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Male Reproductive Tract |
Institute of Human Genetics,4 Hannover Medical School, D-30623 Hannover, Germany
Institute of Human Genetics,5 University of Göttingen, D-37073 Göttingen, Germany
Department of Anatomy and Cell Biology,6 Justus-Liebig-University, D-35385 Giessen, Germany
Institute of Human Genetics, Biocenter,7 University of Würzburg, D-97074 Würzburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, male reproductive tract, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After being first discovered in man [1], TSPY orthologous gene families have subsequently been characterized in many other mammalian species along the primate [12, 13], artiodactyl [14, 15], perissodactyl [16], and rodent [17, 18] lineages. A peculiar situation is encountered in rodents. Although tspy is functional in the rat [17, 19], the Mus musculus-derived laboratory mouse harbors a single-copy pseudogene that is unable to generate a functional transcript [20]. To date, no efforts had been made toward establishing an animal model that would allow experimental investigations of regulation and expression of human TSPY.
Because the laboratory mouse carries the tspy gene in a naturally silenced state, it represents a species in which only the re-establishment of a functional ortholog would be a feasible approach. In the present study, a human Y chromosomal 8.2-kb genomic fragment consisting of a 2.95-kb promoter and 2.8-kb coding region of human TSPY was used to generate TSPY transgenic mice to examine whether and how a TSPY transgene is expressed in animals with otherwise unimpaired male-specific functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An 8.2-kb EcoRI-XmaI genomic fragment containing 2.95 kb of the 5' region of human TSPY, the complete 2.8-kb TSPY coding region, and 2.45 kb of the 3' part were isolated from the human cosmid clone cEMT [2] (derived from the hybrid cell line 3E7) and cloned in the EcoRI-XmaI-restricted pGEM-4Z vector (Promega, Madison, WI), termed pHerbi. The 8.2-kb fragment was characterized by restriction mapping and sequencing with an ABI 310 sequencer (Applied Biosystems, Weiterstadt, Germany). Sequences were analyzed by computer searches performed with BLASTn (NCBI, National Center for Biotechnology Information, AC006335, M98524)).
Generation of Transgenic Mice
DNA from the recombinant plasmid pHerbi was digested with EcoRI-XmaI and separated in an ethidium bromide-free 0.8% agarose gel. The 8.2-kb genomic fragment used as transgene was purified from agarose by QiaExII Gel-extraction kit (Qiagen, Hilden, Germany) and resuspended in buffer containing 10 mM Tris-HCL/0.15 mM EDTA (pH 7.4), to a final concentration of 2.5 ng/µl.
Transgenic mice were generated by microinjection in pronuclei of fertilized one-cell NMRI eggs (Charles River Laboratories, Sulzfeld, Germany) eggs as described elsewhere [21]. Genotyping of transgenic founders was performed on tail genomic DNA by polymerase chain reaction (PCR) and Southern blot analysis. Integration of the human TSPY gene into the genome of three founder animals (M9, M11, and M16) was confirmed by PCR of genomic DNA with human TSPY primers covering the human TSPY promoter region and exons 1 to 6 (Table 1) and sequencing the amplified products. Southern hybridization to TaqI-digested genomic DNA with a TSPY PCR product covering exons 3 to 6 also indicated the presence of human TSPY within the three founders. This probe does not hybridize to DNA of the wild-type mouse. Genomic DNA of all three founders showed exactly the same hybridization pattern as male human DNA.
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RNA Isolation and Northern Blotting
Total RNA was isolated from different tissues using the peqGOLD TriFAST solution (peQLab, Erlangen, Germany) according to the manufacturer's recommendation. Fifteen micrograms RNA were separated by electrophoresis on a 1.2% agarose gel containing formaldehyde, blotted to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Freiburg, Germany), and hybridized with 
-32P-dCTP-labeled PCR product (high prime kit, Roche Diagnostics, Mannheim, Germany) generated with human TSPY primers TDGEX-2 and TD-2 using pEGY9 as a template. pEGY9 contains a human TSPY cDNA fragment covering the complete open reading frame (exons 1, 2, 3, 4, 5, and 6), which was isolated from human total testicular RNA by reverse transcription (RT)-PCR analysis with the primers TDGEX-2 and TD-2. The 927-bp fragment was cloned into modified pGEX2-T (Amersham Pharmacia Biotech). Hybridization was carried out in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto, CA) at 66°C overnight. After hybridization, filters were washed at room temperature for 2 min in 2 x saline sodium citrate (SSC), then for 5 min in 2 x SSC at 65°C, and finally for 3 min in 0.1 x SSC/0.1% SDS at 65°C. Hybridization signals were visualized by x-ray autoradiography. The integrity of RNAs was checked by rehybridization with a rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-cDNA probe.
