Molecular Human Reproduction, Vol. 7, No. 12, 1123-1131,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Targeted expression of SV40 large tumour antigen (TAg) induces a transient enhancement of spermatocyte proliferation and apoptosis
1 Institute of Human Genetics, University of Göttingen, Heinrich Dücker Weg 1237073 Göttingen, 2 Department of Cell Biology and Anatomy, University of Marburg, Robert-Koch-Str.6, 35037 Marburg and 3 Department of Pathology, University of Göttingen, Robert-Koch-Str.40, 37075 Göttingen, Germany
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Abstract |
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In an attempt to determine the susceptibility of spermatocytes to malignant transformation by simian virus 40 (SV40) large tumour antigen (TAg), transgenic mice harbouring a chimeric gene composed of the SV40 TAg gene fused to the 1.4 kb promoter sequence of the human phosphoglycerate kinase 2 (PGK2) gene were generated. Northern blot analysis on RNA from different tissues indicated a specific transcription of TAg in the testis of PGK2-TAg transgenic mice. Reverse transcription–polymerase chain reaction and Western blot analysis on testes at different stages of development revealed that transcription and translation of the TAg gene starts in 12-day-old testis, which coincides with the appearance of pre-leptotene spermatocytes. Germ cells of transgenic mice showed no tendency toward transformation, but in testes of both 18- and 25-day-old transgenic mice, a significantly enhanced number of spermatocytes was found. In contrast, in 42-day-old transgenic mice no differences in the number of spermatocytes and spermatids were observed. The number of Sertoli cells was determined to be equal in transgenic and wild type mice. In-situ end labelling of fragmented DNA revealed a higher rate of apoptosis in testes of 18-day-old transgenic mice as compared with wild type mice. These results indicate that germ cell homeostasis in transgenic mice is maintained by an apoptotic mechanism.
apoptosis/germ cell tumour/spermatogenesis/SV40 large T-antigen/transgenic mice
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Introduction |
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It has been shown that transgenic mice bearing a viral oncogene under the control of a cell-specific promoter offer a model for selectively targeted tumour formation (Van Dyke, 1994
). This process of targeted oncogenesis involves transgenic expression of an immortalizing oncogene such as the commonly used simian virus 40 (SV40) large T-antigen (TAg), capable of transforming the expressing cells and resulting in cell-specific hyperplasia or malignant growth (Garabedian et al., 1998). SV40 TAg is a multifunctional protein of 94 kDa size possessing both immortalizing and transforming capacity in the cells transfected by the virus. In this way TAg is a unique oncogene, since most cellular and viral oncogenes are only capable of either immortalization or transformation. The multiple functions of TAg in vivo include: binding to double-stranded DNA and activation of cellular promoters; activation of autokinase, helicase and non-DNA-dependent ATPase activity; stimulation of G1 progression into S phase; and stimulation of polymerase I transcription for rRNA overproduction (Livingston and Bradley, 1987). The immortalization and transformation of rodent and human cells is usually dependent on persistent TAg expression. The transformation capacity of TAg resides in its ability to bind to the cellular anti-oncoprotein, p53 (Dilworth, 1990).
The first study using TAg to induce tumours in transgenic mice was reported by Brinster et al. (Brinster et al., 1984). They showed that the SV40 early region, i.e. the enhancer element and the large and small T-antigen coding sequences, under the control of a metallothioneine promoter, resulted in transgenic mice with choriod plexus tumours and pathological changes in the thymus and kidney. The upstream region of the rat insulin II gene was the first tissue-specific promoter fragment used to direct TAg expression (Hanahan, 1985). These mice developed pancreatic cell tumours and this study proved that cellular promoters are able to induce high enough expression of TAg to produce tumorigenesis in the target tissue (Hanahan, 1985).
