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Testis |
Department of Science for Laboratory Animal Experimentation,3 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
Institute for Virus Research,4 Kyoto University, Kyoto, Japan
Genome Information Research Center,5 Osaka University, Suita, Osaka, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gene regulation, meiosis, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Suitable promoters for labeling germ cells in the early differentiation phase, from spermatogonia to spermatocytes, especially for study of the switch from mitotic proliferation in spermatogonia to a commitment to meiotic prophase in spermatocytes, have not yet been identified. During this period, chromatin composition in germ cells changes dramatically. This process involves the appearance and subsequent elimination of several histone variants, which are encoded by different genes and are either germ cell specific (H1t, TH2A, TH2B, and TH3) or germ cell enriched (H2A.X and H1a). They are expressed at specific stages of spermatogenesis, mainly during mitosis and meiosis. H2A.X is a member of the histone H2A family, which contains four distinct subfamilies: H2A1, H2A2, H2A.Z, and H2A.X [1]. These proteins are found at high levels in germ cells during the early differentiation phase and in the somatic cells of other tissues, such as spleen and thymus [2, 3]. Because the phosphorylation of H2A.X is correlated with the emergence of DNA double-strand breaks [4], H2A.X may play important roles in both mitotic proliferation of spermatogonia and in homologous recombination in meiotic spermatocytes, or it may act in the switching of these events.
In the present study, we obtained transgenic mouse strains specifically labeled only in the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in the testes. Despite the fact that expression was driven by the H2A.X promoter, type A spermatogonia and somatic cells of other organs were not labeled. Such specific labeling of late-phase spermatogonia and meiotic prophase cells with an enhanced green fluorescent protein (EGFP) will be useful for studying of the mechanism of commitment from spermatogonia to meiosis and the switching from mitosis to meiosis, which are important biological phenomena that have not yet been fully elucidated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNAs were prepared from the testes of C57BL/6 mice (Shizuoka Laboratory Animals Center, Hamamatsu, Japan) at different ages and of busulfan-treated mice and from germ cells (isolated from adult testes) using Trizol Reagent (Invitrogen Corp., Carlsbad, CA). The germ cells of adult testes were prepared as described previously [5]. Busulfan was injected once intraperitoneally into mice at a dose of 40 mg/kg body weight to destroy the spermatogenic cells, and the mice were used after 4 wk. Twenty micrograms of total RNA were electrophoresed on a 1% agarose/formaldehyde gel. After transfer to a Zeta-Probe Blotting Membrane (Bio-Rad Laboratories, Hercules, CA), the RNA was hybridized to 32P-labeled H2A.X cDNA 3' probe.
In Situ Hybridization
Testes were fixed in 4% paraformaldehyde and embedded in methyl methacrylate (MMA) resin. Sections (thickness, 4 µm) were collected on a Superfrost microslide glass with APS coating (Matsunami Glass Ind. Ltd., Osaka, Japan). The MMA resin was removed from the sections, which were then rinsed in alcohol and washed in PBS. The H2A.X probe for in situ hybridization was generated from an ApaI-SacI, 231-base pair (bp) cDNA fragment cloned into pBluescript SK+ (Stratagene, La Jolla, CA). The H2A.X cDNA-pBluescript SK+ construct was digested at the Asp718 or Ecl136II site and used as a template for T7 or T3 RNA polymerase. An antisense probe was generated from an Asp718 digestion product by T7 RNA polymerase, and a sense probe was generated from the Ecl136II digestion product by T3 RNA polymerase. These probes were labeled with dioxigenin-(DIG) uridine triphosphate (Boehringer Mannheim, Mannheim, Germany). In situ hybridization was performed using the TSA Plus DNP System (NEN Life Science Products, Inc., Boston, MA). After hybridization, the bound probe was detected by incubating with anti-DIG-Fab fragments conjugated with peroxidase (Boehringer Mannheim), followed by a color reaction involving 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan). Sections were contrasted with 1% methyl green stain solution (Muto Pure Chemicals Ltd., Tokyo, Japan) and examined under a microscope.
