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Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility Rui-Min Li a, b, 1, Ming-Nan Zhang a, b, 1, 2, Qun-Ye Tang b, c, Man-Gen Song a, b, * a
Institute of Clinical Science, Zhongshan Hospital, Fudan University, Shanghai, 200032, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, 200032, China c Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2020 Accepted 17 March 2020 Available online xxx
The post-transcriptional regulation of gene expression plays an important role in many essential biological processes. The RNA decapping enzyme Dcp2 is a crucial enzyme involved in RNA degradation. Dcp2 proteins are highly expressed in the testis and brain in adult mice. This study aimed to investigate the in vivo functions of Dcp2. An inducible Dcp2 knockout mouse model was established. No obvious health abnormalities were observed after postnatal global deletion of Dcp2 in male mice. However, Dcp2deleted male mice were infertile and showed Sertoli cell vacuolization and germ cell degeneration. Dcp2 deletion resulted in testicular atrophy, reduced number of epididymal sperm, and increased apoptosis in seminiferous tubules. However, spermatocyte-specific deletion of Dcp2 did not compromise the fertility. The findings of this study indicated that Dcp2 was important for spermatogenesis and male fertility. © 2020 Elsevier Inc. All rights reserved.
Keywords: RNA decapping enzyme Dcp2 Spermatogenesis Male infertility Apoptosis
1. Introduction RNA turnover plays an important role in the regulation of gene expression by controlling the RNA level. In eukaryotes, mRNAs typically have two distinct structural characteristics, the 5ʹ 7monomethyl guanosine (m7G) cap [1] and the 3ʹ poly(A) tail [2], which are important for protein translation and stabilization of RNA against ribonuclease hydrolysis. In higher eukaryotic cells, the first step of mRNA decay usually is the removal of 3ʹ poly(A) tail by deadenylases [3]. Subsequently, the deadenylated RNA can be degraded from 5ʹ to 3ʹ or 3ʹ to 5ʹ directions. In the 5ʹ to 3ʹ degradation pathway, the m7G cap structure can be hydrolyzed by the RNA decapping enzymes Dcp2 [4e7] and Nudt16 [8]. Then, the monophosphate RNA is degraded progressively by the exoribonuclease Xrn1 [9]. Although Dcp2 shows decapping activities for all capped RNAs in vitro, it does have substrate selectivity. Dcp2 preferentially binds
* Corresponding author. Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. E-mail address: [email protected] (M.-G. Song). 1 These authors contribute equally to this work. 2 Present address: Department of Immunology and Microbiology, Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
and removes the cap structure of mRNAs with the stem-loop structure in 5ʹ untranslated regions [10,11]. The RNAs with uridine-tract at the 3ʹ end can also be preferentially decapped by Dcp2 [12,13]. The methylation of adenosine in RNAs can also affect the Dcp2 activity. Mauer et al. showed that transcripts with an m7G cap followed by an m6Am or m6A are more stable and resistant to Dcp2 hydrolysis [14]. Dcp2 has been reported to play important roles during growth and development. In yeast, the dcp2D strain has growth defect and grows extremely slowly [4]. An increase in the number of apoptotic cells is also observed in the mutant strain lacking Dcp2 [15]. Dcp2 is required for postembryonic development in Arabidopsis, and the Dcp2 mutant has a lethal phenotype at the seedling cotyledon stage [16]. Dcp2 also plays important roles during oocyte maturation and oocyte-to-zygote transition by the clearance of maternal mRNAs [17e19]. Dcp2 expression is regulated during development. The Dcp2 protein can be detected in nearly all embryonic tissues in mice. However, it is highly expressed only in the testis and brain, expressed to a lesser extent in the spleen and lung, and is not detected in the heart, liver, kidney, and muscle of adults [8]. Thus, Dcp2 can differentially regulate gene expression in a temporal and spatial manner. Previously, attempts were made to generate Dcp2 knockout mice using the gene trap method, but the attempts failed due to the
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Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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leaked expression of Dcp2 protein [8]. Dcp2 conditional knockout mice (Dcp2f/f) were generated to further investigate the functions of Dcp2 in vivo. Dcp2f/f mice were crossed with a ubiquitous Cre recombinase-expressing line (UBC-CreERT2) to delete Dcp2 by tamoxifen administration. No obvious health abnormalities were observed after the global deletion of Dcp2 postnatally in male mice. However, Dcp2 knockout in males caused germ cell degenerative defect and infertility. This study suggested that Dcp2 played an important role in the regulation of spermatogenesis. 2. Materials and methods 2.1. Animal experiments Dcp2 conditional knockout mice were generated by Shanghai Model Organisms Center, Inc. (Shanghai, China) through gene targeting of embryonic stem (ES) cells through homologous recombination in a C57BL/6 background. The targeted mice were crossed with Flp recombinase-expressing mice to remove the neomycin gene. UBC-CreERT2 mice were purchased from Shanghai Model Organisms Center, Inc., and Pgk2-Cre mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Genotypes of mice were determined by polymerase chain reaction (PCR) with primers mDcp2-E-F and mDcp2-F-R for Dcp2f/f mice, Cre-F and Cre-ERT-R for UBC-CreERT2 mice, and Cre-F and CreR for Pgk2-Cre mice. The Dcp2 targeted allele was determined by primers mDcp2-C-F and mDcp2-F-R. The sequences of primers are shown in Table S1. For induced knockout of Dcp2 in mice, tamoxifen (T5648, SigmaeAldrich, MO, USA) was administered to 6-week-old Dcp2f/f and Dcp2f/f; UBC-CreERT2 mice by gavage at a dose of 200 mg/kg in corn oil for five consecutive days. All animal experiments were carried out in accordance with the guidelines for the care and use of experimental animals of the Experimental Animal Ethics Committee of Fudan University in China, and were approved by the Animal Ethics Committee of Zhongshan Hospital of Fudan University.
2.4. RNA isolation, reverse transcription, and real-time quantitative PCR Total RNA was extracted from testes using Tripure Isolation Reagent (Roche) following the manufacturer’s protocols. One microgram total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for quantitative PCR (qPCR; Vazyme Biotech Co., Ltd., Nanjing, China). Then, qPCRs were performed using HieffTM qPCR SYBR Green Master Mix (Yeasen) for individual genes (primer sequences are shown in Table S1). b-actin was used as internal control. 2.5. Enzyme-linked immunosorbent assay All enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Briefly, 5 mL of serums were mixed with 40 mL of PBS (pH 7.4), and then 40 mL of the diluted sample was applied to each well of the antibody-coated plate and incubated with 100 mL of antibodies labeled with horseradish peroxidase for 60 min at 37 C. Substrates A and B were added to each well and incubated for 15 min at 37 C in the dark. The reactions were stopped, and the optical density values at 450 nm were recorded. 2.6. Sperm analysis Epididymides were extracted from male mice. Sperm counts and motility were analyzed using a computer-assisted sperm analysis system (CASAS) (Hamilton Thorne, MA, USA). 2.7. Statistical analysis Statistical analyses were performed using an unpaired t-test with GraphPad Prism 5 (GraphPad Software, CA, USA). All results were performed at least three times independently and indicated as the mean ± standard deviation (SD). A P value less than 0.05 was considered statistically significant. 3. Results
2.2. Western blot analysis
3.1. Dcp2 was not essential for the postnatal survival of male mice
Tissues were lysed in cold lysis buffer [phosphate-buffered saline (PBS) containing 0.25% Triton X-100 and 1 mM dithiothreitol] with a protease inhibitor cocktail (Biotools, Loganholme, Australia) using a homogenizer. Supernatant lysates were harvested after centrifugation at 15,000g for 10 min at 4 C. An anti-Dcp2 antibody was provided by Professor Megerditch Kiledjian (Rutgers University, USA). Anti-caspase-3 (1:1000 dilution, Cat. No. 9662S) and anti-phospho-histone H2A.X (Ser 139) (g-H2AX) (1:1000 dilution, Cat. No. 9718) antibodies were purchased from Cell Signaling Technology (MA, USA). An anti-cleaved caspase-8 (T341) (1:1000 dilution, Cat. No. AP0358) antibody was purchased from Bioworld Technology Inc. (MN, USA). A mouse anti-b-actin antibody (1:5000; Cat. No. 60008-1-Ig) was procured from Proteintech (IL, USA). Protein intensities were visualized using ECL reagent (Yeasen, Shanghai, China).
