|Year : 2020 | Volume
| Issue : 3 | Page : 129-136
Mageb4, a testis-specific gene, is dispensable for mouse spermatogenesis
Sheng Gao, Da-Min Yun, Li-Wei Zhou, Yun-Hao Wu, Deng-Feng Lin, Xiao-Long Wu, Fei Sun
Medical School, Institute of Reproductive Medicine, Nantong University, Nantong 226001, China
|Date of Submission||06-Jun-2020|
|Date of Decision||28-Jun-2020|
|Date of Acceptance||28-Jul-2020|
|Date of Web Publication||29-Sep-2020|
Medical School, Institute of Reproductive Medicine, Nantong University, 19 Qixiu Road, Nantong 226001
Medical School, Institute of Reproductive Medicine, Nantong University, 19 Qixiu Road, Nantong 226001
Source of Support: None, Conflict of Interest: None
Objective: It has recently been shown that the melanoma antigen gene (MAGE) family is expressed in various tumor cell lines but silent in normal tissues, except germ cell lines. Mageb4, a member of the MAGE family, is highly expressed in the testis and homologous in humans and mice. Whole-exome sequencing studies have identified Mageb4 as a possible X-linked cause of inherited male infertility. However, the function of Mageb4 protein remains largely unknown.
Methods: Using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 technology, we generated a Mageb4 knockout mouse model (Mageb4−/Y) to explore the role of this gene in spermatogenesis.
Results: First, immunostaining of testicular cells showed that Mageb4 is localized in the cytoplasm of spermatogonia. Second, Mageb4−/Y male mice displayed significant increases in apoptosis. However, Mageb4−/Y male mice showed normal fertility, including normal sperm concentration, sperm motility, and testicular and epididymal histology.
Conclusions: These findings suggest that, despite testis-exclusive expression, Mageb4 is dispensable for mouse spermatogenesis. Future research should focus on the role of this gene in apoptosis, aiming to provide clinical guidance regarding male infertility.
Keywords: Clustered Regularly Interspaced Palindromic Repeats/CRISPR-Associated Protein 9; Mageb4; Male Infertility; Spermatogenesis
|How to cite this article:|
Gao S, Yun DM, Zhou LW, Wu YH, Lin DF, Wu XL, Sun F. Mageb4, a testis-specific gene, is dispensable for mouse spermatogenesis. Reprod Dev Med 2020;4:129-36
|How to cite this URL:|
Gao S, Yun DM, Zhou LW, Wu YH, Lin DF, Wu XL, Sun F. Mageb4, a testis-specific gene, is dispensable for mouse spermatogenesis. Reprod Dev Med [serial online] 2020 [cited 2022 May 21];4:129-36. Available from: https://www.repdevmed.org/text.asp?2020/4/3/129/296546
| Introduction|| |
Infertility has received increasing research attention and affects approximately 15% of the population, 50% of which are male. Spermatogenesis is a complex, dynamic process that includes mitosis of spermatogonia, meiosis of spermatocytes, transformation of spermatids into spermatozoa, and release of spermatozoa into the seminiferous tubule lumen. More than 2,300 genes are predominantly expressed in the testes and are generally believed to be essential for spermatogenesis; however, the functions of many of these genes remain unknown. Most genes in the melanoma antigen gene (MAGE) family code for cancer/testis antigens, which are commonly observed in tumors but also show special expression in germ cells and placentae. The MAGE family directs the expression of tumor antigens recognized on human melanoma by autologous cytolytic T lymphocytes, making this gene family a powerful target for specific immunotherapy for cancer. The MAGE gene family is divided into the following four main classes based on sequence homology and X chromosome location: MAGE-A, -B, -C, and -D, which are positioned on the X chromosome at Xq28, Xp21.3, Xq26, and Xp11, respectively. Because of high homology with the house mouse, MAGE-A, -B, and -C were isolated and characterized by hybridizing mouse genomic libraries with a MAGE-1 probe. Notably, MAGE-B1 to -B4 are homologous with the expression of 42%–52% of MAGE gene family proteins.
