Xia et al. Cell Death and Disease (2019) 10:229 https://doi.org/10.1038/s41419-019-1455-y Cell Death & Disease ARTICLE Open Access Germline deletion of Cdyl causes teratozoospermia and progressive infertility in male mice Xiaoyu Xia1, Xiaowei Zhou2,YanmeiQuan1,YanqinHu1, Fengying Xing3,ZhengzhengLi1,BufangXu2,ChenXu1 and Aijun Zhang1,2 Abstract Chromodomain Y (CDY) is one of the candidate genes for male dyszoospermia related to Y chromosome microdeletion (YCM). However, the function of CDY in regulating spermatogenesis has not been completely determined. The mouse Cdyl (CDY-like) gene is the homolog of human CDY. In the present study, we generated a germline conditional knockout (cKO) model of mouse Cdyl. Significantly, the CdylcKO male mice suffered from the defects in spermatogonia maintenance and spermatozoon morphogenesis, demonstrating teratozoospermia and a progressive infertility phenotype in early adulthood. Importantly, patterns of specific histone methylation and acetylation were extensively changed, which disturbed the transcriptome in CdylcKO testis. Our findings indicated that Cdyl is crucial for spermatogenesis and male fertility, which provides novel insights into the function of CDY gene, as well as the pathogenesis of YCM-related reproductive failure. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction transmission to the offspring. To date, genes including Since the turn of the millennium, the global incidence DAZ (deleted in azoospermia)2, RBMY (RNA binding rate of infertility has been rising continuously. Among all motif protein, Y-linked)3, USP9Y (ubiquitin specific pep- infertility cases, ~50% of them can be attributed to male tidase 9, Y-linked)4, TSPY (testis-specific protein, Y- factors, such as idiopathic oligozoospermia or azoos- linked)5, and CDY (chromodomain Y)6 have been identi- permia. One of the most frequent molecular genetic fied as candidates for YCM. In contrast to the DAZ gene, causes of spermatogenic failure is deletion of the azoos- whose critical impact upon human spermatogenesis has permia factor region of the Y chromosome. Y chromo- been fully elucidated, the functional role of the CDY gene some microdeletion (YCM) was detected in about 2–5% cluster remains unknown. The deletion or low expression of patients with severe oligozoospermia and in 5–10% of of CDY genes is closely correlated with male dyszoos- – patients with azoospermia1. Studies on YCM are parti- permia7 12, however, the precise molecular mechanism cularly important because of its potential for genetic involved remains to be investigated. The human CDY gene family originated by transposi- tion of an autosomal genomic Cdyl gene in primates6,13. Correspondence: Bufang Xu ([email protected]) or Chen Xu (chenx@shsmu. edu.cn) or Aijun Zhang ([email protected]) In mice, there is no CDY gene on the Y chromosome; 1Department of Histo-Embryology, Genetics and Developmental Biology, instead, the autosomal Cdyl gene is homologous to the Shanghai Jiao Tong University, School of Medicine; Shanghai Key Laboratory of human CDY gene family. The protein products of either Reproductive Medicine, 280 South Chongqing Road, Shanghai 200025, China 2Reproductive Medical Center of Ruijin Hospital, Shanghai Jiao Tong University, human CDY/CDYL or mouse Cdyl genes share high School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, China similarity14. In the present study, we generated a Cdyl Full list of author information is available at the end of the article. germline conditional knockout (CdylcKO) mouse model. These authors contributed equally: Xiaoyu Xia, Xiaowei Zhou, and Yanmei cKO Quan The Cdyl male mice demonstrated a phenotype of Edited by M Agostini © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Xia et al. Cell Death and Disease (2019) 10:229 Page 2 of 13 229 teratozoospermia and progressive infertility. In particular, mating experiments lasted for at least 16 weeks. The date of the patterns of histone methylation and acetylation were delivery and the numbers of litters and pups were recorded. globally altered in CdylcKO testis, which led to tran- scriptomic changes and various spermatogenic defects. RNA extraction and quantitative real-time reverse These findings revealed an essential role of mouse Cdyl in transcription PCR male fertility, and provided novel insights into the Total RNA was extracted using the TRIzol reagent mechanism of YCM-related reproductive failure. according to the manufacturer’s protocols (Invitrogen). Reverse transcription reactions were executed using Pri- Materials and methods meScript RT reagent Kit (Takara). The quantitative real- Animals time PCR reaction was prepared