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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.005801
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BIOLOGY OF REPRODUCTION 68, 553–559 (2003)
DOI: 10.1095/biolreprod.102.005801
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Temporally Controlled Site-Specific Mutagenesis in the Germ Cell Lineage of the Mouse Testis1

Philipp Weber3,a, Michael Schulera, Christelle Gérarda, Manuel Marka, Daniel Metzgera, and Pierre Chambon2,a

a Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collège de France, B.P. 163, 67404 Illkirch-Cedex, Communauté Urbaine de Strasbourg, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have obtained a PrP-Cre-ERT transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ERT recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ERT is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ERT line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.

gametogenesis, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The generation of mouse mutants through homologous recombination in ES cells and transgenesis has become a powerful tool for investigating in vivo gene functions. However, germ line mutations can induce in utero lethality, abnormalities, compensatory mechanisms, or a combination of these during development, thus precluding analysis of the physiological function of a gene in adults. Furthermore, cell-autonomous and non-cell-autonomous functions may not be readily characterized through germ line mutations, particularly for genes whose product or products exert pleiotropic effects. Spatiotemporally controlled somatic mutagenesis might circumvent such problems [1].

Somatic mutations controlled either spatially or temporally have been obtained in mice by cell type-specific or inducible expression of the bacteriophage P1 Cre recombinase [2, 3], which excises DNA segments that are flanked by LoxP recognition sequences ("floxed" sequences; for a review see [4]). Control of floxed DNA excision was achieved by cell-specific expression of the Cre recombinase fused to mutated ligand binding domains of the estrogen receptor (Cre-ERT, [5–12]). Binding of antiestrogens such as tamoxifen or 4-hydroxytamoxifen to such chimeric Cre recombinases leads to their activation, thus allowing temporally controlled ligand-dependent somatic mutagenesis of floxed target genes.

In adult vertebrates, the PrP is highly expressed in the central nervous system (CNS), and to a lesser extent in some other tissues including lung, heart, kidney, spleen, and testis [13–20]. PrP genomic regions were previously used to generate transgenic animals for PrP or reporter gene expression in various regions of the CNS, as well as in skeletal muscle, in epithelial cells of the thymus, and endothelial cells of some capillaries [21–27]. In a previous study we reported the generation of transgenic mouse lines expressing the ligand-inducible Cre-ERT that were under control of a prp genomic fragment 7.5 kilobases (kb) in length [27]. Analysis of these transgenic lines resulted in the identification of "brain selective" lines, and one transgenic line whose ligand-dependent Cre recombinase activity was apparently restricted to the testis. In the present study we have characterized this testis-specific PrP-Cre-ERT transgenic line. We show that it induces efficient ligand-dependent recombination selectively in spermatogonia, spermatocytes, and spermatids. We have also established a transgenic PrP-L-EGFP-L line that expresses a floxed EGFP in the nervous system, as well as in the testis, where it can be used to monitor spatiotemporally controlled Cre-mediated recombination in germ cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

The generation of PrP-Cre-ERT transgenic mice was as described elsewhere [27]. To generate PrP-L-EGFP-L Cre reporter mice, the 736-base pair (bp) EcoRI fragment of pCX-EGFP [28] was blunt-ended and cloned into the filled-in BamHI site of pLox2 [29] resulting in pL-EGFP-L1. To add simian virus 40 (SV40) polyadenylation sequences, the 839-bp EcoRI-NotI fragment isolated from pL-EGFP-L1 was filled in and cloned into EcoRI and blunted BamHI of pGS [30] resulting in pGS-L-EGFP-L1pA. The 1001-bp PstI-NotI fragment of pGS-L-EGFP-L1pA was cloned into the pL-EGFP-L1 PstI-NotI backbone, resulting in pL-EGFP-L1pA. The 1013-bp EcoRV-NotI fragment of pL-EGFP-L1pA was blunt-ended and cloned into the filled-in HindIII site of pUC19, resulting in pL-EGFP-L1pA(pUC19) from which the 1070-bp EcoRI fragment was blunted and cloned into EcoRV and filled-in NotI sites of pL-EGFP-L1 whose XhoI and SalI sites were destroyed. The 1100-bp KpnI fragment from the resulting clone pL-EGFP-L1KK was ligated into the unique KpnI site of pPrPSal-Kpn{Delta} [27], resulting in pPrP-L-EGFP-L. The 8.2-kb NotI-SalI DNA fragment isolated from pPrP-L-EGFP-L was purified and microinjected into fertilized eggs as described [7]. Breeding and maintenance of mice was performed under institutional guidelines. PrP-Cre-ERT transgenic mice were genotyped as described [27]. DNA from PrP-L-EGFP-L transgenic mice was isolated from tail biopsies [31], digested with BamHI, and hybridized after gel electrophoresis with the radiolabeled probe G (839-bp EcoRI-NotI fragment of pL-EGFP-L, Fig. 3A) as described [27]. Probe K (1546-bp HindIII-KpnI fragment of pPrP-Cre-ERT, Fig. 3A and [27]) was used to characterize PrP-L-EGFP-L mice after Cre-mediated DNA excision. In vivo green fluorescence was analyzed with a Leica MZ12 binocular (Paris, France) equipped with a mercury lamp and a GFP Plus Filter set [27].