The ability to express and assay transgenes at specific stages provides a powerful tool for the analysis of cellular and molecular events of spermatogenesis. Spermatogenesis encompasses three phases of germ cell development, namely spermatogonia to spermatocytes to spermatids (Hecht, 1998). The different stages of spermatogenesis are constitutively present in the testis of the adult male and different cell types appear at different times in testicular development. In order to establish a system examining the susceptibility of male germ cells to transformation at various stages of differentiation, we started to generate transgenic mice using stage-specific promoters to analyse the effects of SV40 TAg during germ cell development. Previously, we demonstrated that spermatids show no susceptibility to transformation by SV40 large TAg in transgenic mice (Nayernia et al., 1998). In the present study, we examined the susceptibility of spermatocytes to transformation by targeted expression of SV40 TAg in spermatocytes of transgenic mice using the promoter sequence of the human phosphoglycerate kinase 2 gene (PGK2). Transgenic approaches have been used previously to demonstrate that this promoter region is sufficient to confer spermatocyte-specific expression of a CAT reporter gene (Robinson et al., 1989). We show that spermatocytes do not have the potency to achieve malignant transformation, but expression of TAg is characterized by an enhanced proliferation event of spermatocytes in prepubertal transgenic mice and by a mechanism of apoptosis which counterbalances this abnormal proliferation.
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Materials and methods |
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Plasmid construction and transgenic mice
The basic molecular experiments were performed according to established protocols (Sambrook et al., 1989
). The 5' flanking regulatory sequence of the human PGK2 gene was fused to the SV40 early coding sequences. The 1.4 kb Xho I/Hind III fragment encompassing the regulatory sequences was isolated from a PGK2-CAT plasmid (kindly provided by Dr R.P.Erickson, University of Arizona, USA) and cloned into pBluescript plasmid (Stratagene, La Jolla, CA, USA). From this plasmid, the 1.4 kb PGK2 promoter was isolated using Xba I/Bam HI restriction enzymes and ligated to the LT-BlueI plasmid which contains coding sequences for SV40 large T and small t antigen genes (Reddy et al., 1979). For microinjection, the PGK2-TAg fragment (containing both large T and small t antigen genes) was isolated free of vector sequences by digestion with Sal I/SacI and purified after gel electrophoresis by binding to glass beads (BIO 101, Inc., La Jolla). The fragment was quantified and diluted to a concentration of 3 µg/ml in injection buffer consisting of 10 mmol/l Tris, pH 7.4 and 0.2 mmol/l EDTA as previously described (Hogan et al., 1986). The DNA was injected into the pronuclei of fertilized 1-cell mouse embryos derived from matings of NMRI mice (Hogan et al., 1986). The injected embryos were cultivated overnight and 2-cell embryos were transferred into NMRI pseudopregnant hosts. Transgenic mice harbouring PGK2-TAg sequences were identified by dot blot and Southern blot analyses using a random primed 2.7 kb TAg probe on DNA extracted from mouse tail biopsies as previously described (Hogan et al., 1986). Positive founder animals were bred with non-transgenic NMRI mice. Transgenic progeny of such crosses were identified and bred together to produce homozygous animals.
RNA isolation and analysis
Total RNA was prepared from testes of transgenic mice at different stages of development (5, 10, 11, 12, 13, 15 days and adult) as well as from heart, lung, liver, muscle, brain and uterus of adult transgenic mice by an RNA isolation reagent (BIOMOL, Hamburg, Germany). Total RNA (20 µg) were electrophoresed through a 1% agarose gel containing 5% formaldehyde and transferred to a nylon membrane (Hybond N; Amersham, Braunschweig, Germany) by capillarity in 20x standard saline citrate (SSC) (1x SSC: 0.15 mol/l NaCl and 0.015 mol/l sodium citrate). The filter was UV- irradiated for 2 min, incubated for 2 h at 80°C and hybridized with a random-primed 32P-labelled TAg DNA probe. To determine the integrity of the RNA, the membranes were rehybridized with a cDNA probe for human elongation factor II (hEF) which is expressed ubiquitously (Hanes et al., 1992).
