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Biology of Reproduction 63, 1555-1561 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Immortalization of Murine Male Germ Cells at a Discrete Stage of Differentiation by a Novel Directed Promoter-Based Selection Strategy1

Semi Tascoua, Karim Nayerniaa, Amir Samania, Jörg Schmidtkeb, Tanja Vogelb, Wolfgang Engela, and Peter Burfeind2,4,a

a Institute of Human Genetics, University of Göttingen, 37073 Göttingen, Germany b Institute of Human Genetics, Medical High School of Hannover, Hannover, Germany

ABSTRACT

We developed a novel promoter-based selection strategy that could be used to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.

spermatogenesis, testes

INTRODUCTION

Establishment of somatic and germ cell lines from testis would greatly enhance our ability to study stage-specific cellular interactions and molecular mechanisms underlying specific gene regulation at different stages of spermatogenesis. Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable culture model systems [1]. However, this technique is very labor-intensive and time-consuming. To overcome this problem, we developed a novel promoter-based selection strategy to establish a cell line at a defined differentiation stage of spermatogenesis using cell transfection techniques. Cell transfection with a cassette of immortalizing oncogenes controlled by ubiquitous promoters is the conventional method to establish immortalized cell lines [2]. However, by using these promoters, all cell types in the culture are potentially immortalized; therefore, predetermination of a defined cell type is not possible. Contamination of the selected cell line with other cells is also a severe problem of this method. Other difficulties in the conventional method may arise from the differences between cell types regarding the expression level of the genes controlled by such promoters. This means that immortalizing a given cell type depends on the promoter activity in that cell type.

Murine germ cells are seldom functional for more than a few days under in vitro conditions [3, 4]. Therefore, immortalizing this cell type may be the best way to handle them. We designed a promoter-based selection strategy to specifically select immortalized spermatocytes among various cell types in the testis of the transgenic mouse line TC. In this transgenic line, the CAT (chloramphenicol acetyltransferase) reporter gene is expressed specifically in male germ cells under the control of the proacrosin promoter [5]. For specific selection of spermatocytes, a new fusion gene was constructed using the promoter region of the human gene for phosphoglycerate kinase 2 (PGK2). The human PGK2 promoter has a specific transcriptional activity in male germ cells, and PGK2 transcription has been described to start in mouse preleptotene spermatocytes [6, 7]. Using the promoter-based selection strategy, we established a cell line (GC-4spc) that represents a stage between preleptotene and early pachytene spermatocytes. For this purpose, we analyzed GC-4spc cells for the transcription of the germ cell-specific expressed genes murine Pgk2, proacrosin, and A-myb by using reverse transcription-polymerase chain reaction (RT-PCR). Moreover, transcriptional activity of the germ cell-specific expressed genes TSPY (human testis-specific protein, Y-encoded) and PGK2 was demonstrated using transfection studies. Our results show that all these markers of the spermatogenic lineage are detectable in immortalized GC-4spc cells, and that the transformation process followed by targeted selection with promoters that are activated earlier or later during testicular development might immortalize cells that are more primitive or more differentiated, respectively. The potential uses of the promoter-based selection strategy, combined with the regulatory regions of male germ cell-specific genes, would allow production of a series of cultured spermatogenic cell lines representing sequential stages of differentiation. Our approach could serve as a new tool for studying the cellular and molecular mechanisms of germ cell differentiation and cell-cell interaction in the testis.

MATERIALS AND METHODS

Construction of Fusion Genes

All basic molecular biological techniques for generation of fusion genes were performed according to the method described by Sambrook et al. [8]. The fusion gene PBS/PGK2 (Fig. 1) was constructed through a change in the pMSSVLT vector [9] by substitution of the thymidine kinase (TK) promoter at the EcoRI/BglII site with the 1.65-kilobase (kb) promoter region of the human phosphoglycerate kinase 2 gene [6]. The fusion gene TSPY-CAT was constructed by cloning of the 1.35-kb promoter region of the TSPY gene [10] into the pCAT3-Basic vector (Promega, Madison, WI) at the SmaI site. The fusion gene PGK2-CAT was kindly provided by Dr. R.P. Erickson (University of Arizona). The control vector pSV-ß-galactosidase was obtained from Promega.



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  		FIG. 1. Map of fusion constructs used for establishment of the GC-4spc cell line (PBS/hPGK2) and for promoter analyses (PGK2-CAT and TSPY-CAT). In the promoter-based selection construct, PBS/hPGK2 expression of the SV40 large T-antigen gene (TAg) and neomycin phosphotransferase II gene (NEO) are driven by SV40 early promoter and enhancer (P+E) and human phosphoglycerate kinase 2 (PGK2) promoter, respectively

Cell Isolation and Culture Conditions

The mouse spermatogenic cell line GC-1spg [2] was obtained from the American Type Culture Collection (Rockville, MD) and propagated in Dulbecco's modified Eagle medium (DMEM; Gibco/BRL, Gaithersburg, MD) supplemented with 1x nonessential amino acids, 1% sodium pyruvate, 1% glutamine (200 mM), 1% Pen/Strep, and 10% inactivated fetal calf serum (all supplements purchased from Gibco/BRL).

