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Regular Article |
a Departments of Physiology and
b Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland
c Reproduction & Development Research Group, Blaise Pascal University, CNRS UMR 6547—GEEM, 63177 Aubiere, France
d Research Laboratories of Schering AG, D-13342 Berlin, Germany
ABSTRACT
Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.
epididymis, male sexual function, sperm maturation, spermatid, testes
INTRODUCTION
The male germ cells, spermatozoa, are produced in the testis, but at the time of their transit from testis to epididymis they are still nonmotile and unable to fertilize oocytes in vivo. Mammalian spermatozoa acquire their motility and the ability to fertilize during their transit through the epididymis. These maturation events are believed to be dependent on the local environment provided by the epididymal fluid [1, 2]. The principal components of this environment are the specific proteins synthesized and secreted by the epididymal epithelium in a highly regionalized manner. Some of these proteins are found only in specific regions of the epididymis (i.e., the initial segment, caput, corpus, and cauda [3]). For example, the genes for glutathione peroxidase 5 (GPX5) and murine epididymal retinoic acid-binding protein (mE-RABP) are expressed in caput epididymidis [4, 5] and human epididymal protein type 2 (HE2) is expressed in the caput and proximal corpus epididymidis [6]. The genes for cysteine-rich secretory protein-1 (CRISP-1) and human epididymal protein type 4 (HE4) are expressed in the corpus and cauda regions [7, 8], and human epididymal protein type 5 (HE5) from the distal caput to distal cauda epididymidis, expression being highest in the distal corpus region [6]. This regional expression of epididymal proteins results in a unique luminal fluid environment in each part of the epididymis [9]. The transport of spermatozoa through the epididymis ultimately results in their functional maturation. However, the molecular basis of these maturational events still remains unknown [10]. The aim of the present study was to search for gene regulatory regions capable of directing gene expression into the epididymis of transgenic mice. The promoters of two candidate genes were selected for more detailed study; namely, those of GPX5 and CRISP-1.
GPX5 is a 24-kDa secretory protein that belongs to the glutathione peroxidase family [11, 12]. GPX5 gene encodes a selenium-independent GPX, the expression of which in caput epididymidis is under the control of androgens [13, 14]. Although its role as glutathione peroxidase in the epididymis was not clearly demonstrated, it was shown that despite the absence of the critical selenocysteine residue, a part of the catalytic center of other known animal GPXs, GPX5 behaves in vivo and in vitro as a glutathione peroxidase [15, 16]. The protein was shown to bind to the acrosomic region of spermatozoa during their epididymal transit [17, 18], and therefore, it has been suggested that GPX5 could protect sperm membranes from oxidative damage [3, 13, 15, 16].
In addition to corpus and cauda epididymidis, CRISP-1 is also found to be expressed to a lesser extent in the vas deferens and salivary gland [7, 19]. The CRISP family of secretory proteins also includes CRISP-2 and CRISP-3, which are expressed in testis and salivary gland, respectively [20, 21]. Similarly to GPX5, the expression of CRISP-1 is under the control of androgens [20], and its biological function is unknown [19]. Based on the strong expression of GPX5 and CRISP-1 in epididymis, we evaluated the possibility of using the 5'-flanking regions of these genes to express genes of interest in the epididymis of transgenic mice.
MATERIALS AND METHODS
Gene Constructs
A 5.0-kilobase (kb) fragment of the mouse GPX5 promoter, corresponding to nucleotides -5012 to +24 (transcription initiation site +1), was excised from the Bluescript II KS vector (Stratagene, La Jolla, CA) using the XhoI and SacII restriction enzymes, and ligated to the promoterless pEGFP-1 vector (Clontech, Palo Alto, CA; GenBank accession number U55761) in front of the EGFP reporter gene. Likewise, a 3.8-kb fragment of the mouse CRISP-1 promoter (GenBank accession number Y09162), corresponding to nucleotides -3714 to +138, was excised from the pNEB193 vector (New England BioLabs, Inc., Beverly, MA) using the KpnI and AgeI restriction enzymes, and then ligated to the pEGFP-1 vector. The translation initiation codon (at position +1) was destroyed by site-directed mutagenesis.