An increasing amount of research has focused on MAGE genes. However, previous studies mainly focused on tumors of various histological types, such as melanomas, breast cancers, bladder cancers, and ovarian cancers. As a result, the MAGE-A family has been well studied in tumor cell lines but not in germ cell lines. The expression of MAGE-A proteins can be detected in the nuclei and cytoplasm of spermatogonia and spermatocytes in adult testes, while MAGE-A mRNA is mainly expressed during the first wave of spermatogenesis in juvenile male mice. In male germ cell development, the MAGE-A gene cluster maintains normal testis size, protects germ cells from excessive apoptosis, and activates the Wnt signaling pathway, which is closely associated with mammalian spermatogenesis.Necdin, another member of the MAGE gene family, is a postmitotic neuron-specific growth suppressor that is functionally similar to retinoblastoma protein. Necdin plays an important role in interacting with viral transforming proteins and the cellular transcription factor E2F1.
Mageb4, a member of the MAGE-B gene family located on the mouse X chromosome, shows intensive protein expression in the cytoplasm of premeiotic germ cells but is restricted to prepachytene cells in adult testes. In an analysis of a consanguineous Turkish family, whole-exome sequencing (WES) identified Mageb4 as a possible X-linked cause of inherited male infertility. Multiple genes on the X chromosome are thought to play important roles during the premeiotic stages of mammalian spermatogenesis. During fetal development, high Mageb4 protein expression can be detected in the gonads, especially in germ cells. In females, Mageb4 is highly expressed in the ovaries during the pachytene phase. Such expression patterns suggest that Mageb4 plays a role in reproduction. Mageb4 fixes the position of the sex reversal critical region on chromosome Xp21, which is critical for XX male disorders of sex development (DSD). The results of WES for DSD and high-resolution DSD gene-targeted copy number variations (CNVs) have shown that duplication of the dosage-sensitive sex region containing MAGE-B is related to ambiguous genitalia and ovotestis.
Recent research has revealed that most MAGE genes have an open reading frame located in the last exon of the gene that encodes 320 amino acids. Surprisingly, Mageb4 has a unique feature that most MAGE proteins lack. Besides the standard amino acids, it has a repetitive region consisting of 133 amino acids at the COOH-terminal end, which is considered to be a CNV. In an analysis of a large population, patients with spermatogenic failure were found to have CNV64, CNV67, and CNV69. However, there is insufficient evidence regarding thein vivo function of Mageb4 during spermatogenesis.
In the present study, we investigated the function of Mageb4 during spermatogenesis in mice. To do so, we generated a Mageb4 knockout mouse model using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9. Immunostaining of testicular cells showed that Mageb4 is localized in the cytoplasm of spermatogonia. Mageb4−/Y male mice displayed increased apoptosis, suggesting that the gene may influence the apoptotic response during germ cell development. However, Mageb4−/Y male mice retained normal fertility, including normal sperm concentration, sperm motility, and testicular and epididymal histology. Also, the knockout mice of female, Mageb4−/–, were normal fertility. Overall, this study showed that Mageb4, although a testis-specific gene, is dispensable for mouse spermatogenesis and furthermore, it may prevent the apoptotic response in germ cells.
| Methods|| |
All mice were maintained under specific pathogen-free conditions in the Laboratory Animal Center of Nantong University. Care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Nantong Medical College, Nantong University (approval no. 20171220-005). Euthanasia was performed by cervical dislocation.
Generation of Mageb4 knockout mice and genotyping
Mageb4 knockout mice were generated using CRISPR/Cas9 genome editing technology. First, we used the web tool ChopChop (http://chopchop.cbu.uib.no/) to design single-guide RNAs (sgRNAs) to recruit Cas9 and delete the genomic DNA sequence covering exon 2 to exon 3. C57BL/6 female mice were superovulated with 8 IU of pregnant mare serum gonadotropin (Cat# SYZ (2012) 110044564, Ningbo Sansheng, China) followed by 8 IU of human chorionic gonadotropin (hCG; Ningbo Sansheng). They were mated to C57BL/6 male mice, and fertilized embryos were collected from oviducts post-hCG injection. Then, sgRNA (25 ng/μL) and Cas9 mRNA (50 ng/μL) were mixed and injected into the cytoplasm of the fertilized eggs. The injected zygotes were cultured to the two-cell stage in M16 (Sigma-Aldrich, St. Louis, MO, USA) medium in incubators (Thermo Scientific, Carlsbad, CA, USA) at 37°C and 95% CO2 and transferred into the oviducts of pseudopregnant Institute of Cancer Research females 12 h after mating with vasectomized males. Founders were identified by genomic polymerase chain reaction (PCR) with purified mouse tail DNA using the primer pair 5'-GAAAGGTCAAACAGCTCAGAGG-3' (forward) and 5'-TACAATCATGGCCCAGCAGA-3' (reverse), and further confirmed by Sanger sequencing. After genotype validation, F0 mice underwent serial mating to generate homozygous or heterozygous mutant offspring.