For establishment of the spermatocyte cell line GC-4spc, testes from adult transgenic TC mice [5] were collected aseptically in serum-free culture medium CMRL-1066 (Gibco/BRL). After decapsulation of the testes, interstitial cells were removed by mechanical agitation and washing with the same medium. The isolated seminiferous tubules were treated with 0.1% collagenase and 0.01% trypsin inhibitor (Boehringer, Mannheim, Germany) as described elsewhere [11]. The DNase treatment was avoided to prevent DNA damage during the lipofection step. After a washing step and centrifugation (1000 rpm, 10 min), the cell pellet was suspended to a concentration of 2 x 106 cells/ml in serum-free DMEM containing the lipofection reagent Tfx50 (Promega) and 5-µg construct DNA (PBS/PGK-2, Fig. 1) in a 4:1 v/v ratio. The cell suspension was then transferred to a 60-mm culture flask, and after 2 h, medium was added that was supplemented as described earlier for the GC-1spg cell line. Cells were cultivated for 36 days at 37°C and in 5% CO2, then trypsinized and selected by G418 (Geneticin, Gibco/BRL) at a concentration of 200 µg/ml. Use of the selective medium was continued for 2–3 wk, with frequent changes of medium, until distinct colonies could be visualized. Individual colonies were then trypsinized and transferred to multiwell plates for further propagation in the presence of selective medium. Transient lipofection of PGK2-CAT and TSPY-CAT fusion genes was performed using the Superfect lipofection kit (Quiagen, Hilden, Germany). The day before transfection, 4 x 105 GC-1spg and GC-4spc cells were seeded on 60-mm dishes in 5 ml of appropriate growth medium, respectively. On the day of transfection, cells at a confluency rate of 50%–80% were transfected with 5 µg of construct DNA (PGK2-CAT and TSPY-CAT fusion genes, Fig. 1) and with 5 µg of pSV-ß-galactosidase vector DNA as a control, respectively. Before lipofection, construct DNAs and control DNA were diluted in cell growth medium containing no serum, proteins, or antibiotics to a total volume of 150 µl, and 30 µl of Superfect transfection reagent was added to the DNA solutions. The samples were incubated for 5–10 min at room temperature to allow complex formation. After incubation, 1 ml of cell growth medium (containing serum and antibiotics) was added to the reaction tube containing the transfection complexes, and the total volume was immediately transferred to the cells in the 60-mm dishes. The cells were incubated with the complexes for 2–3 h at 37°C, and the medium containing the remaining complexes was removed from the cells. The cells were washed with 4 ml of phosphate buffered saline, and subsequently, fresh cell growth medium (containing serum and antibiotics) was added. After 48 h, lipofected cells were subjected to RNA isolation and Northern blot analyses.

RNA Isolation and Analysis

Total RNA and Poly(A)+-RNA were extracted from cells and testes using the RNA reagent solution (BIOMOL, Hamburg, Germany) and the mRNA-Isolation Kit (Quiagen) according to the manufacturer's instruction. Genomic DNA contamination was eliminated from RNA by DNase I (Boehringer) treatment. Briefly, 100 µg of total RNA was digested with 10 µl of DNase I (10 U/µl) in a reaction volume of 100 µl for 30 min at 37°C. After DNA digestion, the RNA sample was extracted with an equal volume of phenol/chloroform and precipitated with ethanol. The RT-PCR was performed by using the RT-PCR kit from Amersham Pharmacia Biotech (Freiburg, Germany) and run in a Perkin-Elmer thermocycler (Weiterstadt, Germany). The DNase I-treated total RNA and poly(A)+-RNA (1 µg) was reversed transcribed into cDNA at 42°C for 30 min using the RT-PCR kit from Amersham Pharmacia Biotech and a downstream sequence-specific primer. The PCR step was performed for 40 cycles under the following conditions: 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C.

Primers were designed using published sequences for the mouse Pgk2 gene (upstream primer: 5'-AGG AGA TAC TGC TAC TTG CTG CGC C-3', downstream primer: 5'-GAT GAT GAC AGA ATT AAG ACT TGC T-3') [12], from the mouse proacrosin gene (upstream primer: 5'-GGC CTG GCC CTG GAT GGT CAG-3', downstream primer: 5'-CCC TCC GTC ACT ACG TTG TAT TTC-3') [13], from the mouse A-myb gene (upstream primer: 5'-CAG TGC GTC ACT TAG CGA AGT TGG-3', downstream primer: 5'-TTC AGT TAG CAG TTG AGT CCC TGG-3') [14], from the CAT gene (upstream primer: 5'-CTC ATC CGG AAT TCC GTA TG-3', downstream primer: 5'-GTT GTC CAT ATT GCC CAC GT-3'), from the mouse Insl-3 gene (upstream primer: 5'-TAC TGA TGC TCC TGG CTC TGG-3', downstream primer: 5'-TTA GAC TGT TTG GGA CAC AGG-3') [15], from the mouse transferrin gene (upstream primer: 5'-CTC ACC GTG GGT GCC CTG CTG G-3', downstream primer: 5'-GTG GAA AGT GCA GGC TTC CAG G-3') [16], from the mouse desmin gene (upstream primer: 5'-GTA CCA GGT GTC GCG CAC GTC GGG-3', downstream primer: 5'-GCT CGG AAG GCA GCC AAG TTG TTC-3') [2], and from the mouse lysozyme M gene (upstream primer: 5'-GTG TGT TTA GCT CAG CAC GAG AGC-3', downstream primer: 5'-GCT GAA GTC CTG TGA CTT AGA GGG-3') [17]. To test the RNA in each sample, RT-PCR was performed using ß-actin-specific primers (upstream primer: 5'-GCG GAC TGT TAC TGA GCT GCG T-3', downstream primer: 5'-GAA GCA ATG CTG TCA CCT TCC C-3').

