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DNA Methylation and Alternative Splicing Modulate FBXW11 Gene Expression in Holstein Bull Testis and Are Correlated with Sperm Quality

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DNA Methylation and Alternative Splicing Modulate FBXW11 Gene Expression in Holstein Bull Testis and Are Correlated with Sperm Quality BIOCELL Tech Science Press 2021 45(1): 79-87 DNA methylation and alternative splicing modulate FBXW11 gene expression in Holstein bull testis and are correlated with sperm quality YONG LIU1,2,#;ZHIHUA JU1,#;QIANG JIANG1;WENHAO LIU1;CHUNHONG YANG1;YARAN ZHANG1;XIUGE WANG1;YAPING GAO1; XIAOCHAO WEI1;YAN SUN1;JINPENG WANG1;MINGHAI HOU1;LING YANG2;JINMING HUANG1,* 1 Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan, 250100, China 2 College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, 056021, China Key words: FBXW11, Alternative splicing, DNA methylation, Bull, Sperm quality traits Abstract: F-box and WD-40 domain protein 11 (FBXW11) is an important component of the E3 ubiquitin-ligase enzyme that plays a key role in the ubiquitin-dependent regulation of spermatogenesis. In our previous research, the mRNA expression of FBXW11 in bull sperm with high motility is significantly higher than that with low motility. In the present study, the protein expression levels of FBXW11 in bull testicular tissues with low-performance sperm quality groups were significantly higher than those in normal performance groups. The immunohistochemistry result demonstrated that FBXW11 protein was located in the periphery of Leydig cells and seminiferous tubules. Three splice variants of the FBXW11 gene, namely, FBXW11-tv1, FBXW11-tv2, and FBXW11-tv3, were identified in testicular tissues. The splicing patterns of the three variants are exon skipping. The transcript FBXW11-tv2 expressions were the highest in each sample. The low-performance groups displayed higher FBXW11-tv1 and FBXW11-tv2 transcript expressions than the normal performance groups. Two CpG islands were located within the 5’ UTR and exon 1-2 region of the FBXW11 gene. Bisulfite sequencing PCR results demonstrated that the methylation levels of 11 methylation sites in the CpG island 2 from −99 to −43 in the normal performance groups were significantly lower than those in the low-performance groups. Pearson correlation analysis suggested that the CpG island 2 methylation level was negatively correlated with sperm motility and the transcript FBXW11-tv2 expression level. Our data revealed that alternative splicing and DNA methylation jointly regulated FBXW11 gene expression and were correlated with sperm quality traits during spermatogenesis in Holsteins. Introduction ubiquitination, that is, almost fourfold higher than the others (Rajapurohitam et al., 2002). Spermatogenesis is an extremely complex and precisely The ubiquitin-proteasome system is essential in regulated process. Each stage of the spermatogenesis process spermatogenesis and mainly consists of E1 ubiquitin- is accompanied by complex regulatory mechanisms, activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 including hormone regulation and protein ubiquitination ubiquitin-ligase enzyme. The selectivity and specificity of E3 (Baarends et al., 2000; Sofikitis et al., 2008). The ubiquitin ligase enzyme for substrate proteins are important ubiquitination of proteins occurs in numerous processes in maintaining the stability of spermatogenesis (Sutovsky, needed for the progression of spermatozoa (Manku et al., 2003). E3 ubiquitin ligase enzyme is mainly composed of 2012). Many proteins are degraded by ubiquitination during five proteins, namely, Skp1, Ring-finger, Cullin, Rbxl, E3 spermatogenesis (Liu et al., 2005). The systemic tissue ubiquitin ligase enzyme is mainly composed of five proteins, reports in rats revealed that the testis had the highest rate of namely, Skp1, Ring-finger, Cullin, Rbxl, and F-box proteins (Zheng et al., 2002). F-box and WD-40 domain protein 11 (FBXW11) (also called β-TrCP2) is a member of the F-box protein family and a key component of the E3 ubiquitin *Address correspondence to: Jinming Huang, [email protected] ligase enzyme. FBXW11 can recognize and ubiquitinate #These authors contributed equally to this study specific phosphorylated substrates and regulate the NF-κB Received: 12 August 2020; Accepted: 25 September 2020 signaling pathway, the Wnt signaling pathway, and cell cycle Doi: 10.32604/biocell.2021.013583 www.techscience.com/journal/biocell This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 80 YONG LIU et al. and invasion, thereby affecting cell growth, differentiation, Animals published by the Ministry of Science and apoptosis, and tumor occurrence (Nakayama et al., 2003). Technology, China in 2004 and approved by the Animal FBXW11 is essential for spermatogenesis in mice, and FBXW11 Care and Use Committee from the Dairy Cattle Research gene conditional knockdown disrupts testicular organization Center, Shandong Academy of Agricultural Sciences, (Kanarek et al., 2010; Morohoshi et al., 2019). In our previous Shandong, China. research, we identified differentially expressed genes in semen from bulls with high and low sperm motility by using RNA-seq; Animal