PCR, RT-PCR and Product Cloning
Amplification of genomic DNA, recombinant plasmid DNA, or cDNA was performed by PCR with 10 pmol primer and 1 U of Taq or Pfu polymerase (Qiagen) or Stratagene (Heidelberg, Germany) in a 30-µl reaction volume. Standard conditions were denaturation at 95°C for 5 min, 35 cycles of 1 min at 95°C, 1 min annealing at 55°C, 1- to 3-min incubation at 72°C, and a final extension at 72°C for 10 min.
First-strand cDNA syntheses were performed using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol. PCR- and RT-PCR-amplified products were cloned with the TA Cloning Kit Dual Promoter and Zero Blunt T-P- PCR Cloning Kit (Invitrogen, San Diego, CA).
DNA Sequencing
Sequencing of recombinant plasmid DNA and PCR fragments was carried out with the ABI PRISM 310 genetic analyzer (PE Applied Biosystems, Vaterstetten, Germany), using the ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit (PE Applied Biosystems).
Southern Blotting
Fifteen micrograms genomic male human and mouse DNA were TaqI digested and fractioned in a 0.8% agarose gel in TAE (Tris-acetate-EDTA) buffer, vacuum-transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech), and hybridized with -32P-dCTP-labeled PCR product (High Prime Kit, Roche Diagnostics) generated with human TSPY primers P5 and TV03 (Table 1) using pHerbi as a template. Hybridization was performed under stringent conditions (Church's hybridization at 63°C, buffer 0.25 M NaP04 [pH 7.2], 7% SDS). Blots were washed once with 2 x SSC at room temperature, once with 2 x SSC at 63°C for 3 min, and once in 0.1x SSC, 0.1% SDS at 63°C for 2 min. The membranes were directly exposed to a BAS MP 2040S imaging plate (Fuji, Miyamodai, Japan) for 1 d, and images were read on BAS 1000 PhosphorImager (Fuji). The relative intensity of the radioactive signals at each location was quantified using PCBAS 2 version software (TINA 2.0; Raytest, Straubenhardt, Germany).
Western Blotting
For Western analysis, 50 µg (per lane) protein of testicular tissue were separated on 12% gel in SDS-PAGE, and transferred onto Hybond C membrane (Amersham Pharmacia Biotech) by semidry blotting (Biometra, Göttingen, Germany). Signals were immunodetected with antiserum 837/3 [5], raised against human TSPY peptid, diluted 1/1000, using the ECL-chemiluminescence system (Amersham Pharmacia Biotech). Specificity of the immunostaining was proven by preabsorbing antiserum 837/3 with excess recombinant TSPY protein. Signals were visualized by x-ray autoradiography.
Fluorescence in Situ Hybridization Analysis
To visualize the genomic integration of the TSPY transgene, fluorescence in situ hybridization (FISH) was performed using a human TSPY clone (pHerbi) containing an 8.2-kb insert. DNA was labeled by nick-translation in the presence of biotin-16-dUTP (Roche). Mitotic chromosomes were obtained from in vivo colchicine-treated bone marrow cells of the transgenic mouse (M37) following standard protocols involving hypotonic treatment and methanol:acetic acid (3:1) fixation. FISH was conducted essentially as described previously [20]. The biotinylated probe was denatured and allowed to hybridize to denatured chromosome spreads overnight at 37°C. Hybridization sites on chromosomes were visualized with fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA) followed by signal enhancement through further incubation with biotinylated antiavidin and fluorescein-conjugated avidin. Chromosome preparations were counterstained with diamidino-2-phenylindole (DAPI) and observed with an Axiphot epifluorescence microscope (Carl Zeiss, Göttingen, Germany) equipped with a cooled charged couple device camera. The specific fluorescein signal was overlaid on the digitized DAPI image using the software developed by Applied Spectral Imaging (Easy FISH 1.0; Mannheim, Germany). Specific assignment of the probe was made from the analysis of 10 hybridized metaphases.