Generating the Transgenic Mouse Lines
The CRISP-1-EGFP and GPX5-EGFP transgenes were cleaved from the pEGFP-1 vector by KpnI/DraIII (Fig. 1A) and XhoI/DraIII (Fig. 2A) digestions, respectively. The DNA fragments were purified by a Quick-Pick Electroelution Capsule Kit (Qiagen, Valencia, CA) and Elutip DEAE-columns (Schleicher & Shüell, Dassel, Germany) and diluted to a final concentration of 2 ng/µl. Transgenic founder mice were generated in the genetic background of the FVB/N strain by microinjecting the DNA into the pronuclei of fertilized oocytes using standard techniques [22]. DNA was isolated from tail biopsies by the salting-out method [23], and the integration of the transgenes was verified by polymerase chain reaction (PCR). Approximately 1 µg of genomic DNA was used in a PCR reaction consisting of 35 cycles (1 min at 96°C and 1 min at 61°C for GPX5-EGFP, and at 62°C for CRISP-1-EGFP; and 1.5 min at 72°C) using a primer pair-specific either for the EGFP gene only (GPX5-EGFP mice, 5'-primer: EGFP-1 and 3'-primer: EGFP-2, Table 1) or specific for the CRISP-1 promoter and EGFP-cDNA (5'-primer: CRISP-1 and 3'-primer: EGFP-3, Table 1). The founder mice were mated with nontransgenic FVB/N mice to create specific transgenic mouse lines. The results shown are from F2 to F4 generations of these crossings. The University of Turku Ethical Committee on Use and Care of Animals approved all the procedures using mice. The mice were specific pathogen-free and were fed with complete pelleted chow and tap water ad libitum in a room with controlled light (12L:12D) and temperature (21°C ± 1°C).
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RNA Analyses
Total RNA from various tissues was isolated by using the single-step method [24] and the expression of the endogenous GPX5 (GenBank accession number M68896) and CRISP-1 (GenBank accession number L05559) genes as well as of the GPX5-EGFP and CRISP-1-EGFP transgenes were studied by reverse transcription (RT)-PCR. One microgram of DNase I (Gibco, Paisley, Scotland)-treated total RNA was reverse-transcribed (10 min at 50°C) using AMV reverse transcriptase (Promega, Madison, WI) and amplified using Pfu- (Promega) or Dynazyme II-polymerase (Finnzymes, Espoo, Finland) in the same reaction tube. To amplify the cDNAs in CRISP-1-EGFP mice we used the same primers that were used for genotyping the transgenic mice. For GPX5-EGFP we used the following EGFP specific primers: 5' primer, EGFP-4; and 3' primer, EGFP-5 (Table 1). Similarly, the expression of endogenous GPX5 and CRISP-1 genes were studied by RT-PCR using primers specific for GPX5 (5' primer, GPX5–1; and 3' primer, GPX5–2; Table 1) and for CRISP-1 transcripts (5' primer, CRISP-2; and 3' primer, CRISP-3; Table 1). The mouse ß-actin (GenBank accession number X03672) forward and reverse primers (5' primer, ß-actin-Fw; and 3' primer, ß-actin-Re; Table 1) were used to generate a control for the amount of RNA used in the RT-PCR reactions. Southern Blot was done for the RT-PCR products from the GPX5-EGFP and CRISP-1-EGFP lines.
The gel was denatured, neutralized, and blotted onto a Hybond-XL membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer's instructions. The membranes were baked for 2 h at 80°C and hybridized with either [32P]
CTP labeled full-length EGFP cDNA (CRISP-1-EGFP line) or with [32P]
ATP end-labeled oligonucleotide (EGFP-2; Table 1, GPX5-EGFP line) using standard techniques. Hybridization signals were detected by autoradiography using a Fuji film or a PhosphoImager (Fuji Film Ltd., Tokyo, Japan).