and tissue samples FBXW11 is one of the differentially expressed genes (Wang et Testicular tissue samples were collected from eight healthy al., 2019). The expression level of the FBXW11 gene in sperm adult Chinese Holstein bulls (3–4 years old) at a commercial with high sperm motility is significantly higher than that with slaughterhouse in Jinan, Shandong Province, China. Based low sperm motility, thereby indicating the FBXW11 gene has a on the sperm motility traits of these bulls, the samples were potential effect on bull sperm motility. classified into normal sperm motility (H1, H2, H3, and H4) Alternative splicing is a vital mechanism in regulating gene and low sperm motility group (L1, L2, L3, and L4) (Tab. 1). expression in eukaryotes (Kornblihtt et al., 2013). Approximately The fresh sperm motility assays were performed by the 74% of multiexon genes are alternatively spliced (Johnson et al., Computer-Assisted Semen Analysis (CASA) system. Various 2003). Alternative splicing is predominant in the testis and plays sperm motility parameters were analyzed, including average an important role in spermatogenesis (Elliott and Grellscheid, path velocity (VAP), straight-line velocity (VSL), and 2006). Few studies have examined alternative splicing in curvilinear velocity (VCL). Assessment of sperm motility bovine testes and sperm and reported that such splicing of was performed using criteria: rapid progressive along a reproduction-related genes is one of the influencing factors of linear track. Small pieces of testicular tissues were taken and sperm motility during bull spermatogenesis (Guo et al., 2014; soaked with a 4% paraformaldehyde fixing solution for IHC Zhang et al., 2014). DNA methylation is also an important analysis. The remaining testicular tissues were immediately mechanism for controlling gene expression. DNA methylation frozen in liquid nitrogen. is involved in spermatogenesis (Kropp et al., 2017). Abnormal DNA methylation in genes is associated with human defective Hematoxylin–eosin staining, Western blotting analysis, and sperm quality (Trasler, 2009). immunohistochemistry The bovine FBXW11 gene is a multiexongenethatconsists The hematoxylin–eosin staining (HE-staining) of testicular of 14 exons and 13 introns. We assumed that the expression of the tissues was performed to observe the intact structures and bovine FBXW11 gene may be regulated by alternative splicing and physiological states of the tissues. Western blotting and DNA methylation. Therefore, we analyzed the expression and immunohistochemistry techniques were applied to examine localization of the FBXW11 protein in bull testicular tissues by the expression and localization of the FBXW11 protein in Western blotting (WB) analysis and immunohistochemistry testicular tissues. The total protein of the testicular tissues (IHC). We also identified the splice variants and determined the was extracted using the RIPA lysis buffer. The testicular core promoter region and CpG islands methylation pattern of tissues and samples were embedded in paraffin and the FBXW11 gene in testicular tissues of Holstein bulls with sectioned for HE-staining and IHC. FBXW11 antibodies normal and low-performance sperm quality. were purchased from Proteintech Group. The immunogen of the antibody sequence and the bovine FBXW11 reference Materials and Methods amino acid sequence was aligned to analyze the applicability of the antibody. WB and IHC procedures were performed Ethics statement according to a previously described protocol (Guo et al., This study was carried out according to the Regulations for 2014). Alpha Ease FC 4.0 software was used to quantify the the Administration of Affairs Concerning Experimental band density in WB analysis. Relative protein levels were TABLE 1 Sperm quality traits of eight healthy adult Chinese Holstein bull samples (normal performance [H1, H2, H3, and H4] and low-performance [L1, L2, L3, and L4] groups) Sample Number Ejaculate volume (mL) Sperm density (1 × 108/mL) Sperm motility (%) H1 4.43 ± 0.84 17.77 ± 2.83 65.27 ± 6.37 H2 5.44 ± 1.11 8.92 ± 3.30 63.95 ± 8.22 H3 4.69 ± 0.90 14.54 ± 4.65 63.41 ± 6.17 H4 5.59 ± 1.08 13.33 ± 2.97 62.71 ± 7.93 L1 3.38 ± 0.53 8.93 ± 1.99 56.02 ± 8.77 L2 4.37 ± 1.21 8.88 ± 2.17 55.87 ± 10.17 L3 3.37 ± 1.41 6.36 ± 1.50 46.52 ± 8.08 L4 5.67 ± 1.44 6.53 ± 1.29 44.04 ± 8.10 DNA METHYLATION AND ALTERNATIVE SPLICING MODULATE FBXW11 GENE EXPRESSION 81 quantified using Alpha Ease FC 4.0 software after The murine Leydig tumor cell line (MLTC-1) was normalization with β-actin. cultured in RPMI-1640 medium with 10% fetal bovine serum containing 10 mg/L of penicillin and streptomycin. Identification of splice variants The sub-cultured cells were digested, centrifuged, and added Total RNA was extracted from eight testicular tissues using the to the RPMI-1640 medium without penicillin and RNAsimple Total RNA Kit (Tiangen). RNA quality was streptomycin. The cell suspension was dispensed into a monitored using visualization bands in 1.5% agarose gels after 24-well plate, and cell density was observed under a electrophoresis. The RNA samples were reverse transcribed microscope. The preferred cell density per well was 5 × 104/mL.
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