Mageb4−/Y mice were originally generated from a mixed C57BL/6 background. After sexual maturation, Mageb4−/Y male mice and controls were housed with two or three 6- to 8-week-old wild-type C57BL/6 female mice for at least 8 months in an environment maintained at 23°C ± 2°C with a 12 h dark/light cycle and relative humidity of 45%–55%. For fertility testing, the number of pups was counted at birth. The average litter size for each mouse line was calculated by dividing the total number of pups by the number of litters.
Computer-assisted semen assay
Following fecundity testing, five adult knockout mice and wild-type mice were randomly selected for the assessment of sperm concentration and motility. Sperm were released from the cauda epididymides and dispersed in warm (37°C) Tyrode's solution under 5% CO2(Solarbio T1421, Beijing, China) for 15 min in a constant-temperature incubator. Parameters were measured using the computer-assisted semen assay (CASA) system (Hamilton Throne IVOS II, Lancaster, Pennsylvania, USA).
RNA extraction and real-time reverse transcription-quantitative polymerase chain reaction
Total RNA was extracted from tissues in 1-mL TRIzol reagent (Thermo Fisher Scientific) in 1.5-mL tubes. Following the addition of 200-μL chloroform, the samples were mixed by oscillation for 15 s, allowed to stand for 3 min, and centrifuged for 15 min at 10,000 ×g at 4°C. The supernatants were transferred to new tubes, mixed with 500-μL isopropanol, and centrifuged for 10 min at 10,000 ×g at 4°C to precipitate RNA. The resulting pellets were washed with 75% ethanol and centrifuged twice for 5 min at 7,000 ×g at 4°C. After air drying, the pellets were resuspended in 60-μL RNA-free water. The purity and concentration of RNA was determined using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). A total of 500-ng RNA was reverse transcribed into cDNAs using a PrimeScript™ (Otsu, Japan) 1st Strand cDNA Synthesis Kit according to the manufacturer's protocol. Quantitative reverse transcription-PCR was performed with SYBR green master mix (TaKaRa, Otsu, Japan) on a LightCycler® 96 real-time PCR system (Roche, Basel, Switzerland) under the following cycle conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The relative gene expression was quantified using the DDCT method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Every reaction was performed using technical and biological replicates. The quantitative polymerase chain reaction (qPCR) primers were 5'-GAGGAGAAGGAGGAGGTGGA-3' (forward) and 5'-GTCCTCGAGCCTTGATTGCT-3' for Mageb4; 5'-CCCTTAAG AGGGATGCTGCC-3' (forward) and 5'-TACGGCCAAATCCGTTCACA-3' for GAPDH.
Protein was extracted from the tissues of male and female C57BL/6 mice and suspended in RIPA lysis buffer (50-mmol/L Tris-HCl [pH 7.5], 150-mmol/L NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 5-mmol/L EDTA, and 5-mmol/L NaF) containing 1-mmol/L Phenylmethanesulfonyl fluoride (PMSF), 1-mmol/L Na3 VO4, and a protease inhibitor cocktail (Roche). After incubating on ice for 30 min, the sample lysates were centrifuged at 18,000 ×g for 10 min at 4°C and then boiled in sample buffer containing 2% SDS in a thermostatic metal bath for 10 min. The protein samples were separated in 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, California USA). After blocking with 5% nonfat milk, the membranes were incubated at 4°C overnight with the following primary antibodies: rabbit anti-Mageb4 polyclonal antibody (Proteintech, 12786-2-AP, 1:5,000, Rosemont, IL, USA) and mouse anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:3,000). After three washes in Tris buffered saline tween, the membranes were further incubated with 1 μg/mL of the second antibody Alexa Fluor® 555 goat anti-rabbit IgG (H+L) (Abcam ab150078) and Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Abcam ab150113) for 2 h at room temperature. The resulting signals were detected using Amersham Typhoon5 biomolecular imagers (GE Healthcare Life Sciences, Little Chalfont, UK).