The PCR reaction products were visualized by ethidium bromide staining after agarose gel electrophoresis. The PCR products were subcloned into pGEM-T vectors (Promega) and sequenced with an ABI 377 Sequencer (Applied Biosystems, Weiterstadt, Germany); sequences were analyzed by computer searches performed in the GenBank/EMBL databases (Heidelberg, Germany). For Northern blot analysis, 20 µg RNA samples were denatured in a formaldehyde/formamide buffer and electrophoresed on 1.5% agarose gels. The RNAs were then transferred to nylon membranes (Amersham Pharmacia Biotech) and hybridized with specific DNA probes for the large T antigen, the neomycin gene, ß-galactosidase, and the CAT transgene. Filters were hybridized with 32P-deoxycitidine 5'-triphosphate-labeled (3000 Ci/mmol) DNA probes at 65°C overnight in a solution containing 10% dextran sulfate (w/v), 1x Denhardt solution, 0.1% SDS (w/v), 5x saline sodium citrate (SSC), and single-stranded salmon sperm DNA. After hybridization, filters were washed at room temperature for 15 min in 2x saline sodium citrate (SSC), and then in 0.2x SSC/0.1% SDS at 65°C for 10–30 min. The filters were dried and exposed to x-ray films with an intensifying screen for 16 h at -70°C. For integrity of RNAs, the membranes were rehybridized with a human elongation factor 2 (hEF) cDNA probe [18].

Indirect Immunoperoxidase Staining

Cells were cultured to confluency in Lab-Tek chamber slides (NUNC, Wiesbaden, Germany) with complete DMEM, washed briefly with phosphate buffered saline, and fixed for 5 min with cold (-10°C) methanol. After fixation, cells were immediately washed in three changes of phosphate buffered saline and incubated for 10 min in 0.1% hydrogen peroxidase in phosphate buffered saline to quench endogenous peroxidase activity. Cells were rinsed with phosphate buffered saline and incubated in 1.5% normal blocking serum in phosphate buffered saline (ABC kit, Oncogene Science, Cambridge, MA) for 20 min at room temperature in a humidified chamber. Again, cells were rinsed with phosphate buffered saline and incubated with a primary monoclonal antibody against SV40 large T antigen (Dianova, Hamburg, Germany) for 30 min at room temperature using an antibody concentration of 0.5 µg/ml in phosphate buffered saline containing 1% BSA. After a washing step in three changes of phosphate buffered saline, cells were incubated for 30 min at room temperature with a biotin-conjugated, second-step antibody as provided in the ABC kit. Cells were rinsed in three changes of phosphate buffered saline, incubated with avidin-biotin enzyme reagent for 30 min at room temperature, and washed for 10 min in phosphate buffered saline. Cells were treated with 0.5% Triton X-100/phosphate buffered saline for 30 sec, incubated for 6 min in diaminobenzidine solution (ABC kit), and subsequently rinsed in distilled H2O. Counterstainings were performed with Mayer hematoxylin (Sigma, St. Louis, MO).

RESULTS

Establishment of the Immortalized Spermatocyte Cell Line GC-4spc

Testes from adult male transgenic mice (TC line), which express the CAT reporter gene specifically in male germ cells under the control of the proacrosin promoter [5], were used for preparation of seminiferous tubules, from which a primary cell culture was established. The testicular cells were lipofected with the PBS/PGK2 plasmid (Fig. 1). This plasmid contains both the SV40 large T antigen (TAg) under the control of the early SV40 promoter and enhancer region for immortalization and the neomycin resistance gene under the control of a 1.65-kb human phosphoglycerate kinase 2 (PGK2) promoter region for selection. In transgenic mice, this promoter region of the PGK2 gene can direct the expression of a CAT reporter gene specifically in spermatocytes [6]. In nonselective medium, all lipofected testicular cells were immortalized. After 36 days following lipofection, G418 was added to the culture medium. At weekly intervals, the selective medium was changed until colonies appeared. Cell colonies surviving the selection procedure consisted of immortalized cells that express the neomycin phosphotransferase II gene under control of the PGK2 promoter. Using this strategy, we established the new germ cell line GC-4spc. The GC-4spc cells grow adherently and have integrated the PBS/PGK2 fusion gene in their genome as revealed by Southern blot analysis and chromosomal analysis of the GC-4spc cell line clearly shows that this cell line has a diploid genome (data not shown). This cell line has now been cultured for 15 generations.