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  		FIG. 3. PrP-L-EGFP-L transgenic mice. A) Schematic representation of PrP-L-EGFP-L transgene. Exons I-III of the prp gene are indicated by open boxes. The transcription initiation site is indicated by an arrow. The EGFP coding sequence is indicated by a closed box and LoxP sites are indicated by flags. N, K, and S indicate NotI, KpnI, and SalI restriction sites, respectively. Polyadenylation (pA) sequences are represented by a hatched box, and the locations of probes G and K are indicated. B) EGFP expression in the testis of adult PrP-L-EGFP-L 29.4(tg/0) transgenic mice. EGFP fluorescence in testicular sections of adult PrP-L-EGFP-L 29.4(tg/0) transgenic animals superimposed with DAPI nuclear staining (upper panel, low magnification on the left). Superimposition of EGFP fluorescence and Stra8 immunohistochemical signal is shown on the lower right. Arrows point to one spermatogonia in an individual seminiferous tubule. Note that no EGFP signal could be detected in spermatogonia and in cells of the intertubular region (indicated by asterisks). Bars: low magnification, 300 µm; higher magnification (shown in GFP), 100 µm

RXR{alpha}+/af2(l) mice were genotyped by Southern blotting [32]. Z/AP Cre reporter mice [33] (kindly provided by A. Nagy) and the control Z/AP{Delta} mouse line were as described [27].

Tamoxifen (Sigma, Munich, Germany) was injected i.p. for 5 consecutive days (1 mg/day, [1]) to animals older than 2 mo; these were designated as adult mice.

Analysis of Recombinase Activity at the DNA Level

Cre-ERT-mediated DNA excision in PrP-Cre-ERT(28.8)/RXR{alpha}+/af2(l) double-transgenic mice was determined by Southern blot analysis, performed on genomic DNA isolated 1 day after a 5-day consecutive tamoxifen treatment (1 mg/day). DNA was digested with BamHI and hybridized with probe B [7, 8, 27, 32].

In Situ Hybridization Analysis

Testes of wild-type and transgenic PrP-Cre-ERT mice were freshly frozen in OCT (Sakura, Torrance, CA). Ten-micrometer-thick cryosections were processed for in situ hybridization with a Cre antisense probe [27] as described [34].

Histological Analysis

For Cre immunohistochemistry, PrP-Cre-ERT transgenic mice were treated with tamoxifen and intracardially perfused 1 day after the last ligand injection as described [27]. Testes were embedded in paraffin and 7 µm-thick sections were dewaxed, rehydrated, and incubated in citrate buffer (0.95 mM citric acid, 4.05 mM trisodium citrate dihydrate pH 6.0) and heated in a microwave. Slides were cooled for 45 min at room temperature, washed in PBS, and blocked for 30 min with normal goat serum (5% in PBS and 0.1% Triton X-100), and incubated with a polyclonal antibody directed against Cre (1/3000, [35]) overnight at 4°C. Sections were washed in PBS and incubated with CY3-coupled donkey anti-rabbit secondary antibody. For Stra8 immunohistochemistry and EGFP fluorescence analysis, tissue samples were isolated from intracardially perfused animals at defined time points after tamoxifen administration [36]. Tissues were cryoprotected in 30% sucrose, frozen in OCT (Sakura), and cut in 10-µm-thick cryosections. Sections were postfixed for 20 min in ice-cold methanol, rehydrated in PBS, and incubated with rabbit polyclonal antibody directed against Stra8 as described [37]. Sections were mounted with Vectashield that included 10 µg/ml 4',6'-diamidino-2'-phenylindole (DAPI) and fluorescence was examined using a green fluorescence protein detection filter. Alkaline phosphatase staining was performed on 10-µm-thick cryosections [27]. Sections were mounted with Mowiol (Calbiochem, La Jolla, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cre Recombinase Activity in Testes of PrP-Cre-ERT Transgenic Mice