Immediately after euthanasia, the testes and epididymides of adult wild-type and knockout mice were dissected in CO2 and fixed with 4% paraformaldehyde for up to 24 h. After rinsing in water, dehydrating step wise through an ethanol series (70%, 80%, 90%, 95%, and 100% ethanol), and embedding in paraffin, 5-μm sections were cut using an ultra-thin semiautomatic microtome (Leica, Wetzlar, Germany) and mounted on glass slides. After deparaffinization, the slides were stained with hematoxylin and eosin for histological analysis and imaged with a Leica DM2000 microscope.
For protein immunolocalization, the paraffin sections were deparaffinized twice in xylene and subsequently rehydrated with graded ethanol. Antigen retrieval was achieved by heating sections in a microwave in 10-mmol/L sodium citrate (pH = 6.0) for 10 min. After cooling to room temperature, the sections were treated with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 30 min and blocked with 10% normal donkey serum (Jackson ImmunoResearch, Pennsylvania, USA) for 30 min at room temperature. The sections were then incubated with primary antibodies at 4°C overnight. The next day, the sections were washed in PBS three times and incubated with secondary antibodies at room temperature for 2 h. After three washes in PBS, DAPI (D9542) was used to stain the cell nuclei for 2 min at room temperature. Finally, the slides were mounted in glycerol (ZEISS, Oberkochen, Germany) and imaged using a ZEISS Axiocam 503 mono fluorescence microscope. Antibodies to promyelocytic leukemia zinc finger (PLZF; sc-22839) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); those to synaptonemal complex protein 3 (SYCP3; ab97672) and Mageb4 (12786-2-AP) were bought from Abcam (Cambridge, UK) and Proteintech, respectively.
Terminal deoxynucleotidyl transferase dUTP nick end labeling assay
To detect apoptotic signals, paraffin sections were deparaffinized and rehydrated as described above, and then incubated with protease K at room temperature for 30 min. After incubation according to the One-Step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Apoptosis Assay Kit protocol (Beyotime C1089, Beijing, China), the nuclei were stained with DAPI. Images were obtained with a ZEISS Axiocam 503 mono fluorescence microscope.
The mRNA levels, number of pups/litter, testis/body weight ratio, sperm concentration, and sperm motility were compared between wild-type and knockout mice using the Student's t-test or two-way analysis of variance in Graphpad Prism 8 software (San Diego, USA). Each experiment was repeated at least three times and the results are presented as the mean ± standard error of the mean. Statistical significance was set at P < 0.05.
| Results|| |
Expression of Mageb4 in mouse testes
To investigate Mageb4 mRNA and protein in vivo, we first assessed their expression in different tissues of adult wild-type mice by reverse transcription (RT)-qPCR [Figure 1]a and Western blotting [Figure 1]b. Consistent with a previous study, Mageb4 was expressed specifically in the testes. Earlier studies found that Mageb4 is expressed in fetal testes from 14.5 days postpartum (dpp). Further, we performed RT-qPCR analyses in mouse testes at different times and observed that Mageb4 was expressed from immediately after birth. The expression levels of Mageb4 increased and then decreased gradually, peaking at 14 dpp [Figure 1]c. As Type A and Type B spermatogonia are present by day 8, we speculated that Mageb4 is expressed when germ cells begin to differentiate. To examine the details of the protein location of Mageb4 during germ cell differentiation, we implemented double immunofluorescence with antibodies against Mageb4 protein and PLZF, a marker of spermatogonia. The results revealed that Mageb4 co-localized with PLZF [Figure 1]d. Because of the specificity of PLZF expression, we applied co-immunofluorescence to testis sections from 10 to 28 dpp and found that Mageb4 and PLZF were expressed similarly in germ cells. This showed that Mageb4 and PLZF expression was highest at 14 dpp, which is essentially consistent with the RT-qPCR results [Figure 2]a and [Figure 2]b. Whether Mageb4 is expressed in spermatocytes remains unknown. The co-immunofluorescence results for Mageb4 and SYCP3 showed that Mageb4 was not expressed in pachytene spermatocytes. Interstitial cells also had fluorescence signals, as found in a controlled trial [Figure 1]e. Previous research indicated that Mageb4 is not expressed in Sertoli or interstitial Leydig cells. Collectively, these results suggest that Mageb4, a testis-specific gene, is expressed in spermatogonia.