To further investigate the functions of Dcp2 in vivo, Dcp2 conditional knockout mice were generated to avoid possible embryonic lethality. As shown in Fig. 1A, the exon 2 could be deleted by Cre-Loxemediated gene excision to disrupt the Dcp2 allele (Fig. 1A). Genotypes of the mice were determined by PCR (Fig. 1B). Then, Dcp2f/f; UBC-CreERT2 mice were generated, which ubiquitously expressed recombinase CreER. The 6-week-old male mice were administered with tamoxifen to delete the Dcp2 gene. The resulting mouse strain was referred to as Dcp2D/D. On day 10, Western blot analysis showed that Dcp2 was knocked out in all the tissues tested, although trace amounts of Dcp2 protein could be detected in the Dcp2D/D tissues (Fig. 1C). During the observation period of 6 months, no obvious health abnormalities were found in the Dcp2D/D mice. The body weight of both Dcp2f/f mice and Dcp2D/D mice decreased due to tamoxifen administration, but recovered soon (Fig. 1D). The hair color of the Dcp2D/D male mice also looked fine (data not shown). Thus, it seemed that Dcp2 was not essential for the postnatal survival of adult male mice.
2.3. Histological analysis and TUNEL staining Testes and epididymides were fixed in 4% paraformaldehyde solution overnight, embedded in paraffin, and cut into 5-mm sections. Hematoxylin and eosin (H&E) staining was carried out using standard procedures. TUNEL staining was performed with an InSitu Cell Death Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s protocols.
3.2. Global deletion of Dcp2 caused germ cell degenerative defect in male mice Although the deletion of Dcp2 did not result in health abnormalities in male mice, testicular atrophy occurred as early as 3
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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Fig. 1. Generation of inducible Dcp2 knockout mouse model. (A) Schematic representation of the Dcp2 conditional knockout strategy by homologous recombination in ES cells. (B) Genotyping results of Dcp2 conditional knockout mice. (C) Dcp2 proteins from the indicated tissues of Dcp2f/f (CreER) and Dcp2f/f; UBC-CreERT2 (CreERþ) mice were analyzed by Western blot analysis 10 days after tamoxifen administration. (D) Change in body weight of mice after tamoxifen administration (n ¼ 5).
Fig. 2. Global deletion of Dcp2 in adult male mice resulted in testicular atrophy. (A) Comparison of testis size and testis weight 3 weeks after tamoxifen administration (n ¼ 5, ****P < 0.0001). (B) Expression levels of Dcp2 protein in Dcp2f/f and Dcp2D/D testes were analyzed by Western blot analysis. The results of two pairs of mice were presented. (C) H&E staining of Dcp2f/f and Dcp2D/D testis sections 3 weeks after tamoxifen administration. Arrows indicate elongated spermatids, and asterisks indicate the vacuoles in Sertoli cells. Original magnification 400.
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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weeks after tamoxifen administration (Fig. 2A, left). The testes weight of Dcp2D/D mice was reduced by about 70% compared with Dcp2f/f mice (Fig. 2A, right). The knockout of Dcp2 protein in testes was confirmed by Western blot analysis (Fig. 2B). The testis histological sections of Dcp2D/D mice showed that germ cell loss and Sertoli cell vacuolization occurred in many seminiferous tubules, while the differentiation of spermatozoa was not blocked at a specific stage and a few elongated spermatids could be observed (Fig. 2C). The effect of Dcp2 deletion on spermatogenesis was progressive as the germ cells were almost completely absent in most of the seminiferous tubules, and few sperm could be detected in the caudal epididymis 2 months after Dcp2 deletion (Fig. S1). 3.3. Dcp2-deficient male mice were infertile To further investigate the effect of Dcp2 deletion on spermatogenesis, caudal epididymides were excised out and the sperm counts and motility were analyzed using CASAS 3 weeks after tamoxifen administration. Compared with Dcp2f/f mice, the sperm counts of Dcp2D/D mice decreased to about 10% and the motility of sperm from Dcp2D/D mice also significantly reduced (82 ± 8.0% vs 6.3 ± 9.7%) (Fig. 3A). Only a few spermatozoa could be seen in the caudal epididymis of Dcp2D/D mice, and the debris of germ cells could be detected (Fig. 3B). To investigate whether Dcp2D/D mice were fertile, male Dcp2f/f and Dcp2D/D mice were bred with wild-type (WT) female mice 3 weeks after tamoxifen administration. For 4 months, seven Dcp2f/f mating pairs had 35 litters with an average of 6.7 pups, whereas five Dcp2D/D mating pairs failed to produce any offspring (Table 1). Surprisingly, the copulation capacity of Dcp2D/D male mice was
Table 1 Summary of Dcp2f/f and Dcp2D/D male mice fertility study. Mice
Numbers
No. of plug in
No. of litters
Average No. of pups
Dcp2f/f Dcp2D/D
7 5
35 3
35 0
6.7 ± 0.5 0
significantly impaired. Only three copulation plugs were found for the five Dcp2D/D male mice during the same mating time (Table 1). The analysis of serum testosterone (TT) levels showed that the levels markedly decreased in Dcp2D/D male mice (15.25 ± 0.53 ng/ mL vs 9.35 ± 0.45 ng/mL), although mRNA levels of TT synthesisrelated genes did not decrease (Fig. 3C and D). However, the synthesis of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in Dcp2D/D mice was not compromised. On the contrary, both increased significantly (40.82 ± 1.86 mIU/mL vs 56.21 ± 3.01 mIU/mL for FSH and 5.03 ± 0.22 mIU/mL vs 7.25 ± 0.22 mIU/mL for LH) (Fig. 3E), suggesting that the Dcp2 knockout-caused male infertility might not be regulated through the hypothalamicepituitaryegonadal axis. The findings indicated that Dcp2 deficiency resulted in male sterility.
3.4. Dcp2 was not required for the differentiation of spermatocyte into sperm, but was essential for embryonic development Dcp2 was reported to be expressed in round spermatids in mice [20]. To investigate whether Dcp2 was necessary for the differentiation of spermatids, Dcp2f/f mice were bred with Pgk2-Cre mice [21] to delete the Dcp2 gene in spermatocytes, generating Dcp2SP-KO mice.