We previously generated 10 PrP-Cre-ERT transgenic lines (28.1 to 28.10) and showed that PrP-Cre-ERT transgenic mice from lines 28.4 and 28.6 exhibit tamoxifen-dependent Cre-ERT recombinase activity in broad areas of the nervous system, as well as in some other organs including the testis [27]. To characterize the PrP-Cre-ERT 28.8 line, hemizygous PrP-Cre-ERT 28.8(tg/0) mice were bred with RXR{alpha}+/af2(l) reporter mice that harbor one floxed and one wild-type RXR{alpha} allele [32], and 6-wk-old double hemizygous PrP-Cre-ERT 28.8(tg/0)/RXR{alpha}+/af2(l) transgenic mice were daily treated with 1 mg tamoxifen for 5 days. One day after the last tamoxifen injection about 30% of the floxed alleles were recombined in the testis, whereas no recombination could be detected in other organs, nor in vehicle (oil) treated animals (Fig. 1, and data not shown).



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  		FIG. 1. Analysis of floxed DNA excision in PrP-Cre-ERT 28.8(tg/0) transgenic animals. Southern blot analysis of genomic DNA extracted from tissues of 2-mo-old PrP-Cre-ERT 28.8(tg/0)/RXR{alpha}+/af2(l) double-transgenic mice 1 day after the last tamoxifen (Tam) or sunflower oil (Oil) injection. The position of wild-type (+), floxed [af2(I)], and excised [af2(II)] RXR{alpha} alleles [32] are indicated. The RXR{alpha}+/af2(II) lane corresponds to tail DNA isolated from a mouse carrying one wild-type and one excised af2(II) RXR{alpha} allele. Note the absence of a signal corresponding to the recombined allele in the testis of vehicle (oil) treated animals

Cre-ERT Expression in Testis of PrP-Cre-ERT 28.8(tg/0) Transgenic Mice

The expression pattern of Cre-ERT in testis of transgenic line 28.8 was determined by in situ hybridization (ISH; Fig. 2, A and B) analysis and immunohistochemistry (Fig. 2, C–H). The ISH and immunohistochemical signals were dependent on the stage of the cycle of the seminiferous epithelium: stages I to V exhibited strong signals, whereas tubules at later stages of the cycle (i.e., stages VI to XII) were essentially negative (Fig. 2, A and B; compare with Fig. 2, C and D). These signals were confined to cells located at the periphery of the tubules (ePS; Fig. 2, B, C, E, and F), which were identified as early pachytene spermatocytes (ePS; Fig. 2, C and F). No Cre-ERT transcripts and proteins were detected in pachytene spermatocytes at late stages of their maturation (lPS; Fig. 2, B, G, and H), preleptotene spermatocytes (not shown), nor in round or elongate spermatids (RS and ES in Fig. 2, B, E, and F). Cre-ERT protein was also detected in ovoid nuclei lying in close proximity with and parallel to the basement membrane surrounding the tubules; these nuclei were scarce and scattered, and their presence was apparently not correlated with any specific stage of the seminiferous epithelium cycle, suggesting that they might belong to spermatogonia (SG in Fig. 2, C, G, and H) [38]. No Cre-ERT protein was detected in the somatic cells of the seminiferous tubules; namely, Sertoli (S in Fig. 2, E–H) and myoepithelial cells (M in Fig. 2, G and H). Leydig cells were not labeled above background in ISH experiments (L in Fig. 2A) and showed an unspecific cytoplasmic signal in immunohistochemical experiments (compare L in Fig. 2, C and D; also see Fig. 2H). In agreement with the results obtained with the RXR{alpha}+/af2(l) reporter mice, neither Cre-ERT transcript nor protein was detected in brains from this line (data not shown).