|Figure 1: Expression of melanoma antigen gene-b4 (Mageb4) in mouse testes (a) RT-qPCR analysis of Mageb4 expression levels in adult mouse tissues. (b) Western blot analysis of Mageb4 tissue distribution in adult mice; glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. (c) RT-qPCR using cDNA from 0 dpp (newborn), 7 dpp, 14 dpp, 21 dpp, 28 dpp, 35 dpp, and 70 dpp mice. dpp, days postpartum. (d) Co-immunofluorescence staining with anti-Mageb4 (green) and anti-PLZF (red) antibodies in 18 dpp mouse testes sections. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). (e) Co-immunofluorescence staining with anti-Mageb4 (green) and anti-SYCP3 (red) antibodies in adult mouse testes sections. Nuclei were stained with DAPI (blue). Boxed areas (II–IV) correspond to magnified images on the right, which better depict Mageb4 staining in spermatogonia (white arrowheads). Pachytene spermatocytes are indicated with yellow arrows. Scale bar (D, I; also applies to E, I) = 50 μm. Scale bar (E, II; also applies to D, III, IV; E, II, III, IV) = 20 μm. There is a negative controlled trial at the bottom. RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.|
Click here to view
|Figure 2: Expression of Mageb4 in mice of different ages. (a) Immunofluorescence staining of Mageb4 (green) and PLZF (red) in seminiferous tubules of male mice at 10, 14, 21, and 28 days postpartum. Scale bars, 50 μm. (b) Number of Mageb4 and PLZF cells per seminiferous tubule in mice at the indicated ages as determined from images similar to those in (a). Data are the mean ± standard error of the mean for three mice per group. Statistically significant differences were not detected by two-way analysis of variance.|
Click here to view
Generation of Mageb4 knockout mice
To determine the role of Mageb4 during spermatogenesis, we established a knockout mouse model using the CRISPR/Cas9 system. We used two sgRNAs to target the coding sequence between exon 2 and exon 3 of Mageb4, resulting in a 478 bp residual fragment [Figure 3]a. Genotyping results showed that knockout mice had a lower band compared to wild-type mice [Figure 3]b. This was further confirmed by Sanger sequencing, as shown in [Figure 3]c. These results confirm the successful construction of a mouse model of the Mageb4 null allele.
|Figure 3: Generation of Mageb4 knockout mice. (a) A schematic explanation of the targeting strategy for the production of Mageb4−/Y mice. (b) Genotyping polymerase chain reaction results for wild-type (5,277 bp) and knockout mice (478 bp). M: Marker; WT: Wild type; KO: Mageb4−/Y. (c) Genotype validation of Mageb4−/Y knockout mice by Sanger sequencing. Red triangles represent the cut loci. A: Adenine; G: Guanine; C: Cytosine; T: Thymine.|
Click here to view
Mageb4−/Y mice showed increased apoptosis and normal spermatogenesis
Problems in spermatogenesis usually result in infertility or reduced fertility, reduced testicular weight, and/or abnormal seminiferous tubules. Hence, we carried out a fecundity test to assess fertility in the knockout mouse model [Table 1]. No noticeable difference in fertility was seen between wild-type and Mageb4−/Y mice [Figure 4]a. Furthermore, the size of the testes and epididymides was almost equal between the two groups [Figure 4]b. Subsequently, we weighed the testes and bodies of the mice and compared their ratio, finding no significant difference between the groups [Table 1]. Similarly, CASA showed similar sperm concentration and sperm motility between the two groups [Figure 4]c.
|Figure 4: Mageb4−/Y mice showed normal fertility and spermatogenesis. (a) Number of pups per litter from wild-type and Mageb4−/Y mice. Dots and squares represent the number of pups per litter for WT and Mageb4−/Y mice, respectively. n, number of animals. (b) Gross morphology of the testis and epididymis in wild-type and Mageb4−/Y mice. (c) Sperm motility and concentration in adult wild-type and Mageb4−/Y mice. n = 5. Data were analyzed using the Student's t-test in Graphpad Prism 8 software. NS: No significant difference. Data are presented as the mean ± standard error of the mean. (d) Hematoxylin and eosin staining of the testes and epididymides of adult wild-type and Mageb4−/Y mice. Scale bar (left panel and right panel) = 100 μm. Scale bar (middle panel) = 50 μm.|
Click here to view
To obtain a deeper understanding of the role of Mageb4 during spermatogenesis, hematoxylin and eosin staining of testicular sections was performed. The morphology in the seminiferous tubule was typical, including spermatogonia, spermatocytes, and spermatids, both in wild-type mice and knockout mice [Figure 4]d. Strikingly, the TUNEL assay showed a higher rate of germ cell apoptosis in Mageb4 knockout mice [Figure 5]a and 5]b. Taken together, these experimental results reveal that disruption of Mageb4, a testis-specific and X-linked gene, does not affect fertility and might be involved in apoptosis.