Fig. 3. Effect of Dcp2 deficiency on sperm production and hormone secretion. (A) Number and motility of sperm were analyzed using CASAS (n ¼ 4, ****P < 0.0001). (B) H&E staining of caudal epididymides sections. Arrows indicate the debris of germ cells. Original magnification 400. (C) Serum TT concentrations were determined using ELISA (n ¼ 3, ***P ¼ 0.0001). (D) mRNA levels of Dcp2 and TT synthesis-related genes were analyzed by qPCR. (E) Serum levels of FSH and LH were determined using ELISA (n ¼ 3, P values are shown on the top). All experiments were performed 3 weeks after tamoxifen administration.
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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Dcp2SP-KO male mice were bred with WT female mice. They had offspring with an average of 7.6 pups per litter, which was comparable to control mice with an average of 8.2 pups per litter (Fig. S2A), suggesting that Dcp2SP-KO male mice were fertile. All the offspring of Dcp2SP-KO male mice were confirmed to be Dcp2þ/ genotype (Fig. S2B), suggesting that Dcp2 could be efficiently deleted in spermatocytes. Western blot analysis showed that the expression of Dcp2 was only slightly reduced in Dcp2SP-KO testes than in control testes (Fig. S2C), indicating that Dcp2 was also expressed in other cells of testis besides spermatocytes. These data indicated that the deletion of Dcp2 in spermatocytes did not affect the fertility of male mice. Dcp2þ/ mice appeared healthy, and intercrosses of Dcp2þ/ mice yielded only Dcp2þ/þ and Dcp2þ/ mice in a non-Mendelian manner with more Dcp2þ/þ mice than Dcp2þ/ mice, no live-born Dcp2/ mice were obtained (Table S2), indicating that Dcp2 knockout was embryonically lethal. No Dcp2/ embryos were found when E13.5 embryos were analyzed. However, the ratio of Dcp2þ/þ embryos to Dcp2þ/ embryos was about 1:3 (Table S2), suggesting that part of the Dcp2þ/ embryos were terminated during development. These data demonstrated that Dcp2 was essential for embryonic development. 3.5. Germ cell apoptosis occurred in Dcp2D/D testes The loss of germ cells in Dcp2D/D testes was further confirmed by the analysis of the marker of individual cell types using qPCR. Three
5
weeks after Dcp2 deletion, the spermatogonial cells (Magea4) reduced to about 50%, and the primary spermatocytes (Sycp3) decreased to about 10%. However, the expression level of the Sertoli cell marker, Sox9, increased by about 26-fold possibly due to the increased ratio of Sertoli cells in testes (Fig. 4A). The Sertoli cell vacuolization is a characteristic of endocytosis of apoptotic germ cells, indicating the occurrence of germ cell apoptosis. A TUNEL assay was performed to determine whether the reduced number of germ cells was due to apoptosis. Compared with a few TUNELpositive cells occasionally seen in Dcp2f/f testes, a large number of TUNEL-positive apoptotic germ cells were observed in the seminiferous tubules in Dcp2D/D testes (0.4 ± 1 per tubule vs 4.5 ± 3.9 per tubule) (Fig. 4B). Consistent with the TUNEL results, the cleavage activation of caspase-8 and caspse-3 significantly increased in the extracts of Dcp2D/D testes (Fig. 4C). Furthermore, the expression level of g-H2AX, a marker of DNA double-stranded breaks, also markedly increased in Dcp2D/D testes (Fig. 4C). These results indicated that Dcp2 deletion resulted in germ cell apoptosis.
4. Discussion In this study, an inducible Dcp2 knockout mouse model was generated to investigate the in vivo functions of Dcp2. The postnatal global deletion of Dcp2 caused degenerative spermatogenic defects, leading to male infertility. The reduction of sperm was due to germ cell apoptosis in Dcp2D/D testes. These results provided evidence
Fig. 4. Germ cell apoptosis occurred in Dcp2D/D testes. (A) Quantitative PCR analysis of spermatogonium marker Magea4, spermatocyte marker Sycp3, and Sertoli cell marker Sox9 in Dcp2f/f and Dcp2D/D testes. (B) TUNEL analysis was performed on Dcp2f/f and Dcp2D/D testes. Original magnification 200. The apoptotic cells of 30 tubules were counted, and the results were shown on the right. ****P < 0.0001. (C) Activation of apoptosis-related proteins in Dcp2f/f (CreER) and Dcp2D/D (CreERþ) testes was analyzed by Western blot analysis. The 18-kDa caspase-8 cleaved fragment (Dcasp-8) and 17/19-kDa caspase-3 cleaved fragments (Dcasp-3) were shown. The results of the three pairs of mice were presented. All experiments were performed 3 weeks after tamoxifen administration.
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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that Dcp2 played an important role in regulating spermatogenesis. The type of cells in which Dcp2 is expressed in the testis is not known. Therefore, the phenotype of Dcp2D/D mice could be caused by the deficiency of Dcp2 in germ cells or testicular somatic cells or hypothalamusepituitary axis, or even a combination of all. Three antibodies (antibody used in this study, antibody generated against 179e422 amino acids of mouse Dcp2, and anti-Dcp2 antibody from Thermo Fisher Scientific, PA5-31091) were used, but failed to show the expression localization of Dcp2 in the testis. A Dcp2-IRES-EGFP knock-in mouse model was generated to trace the expression of Dcp2, but failed due to the weak expression of EGFP proteins [22]. To investigate the exact expression pattern of Dcp2 in the testis, specific antibodies that can be used for immunofluorescence should be developed. The lineage tracing technique can also be used by crossing Dcp2 promoter-controlled Cre recombinaseexpressing mice with reporter mice such as Rosa26-LSL-tdTomato. Then, the corresponding Cre mouse lines can be used to delete Dcp2 in specific cells in the testis, such as Stra8-Cre line for germ cells and Amh-Cre line for Sertoli cells. The following findings implied that the apoptosis of germ cells in Dcp2D/D mice might be induced through the extrinsic pathway: (1) Dcp2 deficiency did not block the differentiation of sperm at a specific stage, while the loss of some germ cell-expressed genes could cause germ cell development arrests at the spermatocyte stage, such as TEX11 [23], or during the round spermatid period, such as SOX30 [24]. (2) Mice containing Dcp2-deficient spermatocytes were fertile, and the expression level of Dcp2 protein only slightly reduced when Dcp2 was deleted in spermatocytes, indicating that Dcp2 was expressed mainly in somatic cells of the testis, such as Sertoli cells. (3) The level of activated caspase-8 increased in Dcp2D/D testes, which was usually activated by the extracellular stimuli such as FasL and TNF-a [25], suggesting that germ cells might have been exposed to extracellular stimuli. To determine the pathway responsible for the apoptosis of germ cell in Dcp2D/D mice, Dcp2 should be deleted in specific cell types of the testis. Dcp2 was involved in the negative feedback regulation of innate immune response, and Dcp2 defect upregulated the expression of chemokine CXCL10 in mouse embryonic fibroblast cells [26]. Jiang et al. reported that mumps virus infection could stimulate Sertoli cells to produce chemokine CXCL10, which could induce germ cell apoptosis [27]. Zika virus also induced innate immune responses in Leydig and