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  		FIG. 2. Cre-ERT expression in testis of 3-mo-old PrP-Cre-ERT 28.8(tg/0) transgenic animals. A and B) Cre-ERT transcripts in testicular sections of adult transgenic animals of line 28.8(tg/0) analyzed by radioactive in situ hybridization with a Cre probe. The brightfield and the negative picture of the darkfield were superimposed: the positive signal appears as brown dots corresponding to silver grains, and cell nuclei are stained in blue by hematoxylin. C–H) Detection of Cre-ERT protein in testis of tamoxifen-treated PrP-Cre-ERT 28.8(tg/0) transgenics (C, E–H) and control wild-type (D) mice. Sites of Cre expression were revealed with a Cy3-conjugated secondary antibody and the histological sections were counterstained with DAPI. The immunofluorescence signal of F and H was subtracted in E and G, respectively, in order to identify nuclear morphologies. ES, Early elongating spermatids; L, Leydig cells; M, myoepithelial cells; ePS and lPs, early and late pachytene spermatocytes, respectively; RS, round spermatids; S, Sertoli cells; SG, spermatogonia. Stages of the cycle of the seminiferous epithelium are designated by roman numerals. Bar = 160 µm (A), 55 µm (B), 80 µm (C and D), and 25 µm (E–H)

Generation and Characterization of PrP-L-EGFP-L Transgenic Reporter Lines

To establish a Cre reporter line for the testis and to further analyze the transcriptional activity of the prp genomic fragment in the adult mouse, the coding sequence of the EGFP, flanked by two LoxP sites, was introduced in exon III of the prp gene upstream of the PrP translation initiation codon (construct PrP-L-EGFP-L, see Fig. 3A).

After injection of the PrP-L-EGFP-L transgene into fertilized eggs, two independent transgenic mouse lines (29.1 and 29.4) were identified by Southern blot analysis (data not shown). Newborn offspring of these two transgenic lines exhibited a readily detectable and broadly distributed green fluorescence, which became more restricted with aging (data not shown). In adult PrP-L-EGFP-L transgenic mice EGFP was readily detected in the eye, nose, periphery of the mouth, and tip of the tail and the feet, but not in other parts of the body (not shown). To further characterize the EGFP expression pattern in adult PrP-L-EGFP-L transgenic mice, green fluorescence was analyzed on sections of brain, eye, spinal cord, and sciatic nerve as well as on muscle, heart, spleen, and testis. Animals from both lines exhibited an intense signal in the nervous system, muscle, some cells of the heart, and the testis (Fig. 3B, and data not shown). In the testis of PrP-L-EGFP-L 29.4(tg/0) transgenic mice, EGFP was detected in all seminiferous tubules (Fig. 3B and data not shown). The green fluorescence was strong in meiotic spermatocytes as well as in round and elongated spermatids but was consistently absent in the cell row at the periphery of the tubules, which includes spermatogonia, preleptotene spermatocytes, and the large, basal portion of the Sertoli cells (Fig. 3B). Double-labeling experiments using an antibody to Stra8, a specific marker for premeiotic germ cells [37], confirmed the absence of EGFP in these cells (Fig. 3B). Only a weak EGFP signal was observed in Leydig cells that might correspond to background fluorescence (asterisk in Fig. 3B). To verify the functionality of LoxP sites, PrP-L-EGFPL-L 29.4(tg/0) transgenic mice were crossed with cytomegalovirus-Cre-deletor mice, which efficiently excise floxed DNA segments in germ cells [39]. Cre-mediated recombination induced the loss of EGFP expression by excision of the floxed EGFP cassettes in transgenic line PrP-L-EGFPL-L 29.4(tg/0), and one copy of a transgenic prp genomic fragment remained in the genome (data not shown).

Detection of Recombination at the Cellular Level

PrP-Cre-ERT 28.8(tg/0) transgenic animals were crossed with PrP-L-EGFP-L 29.4(tg/0) reporter mice to further characterize the recombinase activity. Adult PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFPL-L 29.4(tg/0) double-transgenic animals were tamoxifen-treated and EGFP fluorescence was analyzed. The bright signal of EGFP observed in all seminiferous tubules of testis from oil-treated PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFP-L 29.4(tg/0) double-transgenic mice was markedly decreased at the periphery of about half the seminiferous tubules, analyzed 3 wk after tamoxifen treatment (Fig. 4A, compare panels c and d with panels a and b, and data not shown). A sharp boundary between EGFP-positive and EGFP-negative cells was observed, indicating a time-controlled recombination in the germ cells (indicated by arrowheads in Fig. 4A, panel d). In testis analyzed 6 wk and 3 mo after tamoxifen treatment, about half the seminiferous tubules were devoid of EGFP fluorescence, whereas the other tubules exhibited a readily detectable EGFP signal (Fig. 4A, compare panels e and f with panels a and b, and data not shown). The presence of EGFP-negative tubules after more than one complete spermatogenic cycle (lasting about 35 days in the mouse) demonstrates that recombination occurred in most if not all stem spermatogonia of these tubules. However, several seminiferous tubules from tamoxifen-treated PrP-Cre-ERT 28.8(tg/0)/PrP-LEGFPL 29.4(tg/0) double transgenic mice showed mosaic expression of EGFP or were indistinguishable from tubules of oil-treated control mice. This pattern of EGFP expression indicates that excision did not occur in all spermatogonia of all tubules.