For reverse transcription–polymerase chain reaction (RT–PCR) analysis, 500 ng of total RNA prepared from testes of homozygous transgenic mice at different post-natal ages (5–60 days) were subjected to RT and PCR using gene-specific primers in a one Tube system (RT–PCR Beads, Amersham). `No template' controls were run for each experiment to rule out template contamination in reaction components. The amplification profile involved 2 min at 94°C for 1 cycle and 1 min at 94°C, 1 min at 60°C and 1 min at 72°C for 35 cycles. The RT–PCR reactions with PGK2-specific primers (5'-AGG AGA TAC TGC TAC TTG CTG CGC C-3' and 5'-GAT GAT GAC AGA ATT AAG ACT TGC T-3'), TAg-specific primers (5'-GCA GCT AAT GGA CCT TCT AGG-3' and 5'-GCC TCA TCA TCA CTA GAT GGC-3') and actin-specific primers (5'-GCG GAC TGT TAC TGA GCT GCG T-3' and 5'-GAA GCA ATG CTG TCA CCT TCC C-3') were performed at the same conditions to amplify 300, 323 and 482 bp fragments respectively. The PCR reaction products were run on a 2% agarose gel and stained with ethidium bromide.
Protein extraction and Western blot analysis
Proteins were extracted from testes of 11- and 12-day-old and adult transgenic and non-transgenic mice with Sucrose/EDTA/ß-Mercaptoethanol (SEM) buffer (0.32 mol/l sucrose, 1mmol/l EDTA, 0.1% ß-mercaptoethanol). After boiling for 10 min and centrifugation at 500xg, tissue lysates (50 µg/lane) were loaded onto a 6% polyacrylamide gel for electrophoresis. Proteins were transferred to polyvinyldene difluoride membranes (Boehringer, Mannheim, Germany) and TAg protein was detected with the BM chemiluminescence Western blotting detection kit (Boehringer) using an anti-TAg monoclonal antibody (Dianova, Hamburg, Germany). After incubation with anti-mouse immunoglobulin (Ig)G, membranes were washed with TBST (20 mmol/l Tris-HCl, 500 mmol/l NaCl, pH 7.5 and 0.1% Tween-20), incubated in chemiluminescent detection reagents for 1 min at room temperature, and exposed to X-ray films (Eastman Kodak Co.).
Quantification of spermatocytes, spermatids and Sertoli cells and data analysis
Five to six mice from each group (homozygous transgenic and non-transgenic) were killed at post-natal day 18, 25 and 42. Testes were fixed in Bouin's fixative and subsequently embedded in paraffin. Eight testis sections per animal were collected on one glass slide and stained with haematoxylin and eosin. All specimens were assessed at the same magnification (x20 objective) using a Leica DMRE microscope. Data collection always included counting cells from all eight sections on the glass slide and only tubules of approximately the same size (round to slightly ovoid) and diameter were included. For quantification of pachytene spermatocytes (day 18, 25 and 42) tubules were randomly assigned. Fields were sampled with a systematic uniform random scheme by regular movements of the scale from the top left to the bottom right part of a given section. Haematoxylin and eosin-stained pachytene spermatocytes were identified by their characteristic nuclear morphology. For counting of round and elongating spermatids in 42-day-old animals only, tubules at stage VIII of spermatogenesis were assessed. For each treatment a minimum of 8000–10 000 cells were counted. Student's t-test was used for comparing differences in cells numbers between experimental groups using the FPSF software package. All values are expressed as mean ± SEM (n = 5–6 animals/group). A value of P < 0.05 was considered significantly different. The number of Sertoli cells was estimated after immunohistochemical staining with an anti-Vimentin antibody (Santa Cruz, Heidelberg, Germany) and by counting of Sertoli cell nuclei. Vimentin labelling was performed according to the manufacturer's protocol.
Measurement of tubule diameter
Measurement of tubule diameter was performed using a Leitz microscope (Leitz, Wetzlar, Germany) linked to a computer with a professional video adaptor. The Lucia G/Comet Vers. 3.52s software package (Nikon, Germany) was used to measure the diameter of round to slightly ovoid seminiferous tubules of otherwise randomly sampled fields. A minimum of 50 tubules were measured in each experimental group.
In situ end-labelling (ISEL) of apoptotic cells
Five to six mice from each group (homozygous transgenic and non-transgenic) were killed at day 18, 25 and 42. The testes of wildtype and transgenic mice were removed and fixed in Bouin's solution, embedded in paraffin, and cut into 5 µm sections. After deparaffinization, sections were stained histochemically (haematoxylin–eosin) and also by applying in-situ end-labelling (ISEL) of fragmented DNA.