Sertoli cells, but not in spermatogonia, resulting in the production of pro-inflammatory cytokines/chemokines, including IL6 and CXCL10, causing testis damage [28]. The deletion of Dcp2 might deregulate the innate immune response and increase the expression levels of some chemokines in the testis, such as CXCL10, which could induce germ cell apoptosis. Whether Dcp2 deficiency activates the innate immune response in the testis and causes spermatogenic defect needs to be further investigated. The deficiency or mutation of many genes can result in testicular atrophy and male sterility in mice, but does not affect their mating behavior [29e31]. However, the present study found that the global deletion of Dcp2 resulted in impaired mating ability. The serum level of TT significantly decreased in the Dcp2D/D mice, while the expression levels of TT synthesis-related genes did not decrease. Whether the decreased TT level in Dcp2D/D male mice was due to the reduced volumes of testes or Dcp2 deletion in Leydig cells is not clear. The impaired mating ability and the spermatogenic defect in Dcp2D/D male mice limited the potential of Dcp2 as a target for designing male contraceptives. However, Dcp2 may be a potential target for developing animal castration drugs. In summary, the study was novel in demonstrating that Dcp2 did not play an important role as an essential gene for embryonic development in the postnatal survival of male mice, but was necessary for spermatogenesis. The deletion of Dcp2 in adult male
mice resulted in infertility by causing the apoptosis of germ cells. Whether Dcp2 is directly involved in the differentiation of spermatozoa or maintains the immune microenvironment of spermatogenesis needs to be further investigated. This study revealed the potential diagnostic and/or therapeutic significance of Dcp2 for men with infertility. Author contributions Rui-Min Li and Ming-Nan Zhang performed the experiments, and Rui-Min Li wrote the manuscript. Qun-Ye Tang was responsible for mouse maintenance. Man-Gen Song designed the experiments and wrote the manuscript. Funding This study was supported by the National Key R&D Program of China (grant number 2018YFA0107501) and the National Natural Science Foundation of China (grant number 81373148). Declaration of competing interest The authors declare no conflicts of interest. Acknowledgments The authors thank Professor Megerditch Kiledjian (Rutgers University, USA) for supplying the anti-Dcp2 antibody. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.03.101. References [1] A.J. Shatkin, Capping of eucaryotic mRNAs, Cell 9 (4 pt 2) (1976) 645e653. [2] A.B. Sachs, Messenger RNA degradation in eukaryotes, Cell 74 (3) (1993) 413e421. [3] C.J. Decker, R. Parker, A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation, Genes Dev. 7 (8) (1993) 1632e1643. [4] T. Dunckley, R. Parker, The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif, EMBO J. 18 (19) (1999) 5411e5422. [5] E. Van Dijk, N. Cougot, S. Meyer, et al., Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures, EMBO J. 21 (24) (2002) 6915e6924. [6] J. Lykke-Andersen, Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay, Mol. Cell Biol. 22 (23) (2002) 8114e8121. [7] Z. Wang, X. Jiao, A. Carr-Schmid, et al., The hDcp2 protein is a mammalian mRNA decapping enzyme, Proc. Natl. Acad. Sci. U. S. A. 99 (20) (2002) 12663e12668. [8] M.G. Song, Y. Li, M. Kiledjian, Multiple mRNA decapping enzymes in mammalian cells, Mol. Cell. 40 (3) (2010) 423e432. [9] C.L. Hsu, A. Stevens, Yeast cells lacking 5’–>3’ exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5’ cap structure, Mol. Cell Biol. 13 (8) (1993) 4826e4835. [10] Y. Li, M.G. Song, M. Kiledjian, Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein, Mol. Cell Biol. 28 (3) (2008) 939e948. [11] Y. Li, E.S. Ho, S.I. Gunderson, et al., Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5’-end stem-loop structures, Nucleic Acids Res. 37 (7) (2009) 2227e2237. [12] M.G. Song, M. Kiledjian, 3’ Terminal oligo U-tract-mediated stimulation of decapping, RNA 13 (12) (2007) 2356e2365. [13] T.E. Mullen, W.F. Marzluff, Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5’ to 3’ and 3’ to 5’, Genes Dev. 22 (1) (2008) 50e65. [14] J. Mauer, X. Luo, A. Blanjoie, et al., Reversible methylation of m(6)Am in the 5’ cap controls mRNA stability, Nature 541 (7637) (2017) 371e375. [15] K.K. Raju, S. Natarajan, N.S. Kumar, et al., Role of cytoplasmic deadenylation and mRNA decay factors in yeast apoptosis, FEMS Yeast Res. 15 (2) (2015).
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
R.-M. Li et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx [16] J. Xu, J.Y. Yang, Q.W. Niu, et al., Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development, Plant Cell 18 (12) (2006) 3386e3398. [17] J. Ma, M. Flemr, H. Strnad, et al., Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse, Biol. Reprod. 88 (1) (2013) 11. [18] Y. Mishima, Y. Tomari, Pervasive yet nonuniform contributions of Dcp2 and Cnot7 to maternal mRNA clearance in zebrafish, Gene Cell. 22 (7) (2017) 670e678. [19] T. Nishimura, H. Fakim, T. Brandmann, et al., Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs, Nucleic Acids Res. 46 (22) (2018) 12008e12021. [20] F. Shapouri, S. Saeidi, R.U. De Iongh, et al., Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2, Cell Tissue Res. 364 (2) (2016) 443e451. [21] H. Ando, Y. Haruna, J. Miyazaki, et al., Spermatocyte-specific gene excision by targeted expression of Cre recombinase, Biochem. Biophys. Res. Commun. 272 (1) (2000) 125e128. [22] M.N. Zhang, Q.Y. Tang, R.M. Li, et al., MicroRNA-141-3p/200a-3p target and may be involved in post-transcriptional repression of RNA decapping enzyme Dcp2 during renal development, Biosci. Biotechnol. Biochem. 82 (10) (2018) 1724e1732. [23] A.N. Yatsenko, A.P. Georgiadis, A. Ropke, et al., X-linked TEX11 mutations,
[24] [25] [26]
[27]
[28] [29]
[30]
[31]
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meiotic arrest, and azoospermia in infertile men, N. Engl. J. Med. 372 (22) (2015) 2097e2107. C.A. Feng, C. Spiller, D.J. Merriner, et al., SOX30 is required for male fertility in mice, Sci. Rep. 7 (1) (2017) 17619. A. Ashkenazi, V.M. Dixit, Apoptosis control by death and decoy receptors, Curr. Opin. Cell Biol. 11 (2) (1999) 255e260. Y. Li, J. Dai, M. Song, et al., Dcp2 decapping protein modulates mRNA stability of the critical interferon regulatory factor (IRF) IRF-7, Mol. Cell Biol. 32 (6) (2012) 1164e1172. Q. Jiang, F. Wang, L. Shi, et al., C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis, Cell Death Dis. 8 (10) (2017) e3146. W. Ma, S. Li, S. Ma, et al., Zika virus causes testis damage and leads to male infertility in mice, Cell 167 (6) (2016) 1511e1524, e10. M.P. Greenbaum, W. Yan, M.H. Wu, et al., TEX14 is essential for intercellular bridges and fertility in male mice, Proc. Natl. Acad. Sci. U. S. A. 103 (13) (2006) 4982e4987. M.K. O’bryan, B.J. Clark, E.A. Mclaughlin, et al., RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility, PLoS Genet. 9 (7) (2013), e1003628. C. Berthet, A.M. Morera, M.J. Asensio, et al., CCR4-associated factor CAF1 is an essential factor for spermatogenesis, Mol. Cell Biol. 24 (13) (2004) 5808e5820.