Histological Examinations and Immunohistochemistry of Tissue Sections
Five-micrometer sections were obtained after fixation of the tissues in Bouin fixative or 10% buffered formalin fixative (Fischar, Saarbrücken, Germany) and embedding in paraffin. Immunostaining of tissue sections was performed using a modified peroxidase ABC detection protocol [22]. The primary antiserum 837/3 was used at 1:300 dilution ratio. The binding of biotinylated goat anti-rabbit IgG to the primary antibodies was increased by rabbit-peroxidase-linked antiperoxidase antibody (DAKO, Geostrup, Denmark) and detected by ABC-peroxidase reagents (Vectastain Elite ABC kit, Vector Laboratories). For the enzymatic detection, the DAB kit (Vector Laboratories) was used. TSPY-specific immunostaining was assessed using antiserum blocked with excess recombinant TSPY protein.
For histological evaluations, testicular tissue was fixed in Bouin fixative and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) or paraffin according to the manufacturer's recommendation. Testis sections of 2 µm were stained with hematoxylin (Gill) and eosin.
Four mice from each group (nontransgenic wild-type and hemizygous transgenic) were killed at Postnatal Day 18 and 40. Testes were fixed in Bouin fixative and subsequently embedded in Technovit 7100 (Heraeus Kulzer). Six sections per animal were collected on one glass slide and stained with hematoxylin and eosin. Data collection always included counting cells from all six sections on the glass slide, and only tubules with approximately the same size and diameter were included. Within tubules all germ cells with visible nucleus were counted. Per mouse less than 15 tubules were quantified.
Sperm Analysis
Epididymides were removed from 4 adult (3 mo old) transgenic and age-related wild-type mice and dissected in Tyrode medium and then incubated for at least 1 h at 37°C in 5% CO2. An aliquot was diluted (1:500) in M2 (Sigma Chemie, Deisenhofen, Germany), and number of spermatozoa of each epididymis were counted by Neubauer chamber. Five sexually mature TSPY transgenic (2 to 4 mo old) and nontransgenic males of the same age were mated, each with two mature females. Females were checked for the presence of vaginal plugs. Uterus and oviducts from inseminated females were flushed with M2 medium, and number of sperm was counted as described above.
Statistical Analysis
Numerical data are represented as the arithmetic mean ± SEM. In experiments in which sperm number of transgenic and wild-type mice were compared, a Student t-test was performed to analyze the significance of the difference (Table 2). A computer-assisted statistics program (SPSS, Inc., Chicago, IL) was used. In experiments in which the number of pachytene spermatocytes (postnatal day 18) and round and elongated spermatids (postnatal day 40) were counted, values were used for one-way ANOVA. These analyses were conducted using the Statistical Analysis Systems (SAS Institute, Cary, NC). Differences were considered significant if the P values were smaller than 0.05.
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Transgenic Animals
Transgenic mice were kept three to four per cage after weaning at the age of 21 days in a room with controlled light and darkness cycle (12L:12D) and room temperature of 21 ± 1°C. All procedures using mice were approved by the University of Göttingen Ethical Committee on Use and Care of Animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An 8.2-kb EcoRI-XmaI genomic fragment containing 3 kb of the human TSPY promoter region, the complete structural gene, and 2.5 kb of the 3' region of human TSPY was used to generate transgenic mice by pronucleus injection. Three founders were recovered from injection into pronuclei of 900 fertilized eggs; one male, M9; and two females, M11 and M16. After mating of the founder transgenic mice with wild-type mice and genotyping the offspring, it was found that only the male founder, M9, showed a germ-line transmission of the transgene. Both female founders, M11 and M16, were fertile but did not produce transgenic F1 offspring, suggesting that both females were mosaic with respect to germ-line integration. The TSPY line TgTSPY9, founded by male M9 bred to nontransgenic NMRI mice, was used for further studies.
TgTSPY9 Contains Approximately 50 Copies of TSPY on Yq
PCR analyses performed with tail DNA of F1 animals of founder M9 showed that all male offspring were transgenic for human TSPY, whereas no transgenic female F1 mice could be detected, a result compatible with a Y chromosomal integration of the human transgene. The integration site was directly assessed via FISH (Fig. 1). FISH with the biotinylated human TSPY probe (pHerbi) revealed specific signals on a single chromosomal site in more than 90% of metaphases screened, suggesting a single integration site of the transgene. In the TgTSPY9 transgenic mouse, DAPI fluorescence patterns as well as a size comparison allowed identification of the particular chromosome harboring the FISH signal as Y. The hybridization signal comprising all copies of the transgene TSPY was clustered at the distal part of the Y long arm. No cross-hybridization occurred to regions proximal to the centromeric region of the diminutive short arm Yp, in which the murine tspy is localized [20]. In none of the metaphases analyzed were additional signals located on other chromosomes.