Following deparaffinization, sections were digested with proteinase K (Sigma, Deisenhofen, Germany), at a working dilution of 0.7 IU/ml in Tris-buffered saline (TBS), supplemented with 2 mmol/l CaCl2. Slides were rinsed in TBS and then incubated for 60 min at 37°C with 50 µl of the labelling mix [250 IU/ml terminal transferase, 20 µl/ml Digoxigenin-DNA labelling mix at 10x concentration, and 1 mmol/l CaCl2 in reaction buffer for terminal transferase (Roche, Mannheim, Germany)]. After rinsing in TBS (50 mmol/l Tris-HCl; 150 mmol/l NaCl; pH 7.5) sections were blocked with 10% fetal calf serum (Roche) for 15 min. Sections were then incubated for 60 min with a sheep alkaline phosphatase-conjugated F(ab)2 fragment against digoxigenin (Roche). The alkaline phosphatase-conjugated F(ab)2 fragment was applied at a working dilution of 1:250. The nuclear black signals were detected using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and nitro blue tetrazolium as a coupler (Roche) as described previously (Schweyer et al., 2000). Control sections were stained as above, omitting terminal transferase. As positive controls, human lymph nodes with reactive follicular hyperplasia were used.
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Generation of transgenic mice containing the PGK2-TAg fusion gene
The fusion gene PGK2-TAg that was used in these experiments contains 1.4 kb of the 5' flanking region of the human PGK2 gene. This region was previously shown to be expressed in premeiotic male germ cells (Robinson et al., 1989
). This sequence was fused to the early coding region of SV40 containing the small t and the large T-antigens. A total of 139 2-cell stage embryos that had survived the microinjection procedure were transferred to pseudopregnant foster mothers and 26 animals were born. Six animals (23%) were positive for TAg DNA as assayed by dot blot and Southern blot hybridization of tail DNA. Outcrossing of these founder transgenic animals (PL12, PL13, PL14, PL16 and PL18) with wildtype NMRI mice gave rise to three transgenic strains, indicating that only three founder animals (PL13, PL16 and PL18) harboured a transgene integration in their germ line.
Expression of the SV40 TAg in transgenic mice
To determine the profile of TAg transcription in different tissues, total RNA was isolated from heart, lung, liver, kidney, muscle, testis, brain and uterus of homozygous transgenic mice and subjected to Northern blot analysis. As shown in Figure 1, in all transgenic lines, transcription of the TAg gene with a transcript size of 2.3 kb could only be obtained in testicular RNA.
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During post-natal development in male mice, some germ cells in the testicular tubules begin to advance towards the first meiosis at about the same time. The first spermatocytes in meiotic prophase can be observed between days 10–12 after birth. Between days 10–12, the most advanced cells observed are pre-leptotene spermatocytes; at days 14–17, 30% of tubules contain cells in the pachytene stage, and at days 20–22, spermatids are observed for the first time in ~35% of tubules (McCarrey, 1998
). Thus, by analysing the testis at different times during the first 3 weeks of life, an indication can be obtained of the developmental stage at which a transcript is first formed. RT–PCR analysis on RNA isolated from staged prepubertal transgenic males demonstrated TAg transgene transcription starting at post-natal day 12 (Figure 2). Transcription of the PGK2-TAg transgene was consistent with the earliest detected transcription of endogenous mouse Pgk2 analysed by RT–PCR of RNA under the same conditions (Figure 2) and coincides with the earliest appearance of pre-leptotene spermatocytes. In addition, the transcript level continues to increase into adulthood, suggesting further expression in accumulating spermatids. To evaluate the spermatogenic cell type in which the first TAg protein appears, total protein extracts were prepared from testes of homozygous transgenic mice at post-natal ages 11 days, 12 days and adult. These proteins were analysed using a specific anti-TAg antibody in Western blotting assays. TAg protein was first detected in extracts from testes at post-natal day 12 (Figure 3). This result demonstrates that TAg mRNA can be properly translated and TAg mRNA is not subjected to post-translational regulation.