In a first experiment to characterize the GC-4spc cell line, expression studies were performed to analyze proper neomycin and large T-antigen expression, respectively. The spermatogenic cell line GC-1spg [2] was used as a control cell line in the following experiments; this germ cell line corresponds to a stage between spermatogonia type B and primary spermatocytes. Expression of the neomycin gene was detected in GC-4spc cells under the control of the PGK2 promoter as revealed by Northern blot analysis (Fig. 2). The different neomycin transcript sizes in the GC-1spg (2.3 kb) and GC-4spc (1.4 kb) cell lines could be attributed to different transcription start sites directed by TK (thymidine kinase) and PGK2 promoter regions and different polyadenylation sequence lengths in these cell lines, respectively. Expression of the neomycin gene in GC-4spc cells is an evidence for transcriptional activity of the PGK2 promoter in this cell line. The large T antigen was found, appropriately expressed, as revealed by Northern blot analysis (Fig. 2) and by immunhistochemical staining using a monoclonal antibody against the SV40 large T antigen (Fig. 3).



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  		FIG. 2. Transcription of the neomycin phosphotransferase II gene (NEO) and the SV40 large T-antigen gene (TAg) in GC-1spg and GC-4spc cell lines. For integrity of the RNA, the Northern blot was rehybridized with a cDNA for human elongation factor II (hEF), which is expressed ubiquitously



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  		FIG. 3. Immunostaining for the SV40 large T antigen (TAg) using a monoclonal anti-TAg antibody. Binding was revealed using an immunoperoxidase technique. A) Positive staining for TAg protein was obtained in the GC-4spc cell line. B) As a control, staining of fixed GC-4spc cells was performed without the first antibody

Expression of Germ Cell-Specific Markers in the GC-4spc Cell Line

For further characterization of the GC-4spc cell line, RT-PCR was used to analyze endogenous mouse Pgk2 transcription and to study expression of the CAT reporter gene, which is under the control of a 0.9-kb proacrosin promoter fragment in the transgenic mouse line TC. Both RT-PCR products could only be detected in RNA isolated from GC-4spc cells and not in GC-1spg RNA (Fig. 4A). During mouse testicular development, the first Pgk2 transcripts were found in RNA from the testes of 12.5-day-old mice [7]. In the transgenic mouse line TC, the first CAT transcripts could be detected in the testes of 15.5-day-old mice. These stages of mouse testicular development correlate with the first appearance of preleptotene and pachytene spermatocytes, respectively [19]. No CAT enzyme activity was found in the GC4-spc cell line using a CAT assay (data not shown). In the transgenic mouse line TC, the first CAT activity was detected in round spermatids [5]. In addition, expression analysis of the germ cell-specific expressed genes A-myb and proacrosin was performed using poly(A)+-RNA from the GC-4spc cell line and total RNA from adult testis as a positive control. Results of recent studies have shown that in the adult mouse testis, A-myb is highly expressed in a subpopulation of spermatogonia and in primary spermatocytes but is not detectable in spermatids [20], whereas transcription of the proacrosin gene starts in pachytene spermatocytes and continues through the haploid stages [5]. Using gene-specific primers for both A-myb and proacrosin, RT-PCR products were observed at the expected sizes after amplification of total RNA from mouse testis and of poly(A)+-RNA isolated from GC-4spc cells (Fig. 4B). Expression of the A-myb and proacrosin genes, however, could only be observed using poly(A)+-RNA isolated from GC-4spc cells and not with total RNA. These RT-PCR results indicate that expression of these two germ cell-specific genes is reduced in the GC-4spc cell line compared with testicular expression in the transgenic mouse line TC. To verify the specificity of all RT-PCR experiments, PCR products for Pgk2, CAT, A-myb, and proacrosin were subcloned into plasmid vectors and subsequently sequenced; all the sequences analyzed by computer searches corresponded to their respective cDNA sequences.



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  		FIG. 4. Amplification by RT-PCR of Pgk2, CAT, A-myb, and proacrosin on RNA from the GC-4spc cell line (GC-4) and from the testes of transgenic mice TC, respectively. As a control, PCR with the RNAs was performed without reverse transcription to rule out DNA contamination (+, reverse transcription and PCR with RNA; -, PCR with RNA but without the reverse transcription step). A) Amplification of Pgk2 and CAT mRNAs was observed in testes of transgenic mice TC and in the GC-4spc cell line (GC-4). No amplification products were obtained in the GC-1spg cell line (GC-1). As a positive control RT-PCR was performed with RNAs from these cell lines and testicular RNA using ß-actin-specific primers. B) Amplification by RT-PCR of A-myb and proacrosin mRNA using poly(A)+-RNA isolated from GC-4spc cells. Testicular RNA of transgenic mice TC was used as a positive control. Both proacrosin and A-myb RT-PCR products were obtained using poly(A)+-RNA from the GC-4spc cell line. In contrast, A-myb and proacrosin RT-PCR products were not detected in GC-1spg cells (data not shown). M, Standard molecular weight marker

To rule out a concomitant immortalization of somatic testicular cells together with GC-4spc cells, we performed RT-PCR analyses on GC-4spc RNA and testis RNA from adult mice (i.e., positive control) using sequence-specific primers for mouse transferrin (Sertoli cells), for mouse Insl-3 (Leydig cells), for mouse desmin (peritubular cells), and for mouse lysozyme M (macrophages), respectively. All somatic cell markers tested proved not to be expressed in GC-4spc cells, whereas expression of all genes was observed in testis RNA (data not shown). This result verifies the specificity and purity of the germ cell line GC-4spc by using our promoter-based selection strategy.