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Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility Rui-Min Li a, b, 1, Ming-Nan Zhang a, b, 1, 2, Qun-Ye Tang b, c, Man-Gen Song a, b, * a
Institute of Clinical Science, Zhongshan Hospital, Fudan University, Shanghai, 200032, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, 200032, China c Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2020 Accepted 17 March 2020 Available online xxx
The post-transcriptional regulation of gene expression plays an important role in many essential biological processes. The RNA decapping enzyme Dcp2 is a crucial enzyme involved in RNA degradation. Dcp2 proteins are highly expressed in the testis and brain in adult mice. This study aimed to investigate the in vivo functions of Dcp2. An inducible Dcp2 knockout mouse model was established. No obvious health abnormalities were observed after postnatal global deletion of Dcp2 in male mice. However, Dcp2deleted male mice were infertile and showed Sertoli cell vacuolization and germ cell degeneration. Dcp2 deletion resulted in testicular atrophy, reduced number of epididymal sperm, and increased apoptosis in seminiferous tubules. However, spermatocyte-specific deletion of Dcp2 did not compromise the fertility. The findings of this study indicated that Dcp2 was important for spermatogenesis and male fertility. © 2020 Elsevier Inc. All rights reserved.
Keywords: RNA decapping enzyme Dcp2 Spermatogenesis Male infertility Apoptosis
1. Introduction RNA turnover plays an important role in the regulation of gene expression by controlling the RNA level. In eukaryotes, mRNAs typically have two distinct structural characteristics, the 5ʹ 7monomethyl guanosine (m7G) cap [1] and the 3ʹ poly(A) tail [2], which are important for protein translation and stabilization of RNA against ribonuclease hydrolysis. In higher eukaryotic cells, the first step of mRNA decay usually is the removal of 3ʹ poly(A) tail by deadenylases [3]. Subsequently, the deadenylated RNA can be degraded from 5ʹ to 3ʹ or 3ʹ to 5ʹ directions. In the 5ʹ to 3ʹ degradation pathway, the m7G cap structure can be hydrolyzed by the RNA decapping enzymes Dcp2 [4e7] and Nudt16 [8]. Then, the monophosphate RNA is degraded progressively by the exoribonuclease Xrn1 [9]. Although Dcp2 shows decapping activities for all capped RNAs in vitro, it does have substrate selectivity. Dcp2 preferentially binds
* Corresponding author. Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. E-mail address: [email protected] (M.-G. Song). 1 These authors contribute equally to this work. 2 Present address: Department of Immunology and Microbiology, Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
and removes the cap structure of mRNAs with the stem-loop structure in 5ʹ untranslated regions [10,11]. The RNAs with uridine-tract at the 3ʹ end can also be preferentially decapped by Dcp2 [12,13]. The methylation of adenosine in RNAs can also affect the Dcp2 activity. Mauer et al. showed that transcripts with an m7G cap followed by an m6Am or m6A are more stable and resistant to Dcp2 hydrolysis [14]. Dcp2 has been reported to play important roles during growth and development. In yeast, the dcp2D strain has growth defect and grows extremely slowly [4]. An increase in the number of apoptotic cells is also observed in the mutant strain lacking Dcp2 [15]. Dcp2 is required for postembryonic development in Arabidopsis, and the Dcp2 mutant has a lethal phenotype at the seedling cotyledon stage [16]. Dcp2 also plays important roles during oocyte maturation and oocyte-to-zygote transition by the clearance of maternal mRNAs [17e19]. Dcp2 expression is regulated during development. The Dcp2 protein can be detected in nearly all embryonic tissues in mice. However, it is highly expressed only in the testis and brain, expressed to a lesser extent in the spleen and lung, and is not detected in the heart, liver, kidney, and muscle of adults [8]. Thus, Dcp2 can differentially regulate gene expression in a temporal and spatial manner. Previously, attempts were made to generate Dcp2 knockout mice using the gene trap method, but the attempts failed due to the
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Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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leaked expression of Dcp2 protein [8]. Dcp2 conditional knockout mice (Dcp2f/f) were generated to further investigate the functions of Dcp2 in vivo. Dcp2f/f mice were crossed with a ubiquitous Cre recombinase-expressing line (UBC-CreERT2) to delete Dcp2 by tamoxifen administration. No obvious health abnormalities were observed after the global deletion of Dcp2 postnatally in male mice. However, Dcp2 knockout in males caused germ cell degenerative defect and infertility. This study suggested that Dcp2 played an important role in the regulation of spermatogenesis. 2. Materials and methods 2.1. Animal experiments Dcp2 conditional knockout mice were generated by Shanghai Model Organisms Center, Inc. (Shanghai, China) through gene targeting of embryonic stem (ES) cells through homologous recombination in a C57BL/6 background. The targeted mice were crossed with Flp recombinase-expressing mice to remove the neomycin gene. UBC-CreERT2 mice were purchased from Shanghai Model Organisms Center, Inc., and Pgk2-Cre mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Genotypes of mice were determined by polymerase chain reaction (PCR) with primers mDcp2-E-F and mDcp2-F-R for Dcp2f/f mice, Cre-F and Cre-ERT-R for UBC-CreERT2 mice, and Cre-F and CreR for Pgk2-Cre mice. The Dcp2 targeted allele was determined by primers mDcp2-C-F and mDcp2-F-R. The sequences of primers are shown in Table S1. For induced knockout of Dcp2 in mice, tamoxifen (T5648, SigmaeAldrich, MO, USA) was administered to 6-week-old Dcp2f/f and Dcp2f/f; UBC-CreERT2 mice by gavage at a dose of 200 mg/kg in corn oil for five consecutive days. All animal experiments were carried out in accordance with the guidelines for the care and use of experimental animals of the Experimental Animal Ethics Committee of Fudan University in China, and were approved by the Animal Ethics Committee of Zhongshan Hospital of Fudan University.
2.4. RNA isolation, reverse transcription, and real-time quantitative PCR Total RNA was extracted from testes using Tripure Isolation Reagent (Roche) following the manufacturer’s protocols. One microgram total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for quantitative PCR (qPCR; Vazyme Biotech Co., Ltd., Nanjing, China). Then, qPCRs were performed using HieffTM qPCR SYBR Green Master Mix (Yeasen) for individual genes (primer sequences are shown in Table S1). b-actin was used as internal control. 2.5. Enzyme-linked immunosorbent assay All enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Briefly, 5 mL of serums were mixed with 40 mL of PBS (pH 7.4), and then 40 mL of the diluted sample was applied to each well of the antibody-coated plate and incubated with 100 mL of antibodies labeled with horseradish peroxidase for 60 min at 37 C. Substrates A and B were added to each well and incubated for 15 min at 37 C in the dark. The reactions were stopped, and the optical density values at 450 nm were recorded. 2.6. Sperm analysis Epididymides were extracted from male mice. Sperm counts and motility were analyzed using a computer-assisted sperm analysis system (CASAS) (Hamilton Thorne, MA, USA). 2.7. Statistical analysis Statistical analyses were performed using an unpaired t-test with GraphPad Prism 5 (GraphPad Software, CA, USA). All results were performed at least three times independently and indicated as the mean ± standard deviation (SD). A P value less than 0.05 was considered statistically significant. 3. Results
2.2. Western blot analysis
3.1. Dcp2 was not essential for the postnatal survival of male mice
Tissues were lysed in cold lysis buffer [phosphate-buffered saline (PBS) containing 0.25% Triton X-100 and 1 mM dithiothreitol] with a protease inhibitor cocktail (Biotools, Loganholme, Australia) using a homogenizer. Supernatant lysates were harvested after centrifugation at 15,000g for 10 min at 4 C. An anti-Dcp2 antibody was provided by Professor Megerditch Kiledjian (Rutgers University, USA). Anti-caspase-3 (1:1000 dilution, Cat. No. 9662S) and anti-phospho-histone H2A.X (Ser 139) (g-H2AX) (1:1000 dilution, Cat. No. 9718) antibodies were purchased from Cell Signaling Technology (MA, USA). An anti-cleaved caspase-8 (T341) (1:1000 dilution, Cat. No. AP0358) antibody was purchased from Bioworld Technology Inc. (MN, USA). A mouse anti-b-actin antibody (1:5000; Cat. No. 60008-1-Ig) was procured from Proteintech (IL, USA). Protein intensities were visualized using ECL reagent (Yeasen, Shanghai, China).