using the SYBR Premix The mice used in this study were bred on the C57BL/ Ex Taq Kit (Takara) and performed using an ABI 7500 6 × 129 background. All animal experiments were carried System (Applied Biosystems). Gapdh was used as the out in accordance with the guidelines for the Use of internal control. The primers used in this experiment are Animals in Research issued by the Shanghai Jiao Tong listed in Table S2. University, School of Medicine. Protein preparation and western blotting Whole-mount immunofluorescence staining To prepare the proteins from testes and mouse/human Embryonic day 15.5 (E15.5) embryos were dissected sperm, the samples were lysed in Radio- from euthanized pregnant females, and XY embryonic immunoprecipitation assay buffer and then homogenized gonads (EGs) were collected according a previously pub- on ice. The lysates were then centrifuged at 12,000 × g for lished method15. EGs were washed in phosphate-buffered 10 min at 4 °C to collect the supernatant. Protein con- saline (PBS), transferred into 4% paraformaldehyde, and centrations were measured using a BCA Protein Assay Kit fixed overnight at 4 °C on a rocker. The EGs were then (Beyotime). All samples were processed using 10–12% subjected to three 15 min washes with PBS containing sodium dodecyl sulfate polyacrylamide gel electrophoresis 0.1% Triton-X (PBS-T). All the antibodies were diluted in and electrotransferred to polyvinylidene fluoride mem- PBS-T containing 1 mg/ml bovine serum albumin (BSA). branes. Membranes were blocked for 1 h at room tem- The samples were incubated with primary antibodies for perature with 5% BSA and then incubated with primary 2 days at 4 °C, washed with PBS-T, and then incubated antibodies overnight at 4 °C. After incubation with labeled with the Alexa Fluor-conjugated secondary antibodies for secondary antibodies, signals were obtained using a 1 h at room temperature. After washing with PBS-T, the Visualizer Western Blot Detection Kit (Millipore). The EGs were viewed and photographed under a fluorescence primary antibodies are listed in Table S1. microscope (Leica). The antibodies used in this research are listed in Table S1. Histological and immunohistochemistry assays Male mice were killed at 6 weeks, 14 weeks, or 5 months Generation of cKO mouse and genotyping old. The weights of the whole body, testis, and epididymis Conditional Cdyl knockout mice were generated by were recorded. Tissues were fixed in Bouin’ssolution inserting loxP sites flanking the fifth exon of the ubiqui- overnight. Paraffinsections(5μm) were prepared using tously expressed Cdyl transcript. The targeting vector was standard procedures. Periodic acid-Schiff (PAS) staining electroporated into embryonic stem cells (ESCs) to con- was performed according to the manufacturer's instructions + fl struct the heterozygous Cdyl / ox ESCs, followed by injec- (Beyotime). Staging of mouse seminiferous tubule cross- + fl tion of the Cdyl / ox ESCs into mouse blastocysts. After the sections was performed according to a published method16. fl fl standard breeding procedures, homozygous Cdyl ox/ ox For the immunohistochemical assays, antigen retrieval mice were obtained, which were phenotypically normal. was executed by incubation in buffered citrate (pH 6.0) for fl fl The female Cdyl ox/ ox mice were crossed to the male Vasa- 15 min at 105 °C. The sections were blocked in counter- + fl fl Cre transgenic mice to obtain the Vasa-Cre , Cdyl ox/ ox part IgG for 30 min at room temperature and then incu- fl fl + fl mice (CdylcKO). Either Cdyl ox/ ox or Cdyl / ox male mice bated with primary antibodies at 4 °C overnight. On the were used as controls (CdylCtrl) in this study. DNA was following day, the signals were visualized using a extracted from mouse tails and subjected to PCR. The Histostain-Plus Kit (Life Technologies). Images were fl Δ primers used to detect Vasa-Cre, Cdyl ox, Cdyl (Cdyl captured under a microscope BX53F (Olympus). The deletion) in this assay are listed in Table S2. antibodies used in this assay are listed in Table S1. Mating experiment CASA analysis and sperm staining Each CdylcKO or control male mouse was bred with two The cauda epididymis was dissected from an individual wild-type adult females continuously from 6 weeks old. The mouse, and then incubated in Tyrode’s Solution Official journal of the Cell Death Differentiation Association Xia et al. Cell Death and Disease (2019) 10:229 Page 3 of 13 229 (Solarbio) at 37 °C for 15 min to release the sperms. The was 200 μL. Each tube was fully vortexed before collection supernatant was collected, and sperm counts and motility to ensure equal dispersion of cells and beads.