Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Posttranslational lysine 2-hydroxyisobutyrylation of human sperm tail proteins affects motility

Posttranslational lysine 2-hydroxyisobutyrylation of human sperm tail proteins affects motility Abstract STUDY QUESTION Does lysine 2-hydroxyisobutyrylation, a newly identified protein posttranslational modification (PTM), occur in human sperm and affect human sperm function? SUMMARY ANSWER Lysine 2-hydroxyisobutyrylation mainly occurs in human sperm tail proteins, and excessive lysine 2-hydroxyisobutyrylation affects human sperm motility. WHAT IS KNOWN ALREADY PTM is regarded as an important pathway in regulating sperm function since mature sperm are almost transcriptionally silent. However, only phosphorylation was extensively studied in mature sperm to date. Lysine 2-hydroxyisobutyrylation, a newly characterised PTM, is broadly conserved in both eukaryotic and prokaryotic cells. Although histone lysine 2-hydroxyisobutyrylation has been shown to be associated with active gene expression in spermatogenic cells, the presence, regulatory elements and function of lysine 2-hydroxyisobutyrylation have not been characterised in mature sperm. STUDY DESIGN, SIZE, DURATION Sperm samples were obtained from normozoospermic men and asthenozoospermic men who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China, between May 2017 and November 2018. In total, 58 normozoospermic men and 65 asthenozoospermic men were recruited to participate in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays using a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody. The immunofluorescence assay was imaged using super-resolution structured illumination microscopy. Sperm viability was examined by using the eosin staining method, and sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm penetration ability was determined by evaluating the ability of the sperm to penetrate a 1% (w/v) methylcellulose solution. The level of intracellular adenosine triphosphate (ATP) was detected using a rapid bioluminescent ATP assay kit. MAIN RESULTS AND THE ROLE OF CHANCE Lysine 2-hydroxyisobutyrylation was present in several proteins (20–100 kDa) mainly located in the tail of human sperm. Sperm lysine 2-hydroxyisobutyrylation was derived from 2-hydroxyisobutyrate (2-Hib) and was regulated by acyltransferase P300 and nicotinamide adenine dinucleotide-dependent lysine deacylase sirtuins. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and ATP level of human sperm. Interestingly, the level of sperm lysine 2-hydroxyisobutyrylation was higher in asthenozoospermic men than that in normozoospermic men and was negatively correlated with the progressive motility of human sperm. Furthermore, high levels of lysine 2-hydroxyisobutyrylation in asthenozoospermic men accompanied decreased ATP levels. LIMITATIONS, REASONS FOR CAUTION Although the present study indicated the involvement of sperm lysine 2-hydroxyisobutyrylation in regulating human sperm motility, the underlying mechanism needs to be further illustrated. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study provide insight into the novel role of lysine 2-hydroxyisobutyrylation in human sperm and suggest that abnormality of sperm lysine 2-hydroxyisobutyrylation may be one of the causes for asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S) National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). The authors have no conflicts of interest to declare. adenosine triphosphate, asthenozoospermia, lysine 2-hydroxyisobutyrylation, protein posttranslational modifications, sperm motility Introduction Protein posttranslational modifications (PTMs), which regulate cellular processes and diversify the proteome, are the covalent processing events that modulate the physical and chemical properties of target proteins, through either proteolytic cleavage or the addition of a modifying group to amino acids (Venne et al., 2014; Rousseaux and Khochbin, 2015). Among the modified amino acids, lysine is a hot spot for PTMs since it is an alkaline amino acid with an extremely unstable and highly nucleophilic ε-NH2 side chain that can react with various chemical groups (Papanicolaou et al., 2014; Xu et al., 2017). With the development of high-resolution mass spectrometry, more than 100 hundred types of novel protein lysine modifications have been identified (Huang et al., 2015; Sabari et al., 2017; Simithy et al., 2017; Mnatsakanyan et al., 2018), and more importantly, these protein lysine modifications dynamically regulate various biological events and cellular processes (Rousseaux and Khochbin, 2015; Dutta et al., 2016; Sabari et al., 2017; Simithy et al., 2017). Lysine 2-hydroxyisobutyrylation, a newly identified protein lysine modification, is broadly conserved in both eukaryotic and prokaryotic cells (Dai et al., 2014). Lysine 2-hydroxyisobutyrylation neutralises the positive charge of lysine and introduces a hydroxyl group that enables the modified lysine to form hydrogen bonds with other molecules to regulate protein functions (Dai et al., 2014). Proteomic screening of lysine 2-hydroxyisobutyrylation has shown that lysine 2-hydroxyisobutyrylation occurs in both histones and non-histones that participate in a variety of biological functions and processes, including the tricarboxylic acid cycle (Wu et al., 2018), glycolysis/gluconeogenesis (Huang et al., 2017b; Huang et al., 2018) and transcription, among others. (Wu et al., 2018). Of note, lysine 2-hydroxyisobutyrylation especially enriched in mitochondrial proteins is involved in energy metabolic networks (Huang et al., 2017a). In HeLa cells, elements that regulate lysine 2-hydroxyisobutyrylation have already been characterised. Lysine 2-hydroxyisobutyrylation is derived from 2-hydroxyisobutyrate (2-Hib) and 2-hydroxyisobutyryl-coenzyme A (Dai et al., 2014; Huang et al., 2017a). Acyltransferases P300 and lysine acetyltransferase 5 (KAT5), a MYST family acetyltransferase member, act as 2-hydroxyisobutyryltransferases to add 2-hydroxyisobutyryl to the lysine in substrate proteins (Huang et al., 2017a; Huang et al., 2018), while histone deacetylase 2 (HDAC2) and 3 (HDAC3) serve as de-2-hydroxyisobutyrylases to remove lysine 2-hydroxyisobutyrylation (Huang et al., 2017a). Interestingly, a recent study has demonstrated the occurrence of lysine 2-hydroxyisobutyrylation in mouse spermatogenic cells (Dai et al., 2014; Moretti et al., 2016). The distribution of lysine 2-hydroxyisobutyrylation varies during spermatogenesis. Lysine 2-hydroxyisobutyrylation is elevated in spermatogonia and reduced in spermatocytes, indicating histone lysine 2-hydroxyisobutyrylation is closely related to transcriptional activity. In meiotic and post-meiotic cells, the transcriptional repression of sex chromosomes is associated with the depletion of histone lysine 2-hydroxyisobutyrylation, except for the genes on these chromosomes that escape inactivation in round spermatids (Dai et al., 2014; Moretti et al., 2016). Intriguingly, lysine 2-hydroxyisobutyrylation maintains a considerable level in elongating spermatids undergoing histone-to-transition replacement of proteins at the later stage of spermatogenesis (Dai et al., 2014), suggesting the presence of lysine 2-hydroxyisobutyrylation in mature sperm. Unlike spermatogenic cells, mature sperm are almost transcriptionally and translationally silent, since they have a highly condensed chromatin architecture and they undergo several post-meiotic events, such as histone-to-protamine transition (Gur and Breitbart, 2008). Therefore, the role of lysine 2-hydroxyisobutyrylation in mature sperm may be unique and warrants exploration. In this study, we characterised global lysine 2-hydroxyisobutyrylation in normal human sperm using a qualified pan anti-lysine 2-hydroxyisobutyrylation antibody via immunoblotting and immunofluorescence, with super-resolution structured illumination microscopy (SIM). The regulatory elements (regulatory enzymes and cofactor) for lysine 2-hydroxyisobutyrylation were also studied in human sperm. In addition, the present study explored the correlation between sperm lysine 2-hydroxyisobutyrylation and sperm motility, as well as the role that lysine 2-hydroxyisobutyrylation plays in asthenozoospermia, which is a common disorder consisting of sperm motility defects that account for almost half of male infertility cases (Saraswat et al., 2017). Our results may provide new insights into the function of lysine 2-hydroxyisobutyrylation in human sperm. Materials and Methods Chemicals Pan anti-lysine 2-hydroxyisobutyrylation rabbit polyclonal antibody (PTM-801) and pan anti-lysine acetylation rabbit polyclonal antibody (PTM-101) were obtained from PTM BioLabs Inc. (Hangzhou, China). Anti-HDAC2 rabbit polyclonal antibody (16152-1-AP), anti-HDAC3 rabbit polyclonal antibody (10255-1-AP), anti-KAT5 rabbit polyclonal antibody (10827-1-AP), anti-cytochrome c oxidase subunit 6B1 (COX6B1) rabbit polyclonal antibody (11425-1-AP), anti-protamine 2 (PRM2) rabbit polyclonal antibody (14500-1-AP), anti-beta-ACTIN mouse monoclonal antibody (66009-1-Ig) and anti-GAPDH mouse monoclonal antibody (60004-1-Ig) were acquired from Proteintech Group, Inc. (Rosemont, IL, USA). Rabbit (or mouse) IgG, horseradish peroxidase (HRP)-conjugated goat anti-rabbit (or mouse) secondary antibody, DyLight 488-conjugated goat anti-rabbit secondary antibody, minimum essential medium (MEM, 12492013), fetal bovine serum (FBS, 10099133) and antibiotic-antimitotic solution (15240062) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). C646 (HY-13823, purity: >98.0%); nicotinamide (NAM, HY-B0150, purity: >98.0%) and trichostatin A (TSA, HY-15144, purity: >99.53%) were purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA). A normal goat serum (NGS, SL038) and 4′,6-diamidino-2-phenylindole (DAPI, C0065) was purchased from Solarbio Life Sciences (Beijing, China). A human serum albumin (HSA) solution (100 mg/mL, 10064) was purchased from Vitrolife Corporation (Göteborg, Sweden). 2-Hib (164 976, purity: 98.0%), methylcellulose (M0512, viscosity: 4000 cP), phosphate-buffered saline (PBS, P3813) and other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. HeLa cell culture HeLa cells were purchased from Procell Life Science and Technology Co. Ltd. (Wuhan, China). The cells were cultured at 37°C and 5% CO2 for 3 days in MEM, supplemented with 10% FBS and 1% antibiotic-antimitotic solution. Subsequently, the cells were processed by the trypsin digestion method and collected by centrifugation (200g, 3 min). Ethical approval All participants in this study signed the informed consent form. The collection of semen and experiments in this study were approved by the Institutional Ethics Committee on human subjects of Jiangxi Maternal and Child Health Hospital. Semen sample collection Semen samples were collected by masturbation after 3–5 days of sexual abstinence from the participants who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital (Nanchang, China) between May 2017 and November 2018. Totally, 58 normozoospermic men who had known reproductive histories from the past 2 years and normal sperm quality as well as 65 asthenozoospermic men (progressive motility <32%) with normal sperm concentration and morphology were recruited in this study according to the WHO laboratory manual for the examination and processing of human semen (Fifth edition, http://www.who.int/reproductivehealth/publications/infertility/9789241547789/en/). The semen characteristics of the participants are shown in Table I. Semen characteristics of the participants. Table I Semen characteristics of the participants. Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 The reference limit (Reference) is according to WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Routine semen analysis was carried out immediately after liquefaction. SEM: standard error of the mean. *P < 0.05, unpaired t test. Open in new tab Table I Semen characteristics of the participants. Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 The reference limit (Reference) is according to WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Routine semen analysis was carried out immediately after liquefaction. SEM: standard error of the mean. *P < 0.05, unpaired t test. Open in new tab Human sperm treatments After liquefaction, the sperm samples were purified by direct swim-up in human tubal fluid (HTF) medium described in our previous study (Cheng et al., 2019). For chemical treatments, 1-ml aliquots of purified sperm samples from healthy individuals were exposed to the following doses of C646, NAM, TSA and 2-Hib in HTF at 37°C in a 5% CO2 incubator for 1 h: 0, 0.25, 0.5, 1 and 2 μM of C646 (prepared in HTF containing 0.1% DMSO, pH 7.4); 0, 10, 20, 40 and 80 mM of NAM (prepared in HTF, pH 7.4); 0, 10, 20, 40 and 80 μM of 2-Hib (prepared in HTF, pH 7.4); and 0, 1.5, 3, 6 and 12 μM of TSA (prepared in HTF containing 0.1% DMSO, pH 7.4). After chemical treatments, sperm lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays. Viability, total motility, progressive motility and the adenosine triphosphate (ATP) concentrations of human sperm were also assessed. Immunoblotting assay Total proteins from HeLa cells and human sperm were extracted as described previously (Luo et al., 2015). For the isolation of sperm proteins, sperm samples were first treated with somatic cell lysis buffer (0.2% sodium dodecyl sulphate and 0.5% Triton X-100 in HTF medium) to eliminate residual somatic cells. Proteins from sperm mitochondrion, cytoplasm and head proteins were obtained as described previously (Cheng et al., 2019). The protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, 30 μg of proteins was used for western blotting as previous described (Luo et al., 2015). The dilutions used for the primary antibodies were as follows: 1:500 for anti-COX6B1 antibody; 1:1000 for pan anti-lysine 2-hydroxyisobutyrylation antibody, pan anti-lysine acetylation antibody, anti-PMR2 antibody, anti-HDAC2 antibody, anti-HDAC3 antibody and anti-KAT5 antibody; 1:10000 for anti-ACTIN antibody; and 1:20000 for anti-GAPDH antibody. The dilutions used for the secondary antibodies were 1:10000 for HRP-conjugated goat anti-rabbit (or mouse) secondary antibody. The grey values of the target bands detected by the corresponding antibodies were quantified using Image J software (version 1.44, National Institutes of Health), and then statistical analysis of these values was conducted by normalising to the values detected by ACTIN (loading control). For semi-quantifying lysine 2-hydroxyisobutyrylation in different compartments of human sperm, the grey values of lysine 2-hydroxyisobutyrylation were normalised to those of GAPDH (loading control for total and cytoplasmic lysine 2-hydroxyisobutyrylation), COX6B1 (loading control for mitochondrial lysine 2-hydroxyisobutyrylation) and PMR2 (loading control for head lysine 2-hydroxyisobutyrylation), respectively. Each experiment was repeated at least three times. Indirect immunofluorescence assay Immunofluorescence staining was conducted according to our previously published paper (Cheng et al., 2019). Sperm samples and HeLa cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, permeabilised with 0.2% Trion X-100 in PBS and blocked with 10% NGS in PBS, followed by incubation with rabbit IgG (negative control, 1:100) or pan anti-lysine 2-hydroxyisobutyrylation antibody (1:50) and then staining with DyLight 488 conjugated goat anti-rabbit secondary antibody (1:200 dilution) and 0.5 μM DAPI. The stained cells were imaged with the Nikon N-SIM super-resolution microscopy system (Nikon Co. Ltd., Tokyo, Japan) which has twice the resolution of conventional optical microscopes (~115 nm in three-dimensional (3D) SIM mode). To achieve the super-resolution, the images were captured with the Nikon ECLIPSE Ti-E inverted microscope, using a CFI Apochromat TIRF 100x oil objective lens (N.A. 1.49). DyLight 488 was visualised with a 488-nm laser excitation and a BP 515–545-nm emission filter. The DAPI was visualised with a 405-nm laser excitation and a BP 440–485-nm emission filter. The typical images with single sperm that has representative staining and normal morphology, without curly tail, were used for reconstructing the 3D-SIM images with the NIS-Elements Advanced Research microscope imaging software (Nikon). To analyse sperm lysine 2-hydroxyisobutyrylation in detail, sperm tails were divided into 0.5-μm-axial-length regions of interest (ROIs), and the mean lysine 2-hydroxyisobutyrylation fluorescence intensity of each ROI was quantified by NIS-Elements Advanced Research microscope imaging software. In total, 80 ROIs corresponding to 40-μm-axial-length tails from the initial point of midpiece (as 0 μm) to the principle piece were analysed for each stained sperm. The axial distribution of lysine 2-hydroxyisobutyrylation in sperm tails was reflected by an X–Y curve, which takes the axial length as the X-axis (0.5 μm each point), and the values of mean lysine 2-hydroxyisobutyrylation fluorescence intensity of each ROI are taken as the Y-axis. In total, six normozoospermic donors (10 sperm for each donor) for each concentration group in chemical (C646, NAM, TSA and 2-Hib) treatments and five sperm from each clinical sample were analysed for the semi-quantification. Assessment of sperm viability, motility and penetration ability Sperm viability was examined using the eosin staining method as described previously (Zou et al., 2017). A computer-assisted sperm analysis (CASA) system (WLJY-9000, WeiLi Co., Ltd, Beijing, China) was employed to examine sperm total motility and progressive motility. A minimum of 200 sperm were calculated for each assay. To evaluate penetration ability, human sperm were first incubated in capacitating HTF++ medium (HTF plus 25 mM NaHCO3 and 0.4% HSA) for 2 h and then treated with 2-Hib for 1 h. Sperm penetration into a 1% (w/v) methylcellulose solution prepared in HTF++ medium, mimicking the viscous environment of the female reproductive tract, was evaluated as described previously (Zou et al., 2017). ATP measurement in human sperm The ATP level was detected using a rapid bioluminescent ATP assay Kit (KA1661, Abnova Corporation, Taipei, Taiwan). The measurement was performed according to the manufacturer’s instructions. Statistical analysis All the statistical analyses were performed by the statistics software GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± SEM and follow normal distribution measured by Shapiro–Wilk test (P > 0.05). Differences between the lysine 2-hydroxyisobutyrylation level in normozoospermic and asthenozoospermic men were assessed using the unpaired t test, while differences in sperm lysine 2-hydroxyisobutyrylation between the controls and the chemically treated samples were assessed using two-way ANOVA analysis and the Dunnett’s test. Linear regression was performed by GraphPad Prism. Statistically significant differences were determined at P < 0.05. Results Lysine 2-hydroxyisobutyrylation mainly occurs in proteins located in the tails of human sperm To examine whether lysine 2-hydroxyisobutyrylation occurs in human sperm, we performed immunoblotting and immunofluorescence assays with a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody, which specifically recognises 2-hydroxyisobutyrylated lysines in both histone and non-histone substrates, but not an unmodified lysine or other lysine modifications such as acetylation, butyrylation, and β-hydroxybutyrylation (Dai et al., 2014; Huang et al., 2017a; Huang et al., 2018; Wu et al., 2018). The effectiveness of this antibody was validated in HeLa cells (Fig. 1A and Supplementary Fig. S1). Lysine 2-hydroxyisobutyrylation occurred in a wide range of proteins (25–100 kDa) in mature human sperm (Fig. 1A). The immunofluorescence assay demonstrated that lysine 2-hydroxyisobutyrylated proteins were widely distributed along the tail, especially around the median mitochondrial sheath of human sperm, while only a few were situated in the acrosome region (Fig. 1B). A negative control, where pan anti-lysine 2-hydroxyisobutyrylation antibody was replaced with rabbit IgG, showed no fluorescence staining (Supplementary Fig. S1). In addition, immunoblotting assays, with proteins purified from different sperm compartments, showed that lysine 2-hydroxyisobutyrylation was mainly present in proteins located in the cytoplasm and mitochondria (Fig. 1C and Supplementary Fig. S2), consistent with the results of the immunofluorescence assay. The presence of lysine 2-hydroxyisobutyrylation in human sperm. (A) Immunoblotting detection (left panel) of lysine 2-hydroxyisobutyrylation in human sperm (Sperm). In parallel, the effectiveness of pan anti-lysine 2-hydroxyisobutyrylation antibody was confirmed in HeLa cells (HeLa). Right panel shows Coomassie Brilliant Blue staining. (B) The location of 2-hydroxyisobutyrylated proteins in human sperm was determined by super-resolution structured illumination microscopy (SIM). Scale bar: 5 μm. (C) Khib in different compartments of human sperm. Semi-quantification of Khib was performed as described in the ‘Materials and Methods’ section. The error bars denote the mean ± SEM. Khib: lysine 2-hydroxyisobutyrylation; WB: western blot; CBS: Coomassie Brilliant Blue staining; TP: total proteins; Mit: mitochondrial proteins; Head: head proteins; Cyt: cytoplastic proteins. Experiments were replicated in six normozoospermic donors. Figure 1 Open in new tabDownload slide Figure 1 Open in new tabDownload slide Sperm lysine 2-hydroxyisobutyrylation can be modulated by regulatory elements effective in somatic cells In somatic cells, lysine 2-hydroxyisobutyrylation is derived from 2-Hib and can be regulated by P300, KAT5, HDAC2 and HDAC3 (Dai et al., 2014; Huang et al., 2017a; Huang et al., 2018). To explore whether these regulatory elements work efficiently in human sperm, we first examined the presence of P300, KAT5, HDAC2 and HDAC3 in human sperm. In our previous study, the presence of P300 in human sperm had already been confirmed (Cheng et al., 2019). In this study, HDAC2 but not HDAC3 and KAT5 were detected in human sperm (Sperm, Supplementary Fig. S3) although all of them were detected in HeLa cells using immunoblotting assays (HeLa, Supplementary Fig. S3), which was consistent with the published human sperm proteome that contains 6198 proteins (Amaral et al., 2014). Subsequently, inhibitors for P300 (C646) and HDAC class I/II (TSA) were employed to verify the involvement of these proteins in the regulation of human sperm lysine 2-hydroxyisobutyrylation, since a knockdown assay was unfeasible in mature sperm. The immunoblotting assay showed that 0.5, 1 and 2 μM of C646 could not only decrease the global levels of sperm lysine 2-hydroxyisobutyrylation by about 20, 40 and 40%, respectively (Fig. 2A and Supplementary Fig. S4), but it could also lower the level of sperm lysine acetylation (Supplementary Figs S4 and S5). However, TSA did not change human sperm lysine 2-hydroxyisobutyrylation (Fig. 2B and Supplementary Fig. S6), although it increased the global level of lysine acetylation (Supplementary Figs S5 and S6). Consistent with the immunoblotting results, the immunofluorescence assay showed that C646 had an adverse effect on lysine 2-hydroxyisobutyrylation that occurred along the sperm tail (Supplementary Fig. S4D–F), and TSA did not change the level or the localisation of sperm lysine 2-hydroxyisobutyrylation (Supplementary Fig. S6D–F). We also examined the effect of a potent and selective HDAC2 inhibitor, santacruzamate