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Furthermore, the PGK2-TAg transgenic mice were fertile. Litters of transgenic animals ranged from 11–15 neonates, which is typical in our NMRI colony. In addition, no sign of oncogenic transformation in any of these transgenic mice was observed, even with the oldest animals (>20 months). Histopathological examination of testes from transgenic mice did not reveal any abnormalities in the germ cell epithelium or interstitial cells.
Quantification of spermatocytes, spermatids and Sertoli cells
Pachytene spermatocytes were quantified in 18-, 25- and 42-day-old transgenic and wild type mice (Figure 4A–F, Figure 5). The 18-day-old transgenic mice manifested a significant increase in the number of spermatocytes of 54%, compared with that in wild type mice. With advancing age the increased number of spermatocytes in transgenic mice assimilates to that of wild type mice. The 25-day-old transgenic mice showed an increase of 25%, which was also statistically significant. The 42-day-old transgenic mice did not reveal any differences in the number of spermatocytes as compared with wild type mice. Round and elongating spermatids were counted in 42-day-old transgenic and wild type mice in stage VIII of spermatogenesis, and showed no statistical differences.
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Furthermore, the number of Sertoli cells was determined in 18-, 25- and 42-day-old transgenic and wild type mice, by counterstaining with an anti-Vimentin antibody and counting of haematoxylin-stained Sertoli cell nuclei per tubule cross section. No differences were observed in Sertoli cell numbers in transgenic and wild type animals at post-natal days 18, 25 and 42 (Table I
). These data demonstrated that the transient elevation of testicular cell numbers was restricted to spermatocytes in transgenic mice.
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Measurement of tubule diameter
Tubule diameters of transgenic and wild type mice were measured. Round to slightly ovoid seminiferous tubules were used and a minimum of 50 tubules were measured in each experimental group. Diameters of tubules in 18- and 25-day-old transgenic mice were significantly increased compared with those in corresponding wild type mice. Eighteen-day-old SV40 transgenic mice showed an increase of 24% and 25-day-old SV40 transgenic mice showed an increase of 27%. Tubules in 42-day-old transgenic mice showed no statistically significant difference compared with those in corresponding wild type mice (Figure 6
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Detection of apoptotic cells in transgenic and wildtype testis
High numbers of cells showed end-labelling of fragmented DNA in the testis of 18-day-old transgenic mice, whereas only a few labelled cells could be detected in testis of wildtype mice (Figure 7A,B,C
). The difference in the number of apoptotic cells in transgenic and wildtype mice at this age was highly significant (P < 0.0001) (Figure 8). In 25-day-old mice, the number of labelled cells decreased in the testis of transgenic mice and was similar to the number of labelled cells in the testis of wild type mice (Figure 7D,E,F). However, 40% of the values in the group of transgenic mice were slightly higher than the highest value in the group of wild type mice (Figure 8). In 42-day-old mice, low numbers of apoptotic cells were observed in the testes of both transgenic and wild type mice. There was no significant difference in the number of apoptotic cells between 25- or 42-day-old transgenic and wildtype mice (Figure 8).
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Discussion |
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Remarkably little is known about the cellular origin of, or the genes that control susceptibility to, testicular germ cell tumours in humans or mice. Based on a review of available data, the predominant view is that these tumours originate in misplaced primordial germ cells retained in extragonadal sites or in premeiotic germ cells (Skakkebaek et al., 1987
; Grigor and Wylie, 1998). A second theory which is based on genetic properties of human germ cell tumours, e.g. the characteristic cytogenetic abnormality of an isochromosome on chromosome 12, consistent near triploid–tetraploid chromosome numbers and an abundant expression of wild-type p53, suggests that the zygotene–pachytene spermatocyte is the most likely target cell for transformation (Chaganti and Houldsworth, 2000).