Transient Transfection and Luciferase Assay
Six promoter fragments of H2A.X, the 5' ends of which started from -1300, -1000, -600, -300, -150, and 0 bp and the 3' positions of which terminated at the transcription start site, were cloned into the upstream region of the luciferase gene (luc+) using the reporter vector pGL3-Basic (Promega, Madison, WI). The -1000-, -600-, -300-, and -150-bp H2A.X fragments were amplified by polymerase chain reaction (PCR) from the genomic mouse H2A.X clone using forward primers for each position and a common 3' end primer. Each PCR product was inserted into a pGL3-Basic vector at EcoRI and BamHI sites.
The recombinant reporter construct DNAs were transfected into cells using Lipofectamine Plus Reagent (Invitrogen Corp., Carlsbad, CA) as described in the supplier's protocol. F9 cells were plated on 24-well dishes (coated with collagen type I; Asahi Techno Glass Corp., Tokyo, Japan) and grown to a density of 60–70% confluence before transfection. Each plasmid construct (0.3 µg) and 30 ng of pRL-TK vector (Promega) per dish were cotransfected into cells in serum-free medium, and the dishes were incubated for 3 h before feeding with an equal volume of medium containing 10% fetal bovine serum. After 48 h, the growth medium was removed, and each cell population was washed in the culture dish twice with PBS. Then, the luciferase assay was performed with a PicaGene Dual Sea Pansy Luminescence Kit (Toyo, Inc., Tokyo, Japan) standardized with sea pansy as an internal control according to the manufacturer's instructions.
Construction of Transgenes and Generation of Transgenic Mice
An H2A.X promoter fragment containing a 600-bp sequence upstream of the translation start site was subcloned into the EGFP reporter plasmid, pd2EGFP-Enhancer (Clontech Laboratories, Inc., Palo Alto, CA). The -600-bp H2A.X promoter-d2EGFP construct, digested by SalI, was injected into male pronuclei of B6C3F1xB6C3F1 fertilized eggs, which were transferred to pseudopregnant recipient mice. Transgenic mice were identified by PCR with specific primers (forward primer, 5'-CTA CAG AGT GAG TTC CA; reverse primer, 5'-TGA AGC ACT GCA CGC CGT AG) to check for the existence of H2A.X-d2EGFP. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University).
Fluorescent Stereomicroscopic Observation of the Whole Testis and Testicular Cross-Sections of the Transgenic Mouse
The transgenic mouse testis was exposed to ultraviolet (UV) light excitation, photographed with a Leica DC 200 camera (Leica Microscopy System Ltd., Wetzlar, Germany), fixed with 4% paraformaldehyde for 12 h, and then embedded in glycol methacrylate (Technovit 8100; Heraeus Kulzer GmbH, Wehrheim, Germany). Histological sections (thickness, 5 µm) of the whole testis were prepared. After green fluorescence was photographed, sections were stained with hematoxylin and observed under a photomicroscope for a detailed analysis.
