–132 https://doi.org/10.1016/j.bbrc.2020.05.186 pmid: 32703400
31	CJ Klein, Y Wu, P Vogel, HH Goebel, C Bönnemann, K Zukosky, MV Botuyan, X Duan, S Middha, EJ Atkinson, G Mer, PJ Dyck. Ubiquitin ligase defect by DCAF8 mutation causes HMSN2 with giant axons. Neurology 2014; 82(10): 873–878 https://doi.org/10.1212/WNL.0000000000000206 pmid: 24500646
32	LT Gou, P Dai, JH Yang, Y Xue, YP Hu, Y Zhou, JY Kang, X Wang, H Li, MM Hua, S Zhao, SD Hu, LG Wu, HJ Shi, Y Li, XD Fu, LH Qu, ED Wang, MF Liu. Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis. Cell Res 2015; 25(2): 266 https://doi.org/10.1038/cr.2015.14 pmid: 25645811
33	LT Gou, JY Kang, P Dai, X Wang, F Li, S Zhao, M Zhang, MM Hua, Y Lu, Y Zhu, Z Li, H Chen, LG Wu, D Li, XD Fu, J Li, HJ Shi, MF Liu. Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis. Cell 2017; 169(6):1090–1104.e13 https://doi.org/10.1016/j.cell.2017.04.034 pmid: 28552346
34	KJ Livak, TD Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC(T)) method. Methods 2001; 25(4): 402–408 https://doi.org/10.1006/meth.2001.1262 pmid: 11846609
35	D Shechter, HL Dormann, CD Allis, SB Hake. Extraction, purification and analysis of histones. Nat Protoc 2007; 2(6): 1445–1457 https://doi.org/10.1038/nprot.2007.202 pmid: 17545981
36	P Quinn, JF Kerin, GM Warnes. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44(4): 493–498 https://doi.org/10.1016/S0015-0282(16)48918-1 pmid: 3902512
37	Q Chen, H Peng, L Lei, Y Zhang, H Kuang, Y Cao, QX Shi, T Ma, E Duan. Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration. Cell Res 2011; 21(6): 922–933 https://doi.org/10.1038/cr.2010.169 pmid: 21135872
38	MM Matzuk, DJ Lamb. Genetic dissection of mammalian fertility pathways. Nat Cell Biol 2002; 4(Suppl): s41–s49 https://doi.org/10.1038/ncb-nm-fertilityS41 pmid: 12479614
39	D Jamsai, MK O’Bryan. Mouse models in male fertility research. Asian J Androl 2011; 13(1): 139–151 https://doi.org/10.1038/aja.2010.101 pmid: 21057516
40	P Sutovsky. Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 2003; 61(1): 88–102 https://doi.org/10.1002/jemt.10319 pmid: 12672125
41	M Kanatsu-Shinohara, I Onoyama, KI Nakayama, T Shinohara. Skp1-Cullin-F-box (SCF)-type ubiquitin ligase FBXW7 negatively regulates spermatogonial stem cell self-renewal. Proc Natl Acad Sci USA 2014; 111(24): 8826–8831 https://doi.org/10.1073/pnas.1401837111 pmid: 24879440
42	X Hou, W Zhang, Z Xiao, H Gan, X Lin, S Liao, C Han. Mining and characterization of ubiquitin E3 ligases expressed in the mouse testis. BMC Genomics 2012; 13(1): 495 https://doi.org/10.1186/1471-2164-13-495 pmid: 22992278
43	C Yu, YL Zhang, WW Pan, XM Li, ZW Wang, ZJ Ge, JJ Zhou, Y Cang, C Tong, QY Sun, HY Fan. CRL4 complex regulates mammalian oocyte survival and reprogramming by activation of TET proteins. Science 2013; 342(6165): 1518–1521 https://doi.org/10.1126/science.1244587 pmid: 24357321
44	C Yu, YW Xu, QQ Sha, HY Fan. CRL4DCAF1 is required in activated oocytes for follicle maintenance and ovulation. Mol Hum Reprod 2015; 21(2): 195–205 https://doi.org/10.1093/molehr/gau103 pmid: 25371539
45	C Yu, SY Ji, QQ Sha, QY Sun, HY Fan. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation. Nat Commun 2015; 6(1): 8017 https://doi.org/10.1038/ncomms9017 pmid: 26281983
46	SS Suarez, AA Pacey. Sperm transport in the female reproductive tract. Hum Reprod Update 2006; 12(1): 23–37 https://doi.org/10.1093/humupd/dmi047 pmid: 16272225
47	KA Sutton, MK Jungnickel, HM Florman. A polycystin-1 controls postcopulatory reproductive selection in mice. Proc Natl Acad Sci USA 2008; 105(25): 8661–8666 https://doi.org/10.1073/pnas.0800603105 pmid: 18562295
48	J Zhang, YL Zhang, LW Zhao, JX Guo, JL Yu, SY Ji, LR Cao, SY Zhang, L Shen, XH Ou, HY Fan. Mammalian nucleolar protein DCAF13 is essential for ovarian follicle maintenance and oocyte growth by mediating rRNA processing. Cell Death Differ 2019; 26(7): 1251–1266 https://doi.org/10.1038/s41418-018-0203-7 pmid: 30283081

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