In Northern-blot analysis, 20 µg of denatured total RNA were resolved on a 1% denaturing agarose gel and transferred onto nylon membranes (Hybond-XL, Amersham Pharmacia Biotech). The membranes were hybridized with the [32P]CTP labeled full-length cDNA for the EGFP using standard techniques. Hybridization signals were detected by autoradiography using Fuji film or a PhosphoImager.
Detection of EGFP in Tissue Sections
The tissues were fixed for 2 h in 4% paraformaldehyde (PFA) at room temperature, and 70-µm-thick sections were cut with a vibratome (752HA; Campden Instruments Ltd., Sileby, UK). The sections were mounted with PBS and green fluorescent light emitted by the EGFP protein was evaluated using a confocal microscope setup (Leica TCS SP scanner and DMR microscope; Leica Lasertechnik, Heidelberg, Germany). The tissues were exposed to 488 nm excitation light and emissions were obtained at 500–520 nm. Ten images of 2-µm intervals in Z-axis were collected with a confocal scanner equipped with an argon-krypton-ion laser system (Omnichrome, Chino, CA) and coupled with the program LeicaSCANware 4.2a.
Transillumination of CRISP-1-EGFP mice seminiferous tubules and preparation of cell squashes
Transillumination under a stereomicroscope and preparation of cell squashes for phase contrast microscopy were proceeded as described earlier [25, 26]. Briefly, after killing the mice, the testes were removed and decapsulated. The seminiferous tubules were dissected free from the interstitial tissue in a Petri dish containing PBS and subjected to transillumination under a stereomicroscope. Short segments (
1 mm) of seminiferous tubules representing various stages of the seminiferous epithelial cycle were cut out after recognition by transillumination and transferred in a small amount (10 µl) of PBS to microscope slides using a Pasteur pipette. Under continuous monitoring with the 40x phase contrast objective, the medium was carefully blotted with lens paper until the cells showed a slightly flattened monolayer, allowing good resolution that was critical for identifying the different cell types and stages of the seminiferous epithelial cycle. For phase contrast and fluorescence microscopy of the cell monolayer, the coverslip was sealed with paraffin oil and a 100x immersion objective was used. Phase contrast and fluorescent pictures were captured using a black/white video camera (Kappa CF 8/1 FMC CCD, Gleichen, Germany) attached to a research microscope (Leica DM RBE). For fluorescence experiments, tubules and cell monolayers were exposed by UV light through 450–490 nm band pass filter and 510 nm dichroic mirror and the emissions were captured through a 520 nm long pass filter with a Kappa CF 8/1 FMC CCD black/white video camera using 10x, 40x, and 100x objectives.
RESULTS
Generation of GPX5-EGFP and CRISP-1-EGFP Transgenic Mice
The 5.0-kb GPX5 and 3.8-kb CRISP-1 promoter fragments were ligated to the pEGFP-1 vector in front of the EGFP-reporter gene (Figs. 1A and 2A). After pronucleus injection and embryo transfer, four GPX5-EGFP and 10 CRISP-1-EGFP founder mice were generated. Transgene integration was confirmed by Southern blot and PCR analyses on genomic DNA. Transgenic mouse lines were generated from all the GPX5-EGFP founder mice (011, 024, 031) and from 3 out of the 10 CRISP-1-EGFP founders (020, 052, 053) by breeding with wild-type FVB/N mice.
Tissue Distribution of GPX5-EGFP and CRISP-1-EGFP mRNAs
Northern blot analysis of the CRISP-1-EGFP mice indicated that the 3.8-kb-long CRISP-1 promoter was highly expressed only in the testis (data not shown), and this was further confirmed by Southern blot analysis of the RT-PCR products. Among the 11 male and 10 female tissues analyzed in adult 2- to 3-mo-old mice, the mRNA for the reporter gene was expressed highly in the testis, whereas no expression was observed in the epididymis (Fig. 1B). This was surprising because through RT-PCR, expression of the endogenous CRISP-1 gene was confirmed to be highly specific to the epididymis, vas deferens, and salivary gland (Fig. 1C).