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Integration of the entire human TSPY insert into the genome of transgenic mouse line TgTSPY9 was confirmed by probing Southern blots of either TaqI or SacI-digested genomic DNA preparations with different fragments spanning the entire insert of pHerbi (data not shown). To investigate the copy number of human TSPY within the transgenic mouse line TgTSPY9, a quantitative Southern analysis was performed (Fig. 2). A TSPY-specific signal of approximately 2.1 kb in size was detected in TSPY transgenic mice, and the intensity of the signal was consistent with human TSPY being present in approximately 50 copies on the mouse Y chromosome. Thus, the copy number in TgTSPY9 mice mimics the situation in humans.
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Expression of the TSPY Transgene
To identify TSPY transcripts in testicular RNA of TgTSPY9 transgenic mouse, two primers from exon 1 (TD-2) and exon 6 (TDGEX-2), which span the entire length of the coding sequence, were used for poly-dT-RT-PCR. RT-PCR amplification generated cDNA products of the size predicted by the human transcript TSPYmajor, and, in addition, a number of aberrant products (Fig. 3A). The alternatively spliced isoforms were cloned and sequenced. Three of the five identified splice variants correspond to known human TSPY spliced isoforms: a Y-231 equivalent [12], the TSPY A variant [5], and the TSPY C variant that splices out 7 bp of exon 4 using an internal acceptor site (unpublished data). The removal of 261 bp in exon 1 by the use of a cryptic splice donor and splice acceptor site, and this variant in combination with the Y-231-type exon 4–5 border, are novel TSPY splice variants that were identified only in transgenic mice. Northern hybridization of testicular RNA confirmed the presence of TSPY transcripts in the adult transgenic testes (Fig. 3B). Comparisons with the signal intensity in human testis RNA indicated a somewhat lower amount of TSPY transcripts in testicular tissue of transgenic mice but no corrections have been applied to potential differences in the proportion of expressing cell types that may vary between man and transgenic mouse. To determine whether the testis-specific expression of TSPY is also conserved in mice, we performed a Northern analysis with RNA from different tissues of the transgenic line TgTSPY9. A major 1.3-kb transcript was detected in testis only, and cerebrum showed a weak signal, whereas no signal was observed in prostate, epididymis, lung, heart, stomach, large intestine, liver, and spleen (Fig. 3C).
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Western blot analysis of testicular protein extracts derived from human as positive control, the TSPY transgenic mouse, and wild-type mouse as negative control using TSPY specific antiserum 837/3 as a probe confirmed the presence of an immunoreactive transgene and human products in transgenic mouse but not in nontransgenic mouse (data not shown). As in man, two major bands corresponding to proteins of 38 and 33 kDa were detected in the transgenic line TgTSPY9 and both bands were prevented when TSPY antiserum was blocked with the control polypeptide.
Immunohistochemistry for TSPY antigen to sectioned adult testes of TgTSPY9 transgenic mice and wild-type mice was performed to identify the TSPY-expressing cell types by using 837/3 antiserum (Fig. 4). The specificity of TSPY immunostaining was checked using antiserum preabsorbed with excess recombinant TSPY polypeptide. TSPY immunostaining was detectable in spermatogonia and prophase spermatocytes of the leptotene/zygotene stage in TgTSPY9 transgenic mice. The immunohistochemical staining localizes the TSPY epitope mainly to the cytoplasm of spermatogonia and early primary spermatocytes. Most if not all germ cells showed an immunoreaction also in their nucleoplasm. Thus, the transgene expression pattern of cell type specificity and the cellular topology of the human peptide within the transgene testes mirrors TSPY expression in human and bovine testis. No immunoreactions were observed in the negative controls.
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TSPY Hemizygous Male Mice Show Normal Spermatogenesis
The anatomy and histology of TSPY transgenic mice appeared to be normal. Males killed at 8–16 wk after birth did not differ from age-related wild-type mice, with regard to testis size and weight (data not shown). There was no significant difference in epididymal sperm numbers in transgenic and age-matched wild-type controls. Females inseminated by TSPY transgenic or wild-type males of the same age did not significantly differ with regard to the mean number of sperm recovered from uterus and oviduct (Table 2). These observations suggest that spermatogenesis is neither impaired nor enhanced by the transgene. As judged by histological analysis of testes of adult (6 wk to 8 mo) TSPY hemizygous males in comparison with age-matched wild-type males spermatogenesis was normal (Fig. 5). To investigate more closely whether spermatogenesis was affected by the transgene, the numbers of meiotic and postmeiotic germ cells were assessed. Therefore, four nontransgenic wild-type males and hemizygous TSPY transgenic males each were killed at Days 18 and 40. Sectioned testes were stained with hematoxylin and eosin. In comparison with testis sections from wild-type controls lacking the TSPY transgene, TSPY transgenic mice did not significantly differ in number of pachytene spermatocytes (postnatal day 18) and round and elongated spermatids (postnatal day 40), respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We speculate that mouse regulatory sequence elements directing the testicular expression of TSPY are retained owing to selective pressure and that these elements are recognized by the trans-acting transcription factors of the mouse. Tissue-specific expression can be mediated by Sox proteins belonging to the high mobility group box superfamily of DNA-binding proteins found throughout the animal kingdom [26]. We have evidence that human TSPY expression is indeed controlled by two Sox specific consensus motifs within the human TSPY promoter region (B. Skawran, unpublished data). It seems likely that testicular TSPY (expression in TgTSPY9 transgenic mice is also regulated by Sox proteins.