A, on human sperm lysine 2-hydroxyisobutyrylation and found no effect (data not shown). Furthermore, based on the published results that nicotinamide adenine dinucleotide-dependent deacetylase sirtuins (especially sirtuin 5) act as deacylases—rather than deacetylases—in somatic cells (Hirschey and Zhao, 2015; Sabari et al., 2017) and human sperm (Cheng et al., 2019), we examined the effect of NAM (a pharmacological sirtuin inhibitor) on human sperm lysine 2-hydroxyisobutyrylation. We found that 20, 40 and 80 mM of NAM increased both the global level of lysine 2-hydroxyisobutyrylation along the human sperm tail by 30–50% (Fig. 2C and Supplementary Fig. S7) and the global level of lysine acetylation (Supplementary Figs S5 and S7), suggesting the involvement of sirtuins in removing lysine 2-hydroxyisobutyrylation in human sperm. In addition, 20, 40 and 80 μM of 2-Hib elevated the global level of sperm lysine 2-hydroxyisobutyrylation by 70–120% (Fig. 2D and Supplementary Fig. S8), without affecting sperm lysine acetylation (Supplementary Figs S5 and S8), indicating that lysine 2-hydroxyisobutyrylation is also derived from 2-Hib in human sperm. Effects of C646, trichostatin A (TSA), nicotinamide (NAM) and 2-hydroxyisobutyrate (2-Hib) on human sperm lysine 2-hydroxyisobutyrylation (Khib). Khib was semi-quantified as relative grey value (Khib/ACTIN) by normalising the total grey values of all the bands detected by anti-Khib antibody to the value detected by ACTIN antibody (loading control) using Image J software (version 1.44, National Institutes of Health). Human sperm were incubated with C646 (A), TSA (B), NAM (C) and 2-Hib (D), and sperm Khib was detected using an immunoblotting assay and semi-quantified as described in the ‘Materials and Methods’ section. The error bars denote the mean ± SEM. *P < 0.05, two-way ANOVA of variance. Experiments were replicated in six normozoospermic donors. Figure 2 Open in new tabDownload slide Figure 2 Open in new tabDownload slide Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreases sperm motility, penetration ability and the ATP level of human sperm The present study has shown that lysine 2-hydroxyisobutyrylation mainly occurs in sperm tails (Fig. 1), where the motility-related proteins are located. In addition, global characterisation of lysine 2-hydroxyisobutyrylation in somatic cells indicated the involvement of lysine 2-hydroxyisobutyrylation in energy metabolism. Here, we evaluated whether sperm lysine 2-hydroxyisobutyrylation regulated sperm motility and penetration ability, two energy-consuming processes vital for fertilisation. Although C646, NAM and 2-Hib could change the level of sperm lysine 2-hydroxyisobutyrylation (Fig. 2), only 2-Hib was relatively specific since C646 and NAM could also affect other protein lysine modifications in human sperm (Cheng et al., 2019). Therefore, we examined the effect of 2-Hib on sperm motility and penetration ability and found that 20, 40 and 80 μM of 2-Hib reduced the total motility (especially 40 and 80 μM of Hib reducing total motility below the normal level, <40%, Fig. 3A), decreased progressive motility to an asthenozoospermic level (<32%, Fig. 3B) and lowered penetration ability by 20–40% (Fig. 3C and D), without affecting sperm viability (Fig. 3E). In addition, the effect of 2-Hib on the ATP concentration in human sperm was also assessed, showing that 2-Hib lowered the ATP concentration in human sperm by approximately 30% (Fig. 3F). Effects of 2-hydroxyisobutyrate (2-Hib) on viability, motility and adenosine triphosphate (ATP) level of human sperm. Human sperm were incubated with 0, 10, 20, 40 and 80 μM 2-Hib for 1 h. Total motility (A) and progressive motility (B) of human sperm were analysed by computer-aided sperm analysis. (C and D) show penetration ability. Three fields at 1 cm (C) and 2 cm (D) from the base of the tube were counted, and the average cells/field was calculated. The cell densities were normalised to values from parallel, untreated controls (0 μM 2-Hib). (E) Sperm viability was examined using the eosin staining method. (F) Intracellular ATP was detected using a rapid bioluminescent ATP assay. Sperm from 15 normozoospermic donors were analysed in each assay. The error bars denote the mean ± SEM. *P < 0.05, two-way analysis of variance. Figure 3 Open in new tabDownload slide Figure 3 Open in new tabDownload slide Sperm lysine 2-hydroxyisobutyrylation level is enriched in asthenozoospermic men and negatively correlated with progressive motility of human sperm To further validate the correlation between the lysine 2-hydroxyisobutyrylation level and sperm motility, we examined whether sperm lysine 2-hydroxyisobutyrylation was abnormal in asthenozoospermic men compared to normozoospermic men. Firstly, the lysine 2-hydroxyisobutyrylation level in semen samples from 123 males (58 normozoospermic men and 65 asthenozoospermic men) was determined by an immunoblotting assay. A typical example of the immunoblotting assay showed that the level of sperm lysine 2-hydroxyisobutyrylation in asthenozoospermic men was generally higher compared to normozoospermic men (Supplementary Fig. S9A and B). The quantification result confirmed that the mean level of sperm lysine 2-hydroxyisobutyrylation was increased by ~50% in asthenozoospermic men compared to sperm lysine 2-hydroxyisobutyrylation in normozoospermic men (P < 0.05, Fig. 4A). Linear regression analysis of sperm lysine 2-hydroxyisobutyrylation level and progressive motility of the 123 males was conducted to evaluate the relationship between progressive motility and the lysine 2-hydroxyisobutyrylation level of human sperm. The results showed that the sperm lysine 2-hydroxyisobutyrylation level was negatively correlated with progressive motility of human sperm (r2 = 0.81, P < 0.05, Fig. 4B). All these results supported a tight association between lysine 2-hydroxyisobutyrylation and sperm motility. Furthermore, according to the relative levels of sperm lysine 2-hydroxyisobutyrylation in normozoospermic men (0.45–1.75), high lysine 2-hydroxyisobutyrylation samples were classed based on a threshold of 1.75, which corresponded to the highest level of lysine 2-hydroxyisobutyrylation in normozoospermic controls (Fig. 4A); thus, 18 asthenozoospermic men—with a sperm lysine 2-hydroxyisobutyrylation level higher than 1.75—were classed as having high lysine 2-hydroxyisobutyrylation samples (red frame, Fig. 4A) and accounted for 27.69% (18/65) of the asthenozoospermic men. These results were verified by the immunofluorescence assay, showing that enrichment of lysine 2-hydroxyisobutyrylation fluorescence in these 18 asthenozoospermic men was present along the entire tail and the acrosome (Supplementary Fig. S9C and D). We further examined whether the sperm ATP level (a key factor for motility) was affected in these 18 asthenozoospermic men. As a control, 18 normozoospermic men were randomly recruited from the 58 normozoospermic controls and their sperm ATP levels were also measured. The results showed that the asthenozoospermic men with high lysine 2-hydroxyisobutyrylation levels had significantly lower sperm ATP levels compared to normozoospermic controls (P < 0.05, Fig. 4C). Association between lysine 2-hydroxyisobutyrylation and progressive motility of human sperms. (A) Semi-quantification of lysine 2-hydroxyisobutyrylation in 58 normozoospermic men and 65 asthenozoospermic men was performed as described in the ‘Materials and Methods’ section. High-lysine 2-hydroxyisobutyrylation in asthenozoospermic men, with normalised levels of lysine 2-hydroxyisobutyrylation above the threshold of 1.75, are indicated in the red frame (18 men). (B) The correlation between sperm lysine 2-hydroxyisobutyrylation and progressive motility was assessed by linear regression. (C) The ATP levels in normozoospermic men (18 men randomly recruited from 58 normozoospermic men as control) and asthenozoospermic men, with high Khib levels [the 18 men in red frame in panel (A)] were compared. The Error bars denote the mean ± SEM, *P < 0.05, Student’s t test. Khib: lysine 2-hydroxyisobutyrylation; N: normozoospermic men; A: asthenozoospermic men. Figure 4 Open in new tabDownload slide Figure 4 Open in new tabDownload slide Discussion Extensive studies have shown that tyrosine and threonine/serine phosphorylation regulate sperm activities that are essential for fertilisation (Naz and Rajesh, 2004; Chan et al., 2009; Barbonetti et al., 2010; Visconti et al., 2011), indicating that PTM is vital for sperm function. Only a few recent studies have reported protein lysine modification in mature sperm, showing the presence of lysine methylation and lysine acetylation in the histones of mammalian sperm (Brunner et al., 2014; Jana et al., 2015; Kim et al., 2015) and global lysine acetylation in human sperm (Sun et al., 2014; Yu et al., 2015). Our latest study showed that lysine glutarylation in human sperm is associated with progressive motility (Cheng et al., 2019). However, as the most frequent PTM, the functional role of protein lysine modification in mature sperm needs to be further explored. In somatic cells, lysine 2-hydroxyisobutyrylation is not only present in histones to regulate gene expression like lysine methylation and lysine acetylation, but it is also highly enriched in mitochondrial proteins to control energy metabolism (Tan et al., 2014; Huang et al., 2017a). However, in mature human sperm, only ~10% histones are retained and most of histones are replaced by protamines which may be hardly modified to maintain a highly condensed and transcriptionally silent chromosome. Lysine 2-hydroxyisobutyrylation in the retained histones is too small to be detected by immunofluorescent assay, which may explain the findings that little lysine 2-hydroxyisobutyrylation occurs in sperm nucleus where histones are located. These results suggest that lysine 2-hydroxyisobutyrylation in mature sperm may take part in metabolic pathways rather than transcriptional regulation. Interestingly, mature sperm also have functional mitochondria which serve as the energy metabolism centre vital for sperm motility (Piomboni et al., 2012). Therefore, we hypothesised that lysine 2-hydroxyisobutyrylation regulates sperm motility by controlling energy metabolism in mitochondria. All the results in this study indicate the functional role of sperm lysine 2-hydroxyisobutyrylation in sperm motility and ATP metabolism, supporting our hypothesis. However, more asthenozoospermic men should be studied and a big gap in proving a true physiological role for lysine 2-hydroxyisobutyrylation is the need to show that reduction of lysine 2-hydroxyisobutyrylation can actually improve sperm motility and ATP concentration of asthenozoospermic men with the high level of lysine 2-hydroxyisobutyrylation. To fill in the gap, we need to specifically decrease lysine 2-hydroxyisobutyrylation in human sperm. Although we validated that the reported regulatory proteins (P300 and sirtuins) are effective in human sperm, these proteins are not specific for lysine 2-hydroxyisobutyrylation. Thus, regulatory proteins specific for sperm lysine 2-hydroxyisobutyrylation and/or sperm 2-Hib metabolism should be identified and their modulators should be applied. Our next work will utilise a proteomic approach to identify the potential regulatory enzymes responsible for the high level of sperm lysine 2-hydroxyisobutyrylation in asthenozoospermic men, as well as characterise the 2-hydroxyisobutyrylated lysines in target proteins involved in the regulation of sperm motility. Furthermore, the functions of the identified regulatory enzymes of lysine 2-hydroxyisobutyrylation will be validated by knockout mouse models, and the role of 2-hydroxyisobutyrylated lysines in target proteins will be explored by site-specific antibodies in asthenozoospermic men. Recently, we have characterised another lysine modification, lysine glutarylation, in human sperm (Cheng et al., 2019). Like lysine 2-hydroxyisobutyrylation, lysine glutarylation is also a newly identified lysine modification related to energy metabolism (Tan et al., 2014) and is also associated with human sperm motility (Cheng et al., 2019). Different from lysine 2-hydroxyisobutyrylation, lysine glutarylation is positively correlated with the progressive motility of human sperm and was decreased in asthenozoospermic men (Cheng et al., 2019). These results indicate that lysine 2-hydroxyisobutyrylation and lysine glutarylation may regulate human sperm motility by different underlying mechanisms (target proteins, regulatory enzymes and 2-Hib/glutaric acid metabolisms). In conclusion, lysine 2-hydroxyisobutyrylation mainly localises in sperm tails and is modulated by regulatory enzymes and co-factors that are effective in somatic cells. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and the ATP concentration of human sperm. The mean level of sperm lysine 2-hydroxyisobutyrylation was increased in asthenozoospermic men and negatively correlated with the progressive motility of human sperm. Furthermore, asthenozoospermic men with high lysine 2-hydroxyisobutyrylation levels have low ATP concentrations compared to normozoospermic men with normal lysine 2-hydroxyisobutyrylation levels. Together, our findings elucidate the novel role of lysine 2-hydroxyisobutyrylation and provide more evidence for the functional role of protein lysine modification in mature sperm. A schematic summary of the biochemical pathways of lysine 2-hydroxyisobutyrylation in human sperm was shown in Fig. 5. A schematic summary of the biochemical pathways of lysine 2-hydroxyisobutyrylation in human sperm. Proteins in human sperm tail are modified by lysine 2-hydroxyisobutyrylation. Addition of 2-hydroxyisobutyryl from 2-Hib-CoA to the lysines in target proteins is catalysed by regulatory enzymes such as P300. The 2-hydroxyisobutyryl in the modified proteins can be removed by de-2-hydroxyisobutyrylases such as sirtuins. The 2-hydroxyisobutyrylated proteins may be involved in energy metabolism vital for the production of ATP that is essential for sperm motility. In-vitro addition of 2-Hib elevates sperm lysine 2-hydroxyisobutyrylation but decreases sperm motility and ATP concentration in normozoospermic men. In addition, increased sperm lysine 2-hydroxyisobutyrylation accompanies reduced sperm ATP concentration in asthenozoospermic men. 2-Hib: 2-hydroxyisobutyrate; 2-Hib-CoA: 2-hydroxyisobutyryl-coenzyme A, Khib: lysine 2-hydroxyisobutyrylation. Figure 5 Open in new tabDownload slide Figure 5 Open in new tabDownload slide Acknowledgements The authors are extremely grateful to the volunteers for the participation in this study. The authors are grateful for the helpful assistances of immunoblotting assay from Jingjie PTM BioLab (Hangzhou), Co. Ltd (China). Authors’ roles Y.C. and Z.P. conducted the immunoblotting assay. Y.C., T.L. and Z.P. performed the immunofluorescence assay. H.C. acquired and processed all semen samples. F.W. cultivated the HeLa cells. T.P. measured the ATP concentration. X.H. and F.W. assessed sperm viability, motility and penetration ability. T.L. and Y.C. collected the data and performed the statistical analyses. T.L. was responsible for primary study oversight and design, data acquisition, data analysis and interpretation and the primary writing of the manuscript. All the authors made substantial contributions in critically revising the manuscript. All the authors approved the final manuscript for submission. Funding National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). Conflict of interest The authors have no conflicts of interest to declare. References Amaral A , Castillo J, Ramalho-Santos J, Oliva R. The combined human sperm proteome: cellular pathways and implications for basic and clinical science . Hum Reprod Update 2014 ; 20 : 40 – 62 . Google Scholar Crossref Search ADS PubMed WorldCat Barbonetti A , Vassallo MR, Cordeschi G, Venetis D, Carboni A, Sperandio A, Felzani G, Francavilla S, Francavilla F. Protein tyrosine phosphorylation of the human sperm head during capacitation: immunolocalization and relationship with acquisition of sperm-fertilizing ability . Asian J Androl 2010 ; 12 : 853 – 861 . Google Scholar Crossref Search ADS PubMed WorldCat Brunner AM , Nanni P, Mansuy IM. Epigenetic marking of sperm by post-translational modification of histones and protamines . Epigenet Chromatin 2014 ; 7 : 2 . Google Scholar Crossref Search ADS WorldCat Chan CC , Shui HA, Wu CH, Wang CY, Sun GH, Chen HM, Wu GJ. Motility and protein phosphorylation in healthy and asthenozoospermic sperm . J Proteome Res 2009 ; 8 : 5382 – 5386 . Google Scholar Crossref Search ADS PubMed WorldCat Cheng Y-m , Hu X-n, Peng Z, Pan T-t, Wang F, Chen H-y, Chen W-q, Zhang Y, Zeng X-h, Luo T. Lysine glutarylation in human sperm is associated with progressive motility . Hum Reprod 2019 ; 34 : 1186 – 1194 . Google Scholar Crossref Search ADS PubMed WorldCat Dai L , Peng C, Montellier E, Lu Z, Chen Y, Ishii H, Debernardi A, Buchou T, Rousseaux S, Jin F et al. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark . Nat Chem Biol 2014 ; 10 : 365 – 370 . Google Scholar Crossref Search ADS PubMed WorldCat Dutta A , Abmayr SM, Workman JL. Diverse activities of histone acylations connect metabolism to chromatin function . Mol Cell 2016 ; 63 : 547 – 552 . Google Scholar Crossref Search ADS PubMed WorldCat Gur Y , Breitbart H. Protein synthesis in sperm: dialog between mitochondria and cytoplasm . Mol Cell Endocrinol 2008 ; 282 : 45 – 55 . Google Scholar Crossref Search ADS PubMed WorldCat Hirschey MD , Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation . Mol Cell Proteomics 2015 ; 14 : 2308 . Google Scholar Crossref Search ADS PubMed WorldCat Huang H , Lin S, Garcia BA, Zhao Y. Quantitative proteomic analysis of histone modifications . Chem Rev 2015 ; 115 : 2376 – 2418 . Google Scholar Crossref Search ADS PubMed WorldCat Huang H , Luo Z, Qi S, Huang J, Xu P, Wang X, Gao L, Li F, Wang J, Zhao W et al. Landscape of the regulatory elements for lysine 2-hydroxyisobutyrylation pathway . Cell Res 2017a ; 28 : 111 – 125 . Google Scholar Crossref Search ADS WorldCat Huang H , Tang S, Ji M, Tang Z, Shimada M, Liu X, Qi S, Locasale JW, Roeder RG, Zhao Y et al. p300-mediated lysine 2-hydroxyisobutyrylation regulates glycolysis . Mol Cell 2018 ; 70 : 663 – 678.e666 . Google Scholar Crossref Search ADS PubMed WorldCat Huang J , Luo Z, Ying W, Cao Q, Huang H, Dong J, Wu Q, Zhao Y, Qian X, Dai J. 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae . Proc Natl Acad Sci USA 2017b ; 114 : 8782 – 8787 . Google Scholar Crossref Search ADS WorldCat Jana K , Lenka S, Eva P, Soňa L, Stanislav K, Gabriela L, Zbyněk Z, Petra S, Siarhei D, Jan H et al. Post-translational modifications of histones in human sperm . J Cell Biochem 2015 ; 116 : 2195 – 2209 . Google Scholar Crossref Search ADS PubMed WorldCat Kim SK , Jee BC, Kim SH. Histone methylation and acetylation in ejaculated human sperm: effects of swim-up and smoking . Fertil Steril 2015 ; 103 : 1425 – 1431 . Google Scholar Crossref Search ADS PubMed WorldCat Luo T , Li N, He YQ, Weng SQ, Wang T, Zou QX, Zeng XH. Emodin inhibits human sperm functions by reducing sperm [Ca2+]i and tyrosine phosphorylation . Reprod Toxicol 2015 ; 51 : 14 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat Mnatsakanyan R , Shema G, Basik M, Batist G, Borchers CH, Sickmann A, Zahedi RP. Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry . Expert Rev Proteomics 2018 ; 15 : 515 – 535 . Google Scholar Crossref Search ADS PubMed WorldCat Moretti C , Vaiman D, Tores F, Cocquet J. Expression and epigenomic landscape of the sex chromosomes in mouse post-meiotic male germ cells . Epigenet Chromatin 2016 ; 9 : 47 . Google Scholar Crossref Search ADS WorldCat Naz RK , Rajesh PB. Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction . Reprod Biol Endocrinol 2004 ; 2 : 75 . Google Scholar Crossref Search ADS PubMed WorldCat Papanicolaou KN , O'Rourke B, Foster DB. Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria . Front in Physiol 2014 ; 5 : 301 . Google Scholar Crossref Search ADS WorldCat Piomboni P , Focarelli R, Stendardi A, Ferramosca A, Zara V. The role of mitochondria in energy production for human sperm motility . Int J Androl 2012 ; 35 : 109 – 124 . Google Scholar Crossref Search ADS PubMed WorldCat Rousseaux S , Khochbin S. Histone acylation beyond acetylation: Terra incognita in chromatin biology . Cell J 2015 ; 17 : 1 – 6 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Sabari BR , Zhang D, Allis CD, Zhao Y. Metabolic regulation of gene expression through histone acylations . Nat Rev Mol Cell Biol 2017 ; 18 : 90 – 101 . Google Scholar Crossref Search ADS PubMed WorldCat Saraswat M , Joenväärä S, Jain T, Tomar AK, Sinha A, Singh S, Yadav S, Renkonen R. Human spermatozoa quantitative proteomic signature classifies normo- and asthenozoospermia . Mol Cell Proteomics 2017 ; 16 : 57 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat Simithy J , Sidoli S, Yuan Z-F, Coradin M, Bhanu NV, Marchione DM, Klein BJ, Bazilevsky GA, McCullough CE, Magin RS et al. Characterization of histone acylations links chromatin modifications with metabolism . Nat Commun 2017 ; 8 : 1141 . Google Scholar Crossref Search ADS PubMed WorldCat Sun G , Jiang M, Zhou T, Guo Y, Cui Y, Guo X, Sha J. Insights into the lysine acetylproteome of human sperm . J Proteomics 2014 ; 109 : 199 – 211 . Google Scholar Crossref Search ADS PubMed WorldCat Tan M , Peng C, Anderson KA, Chhoy P, Xie Z, Dai L, Park JS, Chen Y, Huang H, Zhang Y. Lysine glutarylation is a protein post-translational modification regulated by SIRT5 . Cell Metab 2014 ; 19 : 605 – 617 . Google Scholar Crossref Search ADS PubMed WorldCat Venne AS , Kollipara L, Zahedi RP. The next level of complexity: crosstalk of posttranslational modifications . Proteomics 2014 ; 14 : 513 – 524 . Google Scholar Crossref Search ADS PubMed WorldCat Visconti PE , Krapf D, de la Vega-Beltran JL, Acevedo JJ, Darszon A. Ion channels, phosphorylation and mammalian sperm capacitation . Asian J Androl 2011 ; 13 : 395 – 405 . Google Scholar Crossref Search ADS PubMed WorldCat Wu Q , Ke L, Wang C, Fan P, Wu Z, Xu X. Global analysis of lysine 2-hydroxyisobutyrylome upon SAHA treatment and its relationship with acetylation and crotonylation . J Proteome Res 2018 ; 17 : 3176 – 3183 . Google Scholar Crossref Search ADS PubMed WorldCat Xu H , Zhou J, Lin S, Deng W, Zhang Y, Xue Y. PLMD: an updated data resource of protein lysine modifications . J Genet Genomics 2017 ; 44 : 243 – 250 . Google Scholar Crossref Search ADS PubMed WorldCat Yu H , Diao H, Wang C, Lin Y, Yu F, Lu H, Xu W, Li Z, Shi H, Zhao S et al. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm) . Mol Cell Proteomics 2015 ; 14 : 1009 – 1023 . Google Scholar Crossref Search ADS PubMed WorldCat Zou QX , Peng Z, Zhao Q, Chen HY, Cheng YM, Liu Q, He YQ, Weng SQ, Wang HF, Wang T et al. Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa . Hum Reprod 2017 ; 32 : 290 – 298 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes " Joint first authors. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected]. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Posttranslational lysine 2-hydroxyisobutyrylation of human sperm tail proteins affects motility

Download PDF
10 pages

Loading next page...
 
/lp/oxford-university-press/posttranslational-lysine-2-hydroxyisobutyrylation-of-human-sperm-tail-9dquEBA70y

References (34)

Publisher
Oxford University Press
Copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected].