To examine the susceptibility of germ cells to transformation at different stages, we started the generation of transgenic mice which express the SV40 large T-antigen stage specifically, using germ cell-specific promoters. A number of genes that are involved in spermatogenesis are specifically expressed in male germ cells (Hecht, 1998). Previously, we described the specific expression of SV40 TAg under the proacrosin promoter in haploid germ cells of transgenic mouse testis (Nayernia et al., 1998). Although the SV40 large T- antigen was detected exclusively in spermatids by immunostaining, no testicular pathology was observed, indicating that spermatids show no susceptibility to transformation (Nayernia et al., 1998). In the present report, we generated transgenic mice, which express TAg at an earlier stage, namely in spermatocytes, using the promoter region of human PGK2. The PGK2-TAg transgenic mice expressed large amounts of TAg mRNA and protein in spermatocytes and spermatids. By using both RT–PCR and Western blot analyses, TAg expression was first detected at post-natal day 12, concurrent with the appearance of pre-leptotene spermatocytes. The expression of TAg in spermatocytes was not tumourigenic and adult PGK2-TAg transgenic testes showed normal morphology, although it is known that TAg has transforming potential and its expression in somatic cells leads to oncogenic transformation (Brinster et al.,1984). Therefore, our observation indicates that spermatocytes also show no susceptibility to malignant transformation. Indeed, our observations are consistent with the hypothesis that male germ cell tumours derive from spermatogonia or primordial germ cells, rather than cells already engaged in spermatogenesis (Skakkebaek et al., 1987; Grigor and Wylie, 1998). Generation of transgenic mice, which express TAg in spermatogonia or primordial germ cells, could offer useful information about the susceptibility of these cells to transformation.
Compared with the fate of stem cells in other tissues, differentiating spermatogonia commit exclusively to one pathway, undergoing the unique process of meiosis and spermiogenesis (Wing and Christensen, 1982). Germ cell numbers in adult males are determined by several factors, including the number of gonocytes at birth, mitotic division of spermatogonia, the number of Sertoli cells (Huckins, 1978) and cell death in spermatogonia and spermatocytes (De Rooiji and Janssen, 1987).
In our experiments regarding the evaluation of germ cell numbers, three different post-natal stages were chosen: 18-day-old transgenic and wild type mice containing all spermatocyte stages, 25-day-old animals harbouring spermatocytes as well as haploid spermatids, and 42-day-old animals carrying each differentiation stage of spermatogenesis from spermatogonia to mature spermatozoa (Hecht, 1998). In the present study, prepubertal transgenic mice showed substantially higher numbers of spermatocytes. Eighteen-day-old transgenic mice showed an elevation of 54% and 25-day-old transgenic mice showed an elevation of 24% in spermatocyte number as compared with wild type mice. However, at later ages the spermatocyte number assimilates to that of wild type mice and adult transgenic mice showed no difference in the number of spermatocytes. The abnormal number of spermatocytes in prepubertal transgenic mice is counterbalanced by apoptosis, as shown by in-situ end labelling of fragmented DNA.
Basically, germ cell number is determined prior to the cells becoming spermatocytes. Therefore, the increase in spermatocyte number in PGK2-TAg transgenic mice implies either an increased proliferation of spermatogonia and/or a decrease in spermatogonial cell death. An effect on proliferation caused by an enhanced amount of Sertoli cells can be ruled out, as their number in transgenic and wild type mice was determined as equal. Thus, our results imply either expression of the transgene in spermatogonia below our detection limits or a mode of feed-back signalling between pachytene spermatocytes and spermatogonia. In the latter case the signalling would be indirect, i.e. through Sertoli cells, as spermatocytes are spatially separated from spermatogonia by tight junctions between Sertoli cells (Russell et al., 1990b). Thus, a plausible explanation for the enhanced proliferation is that TAg expression in prepubertal transgenic mice leads to reduction or inactivation of an intracellular signal from spermatocytes that inhibits proliferation of less mature germ cells. Such a feed-back signal could normally serve to regulate the rate of sperm production, e.g. by inhibiting Sertoli cells from stimulating spermatogonia to mature into spermatocytes. Germ cell–Sertoli cell interactions that modify Sertoli cell behaviour have been reported in previous studies (Skinner et al., 1991; Kierszenbaum, 1994; Grisworld, 1995) and spermatocytes in particular have been shown both in vitro and in vivo to affect Sertoli cell function (Djakiew and Dym, 1998).