To further investigate the functions of Dcp2 in vivo, Dcp2 conditional knockout mice were generated to avoid possible embryonic lethality. As shown in Fig. 1A, the exon 2 could be deleted by Cre-Loxemediated gene excision to disrupt the Dcp2 allele (Fig. 1A). Genotypes of the mice were determined by PCR (Fig. 1B). Then, Dcp2f/f; UBC-CreERT2 mice were generated, which ubiquitously expressed recombinase CreER. The 6-week-old male mice were administered with tamoxifen to delete the Dcp2 gene. The resulting mouse strain was referred to as Dcp2D/D. On day 10, Western blot analysis showed that Dcp2 was knocked out in all the tissues tested, although trace amounts of Dcp2 protein could be detected in the Dcp2D/D tissues (Fig. 1C). During the observation period of 6 months, no obvious health abnormalities were found in the Dcp2D/D mice. The body weight of both Dcp2f/f mice and Dcp2D/D mice decreased due to tamoxifen administration, but recovered soon (Fig. 1D). The hair color of the Dcp2D/D male mice also looked fine (data not shown). Thus, it seemed that Dcp2 was not essential for the postnatal survival of adult male mice.
2.3. Histological analysis and TUNEL staining Testes and epididymides were fixed in 4% paraformaldehyde solution overnight, embedded in paraffin, and cut into 5-mm sections. Hematoxylin and eosin (H&E) staining was carried out using standard procedures. TUNEL staining was performed with an InSitu Cell Death Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s protocols.
3.2. Global deletion of Dcp2 caused germ cell degenerative defect in male mice Although the deletion of Dcp2 did not result in health abnormalities in male mice, testicular atrophy occurred as early as 3
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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Fig. 1. Generation of inducible Dcp2 knockout mouse model. (A) Schematic representation of the Dcp2 conditional knockout strategy by homologous recombination in ES cells. (B) Genotyping results of Dcp2 conditional knockout mice. (C) Dcp2 proteins from the indicated tissues of Dcp2f/f (CreER) and Dcp2f/f; UBC-CreERT2 (CreERþ) mice were analyzed by Western blot analysis 10 days after tamoxifen administration. (D) Change in body weight of mice after tamoxifen administration (n ¼ 5).
Fig. 2. Global deletion of Dcp2 in adult male mice resulted in testicular atrophy. (A) Comparison of testis size and testis weight 3 weeks after tamoxifen administration (n ¼ 5, ****P < 0.0001). (B) Expression levels of Dcp2 protein in Dcp2f/f and Dcp2D/D testes were analyzed by Western blot analysis. The results of two pairs of mice were presented. (C) H&E staining of Dcp2f/f and Dcp2D/D testis sections 3 weeks after tamoxifen administration. Arrows indicate elongated spermatids, and asterisks indicate the vacuoles in Sertoli cells. Original magnification 400.
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
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weeks after tamoxifen administration (Fig. 2A, left). The testes weight of Dcp2D/D mice was reduced by about 70% compared with Dcp2f/f mice (Fig. 2A, right). The knockout of Dcp2 protein in testes was confirmed by Western blot analysis (Fig. 2B). The testis histological sections of Dcp2D/D mice showed that germ cell loss and Sertoli cell vacuolization occurred in many seminiferous tubules, while the differentiation of spermatozoa was not blocked at a specific stage and a few elongated spermatids could be observed (Fig. 2C). The effect of Dcp2 deletion on spermatogenesis was progressive as the germ cells were almost completely absent in most of the seminiferous tubules, and few sperm could be detected in the caudal epididymis 2 months after Dcp2 deletion (Fig. S1). 3.3. Dcp2-deficient male mice were infertile To further investigate the effect of Dcp2 deletion on spermatogenesis, caudal epididymides were excised out and the sperm counts and motility were analyzed using CASAS 3 weeks after tamoxifen administration. Compared with Dcp2f/f mice, the sperm counts of Dcp2D/D mice decreased to about 10% and the motility of sperm from Dcp2D/D mice also significantly reduced (82 ± 8.0% vs 6.3 ± 9.7%) (Fig. 3A). Only a few spermatozoa could be seen in the caudal epididymis of Dcp2D/D mice, and the debris of germ cells could be detected (Fig. 3B). To investigate whether Dcp2D/D mice were fertile, male Dcp2f/f and Dcp2D/D mice were bred with wild-type (WT) female mice 3 weeks after tamoxifen administration. For 4 months, seven Dcp2f/f mating pairs had 35 litters with an average of 6.7 pups, whereas five Dcp2D/D mating pairs failed to produce any offspring (Table 1). Surprisingly, the copulation capacity of Dcp2D/D male mice was
Table 1 Summary of Dcp2f/f and Dcp2D/D male mice fertility study. Mice
Numbers
No. of plug in
No. of litters
Average No. of pups
Dcp2f/f Dcp2D/D
7 5
35 3
35 0
6.7 ± 0.5 0
significantly impaired. Only three copulation plugs were found for the five Dcp2D/D male mice during the same mating time (Table 1). The analysis of serum testosterone (TT) levels showed that the levels markedly decreased in Dcp2D/D male mice (15.25 ± 0.53 ng/ mL vs 9.35 ± 0.45 ng/mL), although mRNA levels of TT synthesisrelated genes did not decrease (Fig. 3C and D). However, the synthesis of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in Dcp2D/D mice was not compromised. On the contrary, both increased significantly (40.82 ± 1.86 mIU/mL vs 56.21 ± 3.01 mIU/mL for FSH and 5.03 ± 0.22 mIU/mL vs 7.25 ± 0.22 mIU/mL for LH) (Fig. 3E), suggesting that the Dcp2 knockout-caused male infertility might not be regulated through the hypothalamicepituitaryegonadal axis. The findings indicated that Dcp2 deficiency resulted in male sterility.
3.4. Dcp2 was not required for the differentiation of spermatocyte into sperm, but was essential for embryonic development Dcp2 was reported to be expressed in round spermatids in mice [20]. To investigate whether Dcp2 was necessary for the differentiation of spermatids, Dcp2f/f mice were bred with Pgk2-Cre mice [21] to delete the Dcp2 gene in spermatocytes, generating Dcp2SP-KO mice.