|Figure 5: Germ cell apoptosis increased in knockout mice. (a) Cell apoptosis was analyzed by TUNEL assay, which was performed on Mageb4 knockout mouse testes at 70 days postpartum. Apoptotic cells are marked red and nuclei were stained with DAPI (blue). Scale bars: 100 μm (50 μm in insets). (b) Results for wild-type mouse testes were used as a control. Scale bar = 100 μm. TUNEL-positive tubules and mean apoptotic cells in TUNEL-positive seminiferous tubules between the two groups. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.|
Click here to view
| Discussion|| |
Despite elaborative experiments, many genes specifically expressed in testes remain unknown. Approximately 400 genes are known to play an indispensable role in male fertility.,, Of course, some of these genes are dispensable for male infertility and spermatogenesis. Regarding the MAGE gene family, a previous work has mainly focused on tumor cell lines to support the rapid development of specific immunotherapy., Studies involving normal cell lines are comparatively less common. Here, we explored and investigated the role of Mageb4 in normal germ cells.
In strong agreement with previous research, our RT-qPCR and Western blot results revealed that Mageb4 is specifically and highly expressed in the testis [Figure 1]a and 1b]. We also performed immunofluorescence experiments to examine Mageb4 protein expression in more detail. Double immunofluorescence with antibodies against Mageb4 and PLZF showed that the Mageb4 protein is expressed in the cytoplasm of spermatogonia, suggesting that it has an effect during the fetal development period. Multiple genes may coordinate to promote efficient cell proliferation and complex differentiation in spermatogonia. Various intrinsic factors regulate transcription during development, such as PLZF and SALL4; these are typically co-expressed in undifferentiated spermatogonia, share thousands of binding sites, and help to control spermatogonial stem cell (SSC) function and spermatogenesis. Several genes and gene families are involved in male infertility; for example, bromodomain testis associated, a classic testis-specific gene in the bromodomain family, plays important roles in regulating chromatin organization, silencing sex chromosomes, and promoting crossover formation during male meiosis. As well as bromodomain-containing 2 has a significant influence on the formation of double-strand breaks and promotes DNA repair. Other gene families within germ cell-specific groups function during germ cell differentiation, such as the testis-expressed (TEX) family, zinc finger, and blood–testis barrier (ZBTB) family.,, WES has revealed that MAGEB4 is involved in inherited male infertility and that Mageb4 has 56% homology with the human MAGEB4 gene. We strongly believe that Mageb4 could be involved in spermatogenesis in mice. Thus, we established Mageb4−/Y mice and confirmed that the gene was disrupted successively [Figure 3]a-3c]. Strikingly, disruption of this gene resulted in a higher rate of germ cell apoptosis, as shown by the TUNEL assay [Figure 5]a. Apoptosis, an essential physiological process, occurs at a high rate in the testes. Proper apoptosis can maintain an appropriate germ cell-to-Sertoli cell ratio, eliminate defective germ cells, and maintain overall quality control during sperm production. RING-finger protein 138 (Rnf138) is highly expressed in spermatogonia and its deficiency in mice results in increased apoptosis in spermatogenic cells and impaired fertility, supporting the important role of Rnf138 in regulating the development of spermatogonia. However, for the Mageb4−/Y mice, it shows normal germ cell count. It is widely believed that SSCs are the most primitive spermatogonia. SSCs have the dual property of continually renewing and undergoing differentiation into a spermatogonial progenitor (potential stem cells) that expands and further differentiates. There is a balance between self-renewal and differentiation in spermatogonia. Under specific conditions, some spermatogonial progenitors can change modes from transient amplification to self-renewal and act as SSCs. For example, the deleting enhancer of zeste homolog 2 (EZH2) promoted spermatogonial differentiation and apoptosis. Hence, we speculated that the disrupting Mageb4, like the deleting of EZH2, may result in spermatogonial differentiation and apoptosis. Furthermore, we hypothesized that the growing undifferentiated cells develop into differentiated spermatogonia to make up the apoptosis cells, which contribute to normal sperm count.