ISSN
0268-1161
eISSN
1460-2350
DOI
10.1093/humrep/dez296
Publisher site
See Article on Publisher Site

Abstract

Abstract STUDY QUESTION Does lysine 2-hydroxyisobutyrylation, a newly identified protein posttranslational modification (PTM), occur in human sperm and affect human sperm function? SUMMARY ANSWER Lysine 2-hydroxyisobutyrylation mainly occurs in human sperm tail proteins, and excessive lysine 2-hydroxyisobutyrylation affects human sperm motility. WHAT IS KNOWN ALREADY PTM is regarded as an important pathway in regulating sperm function since mature sperm are almost transcriptionally silent. However, only phosphorylation was extensively studied in mature sperm to date. Lysine 2-hydroxyisobutyrylation, a newly characterised PTM, is broadly conserved in both eukaryotic and prokaryotic cells. Although histone lysine 2-hydroxyisobutyrylation has been shown to be associated with active gene expression in spermatogenic cells, the presence, regulatory elements and function of lysine 2-hydroxyisobutyrylation have not been characterised in mature sperm. STUDY DESIGN, SIZE, DURATION Sperm samples were obtained from normozoospermic men and asthenozoospermic men who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China, between May 2017 and November 2018. In total, 58 normozoospermic men and 65 asthenozoospermic men were recruited to participate in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays using a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody. The immunofluorescence assay was imaged using super-resolution structured illumination microscopy. Sperm viability was examined by using the eosin staining method, and sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm penetration ability was determined by evaluating the ability of the sperm to penetrate a 1% (w/v) methylcellulose solution. The level of intracellular adenosine triphosphate (ATP) was detected using a rapid bioluminescent ATP assay kit. MAIN RESULTS AND THE ROLE OF CHANCE Lysine 2-hydroxyisobutyrylation was present in several proteins (20–100 kDa) mainly located in the tail of human sperm. Sperm lysine 2-hydroxyisobutyrylation was derived from 2-hydroxyisobutyrate (2-Hib) and was regulated by acyltransferase P300 and nicotinamide adenine dinucleotide-dependent lysine deacylase sirtuins. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and ATP level of human sperm. Interestingly, the level of sperm lysine 2-hydroxyisobutyrylation was higher in asthenozoospermic men than that in normozoospermic men and was negatively correlated with the progressive motility of human sperm. Furthermore, high levels of lysine 2-hydroxyisobutyrylation in asthenozoospermic men accompanied decreased ATP levels. LIMITATIONS, REASONS FOR CAUTION Although the present study indicated the involvement of sperm lysine 2-hydroxyisobutyrylation in regulating human sperm motility, the underlying mechanism needs to be further illustrated. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study provide insight into the novel role of lysine 2-hydroxyisobutyrylation in human sperm and suggest that abnormality of sperm lysine 2-hydroxyisobutyrylation may be one of the causes for asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S) National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). The authors have no conflicts of interest to declare. adenosine triphosphate, asthenozoospermia, lysine 2-hydroxyisobutyrylation, protein posttranslational modifications, sperm motility Introduction Protein posttranslational modifications (PTMs), which regulate cellular processes and diversify the proteome, are the covalent processing events that modulate the physical and chemical properties of target proteins, through either proteolytic cleavage or the addition of a modifying group to amino acids (Venne et al., 2014; Rousseaux and Khochbin, 2015). Among the modified amino acids, lysine is a hot spot for PTMs since it is an alkaline amino acid with an extremely unstable and highly nucleophilic ε-NH2 side chain that can react with various chemical groups (Papanicolaou et al., 2014; Xu et al., 2017). With the development of high-resolution mass spectrometry, more than 100 hundred types of novel protein lysine modifications have been identified (Huang et al., 2015; Sabari et al., 2017; Simithy et al., 2017; Mnatsakanyan et al., 2018), and more importantly, these protein lysine modifications dynamically regulate various biological events and cellular processes (Rousseaux and Khochbin, 2015; Dutta et al., 2016; Sabari et al., 2017; Simithy et al., 2017). Lysine 2-hydroxyisobutyrylation, a newly identified protein lysine modification, is broadly conserved in both eukaryotic and prokaryotic cells (Dai et al., 2014). Lysine 2-hydroxyisobutyrylation neutralises the positive charge of lysine and introduces a hydroxyl group that enables the modified lysine to form hydrogen bonds with other molecules to regulate protein functions (Dai et al., 2014). Proteomic screening of lysine 2-hydroxyisobutyrylation has shown that lysine 2-hydroxyisobutyrylation occurs in both histones and non-histones that participate in a variety of biological functions and processes, including the tricarboxylic acid cycle (Wu et al., 2018), glycolysis/gluconeogenesis (Huang et al., 2017b; Huang et al., 2018) and transcription, among others. (Wu et al., 2018). Of note, lysine 2-hydroxyisobutyrylation especially enriched in mitochondrial proteins is involved in energy metabolic networks (Huang et al., 2017a). In HeLa cells, elements that regulate lysine 2-hydroxyisobutyrylation have already been characterised. Lysine 2-hydroxyisobutyrylation is derived from 2-hydroxyisobutyrate (2-Hib) and 2-hydroxyisobutyryl-coenzyme A (Dai et al., 2014; Huang et al., 2017a). Acyltransferases P300 and lysine acetyltransferase 5 (KAT5), a MYST family acetyltransferase member, act as 2-hydroxyisobutyryltransferases to add 2-hydroxyisobutyryl to the lysine in substrate proteins (Huang et al., 2017a; Huang et al., 2018), while histone deacetylase 2 (HDAC2) and 3 (HDAC3) serve as de-2-hydroxyisobutyrylases to remove lysine 2-hydroxyisobutyrylation (Huang et al., 2017a). Interestingly, a recent study has demonstrated the occurrence of lysine 2-hydroxyisobutyrylation in mouse spermatogenic cells (Dai et al., 2014; Moretti et al., 2016). The distribution of lysine 2-hydroxyisobutyrylation varies during spermatogenesis. Lysine 2-hydroxyisobutyrylation is elevated in spermatogonia and reduced in spermatocytes, indicating histone lysine 2-hydroxyisobutyrylation is closely related to transcriptional activity. In meiotic and post-meiotic cells, the transcriptional repression of sex chromosomes is associated with the depletion of histone lysine 2-hydroxyisobutyrylation, except for the genes on these chromosomes that escape inactivation in round spermatids (Dai et al., 2014; Moretti et al., 2016). Intriguingly, lysine 2-hydroxyisobutyrylation maintains a considerable level in elongating spermatids undergoing histone-to-transition replacement of proteins at the later stage of spermatogenesis (Dai et al., 2014), suggesting the presence of lysine 2-hydroxyisobutyrylation in mature sperm. Unlike spermatogenic cells, mature sperm are almost transcriptionally and translationally silent, since they have a highly condensed chromatin architecture and they undergo several post-meiotic events, such as histone-to-protamine transition (Gur and Breitbart, 2008). Therefore, the role of lysine 2-hydroxyisobutyrylation in mature sperm may be unique and warrants exploration. In this study, we characterised global lysine 2-hydroxyisobutyrylation in normal human sperm using a qualified pan anti-lysine 2-hydroxyisobutyrylation antibody via immunoblotting and immunofluorescence, with super-resolution structured illumination microscopy (SIM). The regulatory elements (regulatory enzymes and cofactor) for lysine 2-hydroxyisobutyrylation were also studied in human sperm. In addition, the present study explored the correlation between sperm lysine 2-hydroxyisobutyrylation and sperm motility, as well as the role that lysine 2-hydroxyisobutyrylation plays in asthenozoospermia, which is a common disorder consisting of sperm motility defects that account for almost half of male infertility cases (Saraswat et al., 2017). Our results may provide new insights into the function of lysine 2-hydroxyisobutyrylation in human sperm. Materials and Methods Chemicals Pan anti-lysine 2-hydroxyisobutyrylation rabbit polyclonal antibody (PTM-801) and pan anti-lysine acetylation rabbit polyclonal antibody (PTM-101) were obtained from PTM BioLabs Inc. (Hangzhou, China). Anti-HDAC2 rabbit polyclonal antibody (16152-1-AP), anti-HDAC3 rabbit polyclonal antibody (10255-1-AP), anti-KAT5 rabbit polyclonal antibody (10827-1-AP), anti-cytochrome c oxidase subunit 6B1 (COX6B1) rabbit polyclonal antibody (11425-1-AP), anti-protamine 2 (PRM2) rabbit polyclonal antibody (14500-1-AP), anti-beta-ACTIN mouse monoclonal antibody (66009-1-Ig) and anti-GAPDH mouse monoclonal antibody (60004-1-Ig) were acquired from Proteintech Group, Inc. (Rosemont, IL, USA). Rabbit (or mouse) IgG, horseradish peroxidase (HRP)-conjugated goat anti-rabbit (or mouse) secondary antibody, DyLight 488-conjugated goat anti-rabbit secondary antibody, minimum essential medium (MEM, 12492013), fetal bovine serum (FBS, 10099133) and antibiotic-antimitotic solution (15240062) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). C646 (HY-13823, purity: >98.0%); nicotinamide (NAM, HY-B0150, purity: >98.0%) and trichostatin A (TSA, HY-15144, purity: >99.53%) were purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA). A normal goat serum (NGS, SL038) and 4′,6-diamidino-2-phenylindole (DAPI, C0065) was purchased from Solarbio Life Sciences (Beijing, China). A human serum albumin (HSA) solution (100 mg/mL, 10064) was purchased from Vitrolife Corporation (Göteborg, Sweden). 2-Hib (164 976, purity: 98.0%), methylcellulose (M0512, viscosity: 4000 cP), phosphate-buffered saline (PBS, P3813) and other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. HeLa cell culture HeLa cells were purchased from Procell Life Science and Technology Co. Ltd. (Wuhan, China). The cells were cultured at 37°C and 5% CO2 for 3 days in MEM, supplemented with 10% FBS and 1% antibiotic-antimitotic solution. Subsequently, the cells were processed by the trypsin digestion method and collected by centrifugation (200g, 3 min). Ethical approval All participants in this study signed the informed consent form. The collection of semen and experiments in this study were approved by the Institutional Ethics Committee on human subjects of Jiangxi Maternal and Child Health Hospital. Semen sample collection Semen samples were collected by masturbation after 3–5 days of sexual abstinence from the participants who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital (Nanchang, China) between May 2017 and November 2018. Totally, 58 normozoospermic men who had known reproductive histories from the past 2 years and normal sperm quality as well as 65 asthenozoospermic men (progressive motility <32%) with normal sperm concentration and morphology were recruited in this study according to the WHO laboratory manual for the examination and processing of human semen (Fifth edition, http://www.who.int/reproductivehealth/publications/infertility/9789241547789/en/). The semen characteristics of the participants are shown in Table I. Semen characteristics of the participants. Table I Semen characteristics of the participants. Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 The reference limit (Reference) is according to WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Routine semen analysis was carried out immediately after liquefaction. SEM: standard error of the mean. *P < 0.05, unpaired t test. Open in new tab Table I Semen characteristics of the participants. Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 Test . Normozoospermic men (n = 58) . Asthenozoospermic men (n = 65) . Reference . Mean ± SEM (range) . Mean ± SEM (range) . Age (years old) 26.4 ± 0.7 (20.0–35.0) 28.4 ± 0.5 (19.0–38.0) Reproductive age Semen volume (mL) 2.7 ± 0.1 (1.5–5.2) 2.8 ± 0.1 (1.5–6) 1.5–7 liquefaction time (min) <60 <60 <60 pH 7.5 ± 0.03 (7.2–8.0) 7.5 ± 0.02 (7.2–8.0) 7.2–8.0 Sperm count (106 cell/mL) 55.4 ± 4.0 (16.2–162.6) 58.1 ± 3.4 (16.4–159.6) >15 Viability (%) 74.7 ± 0.4 (70.0–82.5) 76.5 ± 0.6 (66.1–83.6) >50 Total motility (%) 57.8 ± 0.7 (49.0–68.9) 30.7 ± 1.2* (17.0–45.6) >40 Progressive motility (%) 40.7 ± 0.7 (33.5–48.0) 24.0 ± 0.8* (8.9–31.5) >32 Morphology (% normal) 8.1 ± 0.3 (6.0–12.0) 8.5 ± 0.2 (5.0–12.0) >4 The reference limit (Reference) is according to WHO laboratory manual for the examination and processing of human semen (WHO, 2010). Routine semen analysis was carried out immediately after liquefaction. SEM: standard error of the mean. *P < 0.05, unpaired t test. Open in new tab Human sperm treatments After liquefaction, the sperm samples were purified by direct swim-up in human tubal fluid (HTF) medium described in our previous study (Cheng et al., 2019). For chemical treatments, 1-ml aliquots of purified sperm samples from healthy individuals were exposed to the following doses of C646, NAM, TSA and 2-Hib in HTF at 37°C in a 5% CO2 incubator for 1 h: 0, 0.25, 0.5, 1 and 2 μM of C646 (prepared in HTF containing 0.1% DMSO, pH 7.4); 0, 10, 20, 40 and 80 mM of NAM (prepared in HTF, pH 7.4); 0, 10, 20, 40 and 80 μM of 2-Hib (prepared in HTF, pH 7.4); and 0, 1.5, 3, 6 and 12 μM of TSA (prepared in HTF containing 0.1% DMSO, pH 7.4). After chemical treatments, sperm lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays. Viability, total motility, progressive motility and the adenosine triphosphate (ATP) concentrations of human sperm were also assessed. Immunoblotting assay Total proteins from HeLa cells and human sperm were extracted as described previously (Luo et al., 2015). For the isolation of sperm proteins, sperm samples were first treated with somatic cell lysis buffer (0.2% sodium dodecyl sulphate and 0.5% Triton X-100 in HTF medium) to eliminate residual somatic cells. Proteins from sperm mitochondrion, cytoplasm and head proteins were obtained as described previously (Cheng et al., 2019). The protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, 30 μg of proteins was used for western blotting as previous described (Luo et al., 2015). The dilutions used for the primary antibodies were as follows: 1:500 for anti-COX6B1 antibody; 1:1000 for pan anti-lysine 2-hydroxyisobutyrylation antibody, pan anti-lysine acetylation antibody, anti-PMR2 antibody, anti-HDAC2 antibody, anti-HDAC3 antibody and anti-KAT5 antibody; 1:10000 for anti-ACTIN antibody; and 1:20000 for anti-GAPDH antibody. The dilutions used for the secondary antibodies were 1:10000 for HRP-conjugated goat anti-rabbit (or mouse) secondary antibody. The grey values of the target bands detected by the corresponding antibodies were quantified using Image J software (version 1.44, National Institutes of Health), and then statistical analysis of these values was conducted by normalising to the values detected by ACTIN (loading control). For semi-quantifying lysine 2-hydroxyisobutyrylation in different compartments of human sperm, the grey values of lysine 2-hydroxyisobutyrylation were normalised to those of GAPDH (loading control for total and cytoplasmic lysine 2-hydroxyisobutyrylation), COX6B1 (loading control for mitochondrial lysine 2-hydroxyisobutyrylation) and PMR2 (loading control for head lysine 2-hydroxyisobutyrylation), respectively. Each experiment was repeated at least three times. Indirect immunofluorescence assay Immunofluorescence staining was conducted according to our previously published paper (Cheng et al., 2019). Sperm samples and HeLa cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, permeabilised with 0.2% Trion X-100 in PBS and blocked with 10% NGS in PBS, followed by incubation with rabbit IgG (negative control, 1:100) or pan anti-lysine 2-hydroxyisobutyrylation antibody (1:50) and then staining with DyLight 488 conjugated goat anti-rabbit secondary antibody (1:200 dilution) and 0.5 μM DAPI. The stained cells were imaged with the Nikon N-SIM super-resolution microscopy system (Nikon Co. Ltd., Tokyo, Japan) which has twice the resolution of conventional optical microscopes (~115 nm in three-dimensional (3D) SIM mode). To achieve the super-resolution, the images were captured with the Nikon ECLIPSE Ti-E inverted microscope, using a CFI Apochromat TIRF 100x oil objective lens (N.A. 1.49). DyLight 488 was visualised with a 488-nm laser excitation and a BP 515–545-nm emission filter. The DAPI was visualised with a 405-nm laser excitation and a BP 440–485-nm emission filter. The typical images with single sperm that has representative staining and normal morphology, without curly tail, were used for reconstructing the 3D-SIM images with the NIS-Elements Advanced Research microscope imaging software (Nikon). To analyse sperm lysine 2-hydroxyisobutyrylation in detail, sperm tails were divided into 0.5-μm-axial-length regions of interest (ROIs), and the mean lysine 2-hydroxyisobutyrylation fluorescence intensity of each ROI was quantified by NIS-Elements Advanced Research microscope imaging software. In total, 80 ROIs corresponding to 40-μm-axial-length tails from the initial point of midpiece (as 0 μm) to the principle piece were analysed for each stained sperm. The axial distribution of lysine 2-hydroxyisobutyrylation in sperm tails was reflected by an X–Y curve, which takes the axial length as the X-axis (0.5 μm each point), and the values of mean lysine 2-hydroxyisobutyrylation fluorescence intensity of each ROI are taken as the Y-axis. In total, six normozoospermic donors (10 sperm for each donor) for each concentration group in chemical (C646, NAM, TSA and 2-Hib) treatments and five sperm from each clinical sample were analysed for the semi-quantification. Assessment of sperm viability, motility and penetration ability Sperm viability was examined using the eosin staining method as described previously (Zou et al., 2017). A computer-assisted sperm analysis (CASA) system (WLJY-9000, WeiLi Co., Ltd, Beijing, China) was employed to examine sperm total motility and progressive motility. A minimum of 200 sperm were calculated for each assay. To evaluate penetration ability, human sperm were first incubated in capacitating HTF++ medium (HTF plus 25 mM NaHCO3 and 0.4% HSA) for 2 h and then treated with 2-Hib for 1 h. Sperm penetration into a 1% (w/v) methylcellulose solution prepared in HTF++ medium, mimicking the viscous environment of the female reproductive tract, was evaluated as described previously (Zou et al., 2017). ATP measurement in human sperm The ATP level was detected using a rapid bioluminescent ATP assay Kit (KA1661, Abnova Corporation, Taipei, Taiwan). The measurement was performed according to the manufacturer’s instructions. Statistical analysis All the statistical analyses were performed by the statistics software GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± SEM and follow normal distribution measured by Shapiro–Wilk test (P > 0.05). Differences between the lysine 2-hydroxyisobutyrylation level in normozoospermic and asthenozoospermic men were assessed using the unpaired t test, while differences in sperm lysine 2-hydroxyisobutyrylation between the controls and the chemically treated samples were assessed using two-way ANOVA analysis and the Dunnett’s test. Linear regression was performed by GraphPad Prism. Statistically significant differences were determined at P < 0.05. Results Lysine 2-hydroxyisobutyrylation mainly occurs in proteins located in the tails of human sperm To examine whether lysine 2-hydroxyisobutyrylation occurs in human sperm, we performed immunoblotting and immunofluorescence assays with a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody, which specifically recognises 2-hydroxyisobutyrylated lysines in both histone and non-histone substrates, but not an unmodified lysine or other lysine modifications such as acetylation, butyrylation, and β-hydroxybutyrylation (Dai et al., 2014; Huang et al., 2017a; Huang et al., 2018; Wu et al., 2018). The effectiveness of this antibody was validated in HeLa cells (Fig. 1A and Supplementary Fig. S1). Lysine 2-hydroxyisobutyrylation occurred in a wide range of proteins (25–100 kDa) in mature human sperm (Fig. 1A). The immunofluorescence assay demonstrated that lysine 2-hydroxyisobutyrylated proteins were widely distributed along the tail, especially around the median mitochondrial sheath of human sperm, while only a few were situated in the acrosome region (Fig. 1B). A negative control, where pan anti-lysine 2-hydroxyisobutyrylation antibody was replaced with rabbit IgG, showed no fluorescence staining (Supplementary Fig. S1). In addition, immunoblotting assays, with proteins purified from different sperm compartments, showed that lysine 2-hydroxyisobutyrylation was mainly present in proteins located in the cytoplasm and mitochondria (Fig. 1C and Supplementary Fig. S2), consistent with the results of the immunofluorescence assay. The presence of lysine 2-hydroxyisobutyrylation in human sperm. (A) Immunoblotting detection (left panel) of lysine 2-hydroxyisobutyrylation in human sperm (Sperm). In parallel, the effectiveness of pan anti-lysine 2-hydroxyisobutyrylation antibody was confirmed in HeLa cells (HeLa). Right panel shows Coomassie Brilliant Blue staining. (B) The location of 2-hydroxyisobutyrylated proteins in human sperm was determined by super-resolution structured illumination microscopy (SIM). Scale bar: 5 μm. (C) Khib in different compartments of human sperm. Semi-quantification of Khib was performed as described in the ‘Materials and Methods’ section. The error bars denote the mean ± SEM. Khib: lysine 2-hydroxyisobutyrylation; WB: western blot; CBS: Coomassie Brilliant Blue staining; TP: total proteins; Mit: mitochondrial proteins; Head: head proteins; Cyt: cytoplastic proteins. Experiments were replicated in six normozoospermic donors. Figure 1 Open in new tabDownload slide Figure 1 Open in new tabDownload slide Sperm lysine 2-hydroxyisobutyrylation can be modulated by regulatory elements effective in somatic cells In somatic cells, lysine 2-hydroxyisobutyrylation is derived from 2-Hib and can be regulated by P300, KAT5, HDAC2 and HDAC3 (Dai et al., 2014; Huang et al., 2017a; Huang et al., 2018). To explore whether these regulatory elements work efficiently in human sperm, we first examined the presence of P300, KAT5, HDAC2 and HDAC3 in human sperm. In our previous study, the presence of P300 in human sperm had already been confirmed (Cheng et al., 2019). In this study, HDAC2 but not HDAC3 and KAT5 were detected in human sperm (Sperm, Supplementary Fig. S3) although all of them were detected in HeLa cells using immunoblotting assays (HeLa, Supplementary Fig. S3), which was consistent with the published human sperm proteome that contains 6198 proteins (Amaral et al., 2014). Subsequently, inhibitors for P300 (C646) and HDAC class I/II (TSA) were employed to verify the involvement of these proteins in the regulation of human sperm lysine 2-hydroxyisobutyrylation, since a knockdown assay was unfeasible in mature sperm. The immunoblotting assay showed that 0.5, 1 and 2 μM of C646 could not only decrease the global levels of sperm lysine 2-hydroxyisobutyrylation by about 20, 40 and 40%, respectively (Fig. 2A and Supplementary Fig. S4), but it could also lower the level of sperm lysine acetylation (Supplementary Figs S4 and S5). However, TSA did not change human sperm lysine 2-hydroxyisobutyrylation (Fig. 2B and Supplementary Fig. S6), although it increased the global level of lysine acetylation (Supplementary