Fig. 3. Effect of Dcp2 deficiency on sperm production and hormone secretion. (A) Number and motility of sperm were analyzed using CASAS (n ¼ 4, ****P < 0.0001). (B) H&E staining of caudal epididymides sections. Arrows indicate the debris of germ cells. Original magnification 400. (C) Serum TT concentrations were determined using ELISA (n ¼ 3, ***P ¼ 0.0001). (D) mRNA levels of Dcp2 and TT synthesis-related genes were analyzed by qPCR. (E) Serum levels of FSH and LH were determined using ELISA (n ¼ 3, P values are shown on the top). All experiments were performed 3 weeks after tamoxifen administration.
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Dcp2SP-KO male mice were bred with WT female mice. They had offspring with an average of 7.6 pups per litter, which was comparable to control mice with an average of 8.2 pups per litter (Fig. S2A), suggesting that Dcp2SP-KO male mice were fertile. All the offspring of Dcp2SP-KO male mice were confirmed to be Dcp2þ/ genotype (Fig. S2B), suggesting that Dcp2 could be efficiently deleted in spermatocytes. Western blot analysis showed that the expression of Dcp2 was only slightly reduced in Dcp2SP-KO testes than in control testes (Fig. S2C), indicating that Dcp2 was also expressed in other cells of testis besides spermatocytes. These data indicated that the deletion of Dcp2 in spermatocytes did not affect the fertility of male mice. Dcp2þ/ mice appeared healthy, and intercrosses of Dcp2þ/ mice yielded only Dcp2þ/þ and Dcp2þ/ mice in a non-Mendelian manner with more Dcp2þ/þ mice than Dcp2þ/ mice, no live-born Dcp2/ mice were obtained (Table S2), indicating that Dcp2 knockout was embryonically lethal. No Dcp2/ embryos were found when E13.5 embryos were analyzed. However, the ratio of Dcp2þ/þ embryos to Dcp2þ/ embryos was about 1:3 (Table S2), suggesting that part of the Dcp2þ/ embryos were terminated during development. These data demonstrated that Dcp2 was essential for embryonic development. 3.5. Germ cell apoptosis occurred in Dcp2D/D testes The loss of germ cells in Dcp2D/D testes was further confirmed by the analysis of the marker of individual cell types using qPCR. Three
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weeks after Dcp2 deletion, the spermatogonial cells (Magea4) reduced to about 50%, and the primary spermatocytes (Sycp3) decreased to about 10%. However, the expression level of the Sertoli cell marker, Sox9, increased by about 26-fold possibly due to the increased ratio of Sertoli cells in testes (Fig. 4A). The Sertoli cell vacuolization is a characteristic of endocytosis of apoptotic germ cells, indicating the occurrence of germ cell apoptosis. A TUNEL assay was performed to determine whether the reduced number of germ cells was due to apoptosis. Compared with a few TUNELpositive cells occasionally seen in Dcp2f/f testes, a large number of TUNEL-positive apoptotic germ cells were observed in the seminiferous tubules in Dcp2D/D testes (0.4 ± 1 per tubule vs 4.5 ± 3.9 per tubule) (Fig. 4B). Consistent with the TUNEL results, the cleavage activation of caspase-8 and caspse-3 significantly increased in the extracts of Dcp2D/D testes (Fig. 4C). Furthermore, the expression level of g-H2AX, a marker of DNA double-stranded breaks, also markedly increased in Dcp2D/D testes (Fig. 4C). These results indicated that Dcp2 deletion resulted in germ cell apoptosis.
4. Discussion In this study, an inducible Dcp2 knockout mouse model was generated to investigate the in vivo functions of Dcp2. The postnatal global deletion of Dcp2 caused degenerative spermatogenic defects, leading to male infertility. The reduction of sperm was due to germ cell apoptosis in Dcp2D/D testes. These results provided evidence
Fig. 4. Germ cell apoptosis occurred in Dcp2D/D testes. (A) Quantitative PCR analysis of spermatogonium marker Magea4, spermatocyte marker Sycp3, and Sertoli cell marker Sox9 in Dcp2f/f and Dcp2D/D testes. (B) TUNEL analysis was performed on Dcp2f/f and Dcp2D/D testes. Original magnification 200. The apoptotic cells of 30 tubules were counted, and the results were shown on the right. ****P < 0.0001. (C) Activation of apoptosis-related proteins in Dcp2f/f (CreER) and Dcp2D/D (CreERþ) testes was analyzed by Western blot analysis. The 18-kDa caspase-8 cleaved fragment (Dcasp-8) and 17/19-kDa caspase-3 cleaved fragments (Dcasp-3) were shown. The results of the three pairs of mice were presented. All experiments were performed 3 weeks after tamoxifen administration.
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that Dcp2 played an important role in regulating spermatogenesis. The type of cells in which Dcp2 is expressed in the testis is not known. Therefore, the phenotype of Dcp2D/D mice could be caused by the deficiency of Dcp2 in germ cells or testicular somatic cells or hypothalamusepituitary axis, or even a combination of all. Three antibodies (antibody used in this study, antibody generated against 179e422 amino acids of mouse Dcp2, and anti-Dcp2 antibody from Thermo Fisher Scientific, PA5-31091) were used, but failed to show the expression localization of Dcp2 in the testis. A Dcp2-IRES-EGFP knock-in mouse model was generated to trace the expression of Dcp2, but failed due to the weak expression of EGFP proteins [22]. To investigate the exact expression pattern of Dcp2 in the testis, specific antibodies that can be used for immunofluorescence should be developed. The lineage tracing technique can also be used by crossing Dcp2 promoter-controlled Cre recombinaseexpressing mice with reporter mice such as Rosa26-LSL-tdTomato. Then, the corresponding Cre mouse lines can be used to delete Dcp2 in specific cells in the testis, such as Stra8-Cre line for germ cells and Amh-Cre line for Sertoli cells. The following findings implied that the apoptosis of germ cells in Dcp2D/D mice might be induced through the extrinsic pathway: (1) Dcp2 deficiency did not block the differentiation of sperm at a specific stage, while the loss of some germ cell-expressed genes could cause germ cell development arrests at the spermatocyte stage, such as TEX11 [23], or during the round spermatid period, such as SOX30 [24]. (2) Mice containing Dcp2-deficient spermatocytes were fertile, and the expression level of Dcp2 protein only slightly reduced when Dcp2 was deleted in spermatocytes, indicating that Dcp2 was expressed mainly in somatic cells of the testis, such as Sertoli cells. (3) The level of activated caspase-8 increased in Dcp2D/D testes, which was usually activated by the extracellular stimuli such as FasL and TNF-a [25], suggesting that germ cells might have been exposed to extracellular stimuli. To determine the pathway responsible for the apoptosis of germ cell in Dcp2D/D mice, Dcp2 should be deleted in specific cell types of the testis. Dcp2 was involved in the negative feedback regulation of innate immune response, and Dcp2 defect upregulated the expression of chemokine CXCL10 in mouse embryonic fibroblast cells [26]. Jiang et al. reported that mumps virus infection could stimulate Sertoli cells to produce chemokine CXCL10, which could induce germ cell apoptosis [27]. Zika virus also induced innate immune responses in Leydig and Sertoli cells, but not in spermatogonia, resulting in the production of