However, our Mageb4 knockout mice showed normal fertility [Figure 4]a-4d]. A possible explanation for this phenomenon is functional redundancy. Ubqln3, Lypd9, and Dpep3 are all testis-specific genes, although the disruption of these genes in mice results in normal male fertility and spermatogenesis.,Mageb1, b2, and b3 have exceedingly similar expression patterns to Mageb4. When Mageb4 is disrupted, other clusters of Mageb genes may compensate. In addition, Mageb4−/Y mice in the present study were born and grew up in a standard laboratory environment. Without an external stimulus, knockout mice can develop and sire normally. Perhaps, there exist some other phenotypes that we cannot detect through conventional technology, especially related to the proliferation of tumor cell lines. When the Magea gene family (Magea1, a2, a3, a5, a6, and a8) is disrupted simultaneously, Magea male knockout mice show smaller testes with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. In conclusion, the present study provides a direction for future studies to explore the function of Mageb4 in apoptosis.
Financial support and sponsorship
This work was supported by the National Key Research and Development Program of China (No. 2018YFC1003500 to F.S) and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX19_2027).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dunson DB, Baird DD, Colombo B. Increased infertility with age in men and women. Obstet Gynecol 2004;103:51-6. doi: 10.1097/01.AOG.0000100153.24061.45.
Li L, Li H, Wang L, Wu S, Lv L, Tahir A, et al
. Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis. Crit Rev Biochem Mol Biol 2020;55:71-87. doi: 10.1080/10409238.2020.1742091.
O'Donnell L. Mechanisms of spermiogenesis and spermiation and how they are disturbed. Spermatogenesis 2014;4:e979623. doi: 10.4161/21565562.2014.979623.
Ramm SA, Schärer L, Ehmcke J, Wistuba J. Sperm competition and the evolution of spermatogenesis. Mol Hum Reprod 2014;20:1169-79. doi: 10.1093/molehr/gau070.
Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl Acad Sci U S A 2003;100:12201-6. doi: 10.1073/pnas.1635054100.
Chomez P, De Backer O, Bertrand M, De Plaen E, Boon T, Lucas S. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res 2001;61:5544-51.
Liu Y, Wen L, Ma L, Kang Y, Liu KY, Huang XJ, et al
. MAGE genes: Prognostic indicators in AL amyloidosis patients. J Cell Mol Med 2019;23:5672-8. doi: 10.1111/jcmm.14475.
Hofmann O, Caballero OL, Stevensond BJ, Chen YT, Cohen T, Chua R, et al
. Genome-wide analysis of cancer/testis gene expression. Proc Natl Acad Sci USA 2008;105:20422-7. doi: 10.1073/pnas.0810777105.
De Plaen E, Arden K, Traversari C, Gaforio JJ, Szikora JP, De Smet C, Brasseur F, et al
. Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 1994;40:360-9. doi: 10.1007/BF01246677.
Tacer KF, Montoya MC, Oatley MJ, Lord T, Oatley JM, Klein J, et al
. MAGE cancer-testis antigens protect the mammalian germline under environmental stress. Sci Adv 2019;5:eaav4832. doi: 10.1126/sciadv.aav4832.
Lucas S, De Smet C, Arden KC, Viars CS, Lethé B, Lurquin C,et al
. Identification of a new MAGE gene with tumor-specific expression by representational difference analysis. Cancer Res 1998;58:743-52.
Põld M, Zhou J, Chen GL, Hall JM, Vescio RA, Berenson JR. Identification of a new, unorthodox member of the MAGE gene family. Genomics 1999;59:161-7. doi: 10.1006/geno.1999.5870.
Lucas S, Brasseur F, Boon T. A new MAGE gene with ubiquitous expression does not code for known MAGE antigens recognized by T cells. Cancer Res 1999;59:4100-3.
Osterlund C, Töhönen V, Forslund KO, Nordqvist K. Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation. Cancer Res 2000;60:1054-61.
Okutman O, Muller J, Skory V, Garnier JM, Gaucherot A, Baert Y, et al
. A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family. J Assist Reprod Genet 2017;34:683-94. doi: 10.1007/s10815-017-0900-z.
Khil PP, Smirnova NA, Romanienko PJ, Camerini-Otero RD. The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation. Nat Genet 2004;36:642-6. doi: 10.1038/ng1368.
Lo Giacco D, Chianese C, Ars E, Ruiz-Castañé E, Forti G, Krausz C. Recurrent X chromosome-linked deletions: Discovery of new genetic factors in male infertility. J Med Genet 2014;51:340-4. doi: 10.1136/jmedgenet-2013-101988.