Transcriptional Activity of Germ Cell-Specific Promoters in the GC-4spc Cell Line

To examine transcriptional activities of meiotic-specific promoter regions in GC-1spg and GC-4spc cell lines, these cell lines were transiently lipofected with CAT constructs containing 1.35 kb of the human TSPY and 1.65 kb of the human PGK2 promoter regions, respectively (Fig. 1). The human TSPY gene is specifically expressed in the testis, and by immunostaining, TSPY protein was detected specifically in distinct subsets of spermatogonia. In addition, a trace amount of TSPY protein was also found in primary spermatocytes [10]. For control experiments, these cell lines were also transfected with pCAT3-Basic. Endogenous CAT transcripts in the GC-4spc cell line could only be detected by RT-PCR and not by Northern blot analysis. As a control for transfection efficiency, PGK2-CAT and TSPY-CAT constructs were cotransfected with the pSV-ß-Galactosidase vector in each experiment. Figure 5 shows the results of this promoter study. Whereas a high transcriptional activity of the PGK2 promoter was observed in the GC-4spc cell line, no activity of this promoter could be detected in the GC-1spg cell line. In contrast, the TSPY promoter showed a higher transcriptional activity in the GC-1spg cell line than in the GC-4spc cell line.



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  		FIG. 5. Transcriptional activities of TSPY and PGK2 promoters in GC-1spg and GC-4spc cell lines. These cell lines were transfected transiently with ß-galactosidase (ß-Gal, positive control), TSPY/CAT (TSPY), and PGK2/CAT (PGK2) plasmids. The relative CAT transcriptional activities were determined from the Northern blots densitometrically as a percentage of the amount of ß-Gal transcripts. The Northern blots were rehybridized with a human elongation factor II cDNA probe (HEF) to verify equal amounts and integrity of endogenous RNAs. The relative CAT transcriptional activity represents at least three independent lipofections. For negative controls, these cells were lipofected with the pCAT3-basic plasmid (pCAT3)

DISCUSSION

In the present study, we established a novel promoter-based strategy that can immortalize a differentiated cell line from a particular tissue, and we demonstrated that immortalization and selection can be targeted not only spatially but temporally as well. This approach could allow generation of cell lines that are representative of discrete stages of development within cell lineages that occur transiently within complex tissues and otherwise might be inaccessible. The 5'-flanking regions of genes expressed at defined stages of spermatogenesis can be used to direct expression of a selective marker protein (Neor) in a distinct cell type. First, immortalization of cells was achieved using oncogene expression for initiation of the transformation process. Subsequently, with selection of cells at the stage when the regulatory region becomes active, it was possible to establish a cell lineage in a defined stage of germ cell development. As an example for this approach, we used the regulatory region of the human PGK2 gene to select the immortalized spermatocytes from murine testis.

The human PGK2 promoter had a specific transcriptional activity in male germ cells, and the first PGK2 transcripts were detected in preleptotene spermatocytes [6, 7]. Moreover, in a different study [12], a human PGK2 transgene, a PGK2/CAT transgene, and the endogenous mouse Pgk2 gene all displayed similar patterns and levels of expression, which is consistent with the conclusion that transcription starts at Postnatal Day 13 and that peak RNA accumulation occurs in pachytene spermatocytes. In a more recent study [21], direct evidence was provided that even the pattern for expression of the PGK2 gene in human spermatogenic cells is similar to that in the mouse, both at the transcriptional and the translational level. These data and our own results concerning expression of the mouse Pgk2 gene provided in this study indicate that the immortalized GC-4spc cell line originated from spermatogenic cells.