Northern analysis showed that TSPY is mainly expressed in testis and to a much lesser extent the cerebrum of the transgenic mouse (Fig. 3C). Su et al. [27] investigated the expression pattern from 91 human and mouse tissue samples and cell lines by using GENECHIP 3.2 (Affymetrix, Santa Clara, CA) and detected TSPY transcripts exclusively in human testis. Because transgenes may be influenced by their site of integration in the host genome [28], a deviating expression pattern of human TSPY in transgenic mice may be caused by a chromosomal position effect on transgene expression. Additional possibilities are incomplete conservation of cis-acting or trans-acting regulatory elements or both. Furthermore, it is possible that the 8.2-kb fragment used for transgenesis did not contain all cis-acting elements (e.g., as silencer) that may repress TSPY expression in brain.
In the present study, a genomic human TSPY sequence was used for generation of transgenic mice because the expression efficiency has been shown to be increased by intronic sequences [29]. We demonstrated that transcription and splicing of TSPY transgene essentially followed the pattern in humans. Besides the main transcript (TSPYmajor) that dominated within the transgene testis, aberrant TSPY splice variants were observed, and most of them corresponded to known human TSPY spliced isoforms. Therefore, human cis-acting elements that regulate processing of the pre-mRNAs transcribed from the human transgene can be recognized by trans-acting splice factors of mouse. Other studies have also demonstrated that mouse-splicing machinery is interacting with human gene sequences in a manner similar to the human cellular machinery [29, 30].
One of the major problems regarding pronucleus microinjection is that the injected DNA fragment integrates randomly into chromosomal DNA. Furthermore, high copy number of the transgene can induce silencing of the transgene [31]. However, in TSPY transgenic mice, the transgene is integrated in approximately 50 copies in one insertion site on the long arm of the mouse Y chromosome and Northern blot analysis revealed a proper amount of human TSPY transcripts in transgenic testis. The peculiar integration pattern of the transgene—a large cluster of multiple copies at a single chromosomal site rather than integration of a few copies at random locations—is astonishing but not without precedence. Recently Devgan et al. [32] generated transgenic mice via microinjection harboring approximately 100 copies of the enhanced green fluorescent protein gene at a single site. That transgenic DNA fragments integrated as large tandem repeats in a single integration site was also demonstrated by Tacken et al. [33], who established two human very low-density lipoprotein receptor transgenic strains carrying at least 44 and 64 DNA fragments, respectively. It is nevertheless tempting to speculate that clustered integration reflects an intrinsic property of a typical TSPY, the only mammalian protein-coding gene known to normally occur as a tandem array of multiple copies [2, 34]. Our approach has the general potential to be exploited systematically to study the fate of regulatory networks following loss or functional silencing of structural genes, in particular those on the decaying Y chromosome [35, 36].
In the TSPY transgenic mouse, spermatogenesis seems not to be affected by the transgene. The strategy to introduce a human TSPY gene into the mouse is indeed not expected to lead to a readily identifiable phenotype per se because, in this species, the original function of TSPY must have become dispensable or may have been taken over by one or more other genes, not identified so far. One could speculate that the human transgene and its yet-unknown counterpart both fulfill the same function and interact and compete with each other for the same partners within the germ cells. Thus, under normal conditions, mouse spermatogenesis is not visibly influenced by human TSPY, the function of which may only become apparent in combination with mutations affecting spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jörg Schmidtke, Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str.1, D-30623 Hannover, Germany. FAX: 0049 511 532 5865; schmidtke.joerg{at}mh-hannover.de
3 Current address: Laboratory of Cytogenetic and Molecular Genetics, D-31134 Hildesheim, Germany
Received: 18 February 2003.
First decision: 16 March 2003.
Accepted: 7 May 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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