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  		FIG. 4. EGFP and alkaline phosphatase staining in the testis of tamoxifen-treated PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFP-L 29.4(tg/0) and PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals. A) EGFP in testicular sections of PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFP-L 29.4(tg/0) double-transgenic animals 6 wk after oil injection (a and b), 3 wk after the last tamoxifen injection (c and d), and 6 wk after the last tamoxifen injection (e and f). Note that about 50% EGFP-positive tubules are observed in testicular sections of PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFP-L 29.4(tg/0) double-transgenic animals even 6 wk after tamoxifen treatment (indicated by arrows in e). Arrowheads point to the limit of EGFP-positive cells of the seminiferous tubules. Note the remaining signal of EGFP signal close to the lumen (lu) in the seminiferous tubules (d). Nuclei are stained with DAPI. Bars = 300 µm in a, c, and e and 100 µm in b, d, and f. B) Alkaline phosphatase (hAP) staining of testicular sections of an adult control Z/AP{Delta} animal (a and b) and of an adult PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals 1 day after the end of tamoxifen treatment (c and d). Note that some tubules in PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals are only weakly stained for hAP (indicated by arrows in c). Arrowheads in b point to hAP-positive elongate and condensed spermatids. Note the absence of hAP staining in Leydig cells of tamoxifen-treated PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals when compared with that in Z/AP{Delta} control mice (indicated by asterisks in b and d). No alkaline phosphatase staining was observed on testicular sections of oil-treated PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals (not shown). Lu indicates lumen of seminiferous tubules (shown in b and d). Bars = 300 µm in a and c and 100 µm in b and d

PrP-Cre-ERT transgenic animals of line 28.8 were also bred with Z/AP Cre-reporter mice in which expression of ß-galactosidase is abolished after Cre-mediated recombination, whereas the expression of human alkaline phosphatase (hAP) is induced [33]. This Cre reporter line was selected because 1) a robust LacZ expression was detected in all the seminiferous tubules and in the interstitial spaces in testes of adult Z/AP mice (data not shown), and 2) alkaline phosphatase activity was detected in all seminiferous tubules and Leydig cells in the adult testis of control Z/AP{Delta} mice (Fig. 4B; panels a and b) in which the floxed LacZ cassette is excised in the germ line [27]. One day after the last tamoxifen administration to PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic animals hAP staining was detected in approximately 75% of the seminiferous tubule cross-sections (Fig. 4B; panels c and d), including spermatocytes, and round and elongated spermatids, whereas no AP staining was observed in oil-treated double-transgenic mice (data not shown). Note that no hAP signal was detected in Leydig cells, thus excluding this cell type as Cre-ERT expressing cells (asterisks in Fig. 4B, panel d). Six weeks after tamoxifen treatment approximately 50% of the seminiferous tubule cross-sections were still hAP positive, thus confirming recombination in spermatogonia.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here the generation and characterization of transgenic mice that express the ligand-inducible Cre-ERT or a "floxed" EGFP reporter gene under the control of a prp genomic fragment. We previously described two PrP-Cre-ERT transgenic lines that express the ligand-inducible Cre-ERT not only in neuronal structures, but also in the testis [27]. The observed testicular activity of the PrP promoter is in agreement with the reported expression of the endogenous PrP gene in seminiferous tubules of the rat testis [20] and in mature sperm cells [19]. We have characterized here a PrP-Cre-ERT transgenic line (line 28.8) that expresses Cre-ERT selectively in the testis, and in which tamoxifen efficiently induced excision of a LoxP-flanked DNA segment in this organ. In this PrP-Cre-ERT transgenic line, the absence of detectable transgene expression and activity in other tissues of the mouse, including the brain, is most probably due to the site of integration of the transgene into the genome.