pro-inflammatory cytokines/chemokines, including IL6 and CXCL10, causing testis damage [28]. The deletion of Dcp2 might deregulate the innate immune response and increase the expression levels of some chemokines in the testis, such as CXCL10, which could induce germ cell apoptosis. Whether Dcp2 deficiency activates the innate immune response in the testis and causes spermatogenic defect needs to be further investigated. The deficiency or mutation of many genes can result in testicular atrophy and male sterility in mice, but does not affect their mating behavior [29e31]. However, the present study found that the global deletion of Dcp2 resulted in impaired mating ability. The serum level of TT significantly decreased in the Dcp2D/D mice, while the expression levels of TT synthesis-related genes did not decrease. Whether the decreased TT level in Dcp2D/D male mice was due to the reduced volumes of testes or Dcp2 deletion in Leydig cells is not clear. The impaired mating ability and the spermatogenic defect in Dcp2D/D male mice limited the potential of Dcp2 as a target for designing male contraceptives. However, Dcp2 may be a potential target for developing animal castration drugs. In summary, the study was novel in demonstrating that Dcp2 did not play an important role as an essential gene for embryonic development in the postnatal survival of male mice, but was necessary for spermatogenesis. The deletion of Dcp2 in adult male
mice resulted in infertility by causing the apoptosis of germ cells. Whether Dcp2 is directly involved in the differentiation of spermatozoa or maintains the immune microenvironment of spermatogenesis needs to be further investigated. This study revealed the potential diagnostic and/or therapeutic significance of Dcp2 for men with infertility. Author contributions Rui-Min Li and Ming-Nan Zhang performed the experiments, and Rui-Min Li wrote the manuscript. Qun-Ye Tang was responsible for mouse maintenance. Man-Gen Song designed the experiments and wrote the manuscript. Funding This study was supported by the National Key R&D Program of China (grant number 2018YFA0107501) and the National Natural Science Foundation of China (grant number 81373148). Declaration of competing interest The authors declare no conflicts of interest. Acknowledgments The authors thank Professor Megerditch Kiledjian (Rutgers University, USA) for supplying the anti-Dcp2 antibody. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.03.101. References [1] A.J. Shatkin, Capping of eucaryotic mRNAs, Cell 9 (4 pt 2) (1976) 645e653. [2] A.B. Sachs, Messenger RNA degradation in eukaryotes, Cell 74 (3) (1993) 413e421. [3] C.J. Decker, R. Parker, A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation, Genes Dev. 7 (8) (1993) 1632e1643. [4] T. Dunckley, R. Parker, The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif, EMBO J. 18 (19) (1999) 5411e5422. [5] E. Van Dijk, N. Cougot, S. Meyer, et al., Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures, EMBO J. 21 (24) (2002) 6915e6924. [6] J. Lykke-Andersen, Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay, Mol. Cell Biol. 22 (23) (2002) 8114e8121. [7] Z. Wang, X. Jiao, A. Carr-Schmid, et al., The hDcp2 protein is a mammalian mRNA decapping enzyme, Proc. Natl. Acad. Sci. U. S. A. 99 (20) (2002) 12663e12668. [8] M.G. Song, Y. Li, M. Kiledjian, Multiple mRNA decapping enzymes in mammalian cells, Mol. Cell. 40 (3) (2010) 423e432. [9] C.L. Hsu, A. Stevens, Yeast cells lacking 5’–>3’ exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5’ cap structure, Mol. Cell Biol. 13 (8) (1993) 4826e4835. [10] Y. Li, M.G. Song, M. Kiledjian, Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein, Mol. Cell Biol. 28 (3) (2008) 939e948. [11] Y. Li, E.S. Ho, S.I. Gunderson, et al., Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5’-end stem-loop structures, Nucleic Acids Res. 37 (7) (2009) 2227e2237. [12] M.G. Song, M. Kiledjian, 3’ Terminal oligo U-tract-mediated stimulation of decapping, RNA 13 (12) (2007) 2356e2365. [13] T.E. Mullen, W.F. Marzluff, Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5’ to 3’ and 3’ to 5’, Genes Dev. 22 (1) (2008) 50e65. [14] J. Mauer, X. Luo, A. Blanjoie, et al., Reversible methylation of m(6)Am in the 5’ cap controls mRNA stability, Nature 541 (7637) (2017) 371e375. [15] K.K. Raju, S. Natarajan, N.S. Kumar, et al., Role of cytoplasmic deadenylation and mRNA decay factors in yeast apoptosis, FEMS Yeast Res. 15 (2) (2015).
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101
R.-M. Li et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx [16] J. Xu, J.Y. Yang, Q.W. Niu, et al., Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development, Plant Cell 18 (12) (2006) 3386e3398. [17] J. Ma, M. Flemr, H. Strnad, et al., Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse, Biol. Reprod. 88 (1) (2013) 11. [18] Y. Mishima, Y. Tomari, Pervasive yet nonuniform contributions of Dcp2 and Cnot7 to maternal mRNA clearance in zebrafish, Gene Cell. 22 (7) (2017) 670e678. [19] T. Nishimura, H. Fakim, T. Brandmann, et al., Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs, Nucleic Acids Res. 46 (22) (2018) 12008e12021. [20] F. Shapouri, S. Saeidi, R.U. De Iongh, et al., Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2, Cell Tissue Res. 364 (2) (2016) 443e451. [21] H. Ando, Y. Haruna, J. Miyazaki, et al., Spermatocyte-specific gene excision by targeted expression of Cre recombinase, Biochem. Biophys. Res. Commun. 272 (1) (2000) 125e128. [22] M.N. Zhang, Q.Y. Tang, R.M. Li, et al., MicroRNA-141-3p/200a-3p target and may be involved in post-transcriptional repression of RNA decapping enzyme Dcp2 during renal development, Biosci. Biotechnol. Biochem. 82 (10) (2018) 1724e1732. [23] A.N. Yatsenko, A.P. Georgiadis, A. Ropke, et al., X-linked TEX11 mutations,
[24] [25] [26]
[27]
[28] [29]
[30]
[31]
7
meiotic arrest, and azoospermia in infertile men, N. Engl. J. Med. 372 (22) (2015) 2097e2107. C.A. Feng, C. Spiller, D.J. Merriner, et al., SOX30 is required for male fertility in mice, Sci. Rep. 7 (1) (2017) 17619. A. Ashkenazi, V.M. Dixit, Apoptosis control by death and decoy receptors, Curr. Opin. Cell Biol. 11 (2) (1999) 255e260. Y. Li, J. Dai, M. Song, et al., Dcp2 decapping protein modulates mRNA stability of the critical interferon regulatory factor (IRF) IRF-7, Mol. Cell Biol. 32 (6) (2012) 1164e1172. Q. Jiang, F. Wang, L. Shi, et al., C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis, Cell Death Dis. 8 (10) (2017) e3146. W. Ma, S. Li, S. Ma, et al., Zika virus causes testis damage and leads to male infertility in mice, Cell 167 (6) (2016) 1511e1524, e10. M.P. Greenbaum, W. Yan, M.H. Wu, et al., TEX14 is essential for intercellular bridges and fertility in male mice, Proc. Natl. Acad. Sci. U. S. A. 103 (13) (2006) 4982e4987. M.K. O’bryan, B.J. Clark, E.A. Mclaughlin, et al., RBM5 is a male germ cell splicing factor and is required for spermatid differentiation and male fertility, PLoS Genet. 9 (7) (2013), e1003628. C. Berthet, A.M. Morera, M.J. Asensio, et al., CCR4-associated factor CAF1 is an essential factor for spermatogenesis, Mol. Cell Biol. 24 (13) (2004) 5808e5820.
Please cite this article as: R.-M. Li et al., Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.101