De Plaen E, De Backer O, Arnaud D, Bonjean B, Chomez P, Martelange V, et al
. A new family of mouse genes homologous to the human MAGE genes. Genomics 1999;55:176-84. doi: 10.1006/geno998.5638.
Mueller JL, Mahadevaiah SK, Park PJ, Warburton PE, Page DC, Turner JM. The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression. Nat Genet 2008;40:794-9. doi: 10.1038/ng26.
Takasaki N, Tachibana K, Ogasawara S, Matsuzaki H, Hagiuda J, Ishikawa H, et al
. A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility. Proc Natl Acad Sci U S A 2014;111:1120-5. doi: 10.1073/pnas.1310777111.
Sharma M, Braun RE. Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis. Development 2018;145:dev151555. doi: 10.1242/dev.151555.
Ben Khelifa M, Coutton C, Zouari R, Karaouzène T, Rendu J, Bidart M, et al
. Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella. Am J Hum Genet 2014;94:95-104. doi: 10.1016/j.ajhg.2013.11.017.
Yazarlou F, Mowla SJ, Oskooei VK, Motevaseli E, Tooli LF, Afsharpad M, et al
. Urine exosome gene expression of cancer-testis antigens for prediction of bladder carcinoma. Cancer Manag Res 2018;10:5373-81. doi: 10.2147/cmar.S180389.
Kaufmann J, Wentzensen N, Brinker TJ, Grabe N. Large-scale in-silico identification of a tumor-specific antigen pool for targeted immunotherapy in triple-negative breast cancer. Oncotarget 2019;10:2515-29. doi: 10.18632/oncotarget.26808.
Lovelace DL, Gao Z, Mutoji K, Song YC, Ruan J, Hermann BP. The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia. Development 2016;143:1893-906. doi: 10.1242/dev32761.
Manterola M, Brown TM, Oh MY, Garyn C, Gonzalez BJ, Wolgemuth DJ. BRDT is an essential epigenetic regulator for proper chromatin organization, silencing of sex chromosomes and crossover formation in male meiosis. PLoS Genet 2018;14:e1007209. doi: 10.1371/journal.pgen.1007209.
Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, et al
. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004;36:647-52. doi: 10.1038/ng1366.
Greenbaum MP, Yan W, Wu MH, Lin YN, Agno JE, Sharma M, et al
. TEX14 is essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci U S A 2006;103:4982-7. doi: 10.1073/pnas505123103.
Kwon JT, Jin S, Choi H, Kim J, Jeong J, Kim J, et al
. TEX13 is a novel male germ cell-specific nuclear protein potentially involved in transcriptional repression. FEBS Lett 2016;590:3526-37. doi: 10.1002/1873-3468.12433.
Shukla KK, Rajender S. Apoptosis, spermatogenesis and male infertility. Frontiers Biosci 2012;4:746-54. doi: 10.2741/415.
Xu L, Lu Y, Han D, Yao R, Wang H, Zhong S, et al
. Rnf138 deficiency promotes apoptosis of spermatogonia in juvenile male mice. Cell Death Dis 2017;8:e2795. doi: 10.1038/cddis.2017.110.
Song HW, Wilkinson MF. Transcriptional control of spermatogonial maintenance and differentiation. Semin Cell Dev Biol 2014;30:14-26. doi: 10.1016/j.semcdb.2014.02.005.
Jin C, Zhang Y, Wang ZP, Wang XX, Sun TC, Li XY, et al
. EZH2 deletion promotes spermatogonial differentiation and apoptosis. Reproduction 2017;154:615-25. doi: 10.1530/rep-17-0302.
Yuan S, Qin W, Riordan CR, McSwiggin H, Zheng H, Yan W. Ubqln3, a testis-specific gene, is dispensable for embryonic development and spermatogenesis in mice. Mol Reprod Dev 2015;82:266-7. doi: 10.1002/mrd.22475.
Feng J, Liang P, Chen Y, Zhang X, Songyang Z, Zheng H, et al
. Testis-specific Lypd9 is dispensable for spermatogenesis in mouse. Mol Reprod Dev 2018;85:87-9. doi: 10.1002/mrd.22942.
Hou S, Xian L, Shi P, Li C, Lin Z, Gao X. The Magea gene cluster regulates male germ cell apoptosis without affecting the fertility in mice. Sci Rep 2016;6:26735. doi: 10.1038/srep26735.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]