Figs S5 and S6). Consistent with the immunoblotting results, the immunofluorescence assay showed that C646 had an adverse effect on lysine 2-hydroxyisobutyrylation that occurred along the sperm tail (Supplementary Fig. S4D–F), and TSA did not change the level or the localisation of sperm lysine 2-hydroxyisobutyrylation (Supplementary Fig. S6D–F). We also examined the effect of a potent and selective HDAC2 inhibitor, santacruzamate A, on human sperm lysine 2-hydroxyisobutyrylation and found no effect (data not shown). Furthermore, based on the published results that nicotinamide adenine dinucleotide-dependent deacetylase sirtuins (especially sirtuin 5) act as deacylases—rather than deacetylases—in somatic cells (Hirschey and Zhao, 2015; Sabari et al., 2017) and human sperm (Cheng et al., 2019), we examined the effect of NAM (a pharmacological sirtuin inhibitor) on human sperm lysine 2-hydroxyisobutyrylation. We found that 20, 40 and 80 mM of NAM increased both the global level of lysine 2-hydroxyisobutyrylation along the human sperm tail by 30–50% (Fig. 2C and Supplementary Fig. S7) and the global level of lysine acetylation (Supplementary Figs S5 and S7), suggesting the involvement of sirtuins in removing lysine 2-hydroxyisobutyrylation in human sperm. In addition, 20, 40 and 80 μM of 2-Hib elevated the global level of sperm lysine 2-hydroxyisobutyrylation by 70–120% (Fig. 2D and Supplementary Fig. S8), without affecting sperm lysine acetylation (Supplementary Figs S5 and S8), indicating that lysine 2-hydroxyisobutyrylation is also derived from 2-Hib in human sperm. Effects of C646, trichostatin A (TSA), nicotinamide (NAM) and 2-hydroxyisobutyrate (2-Hib) on human sperm lysine 2-hydroxyisobutyrylation (Khib). Khib was semi-quantified as relative grey value (Khib/ACTIN) by normalising the total grey values of all the bands detected by anti-Khib antibody to the value detected by ACTIN antibody (loading control) using Image J software (version 1.44, National Institutes of Health). Human sperm were incubated with C646 (A), TSA (B), NAM (C) and 2-Hib (D), and sperm Khib was detected using an immunoblotting assay and semi-quantified as described in the ‘Materials and Methods’ section. The error bars denote the mean ± SEM. *P < 0.05, two-way ANOVA of variance. Experiments were replicated in six normozoospermic donors. Figure 2 Open in new tabDownload slide Figure 2 Open in new tabDownload slide Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreases sperm motility, penetration ability and the ATP level of human sperm The present study has shown that lysine 2-hydroxyisobutyrylation mainly occurs in sperm tails (Fig. 1), where the motility-related proteins are located. In addition, global characterisation of lysine 2-hydroxyisobutyrylation in somatic cells indicated the involvement of lysine 2-hydroxyisobutyrylation in energy metabolism. Here, we evaluated whether sperm lysine 2-hydroxyisobutyrylation regulated sperm motility and penetration ability, two energy-consuming processes vital for fertilisation. Although C646, NAM and 2-Hib could change the level of sperm lysine 2-hydroxyisobutyrylation (Fig. 2), only 2-Hib was relatively specific since C646 and NAM could also affect other protein lysine modifications in human sperm (Cheng et al., 2019). Therefore, we examined the effect of 2-Hib on sperm motility and penetration ability and found that 20, 40 and 80 μM of 2-Hib reduced the total motility (especially 40 and 80 μM of Hib reducing total motility below the normal level, <40%, Fig. 3A), decreased progressive motility to an asthenozoospermic level (<32%, Fig. 3B) and lowered penetration ability by 20–40% (Fig. 3C and D), without affecting sperm viability (Fig. 3E). In addition, the effect of 2-Hib on the ATP concentration in human sperm was also assessed, showing that 2-Hib lowered the ATP concentration in human sperm by approximately 30% (Fig. 3F). Effects of 2-hydroxyisobutyrate (2-Hib) on viability, motility and adenosine triphosphate (ATP) level of human sperm. Human sperm were incubated with 0, 10, 20, 40 and 80 μM 2-Hib for 1 h. Total motility (A) and progressive motility (B) of human sperm were analysed by computer-aided sperm analysis. (C and D) show penetration ability. Three fields at 1 cm (C) and 2 cm (D) from the base of the tube were counted, and the average cells/field was calculated. The cell densities were normalised to values from parallel, untreated controls (0 μM 2-Hib). (E) Sperm viability was examined using the eosin staining method. (F) Intracellular ATP was detected using a rapid bioluminescent ATP assay. Sperm from 15 normozoospermic donors were analysed in each assay. The error bars denote the mean ± SEM. *P < 0.05, two-way analysis of variance. Figure 3 Open in new tabDownload slide Figure 3 Open in new tabDownload slide Sperm lysine 2-hydroxyisobutyrylation level is enriched in asthenozoospermic men and negatively correlated with progressive motility of human sperm To further validate the correlation between the lysine 2-hydroxyisobutyrylation level and sperm motility, we examined whether sperm lysine 2-hydroxyisobutyrylation was abnormal in asthenozoospermic men compared to normozoospermic men. Firstly, the lysine 2-hydroxyisobutyrylation level in semen samples from 123 males (58 normozoospermic men and 65 asthenozoospermic men) was determined by an immunoblotting assay. A typical example of the immunoblotting assay showed that the level of sperm lysine 2-hydroxyisobutyrylation in asthenozoospermic men was generally higher compared to normozoospermic men (Supplementary Fig. S9A and B). The quantification result confirmed that the mean level of sperm lysine 2-hydroxyisobutyrylation was increased by ~50% in asthenozoospermic men compared to sperm lysine 2-hydroxyisobutyrylation in normozoospermic men (P < 0.05, Fig. 4A). Linear regression analysis of sperm lysine 2-hydroxyisobutyrylation level and progressive motility of the 123 males was conducted to evaluate the relationship between progressive motility and the lysine 2-hydroxyisobutyrylation level of human sperm. The results showed that the sperm lysine 2-hydroxyisobutyrylation level was negatively correlated with progressive motility of human sperm (r2 = 0.81, P < 0.05, Fig. 4B). All these results supported a tight association between lysine 2-hydroxyisobutyrylation and sperm motility. Furthermore, according to the relative levels of sperm lysine 2-hydroxyisobutyrylation in normozoospermic men (0.45–1.75), high lysine 2-hydroxyisobutyrylation samples were classed based on a threshold of 1.75, which corresponded to the highest level of lysine 2-hydroxyisobutyrylation in normozoospermic controls (Fig. 4A); thus, 18 asthenozoospermic men—with a sperm lysine 2-hydroxyisobutyrylation level higher than 1.75—were classed as having high lysine 2-hydroxyisobutyrylation samples (red frame, Fig. 4A) and accounted for 27.69% (18/65) of the asthenozoospermic men. These results were verified by the immunofluorescence assay, showing that enrichment of lysine 2-hydroxyisobutyrylation fluorescence in these 18 asthenozoospermic men was present along the entire tail and the acrosome (Supplementary Fig. S9C and D). We further examined whether the sperm ATP level (a key factor for motility) was affected in these 18 asthenozoospermic men. As a control, 18 normozoospermic men were randomly recruited from the 58 normozoospermic controls and their sperm ATP levels were also measured. The results showed that the asthenozoospermic men with high lysine 2-hydroxyisobutyrylation levels had significantly lower sperm ATP levels compared to normozoospermic controls (P < 0.05, Fig. 4C). Association between lysine 2-hydroxyisobutyrylation and progressive motility of human sperms. (A) Semi-quantification of lysine 2-hydroxyisobutyrylation in 58 normozoospermic men and 65 asthenozoospermic men was performed as described in the ‘Materials and Methods’ section. High-lysine 2-hydroxyisobutyrylation in asthenozoospermic men, with normalised levels of lysine 2-hydroxyisobutyrylation above the threshold of 1.75, are indicated in the red frame (18 men). (B) The correlation between sperm lysine 2-hydroxyisobutyrylation and progressive motility was assessed by linear regression. (C) The ATP levels in normozoospermic men (18 men randomly recruited from 58 normozoospermic men as control) and asthenozoospermic men, with high Khib levels [the 18 men in red frame in panel (A)] were compared. The Error bars denote the mean ± SEM, *P < 0.05, Student’s t test. Khib: lysine 2-hydroxyisobutyrylation; N: normozoospermic men; A: asthenozoospermic men. Figure 4 Open in new tabDownload slide Figure 4 Open in new tabDownload slide Discussion Extensive studies have shown that tyrosine and threonine/serine phosphorylation regulate sperm activities that are essential for fertilisation (Naz and Rajesh, 2004; Chan et al., 2009; Barbonetti et al., 2010; Visconti et al., 2011), indicating that PTM is vital for sperm function. Only a few recent studies have reported protein lysine modification in mature sperm, showing the presence of lysine methylation and lysine acetylation in the histones of mammalian sperm (Brunner et al., 2014; Jana et al., 2015; Kim et al., 2015) and global lysine acetylation in human sperm (Sun et al., 2014; Yu et al., 2015). Our latest study showed that lysine glutarylation in human sperm is associated with progressive motility (Cheng et al., 2019). However, as the most frequent PTM, the functional role of protein lysine modification in mature sperm needs to be further explored. In somatic cells, lysine 2-hydroxyisobutyrylation is not only present in histones to regulate gene expression like lysine methylation and lysine acetylation, but it is also highly enriched in mitochondrial proteins to control energy metabolism (Tan et al., 2014; Huang et al., 2017a). However, in mature human sperm, only ~10% histones are retained and most of histones are replaced by protamines which may be hardly modified to maintain a highly condensed and transcriptionally silent chromosome. Lysine 2-hydroxyisobutyrylation in the retained histones is too small to be detected by immunofluorescent assay, which may explain the findings that little lysine 2-hydroxyisobutyrylation occurs in sperm nucleus where histones are located. These results suggest that lysine 2-hydroxyisobutyrylation in mature sperm may take part in metabolic pathways rather than transcriptional regulation. Interestingly, mature sperm also have functional mitochondria which serve as the energy metabolism centre vital for sperm motility (Piomboni et al., 2012). Therefore, we hypothesised that lysine 2-hydroxyisobutyrylation regulates sperm motility by controlling energy metabolism in mitochondria. All the results in this study indicate the functional role of sperm lysine 2-hydroxyisobutyrylation in sperm motility and ATP metabolism, supporting our hypothesis. However, more asthenozoospermic men should be studied and a big gap in proving a true physiological role for lysine 2-hydroxyisobutyrylation is the need to show that reduction of lysine 2-hydroxyisobutyrylation can actually improve sperm motility and ATP concentration of asthenozoospermic men with the high level of lysine 2-hydroxyisobutyrylation. To fill in the gap, we need to specifically decrease lysine 2-hydroxyisobutyrylation in human sperm. Although we validated that the reported regulatory proteins (P300 and sirtuins) are effective in human sperm, these proteins are not specific for lysine 2-hydroxyisobutyrylation. Thus, regulatory proteins specific for sperm lysine 2-hydroxyisobutyrylation and/or sperm 2-Hib metabolism should be identified and their modulators should be applied. Our next work will utilise a proteomic approach to identify the potential regulatory enzymes responsible for the high level of sperm lysine 2-hydroxyisobutyrylation in asthenozoospermic men, as well as characterise the 2-hydroxyisobutyrylated lysines in target proteins involved in the regulation of sperm motility. Furthermore, the functions of the identified regulatory enzymes of lysine 2-hydroxyisobutyrylation will be validated by knockout mouse models, and the role of 2-hydroxyisobutyrylated lysines in target proteins will be explored by site-specific antibodies in asthenozoospermic men. Recently, we have characterised another lysine modification, lysine glutarylation, in human sperm (Cheng et al., 2019). Like lysine 2-hydroxyisobutyrylation, lysine glutarylation is also a newly identified lysine modification related to energy metabolism (Tan et al., 2014) and is also associated with human sperm motility (Cheng et al., 2019). Different from lysine 2-hydroxyisobutyrylation, lysine glutarylation is positively correlated with the progressive motility of human sperm and was decreased in asthenozoospermic men (Cheng et al., 2019). These results indicate that lysine 2-hydroxyisobutyrylation and lysine glutarylation may regulate human sperm motility by different underlying mechanisms (target proteins, regulatory enzymes and 2-Hib/glutaric acid metabolisms). In conclusion, lysine 2-hydroxyisobutyrylation mainly localises in sperm tails and is modulated by regulatory enzymes and co-factors that are effective in somatic cells. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and the ATP concentration of human sperm. The mean level of sperm lysine 2-hydroxyisobutyrylation was increased in asthenozoospermic men and negatively correlated with the progressive motility of human sperm. Furthermore, asthenozoospermic men with high lysine 2-hydroxyisobutyrylation levels have low ATP concentrations compared to normozoospermic men with normal lysine 2-hydroxyisobutyrylation levels. Together, our findings elucidate the novel role of lysine 2-hydroxyisobutyrylation and provide more evidence for the functional role of protein lysine modification in mature sperm. A schematic summary of the biochemical pathways of lysine 2-hydroxyisobutyrylation in human sperm was shown in Fig. 5. A schematic summary of the biochemical pathways of lysine 2-hydroxyisobutyrylation in human sperm. Proteins in human sperm tail are modified by lysine 2-hydroxyisobutyrylation. Addition of 2-hydroxyisobutyryl from 2-Hib-CoA to the lysines in target proteins is catalysed by regulatory enzymes such as P300. The 2-hydroxyisobutyryl in the modified proteins can be removed by de-2-hydroxyisobutyrylases such as sirtuins. The 2-hydroxyisobutyrylated proteins may be involved in energy metabolism vital for the production of ATP that is essential for sperm motility. In-vitro addition of 2-Hib elevates sperm lysine 2-hydroxyisobutyrylation but decreases sperm motility and ATP concentration in normozoospermic men. In addition, increased sperm lysine 2-hydroxyisobutyrylation accompanies reduced sperm ATP concentration in asthenozoospermic men. 2-Hib: 2-hydroxyisobutyrate; 2-Hib-CoA: 2-hydroxyisobutyryl-coenzyme A, Khib: lysine 2-hydroxyisobutyrylation. Figure 5 Open in new tabDownload slide Figure 5 Open in new tabDownload slide Acknowledgements The authors are extremely grateful to the volunteers for the participation in this study. The authors are grateful for the helpful assistances of immunoblotting assay from Jingjie PTM BioLab (Hangzhou), Co. Ltd (China). Authors’ roles Y.C. and Z.P. conducted the immunoblotting assay. Y.C., T.L. and Z.P. performed the immunofluorescence assay. H.C. acquired and processed all semen samples. F.W. cultivated the HeLa cells. T.P. measured the ATP concentration. X.H. and F.W. assessed sperm viability, motility and penetration ability. T.L. and Y.C. collected the data and performed the statistical analyses. T.L. was responsible for primary study oversight and design, data acquisition, data analysis and interpretation and the primary writing of the manuscript. All the authors made substantial contributions in critically revising the manuscript. All the authors approved the final manuscript for submission. Funding National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). Conflict of interest The authors have no conflicts of interest to declare. References Amaral A , Castillo J, Ramalho-Santos J, Oliva R. The combined human sperm proteome: cellular pathways and implications for basic and clinical science . Hum Reprod Update 2014 ; 20 : 40 – 62 . Google Scholar Crossref Search ADS PubMed WorldCat Barbonetti A , Vassallo MR, Cordeschi G, Venetis D, Carboni A, Sperandio A, Felzani G, Francavilla S, Francavilla F. Protein tyrosine phosphorylation of the human sperm head during capacitation: immunolocalization and relationship with acquisition of sperm-fertilizing ability . Asian J Androl 2010 ; 12 : 853 – 861 . Google Scholar Crossref Search ADS PubMed WorldCat Brunner AM , Nanni P, Mansuy IM. Epigenetic marking of sperm by post-translational modification of histones and protamines . Epigenet Chromatin 2014 ; 7 : 2 . Google Scholar Crossref Search ADS WorldCat Chan CC , Shui HA, Wu CH, Wang CY, Sun GH, Chen HM, Wu GJ. Motility and protein phosphorylation in healthy and asthenozoospermic sperm . J Proteome Res 2009 ; 8 : 5382 – 5386 . Google Scholar Crossref Search ADS PubMed WorldCat Cheng Y-m , Hu X-n, Peng Z, Pan T-t, Wang F, Chen H-y, Chen W-q, Zhang Y, Zeng X-h, Luo T. Lysine glutarylation in human sperm is associated with progressive motility . Hum Reprod 2019 ; 34 : 1186 – 1194 . Google Scholar Crossref Search ADS PubMed WorldCat Dai L , Peng C, Montellier E, Lu Z, Chen Y, Ishii H, Debernardi A, Buchou T, Rousseaux S, Jin F et al. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark . Nat Chem Biol 2014 ; 10 : 365 – 370 . Google Scholar Crossref Search ADS PubMed WorldCat Dutta A , Abmayr SM, Workman JL. Diverse activities of histone acylations connect metabolism to chromatin function . Mol Cell 2016 ; 63 : 547 – 552 . Google Scholar Crossref Search ADS PubMed WorldCat Gur Y , Breitbart H. Protein synthesis in sperm: dialog between mitochondria and cytoplasm . Mol Cell Endocrinol 2008 ; 282 : 45 – 55 . Google Scholar Crossref Search ADS PubMed WorldCat Hirschey MD , Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation . Mol Cell Proteomics 2015 ; 14 : 2308 . Google Scholar Crossref Search ADS PubMed WorldCat Huang H , Lin S, Garcia BA, Zhao Y. Quantitative proteomic analysis of histone modifications . Chem Rev 2015 ; 115 : 2376 – 2418 . Google Scholar Crossref Search ADS PubMed WorldCat Huang H , Luo Z, Qi S, Huang J, Xu P, Wang X, Gao L, Li F, Wang J, Zhao W et al. Landscape of the regulatory elements for lysine 2-hydroxyisobutyrylation pathway . Cell Res 2017a ; 28 : 111 – 125 . Google Scholar Crossref Search ADS WorldCat Huang H , Tang S, Ji M, Tang Z, Shimada M, Liu X, Qi S, Locasale JW, Roeder RG, Zhao Y et al. p300-mediated lysine 2-hydroxyisobutyrylation regulates glycolysis . Mol Cell 2018 ; 70 : 663 – 678.e666 . Google Scholar Crossref Search ADS PubMed WorldCat Huang J , Luo Z, Ying W, Cao Q, Huang H, Dong J, Wu Q, Zhao Y, Qian X, Dai J. 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae . Proc Natl Acad Sci USA 2017b ; 114 : 8782 – 8787 . Google Scholar Crossref Search ADS WorldCat Jana K , Lenka S, Eva P, Soňa L, Stanislav K, Gabriela L, Zbyněk Z, Petra S, Siarhei D, Jan H et al. Post-translational modifications of histones in human sperm . J Cell Biochem 2015 ; 116 : 2195 – 2209 . Google Scholar Crossref Search ADS PubMed WorldCat Kim SK , Jee BC, Kim SH. Histone methylation and acetylation in ejaculated human sperm: effects of swim-up and smoking . Fertil Steril 2015 ; 103 : 1425 – 1431 . Google Scholar Crossref Search ADS PubMed WorldCat Luo T , Li N, He YQ, Weng SQ, Wang T, Zou QX, Zeng XH. Emodin inhibits human sperm functions by reducing sperm [Ca2+]i and tyrosine phosphorylation . Reprod Toxicol 2015 ; 51 : 14 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat Mnatsakanyan R , Shema G, Basik M, Batist G, Borchers CH, Sickmann A, Zahedi RP. Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry . Expert Rev Proteomics 2018 ; 15 : 515 – 535 . Google Scholar Crossref Search ADS PubMed WorldCat Moretti C , Vaiman D, Tores F, Cocquet J. Expression and epigenomic landscape of the sex chromosomes in mouse post-meiotic male germ cells . Epigenet Chromatin 2016 ; 9 : 47 . Google Scholar Crossref Search ADS WorldCat Naz RK , Rajesh PB. Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction . Reprod Biol Endocrinol 2004 ; 2 : 75 . Google Scholar Crossref Search ADS PubMed WorldCat Papanicolaou KN , O'Rourke B, Foster DB. Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria . Front in Physiol 2014 ; 5 : 301 . Google Scholar Crossref Search ADS WorldCat Piomboni P , Focarelli R, Stendardi A, Ferramosca A, Zara V. The role of mitochondria in energy production for human sperm motility . Int J Androl 2012 ; 35 : 109 – 124 . Google Scholar Crossref Search ADS PubMed WorldCat Rousseaux S , Khochbin S. Histone acylation beyond acetylation: Terra incognita in chromatin biology . Cell J 2015 ; 17 : 1 – 6 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Sabari BR , Zhang D, Allis CD, Zhao Y. Metabolic regulation of gene expression through histone acylations . Nat Rev Mol Cell Biol 2017 ; 18 : 90 – 101 . Google Scholar Crossref Search ADS PubMed WorldCat Saraswat M , Joenväärä S, Jain T, Tomar AK, Sinha A, Singh S, Yadav S, Renkonen R. Human spermatozoa quantitative proteomic signature classifies normo- and asthenozoospermia . Mol Cell Proteomics 2017 ; 16 : 57 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat Simithy J , Sidoli S, Yuan Z-F, Coradin M, Bhanu NV, Marchione DM, Klein BJ, Bazilevsky GA, McCullough CE, Magin RS et al. Characterization of histone acylations links chromatin modifications with metabolism . Nat Commun 2017 ; 8 : 1141 . Google Scholar Crossref Search ADS PubMed WorldCat Sun G , Jiang M, Zhou T, Guo Y, Cui Y, Guo X, Sha J. Insights into the lysine acetylproteome of human sperm . J Proteomics 2014 ; 109 : 199 – 211 . Google Scholar Crossref Search ADS PubMed WorldCat Tan M , Peng C, Anderson KA, Chhoy P, Xie Z, Dai L, Park JS, Chen Y, Huang H, Zhang Y. Lysine glutarylation is a protein post-translational modification regulated by SIRT5 . Cell Metab 2014 ; 19 : 605 – 617 . Google Scholar Crossref Search ADS PubMed WorldCat Venne AS , Kollipara L, Zahedi RP. The next level of complexity: crosstalk of posttranslational modifications . Proteomics 2014 ; 14 : 513 – 524 . Google Scholar Crossref Search ADS PubMed WorldCat Visconti PE , Krapf D, de la Vega-Beltran JL, Acevedo JJ, Darszon A. Ion channels, phosphorylation and mammalian sperm capacitation . Asian J Androl 2011 ; 13 : 395 – 405 . Google Scholar Crossref Search ADS PubMed WorldCat Wu Q , Ke L, Wang C, Fan P, Wu Z, Xu X. Global analysis of lysine 2-hydroxyisobutyrylome upon SAHA treatment and its relationship with acetylation and crotonylation . J Proteome Res 2018 ; 17 : 3176 – 3183 . Google Scholar Crossref Search ADS PubMed WorldCat Xu H , Zhou J, Lin S, Deng W, Zhang Y, Xue Y. PLMD: an updated data resource of protein lysine modifications . J Genet Genomics 2017 ; 44 : 243 – 250 . Google Scholar Crossref Search ADS PubMed WorldCat Yu H , Diao H, Wang C, Lin Y, Yu F, Lu H, Xu W, Li Z, Shi H, Zhao S et al. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm) . Mol Cell Proteomics 2015 ; 14 : 1009 – 1023 . Google Scholar Crossref Search ADS PubMed WorldCat Zou QX , Peng Z, Zhao Q, Chen HY, Cheng YM, Liu Q, He YQ, Weng SQ, Wang HF, Wang T et al. Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa . Hum Reprod 2017 ; 32 : 290 – 298 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes " Joint first authors. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected]. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Human Reproduction – Oxford University Press

Published: Mar 27, 2020

There are no references for this article.