For additional germ cell-specific markers, we analyzed the transcriptional activity of the mouse A-myb and the proacrosin gene in GC-4spc cells. The A-myb mRNA was detectable specifically in spermatogenic cells, and its expression increased postnatally at Day 10, when primary spermatocytes first appear. In the adult mouse, A-myb RNA was highly expressed in a subpopulation of spermatogonia and in primary spermatocytes but was not detectable in spermatids [20]. Moreover, mice that are homozygous for a germline mutation in A-myb show male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- male mice revealed that the germ cells enter meiotic prophase and arrest at pachytene [22]. During mouse and rat spermatogenesis, proacrosin gene transcription is first observed in pachytene spermatocytes, which resemble diploid spermatogenic cells [23]. These results were supported by those of transgenic approaches, which demonstrated that 2.3 kb of the proacrosin 5'-flanking sequence is not only sufficient for germ cell-specific expression of a CAT reporter gene but also for correct temporal expression of the proacrosin/CAT transgene in pachytene spermatocytes [24]. For detailed analysis, additional transgenic lines were generated that included deletions in the 5'-flanking region [5]. Results of analyses of transgenic lines (transgenic mouse line TC) harboring 900 base pairs of the 5'-flanking region of the proacrosin gene demonstrated that the spatial and temporal expression of this transgene mimics expression of the transgene that contains 2.3 kb of the 5'-flanking region. The germ cell-derived cell line GC-4spc was generated from testes of the latter transgenic mouse line TC, thus providing us with an additional marker gene, the proacrosin/CAT transgene. All the previously mentioned germ cell-specific expressed genes, as well as the proacrosin/CAT transgene, were detected in the immortalized GC-4spc cell line. Taken together, the expression pattern of these germ cell-specific genes in GC-4spc cells clearly demonstrates that this cell line represents the differentiation stages between preleptotene and early pachytene spermatocytes—the stage of differentiation at which the promoter of Pgk2 is first activated.

Results of our promoter studies indicate that GC-4spc cells are transfectable, and that transcription factors, which are necessary for the induction of TSPY and PGK2 expression, are present in the GC-4spc cell line, but with a different pattern of activity. Whereas high transcriptional activity of the TSPY gene could be demonstrated in the spermatogonia-derived cell line GC-1spg, reduced expression of TSPY was observed in the GC-4spc cells. Results of recent studies have shown by RNA in situ analysis and immunostaining that in both humans and cattle, TSPY is expressed in spermatogonia and, to a lesser extent, in primary spermatocytes [10, 25, 26]. In contrast, expression of the PGK2/CAT construct was exclusively observed in transfected GC-4spc cells and not in transfected GC-1spg cells. In summary, the results from our expression studies of germ cell-specific expressed genes and from our transfection analyses support the idea that GC-4spc cells derive from germ cells at the spermatocyte stage and not from premeiotic spermatogonia or postmeiotic spermatids.

We observed a lower expression of these germ cell-specific genes, however, in immortalized GC-4spc cells than in the testis of the transgenic mouse line TC. In addition, expression of the PGK2/CAT construct was higher in transfected GC-4spc cells compared with that of endogenous Pgk2 in this cell line. Another transformed spermatogenic cell line, GC-2spd(ts), encompassing germ cells from mid to late meiosis and generated by Hofmann et al. [27] was tested for the presence of several mRNAs encoded by genes transcribed specifically in the testis and at precise stages of spermatogenesis. In that study, mRNAs for the stage-specific marker proteins LDH-C4 (preleptotene), acrosin (premeiotic), protamine-2 (postmeiotic), and SP-10 (postmeiotic round spermatid stage) were not detected in GC-2spd (ts) cells [28]. On the other hand, results of more recent studies have demonstrated that GC-2spd (ts) cells express the testis-specific CREB splice variant containing exon W [29] and mitochondrial differentiation markers hsp60, SOx, and COx-IV [30]. Further expression studies are needed to reveal whether the influence of paracrine factors on the expression of germ cell-specific genes in these cell lines is comparable to that in vivo.

Two possibilities for low expression of the genes analyzed in GC-4spc cells are proposed. One reason could be the maintenance of gene expression through Sertoli cells in vivo, which are in close contact with spermatocytes in the functional testis [31, 32]. Results of in vitro studies have shown that Sertoli cells secrete several proteins that exert their effect on the regulation of specific genes in germ cells. For example, cultured Sertoli cells express insulin-like growth factor II, and this growth factor can alter the expression of c-fos in spermatogenic cells isolated from murine testis [33]. In a recent study, it was demonstrated in coculture experiments that transcription of the haploid expressed gene THEG is maintained in spermatids only in the presence of Sertoli cells at a high level [34]. In contrast, if isolated spermatids were cultivated for 16 h alone, the expression of THEG was downregulated. Therefore, our future experiments will include Sertoli cell/GC-4spc coculture studies to test this hypothesis.

A second possibility is the change of accessibility of cis-acting elements in the promoter region for transcription factors in the succession of chromatin remodeling. Chromatin structure in germ cells affects the specific gene expression in these cells [35, 36]. Thus, after isolation of germ cells from the testis, changes in chromatin structures may possibly occur that subsequently influence the accessibility of a promoter region for transcription factors and, consequently, change gene expression. The observation presented in our study may support this hypothesis, because expression of endogenous murine Pgk2 in GC-4spc cells is lower than that of the lipofected PGK2/CAT fusion gene. This difference could be explained by accessibility of the promoter region in the PGK2/CAT fusion gene for transcription factors and changes of the chromatin structure in isolated, immortalized spermatocytes and, consequently, reduction of endogenous Pgk2 gene expression.