In newborn mice, development of the initial cohort of gonocytes into spermatozoa is known as the first wave of spermatogenesis and is accompanied by extensive germ cell apoptosis, which in mice peaks at ~2 weeks after birth (Rodriguez et al., 1997; Wang et al., 1998). It reflects an adjustment in the number of germ cells that can be maintained by Sertoli cells. Throughout spermatogenesis, each germ cell is enfolded by one or more somatic Sertoli cells which provide nutrition, adhesion, transport functions and essential factors for normal germ cell maturation (Russell et al., 1990a). The ratio of the different stages of germ cells to Sertoli cells remains relatively constant in mammalian spermatogenesis and control of this ratio is a critical requirement during testis differentiation (De Kretser et al., 1998; Print and Loveland, 2000). In support of this concept, induction of hypothyroidism for the first 30 days of life in the rat results in excessive Sertoli cell proliferation and an increase in spermatogenic output (Van Haaster et al., 1992). Conversely, neonatal hyperthyroidism decreases Sertoli cell proliferation and causes the opposite effect (Cooke et al., 1994). Experimental reduction of Sertoli cell number in immature rat testes causes a proportionate reduction in the number of round spermatids in the adult animal (Orth et al., 1998). The assimilation of the enhanced number of spermatocytes in prepubertal transgenic mice to the number in wildtype mice with increasing age supports the theory that there is a finite numerical relationship between germ cells and Sertoli cells in vivo. The apoptotic regulation in prepubertal transgenic mice maintains germ cell homeostasis and reflects the adjustment of the critical ratio between germ cells and Sertoli cells.
The present study demonstrates the potency of viral oncogenes to induce proliferation as well as apoptosis. Previous in-vitro studies have shown that the SV40 large T antigen is able to cause apoptosis directly in immortalized human epithelial cells through an p53-independent pathway (Tsao et al., 1998). Transgenic mice that developed hepatocarcinoma in response to SV40 large T antigen expression controlled by regulatory sequences of human antithrombin III, have been shown to counterbalance the elevated proliferation of hepatocytes by an apoptotic mechanism at later ages (Allemand et al., 1995). The same effect has been observed in transgenic mice expressing the HBx protein of hepatitis B virus (Koike et al., 1998). How the molecular mechanisms of these counteracting effects of proliferation and apoptosis function in detail are not fully understood. We hypothesize that in PGK2-TAg transgenic mice, proteins of the Bcl-2 family are involved in the cellular response. These proteins provide one signalling pathway which appears to be essential for male germ cell homeostasis. Some members of this protein family promote cell survival (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1) while others antagonize it (e.g. Bax, Bak and Bim) (Print and Loveland, 2000). The competitive interactions of the pro- and anti-survival proteins in the Bcl2 family are crucial for germ cell survival and apoptosis, respectively.
Transgenic mice expressing high levels of the Bcl-xL or Bcl2 proteins in male germinal cells show a highly abnormal rate of adult spermatogenesis accompanied by sterility (Rodriguez et al., 1997). This appears to result from the prevention of the early, massive wave of apoptosis, which occurs in the testis among germinal cells during the first round of spermatogenesis, presumably to maintain the critical ratio between some defined cell stages and Sertoli cells. Crossbreeding of Bax (–/–) mice with C3(1)/SV40-TAg transgenic mice leads to accelerated mammary tumour development and a reduction in protective apoptotic response at the pre-neoplastic stage (Shibata et al., 1999). Recently, it was reported that the SV40 large T antigen binds to p193, a novel Bcl2 homology domain 3 pro-apoptosis protein, suggesting an anti-apoptotic activity of TAg independent of p53 sequestration (Tsai et al., 2000).
Combined conclusions of the present study and of our previous study of transgenic mice expressing the SV40 TAg in spermatids (Nayernia et al., 1998), suggest that neither spermatids nor spermatocytes show a susceptibility to malignant transformation. Furthermore, we provide a novel mouse model showing an enhanced proliferation of spermatocytes in prepubertal mice, accompanied by transient apoptosis to maintain germ cell homeostasis.
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This work was supported by the Grant SFB500/A3 from the German Research Council (Deutsche Forschungsgemeinschaft) to K.N. and W.E. We thank Professor R.P.Erickson (University of Arizona) for providing the plasmid PGK2-CAT and Seema Singh for technical assistance.
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4 To whom correspondence should be addressed. E-mail: pburfei{at}gwdg.de
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References |
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Submitted on April 23, 2001; accepted on September 27, 2001.
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