In contrast to CRISP-1, RT-PCR analysis of the GPX5-EGFP mice indicated that the 5.0-kb-long GPX5 promoter was able to direct the reporter gene expression into the epididymis (Fig. 2B). In these mice, the strongest signal for EGFP mRNA among all tissues analyzed was found in the epididymis, but using Southern blot analysis of the RT-PCR products, a variable level of reporter gene expression was detected in all tissues studied, except in the seminal vesicle (Fig. 2B). Similar results were found when endogenous GPX5 expression was analyzed. The expression of GPX5 mRNA was highest in the epididymis but low levels of mRNA were detected in all other tissues (Fig. 2C).
Cell-Specific Expression of CRISP-1-EGFP and GPX5-EGFP
Fluorescent light emitted by the EGFP protein was analyzed in tissues where the reporter gene expression was detected by RT-PCR. In all the CRISP-1-EGFP mouse lines, the EGFP fluorescence showed an identical pattern, with a strong signal in tubular compartment of the testis (Fig. 3, A and B). EGFP fluorescence was detected in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16, as summarized in Figure 3G. The highest fluorescence intensity was observed in spermatids at steps 10–16 (Fig. 3, C and D). Stage VII pachytene spermatocytes (Fig. 3, E and F) showed a low level of EGFP fluorescence, whereas stages X–XI pachytene spermatocytes (Fig. 3, C and D) showed an increasingly EGFP fluorescence. No specific EGFP fluorescence could be detected in Sertoli cells, Leydig cells, stem cells, preleptotene, leptotene, zygotene, or early pachytene spermatocytes.
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Of all of the tissues analyzed from the GPX5-EGFP mouse lines, EGFP fluorescence could only be detected in the epididymis. Therefore, determination of the cellular localization of the low level of reporter gene expression present in a variety of other tissues could not be carried out. Thus, the data indicate that similarly to endogenous GPX5, the reporter gene driven by the 5.0-kb 5'-flanking region of the GPX5 gene is highly expressed in the epididymis. All the GPX5-EGFP mouse lines showed strong EGFP expression in principal cells of the distal caput region, corresponding to segment 4 (Fig. 4, A–D), as defined previously by Abou-Haila and Fain-Maurel [27], whereas only a few cells expressed EGFP in the cauda region (data not shown). EGFP fluorescence could not be detected in the corpus region. However, intensity of the EGFP fluorescence and number of EGFP-positive principal cells in the distal caput and cauda epididymides varied between the different mouse lines generated. EGFP expression in the distal caput epididymis was highest in line 031, where all the tubulae in segment 4 contained a number of fluorescent cells (Fig. 4, B and C). In line 024, the expression pattern was similar, although the number of fluorescent cells was lower (Fig. 4D), and in mouse line 011, only a few fluorescent cells were detected in the distal caput region. In all the mouse lines, only a few EGFP-positive cells (<10 cells/cauda) were present in the cauda epididymis (data not shown).
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DISCUSSION
Searching for tissue-specific promoters to drive transgenes to specific target organs of interest is of key importance for the development of specific disease models and models for drug development. Our studies are directed toward understanding the physiology of the epididymis in order to develop novel strategies for male contraception based on inhibition of post-testicular sperm maturation. In the present study, we evaluated whether the 5.0-kb- and 3.8-kb-long 5'-flanking regions of GPX5 and CRISP-1 genes, respectively, could be used to direct transgene expression into the different regions of the epididymis.
The mouse CRISP-1 gene is expressed in the corpus and cauda epididymides, but also to a lesser extent in the male salivary gland and vas deferens [7, 19], as was also confirmed by our RT-PCR results. Because of the specific expression pattern of the endogenous mouse CRISP-1, it was surprising to find that, instead of the epididymis, the 3.8-kb-long 5'-flanking segment of the CRISP-1 gene directed the reporter gene expression into late meiotic and postmeiotic germ cells in the testis. This indicates that the 3.8-kb-long promoter fragment used does not contain the regulatory elements needed for epididymis-specific gene expression. It is interesting that another member of this gene family, CRISP-2, is specifically expressed in male haploid germ cells [20]. By aligning the 5'-flanking regions of the CRISP-1 and CRISP-2 genes, no significant sequence similarity between DNA sequences could be identified [28]. Therefore, the reason for the germ cell-specific expression of the 3.8-kb-long CRISP-1 promoter used remains unknown. Because of the high specificity of the CRISP-1 promoter it is suggested to be a good tool in further studies to express transgenes in late meiotic and postmeiotic germ cells in a stage-specific manner. Other promoters suitable for germ cell-specific transgene expression in male mice include those of mouse acrosomal protein SP10 gene, which can be used to direct the transgene expression into round spermatids [29], protamine 1 (Prm-1), which directs transgene expression into round and elongated spermatids [30], and phosphoglycerate kinase 2 promoter (Pgk-2), which directs gene expression into spermatocytes and spermatids [31].