Although Pgk2 expression starts in preleptotene spermatocytes and continues through meiosis to the early stages of spermatid differentiation, only spermatocytes could be immortalized and selected by our strategy. The question remains whether the immortalization event occurs during development, freezing the cells in a discrete stage of differentiation, or whether the transformation event occurs in more differentiated cells, which then differentiate during the transformation process. Results of chromosomal analyses and culture studies of GC-4spc cells clearly show that this cell line has a diploid genome and appears to be unable to differentiate in vitro, respectively (data not shown). The SV40 large T antigen may possibly not be able to immortalize postmeiotic germ cells [37], and/or expression of this oncogene during early meiotic stages of germ cells may prevent their differentiation into later meiotic stages. An excess of wild-type p53 can abolish the proliferative function of SV40 large T antigen, enabling immortalized germ cells to undergo meiosis in vitro. Therefore, our strategy could be improved by using steps with conditionally inactivated SV40 large T antigen [27, 38]. Under these conditions, the cells can express genes that are involved in the differentiated functions of the cultured cells, but whose expression is suppressed during permanent growth to differentiate immortalized germ cell lines along the pathway of meiosis. Thus, this strategy could enable us to obtain germ cell lines that are representative of each stage of spermatogenesis by using the regulatory regions from stage-specific genes, an important approach to studying the molecular and cellular mechanisms involved in mammalian spermatogenesis.

ACKNOWLEDGMENTS

We would like to thank Dr. M. Schmid for chromosomal analysis of the GC-1spg and GC-4spc cell lines.

FOOTNOTES

First decision: 10 May 2000.

1 Supported by the Deutsche Forschungsgemeinschaft (grant SFB500/A3 to K.N. and W.E.). Back

2 Correspondence: Peter Burfeind, Institute of Human Genetics, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany. FAX: 49 551 399303; pburfei{at}gwdg.de Back

3 The first two authors contributed equally to this work. Back

Accepted: July 7, 2000.

Received: April 12, 2000.