Cre-ERT transcripts were readily detected in the periphery of seminiferous tubules and were present at specific stages of the seminiferous epithelium cycle. Moreover, the Cre-ERT protein was revealed in some spermatogonia and in early pachytene spermatocytes. In testes of tamoxifen-treated PrP-Cre-ERT 28.8(tg/0)/PrP-L-EGFP-L 29.4(tg/0) double-transgenic animals, no decrease in the number of EGFP-positive spermatocytes was observed 1 wk after tamoxifen treatment, which most likely reflects a high stability of EGFP protein in these cells. However, the number of EGFP-positive germ cells in the seminiferous tubules decreased with time due to maturation-dependent migration of these cells toward the lumen. After more than one spermatogenic cycle (>=6 wk), EGFP-expressing cells had disappeared in ~50% of tubules, thus demonstrating that Cre-ERT efficiently induces recombination in spermatogonia.

Tamoxifen-treated PrP-Cre-ERT 28.8(tg/0)/Z/AP double-transgenic mice revealed that tamoxifen also efficiently induced recombination in spermatocytes, because conditional expression of hAP was detected in these cells as early as 1 day after the end of tamoxifen treatment. The detection of hAP activity in round and elongated spermatids at this time point cannot be accounted by the continuous migration of germ cells toward the lumen of the seminiferous tubules because it takes pachytene spermatocytes 2 wk to progress to elongated spermatids. Instead, it probably reflects the presence of low levels of Cre-ERT protein in spermatids, and demonstrates that tamoxifen (or its active metabolite, hydroxytamoxifen, or both) can pass the blood-testis barrier. When testes were analyzed 6 wk after the last application of tamoxifen, a ratio of 50% of hAP-positive tubules was observed. This is in agreement with the above EGFP results and those obtained with the floxed TIF1ß gene in which we observed altered protein expression and tubular degeneration in ~50% of the tubules [40].

No recombination was found in Leydig cells, because no Cre-ERT protein nor hAP activity was detected in these cells after administration of tamoxifen to double-hemizygous mice. No Cre-ERT protein was detected in Sertoli cells, which is also in agreement with the observation that expression of the floxed TIF1ß gene was not impaired in these cells by tamoxifen treatment of double transgenic mice [40].

The testis-specific expression of the ligand-inducible Cre-ERT in PrP-Cre-ERT transgenic line 28.8 with its germ cell lineage-specific recombination, will allow study of the function of genes involved in spermatogenesis. Because the PrP-Cre-ERT transgene is expressed at specific stages of the spermatogenic cycle, the overall excision rate in germ cells is lower than in previously described transgenic lines that express Cre in a constitutive manner [41, 42]. However, the PrP-Cre-ERT 28.8 mouse line provides an interesting tool for which to discriminate in a conditional manner between Sertoli cell dysfunction and intrinsic germ cell defects that lead to germ cell degeneration [43], and might circumvent chromosomal rearrangements that occurred in transgenic mice expressing constitutively high levels of Cre in the germ cells [44]. Finally, it is noteworthy that the present Cre-ERT-expressing mice can be used not only for conditional inactivation of genes in stem cells of the mouse testis, but also for conditional expression of transgenes in the mouse testis.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Ch. Weissmann and Dr. A. Cozzio for providing plasmid pProHGPrPSal2, Dr. M. Okabe for plasmid pCX-EGFP, Dr. G Schütz for Cre antibodies, as well as Dr. A. Nagy for providing Z/AP Cre reporter mice. We thank R. Lorenz, S. Bronner, J.-L. Vonesch, and the animal facility staff for excellent technical assistance.


   FOOTNOTES
 
1 This work was supported by funds from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Collège de France, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Médicale, Human Frontier Science Program, Ministère de l'Education Nationale de la Recherche et de la Technologie, and the European Community. P.W. was supported by a fellowship (823A-056725) from the Swiss National Science Foundation. M.S. was supported by a Marie Curie Individual Fellowship (1999-01507). Back

2 Correspondence: Pierre Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale/Centre National de la Recherche Scientifique/Université Louis Pasteur, Boîte Postale 163, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France. FAX: 33 3 88 65 32 03; chambon{at}igbmc.u-strasbg.fr Back

3 Current address: Brain Research Institute, University and ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Back

Received: 25 March 2002.

First decision: 10 April 2002.

Accepted: 28 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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