Previous studies have shown that the mouse GPX5 gene is expressed at a high level in caput epididymidis [15], but also at low levels in the kidney and liver [32]. We confirmed the wide expression of low amounts of GPX5 in almost all mouse tissues. Similarly, a low level of reporter gene expression was detected in almost all the tissues analyzed. As the endogenous GPX5 mRNA was detected by RT-PCR in the various tissues, the leakage of the transgene outside the epididymis was not surprising, and not a flaw of the transgene construct. However, similar to the endogenous gene, the reporter gene driven by the 5.0-kb 5'-flanking fragment of the GPX5 gene was found to be expressed at the highest level by far in the caput epididymis. The endogenous GPX5 gene is expressed throughout the entire caput region and its expression decreases thereafter [4, 18]. The results we have obtained with the 5.0-kb promoter-driven EGFP showed that in the caput epididymis EGFP, expression is restricted to the distal part, especially in segment 4 [27]. This suggests that all the key regulatory regions involved in the control of the GPX5 gene expression in vivo in the caput are not present in the promoter fragment used. It also brings forward the idea that in addition to tissue-, region-, and cell-specific control regions there are segment-specific cis-acting regulatory domains within the GPX5 sequence. Furthermore, the level of expression and the number of EGFP-positive cells varied between the different GPX5-EGFP lines generated. This indicates that the expression of EGFP under the 5.0-kb fragment used may partially be dependent on the integration site.
During the course of this study, the promoter of the murine epididymal retinoic acid-binding protein (mE-RABP) was shown to direct transgene expression in caput epididymis [33]. However, the GPX5 and mE-RABP promoters seem to direct transgene expression differentially in the caput epididymis. Similarly to the endogenous gene, the 5.0-kb 5' fragment of the mE-RABP gene directs transgene expression into segments 2 to 5, the expression being highest in the distal part of segment 3 and segment 4 [33]. The 5.0-kb GPX5 promoter directs transgene expression into segment 4 of the caput epididymis but transgene is also expressed at low levels in cauda epididymis and in various other tissues. In summary, we have shown in the present study that the 5.0-kb-long GPX5 promoter drives reporter gene expression in transgenic mice to the epididymis and the expression is highest in segment 4 of the distal caput epididymidis. In contrast, for unknown reasons, the 3.8-kb-long CRISP-1 promoter directs reporter gene expression specifically to late meiotic and postmeiotic germ cells in the testis. This finding is in contrast to the epididymis-specific expression of the endogenous CRISP-1 gene. The data suggest that the 5.0-kb 5'-flanking segment of GPX5 gene is a valuable tool for expressing genes of interest, especially in the distal caput region of epididymis in transgenic mice. In addition, the 3.8-kb 5'-flanking region of CRISP-1 promoter may be used for expressing genes in the late meiotic and postmeiotic spermatogenic cells.
ACKNOWLEDGMENTS
We thank Nina Messer for the DNA microinjection and animal handling, Maritta Forsblom and Birgitta Helle for animal husbandry, and Johanna Vesa for the technical assistance with the histology specimens.
FOOTNOTES
1 This work was founded by grants from the Rockefeller and Ernst-Schering Foundations, the Academy of Finland (project 42145 and 43745), Turku University Foundation, and Turku Graduate School of Biomedical Sciences. 
2 Correspondence: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. FAX: 358 2 2502610; matti.poutanen{at}utu.fi
Accepted: November 13, 2000.
Received: April 12, 2000.
REFERENCES
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