REFERENCES

  1. Hanahan D. Transgenic mice as probes into complex systems. Science 1989; 246:1265–1275.[Abstract/Free Full Text]
  2. Hofmann MC, Narisawa S, Hess RA, Millan JL. Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen. Exp Cell Res 1992; 201:417–435.[CrossRef][Medline]
  3. Guillou F. Characterization of a new superfusion, two-compartment culture system for Sertoli cells: influence of extracellular matrix on the cell permeability and dynamics of transferrin secretion. J Androl 1990; 11:182–194.[Abstract/Free Full Text]
  4. Rassoulzadegan M, Paquis-Flucklinger V, Bertino B, Sage J, Jasin M, Miyagawa K, van Heyningen V, Besmer P, Cuzin F. Transmeiotic differentiation of male germ cells in culture. Cell 1993; 75:997–1006.[CrossRef][Medline]
  5. Nayernia K, Nieter S, Kremling H, Oberwinkler H, Engel W. Functional and molecular characterization of the transcriptional regulatory region of the proacrosin gene. J Biol Chem 1994; 269:32181–32186.[Abstract/Free Full Text]
  6. Robinson MO, McCarrey JR, Simon MI. Transcriptional regulatory regions of testis–specific PGK2 defined in transgenic mice. Proc Natl Acad Sci USA 1989; 86:8437–8441.[Abstract/Free Full Text]
  7. McCarrey JR, Berg WM, Paragioudakis SJ, Zhang PL, Dilworth DD, Arnold BL, Rossi JJ. Differential transcription of Pgk genes during spermatogenesis in the mouse. Dev Biol 1992; 154:160–168.[CrossRef][Medline]
  8. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning 1989: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  9. Schuermann M. An expression vector system for stable expression of oncogenes. Nucleic Acids Res 1990; 18:4945–4946.[Free Full Text]
  10. Schnieders F, Dörk T, Arnemann J, Vogel T, Werner M, Schmidtke J. Testis-specific protein, Y-encoded (TSPY) expression in testicular tissues. Hum Mol Genet 1996; 5:1801–1807.[Abstract/Free Full Text]
  11. Romrell LJ, Bellve AR, Fawcett DW. Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dev Biol 1976; 49:119–131.[CrossRef][Medline]
  12. Robinson MO, Simon MI. Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis. Nucleic Acids Res 1991; 19:1557–1562.[Abstract/Free Full Text]
  13. Kremling H, Keime S, Wilhelm K, Adham I, Hameister H, Engel W. Mouse proacrosin gene: nucleotide sequence, diploid expression, and chromosomal localization. Genomics 1991; 11:828–834.[CrossRef][Medline]
  14. Trauth K, Mutschler B, Jenkins NA, Gilbert DJ, Copeland NG, Klempnauer KH. Mouse A-myb encodes a trans-activator and is expressed in mitotically active cells of the developing central nervous system, adult testis and B-lymphocytes. EMBO J 1994; 13:5994–6005.[Medline]
  15. Zimmermann S, Schöttler P, Engel W, Adham IM. Mouse Leydig Insulin-like (Ley I-L) gene: structure and expression during testis and ovary development. Mol Reprod Dev 1997; 47:30–38.[CrossRef][Medline]
  16. Idzerda RL, Behringer RR, Theisen M, Huggenvik J, McKnight GS, Brinster RL. Expression from the transferrin gene promoter in transgenic mice. Mol Cell Biol 1989; 9:5154–5162.[Abstract/Free Full Text]
  17. Cross M, Mangelsdorf I, Wedel A, Renkawitz R. Mouse lysozyme M gene: isolation, characterization, and expression studies. Proc Natl Acad Sci U S A 1988; 85:6232–6236.[Abstract/Free Full Text]
  18. Hanes J, Freudenstein J, Rapp G, Scheit KH. Construction of a plasmid containing the complete coding region of human elongation factor-2. Biol Chem Hoppe-Seyler 1992; 373:201–204.[Medline]
  19. Bellve AR, Caviccnia JC, Millette CF, O'Brien DA, Bhatnager YM, Dym M. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol 1977; 74:68–85.[Abstract/Free Full Text]
  20. Latham KE, Litvin J, Orth JM, Patel B, Mettus R, Reddy EP. Temporal patterns of A-myb and B-myb gene expression during testis development. Oncogene 1996; 13:1161–1168.[Medline]
  21. McCarrey JR, Kumari M, Aivaliotis MJ, Wang Z, Zhang P, Marshall F, Vandeberg JL. Analysis of the cDNA and encoded protein of the human testis-specific PGK-2 gene. Dev Genet 1996; 19:321–332.[CrossRef][Medline]
  22. Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth J, Hatton KS, Reddy EP. Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 1997; 386:713–717.[CrossRef][Medline]
  23. Nayernia K, Reim K, Oberwinkler H, Engel W. Diploid expression and translational regulation of rat proacrosin gene. Biochem Biophys Res Commun 1994; 202:88–93.[CrossRef][Medline]
  24. Nayernia K, Burkhardt E, Beimesche S, Keime S, Engel W. Germ cell-specific expression of a proacrosin-CAT fusion gene in transgenic mouse testis. Mol Reprod Dev 1992; 31:241–248.[CrossRef][Medline]
  25. Chanley AC, Cooke HJ. Human male fertility-Y-linked genes and spermatogenesis. Hum Mol Genet 1994; 3:1449–1452.[Abstract]
  26. Vogel T, Dechend F, Manz E, Jung C, Jakubiczka S, Fehr S, Schmidtke J, Schnieders F. Organization and expression of bovine TSPY. Mamm Genome 1997; 8:491–496.[CrossRef][Medline]
  27. Hofmann MC, Hess RA, Goldberg E, Millan JL. Immortalized germ cells undergo meiosis in vitro. Proc Natl Acad Sci U S A 1994; 91:5533–5537.[Abstract/Free Full Text]
  28. Wolkowicz MJ, Coonrod SM, Reddi PP, Millan JL, Hofmann MC, Herr JC. Refinement of the differentiated phenotype of the spermatogenic cell line GC-2spd(ts). Biol Reprod 1996; 55:923–932.[Abstract]
  29. Sanborn BM, Millan JL, Meistrich ML, Moore LC. Alternative splicing of CREB and CREM mRNAs in an immortalized germ cell line. J Androl 1997; 18:62–70.[Abstract/Free Full Text]
  30. Meinhardt A, Renneberg H, Dersch A, Wennemuth G, Millan JL, Aumüller G, Seitz J. The immortalized mouse germ cell lines GC-1spg and GC-2spd as a model for mitochondrial differentiation during meiosis. Adv Exp Med Biol 1997; 424:61–63.[Medline]
  31. Skinner MK. Cell-cell interactions in the testis. Endocr Rev 1991, 12:45–77.
  32. Syed V, Hecht NB. Up-regulation and down-regulation of genes expressed in cocultures of rat Sertoli cells and germ cells. Mol Reprod Dev 1997; 47:380–389.[CrossRef][Medline]
  33. Tsuruta JK, O'Brien DA. Sertoli cell-spermatogenic cell interaction: the insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor mediates changes in spermatogenic cell gene expression in mice. Biol Reprod 1995; 53:1454–1464.[Abstract]
  34. Nayernia K, von Mering MHP, Kraszucka K, Burfeind P, Wehrend A, Köhler M, Schmid M, Engel W. A novel testicular haploid expressed gene (THEG) involved in mouse spermatid-Sertoli cell interaction. Biol Reprod 1999; 60:1488–1495.[Abstract/Free Full Text]
  35. Choi YC, Aizawa A, Hecht NB. Genomic analysis of the mouse protamine 1, protamine 2, and transition protein 2 gene cluster reveals hypermethylation in expressing cells. Mamm Genome 1997; 8:317–323.[CrossRef][Medline]
  36. Kumari M, Stroud JC, Anji A, McCarrey JR. Differential appearance of DNase I-hypersensitive sites correlates with differential transcription of Pgk genes during spermatogenesis in the mouse. J Biol Chem 1996; 271:14390–14397.[Abstract/Free Full Text]
  37. Nayernia K, Samani AA, Klaproth S, Engel W. Haploid male germ cells show no susceptibility to transformation by simian virus 40 large T antigen in transgenic mice. Cell Biol Int 1999; 22:437–443.
  38. Walter N, Jansen M, Ergün S, Kascheike B, Ivell R. Sertoli cell lines established from H-2Kb-tsA58 transgenic mice differentially regulate the expression of cell-specific genes. Exp Cell Res 1996; 225:411–421.[CrossRef][Medline]



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