Fluorescence-Activated Cell Sorting Analysis
Single-cell suspensions from the H2A.X transgenic mouse testes were prepared by enzymatic digestion [6] and washed twice with PBS containing 0.5% fetal bovine serum (PBS/FBS). Cells were suspended in PBS/FBS containing 5 µg/ml of Hoechst 33342 (Dojindo) and incubated for 45 min at 37°C. Then, the cells were filtered through a 35-µm nylon screen (BD Falcon, Franklin Lakes, NJ) and kept in the dark on ice until they were analyzed with a dual-laser Fluorescence-Activated Cell Sorting (FACS) Vantage (Becton Dickinson, Franklin Lakes, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression pattern of mouse histone H2A.X mRNA in the developmental stages of mouse testis was first examined by Northern blot analysis using a probe of the 3' noncoding region of mouse histone H2A.X cDNA. In adult testis, two bands, one strong 0.5-kilobase pair (kbp) band and a weaker 1.4-kbp band, were detected, as previously reported (Fig. 1) [3]. Although chronological observations showed a small difference in the intensity of these two bands, all of the testicular samples, except busulfan-treated testis, showed a similar blotting pattern. In the testes of busulfan-treated mice with no germ cells [6, 7], the 0.5-kbp mRNA band could not be identified, whereas this band was strongly observed in the purified germ cell fraction. The 1.4-kbp mRNA was observed in all lanes, including busulfan-treated testis and germ cells, though the expression level was low, especially in adult testis (Fig. 1B). Thus, the 0.5-kbp mRNA was exclusively expressed in germ cells of mouse testis, whereas the 1.4-kbp mRNA was transcribed in both germ and somatic cells.
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To confirm the localization of H2A.X mRNAs in testicular germ cells, we performed in situ hybridization (Fig. 2). Specific antisense and sense probes were used to detect both 1.4-kbp and 0.5-kbp mRNA (Fig. 1A). Specific staining was observed, at a high level, from type A spermatogonia to meiotic prophase spermatocytes, but not in somatic cells, which express 1.4-kbp mRNA at a low level in adult mouse testis. The pattern of mRNA expression observed in the in situ hybridization experiment was consistent with previous results of H2A.X protein expression [2].
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Promoter Activity of H2A.X Gene Analyzed by Transient Transfection of Promoter-Luciferase Reporter DNA into Cell Lines
To determine the active region of the H2A.X promoter in mouse testis, pGL3 vectors with various truncated upstream regions of the H2A.X gene were generated and transfected into teratocarcinoma F9 cells. The longest construct, the 1300-bp region upstream of the H2A.X gene, showed a lower level of promoter activity than did the 1000-bp construct, indicating that some negative control elements exist in the 300-bp region furthest upstream. In contrast, constructs having -300- or -600-bp promoter fragments showed a similar maximal level of luciferase activity (Fig. 3). Constructs with less than 300 bp had little promoter activity. These results indicated that the promoter activity in F9 cells exists in the 300- to 600-bp upstream region of the gene H2A.X.
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Establishing H2A.X/d2EGFP Transgenic Mouse Lines and Germ Cell Specific Expression of EGFP
We generated eight lines from the founder transgenic mouse (F0) by injecting d2EGFP reporter gene, under the control of the -600-bp fragment of the H2A.X upstream region, into fertilizing oocytes (Fig. 4A). Half of them, four independent transgenic mouse lines, showed detectable fluorescence in the testis. However, the intensity of fluorescence varied among the lines (Table 1).
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For further detailed analyses, a representative transgenic mouse line (no. 30) was used. Various organs were examined under UV light for reporter gene expression at 8–10 wk of age. The EGFP fluorescence was specifically detectable in the seminiferous tubules of the testis (Fig. 4, Ba and Bb), but not in any squashed preparations of brain, lung, kidney, liver, skeletal muscle, heart, thymus, and spleen from the transgenic mouse. The exclusive expression of EGFP in the testis was also confirmed by Northern blot analysis (data not shown).
To examine the EGFP-labeled cells in the testis, histological sections were observed under fluorescent microscopy. Cross-sections of the testis showed that the EGFP-labeled cells could be identified as intermediate spermatogonia to meiotic prophase spermatocytes by observing the seminiferous tubules at several stages (Fig. 4, Be–Bl). No fluorescence was observed in postmeiotic germ cells and Sertoli cells (Fig. 4, Bc and Bd). Although it was expected that EGFP expression would mimic the localization of H2A.X mRNA and protein expression (Fig. 2) [2], detailed analyses of expression patterns in the transgenic mouse showed some differences, with EGFP expression being restricted to differentiated spermatogonia and to premeiotic and meiotic prophase cells. Living EGFP-labeled cells were confirmed, using FACS, as cells with 2N and 4N DNA content (Fig. 5). Approximately 14% of total testicular cells were labeled with EGFP reporter protein, and 77% and 23% of them showed 2N and 4N DNA content, indicating differentiated spermatogonia and meiotic prophase spermatocytes, respectively. Differentiated spermatogonia and premeiotic and meiotic prophase cells, which were easily separated from this transgenic mouse testis, would provide a good tool for the biochemical study of the commitment of germ cell differentiation to meiosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The chromatin structures in each differentiated germ cell change during spermatogenesis, and histone variants arise in the change from mitotic to meiotic cells during spermatogenesis [2]. Histone H2A.X is a chromatin component in all eukaryotes, including yeast [12], but it also plays roles in repairing DNA damage and in DNA recombination in meiosis [13–15]. Because the H2A.X gene is highly expressed in spermatogonia to meiotic prophase cells (Figs. 1 and 2) [2], the promoter region is suitable for labeling premeiotic and meiotic prophase cells. Using a part of the promoter region, transgenic mouse lines, specifically labeled with EGFP at differentiated spermatogonia committed to meiosis and meiotic prophase cell stages, were prepared. Because d2EGFP was used, care was taken to avoid labeling for too long to reflect gene expression, given the short half-life of EGFP proteins. The transgenic mouse expressed d2EGFP protein in intermediate spermatogonia to meiotic prophase cell stages, but not in mitotic type A spermatogonia or in the spleen or thymus, which was different from the expression pattern of the endogenous H2A.X gene [3]. Although the 300-nucleotide upstream region of the H2A.X gene may be sufficient for basic transcription of the H2A.X gene in somatic cell culture (Fig. 3) [16], the 600-nucleotide construct was chosen to raise transgenic mice for specific expression of EGFP protein in specific stages of germ cell differentiation. Transgenic mice of four independent strains, showing a similar specific expression of reporter gene exclusively in germ cells, were successfully raised (data not shown). Thus, the data obtained in the present study demonstrated that the regulatory region for differentiated spermatogonia to meiotic prophase-specific transcription was localized in the 600-bp upstream region of the H2A.X gene. Furthermore, the regulatory element was not sufficient for all the promoter activity of the H2A.X gene which showed wider expression in more immature spermatogonia and in somatic cells. Thus, the transgenic mouse raised in the present study did not precisely mimic the specific expression of the H2A.X gene. It may, however, be useful for the further study of germ cell differentiation and spermatogonial cell commitment.
By using the methods in the present study, as compared with the former, nonspecific fractionation techniques [8, 9], testicular germ cells could be available at almost any stage of differentiation and with a relatively high degree of separation. Some transcription factors controlling the activation of the element should be exclusively expressed at the step of commitment in meiotic prophase. It would be helpful to pursue these factors. They would differ from the factors in somatic cells, which were characterized in a previous report [16].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp
Received: 2 May 2003.
First decision: 21 May 2003.
Accepted: 28 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-H2AX signaling in the testis. Biol Reprod 2003 68:628-634This article has been cited by other articles:
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  A. Chicheportiche, J. Bernardino-Sgherri, B. de Massy, and B. Dutrillaux Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse J. Cell Sci., May 15, 2007; 120(10): 1733 - 1742. [Abstract] [Full Text] [PDF]
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E. M. Neuhaus, A. Mashukova, J. Barbour, D. Wolters, and H. Hatt Novel function of {beta}-arrestin2 in the nucleus of mature spermatozoa J. Cell Sci., August 1, 2006; 119(15): 3047 - 3056. [Abstract] [Full Text] [PDF]
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 S. Han, W. Xie, S. H. Kim, L. Yue, and J. DeJong A Short Core Promoter Drives Expression of the ALF Transcription Factor in Reproductive Tissues of Male and Female Mice Biol Reprod, September 1, 2004; 71(3): 933 - 941. [Abstract] [Full Text] [PDF]
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