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Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Miles F. Wilkinson, Department of Immunology, Box 180, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: mwilkins{at}mail.mdanderson.org.
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Abstract |
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Introduction |
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In this communication, we provide evidence that Pem, a member of the homeobox transcription factor family expressed in Sertoli cells (4, 5, 6, 7), regulates events associated with germ cell maturation in the adult testis. Homeobox transcription factors are best known for regulating embryonic developmental events, including axis formation and the specification of segment identity (8). In contrast, little is known about their role in postnatal and adult development. For example, although many Hox homeobox transcription factors are expressed in germ cells in the adult testis, it is not clear whether they function there (9, 10). Among all known homeobox proteins expressed in the adult testis, Pem is unique in that it is expressed exclusively in the somatic Sertoli cells (4, 5, 7). Pem is the founding member of the recently defined PEPP homeobox subfamily, named after the members that have so far been identified: Pem, Esx-1 (Spx-1), Psx-1, and Psx-2 (Gpbox) (10). All members of the PEPP family share related homeodomains interrupted by two introns at signature positions, are located on the X chromosome, and are preferentially expressed in reproductive tissues (11, 12, 13, 14, 15).
Pem mRNA expression in Sertoli cells first occurs when the adjacent germ cells initiate meiosis during the first wave of mouse spermatogenesis (between postpartum d 8 and 9). In adult mice testes, Pem mRNA and protein are expressed in a stage-specific manner in Sertoli cells; expression initiates in stages IV–VI of the seminiferous epithelial cycle, peaks at stage VII, and precipitously declines during late stage VIII (4, 5). Pem expression during these androgen-dependent stages of the seminiferous epithelial cycle (16) is consistent with our studies demonstrating that Pem’s expression in Sertoli and epididymal cells depends on androgen (4, 5, 12, 17). The androgen-dependent and stage-specific expression pattern of Pem suggests that it is a good candidate to regulate secondary androgen-response genes important for stage-specific events in the testis.
Pitman et al. (6) (1998) created Pem-null mice to analyze the protein’s function, but they did not uncover a functional role for Pem in these mice. Therefore, we elected to use a gain-of-function approach to examine Pem’s function in the testis in vivo. We hypothesized that overriding the tight regulatory control of Pem expression by constitutively expressing Pem in Sertoli cells in all stages of the seminiferous epithelial cycle would enable us to discern one or more of its roles in spermatogenesis. We discovered that this dramatically increased the number of DNA-strand breaks in specific germ cell populations that normally acquire DNA strand breaks. Our observations suggest that Pem regulates the expression of Sertoli-cell proteins that control DNA modification events in germ cells.
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Materials and Methods |
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Transgenic mice were generated, according to standard procedures, by microinjection of gel-purified DNA (20) into fertilized F1 (B6 x D2) eggs by the University of Texas M. D. Anderson Cancer Center Transgenic Mouse Facility. Founder Pem-121 mice were analyzed for the presence of the Pem-121 transgene by PCR of tail DNA with primers corresponding to the tet promoter (5'-GTT TAG TGA ACC GTC AGA TCG-3') and exon 3 of the Pem gene (5'-TTC CGA GTC TTC CTT GAC TC-3'). The resulting PCR product was 750 bp in size. Animals were housed in standard lighting (12 h of light, 12 h of darkness) and allowed food and water ad libitum. They were maintained in facilities approved by the American Association for the Accreditation of Laboratory Animal Care.
Histological examination, immunohistochemistry, and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining
Testes were collected and fixed in Bouin’s solution (Sigma, St. Louis, MO) overnight, washed in a saturated solution of Li2CO3 in 80% ethanol to remove Bouin’s fixative until dye was no longer visible in the wash solution, dehydrated, embedded in paraffin, and sectioned following standard histological procedures. Sections (10 µm) were mounted on siliconized slides and were stained for histological examination with either hematoxylin-and-eosin or periodic acid/Schiff stain. Immunohistochemical staining of testis sections was carried out as described previously (6, 21) using a polyclonal antibody specific to mouse Pem (21). For TUNEL assessment, we used the ApopTag Plus peroxidase in situ apoptosis detection kit (S-7101; Intergen, Purchase, NY) following the manufacturer’s instructions. Sections for immunohistochemistry and TUNEL were counterstained with Harris’ hematoxylin. Counts of TUNEL-positive cells in all round tubules were made on testis sections of at least three animals from each founder group.
RNA purification and analysis
Total cellular RNA from tissues was prepared by guanidinium isothiocyanate lysis and centrifugation over a cesium chloride cushion (22) and quantified by measuring optical density. [32P]uridine 5'-triphosphate-labeled RNA probes were prepared for RPA by an in vitro transcription protocol as described previously (23). The probes were generated from sequenced cDNA fragments as follows: Probe A was generated from a DNA template prepared from the Pem-121 transgene vector digested with NdeI. It contains a fragment (approximately 0.3-kb) containing 61 nucleotides (nt) of the 3' end of exon 6 and approximately 250 nt of the bGH poly(A) region. The ß-actin probe, which contains 34 nt of human ß-actin exon 3, was prepared by linearizing plasmid G-98, which contains human ß-actin (accession no. X00351), with BanI. It was used as a positive control in annealing reactions for RPA. A century ladder template (Ambion, Inc., Austin, TX) was used to generate size markers for RNA. Probes were purified on a 6% polyacrylamide denaturing gel as described previously (9).
RPA was performed as described previously (9, 23), with minor modifications. Briefly, sample RNAs were coethanol-precipitated with the appropriate gel-purified [32P]uridine 5'-triphosphate-labeled probe, and the pellet was resuspended in 10 µl annealing buffer [40 mM piperazine diethanesulfonic acid (pH 6.4), 0.4 M NaCl, 1 mM EDTA, 80% formamide] and allowed to hybridize overnight at 44 C. Unhybridized RNA was digested with RNase A (25 µg/ml) and RNase T1 (4 µg/ml) for 40 min at 30 C. The RNases were then inactivated with proteinase K treatment and extracted with phenol/chloroform/iso-amyl alcohol. After ethanol precipitation, the RNA pellet was resuspended in 90% formamide loading buffer, denatured at 85 C, electrophoresed on a 6% polyacrylamide denaturing gel, and analyzed by autoradiography and by InstantImager electronic autoradiography analysis (Packard Instruments Co., Inc., Meriden, CT).
Northern blot analysis was carried out by electrophoresis of RNA on 1% agarose denaturing (formaldehyde) gels as described previously (9), with minor modifications. Briefly, after transfer and UV cross-linking, blots were prehybridized in prehybridization buffer (50% formamide, 5-strength Denhardt’s solution, 5-strength SSPE (sodium chloride-sodium phosphate-EDTA), 0.5% sodium dodecyl sulfate, and 100 µg/ml sheared fish-sperm DNA) for 30–60 min at 42 C. Blots were then hybridized with random oligomer-primed 32P-labeled cloned cDNAs (prepared with a random-primer labeling kit from Roche, Indianapolis, IN) in hybridization buffer (prehybridization buffer plus 10% dextran sulfate) overnight at 42 C. The Pem probe consisted of full-length mouse Pem cDNA. Full-length cyclophilin cDNA (24) was used as a loading control.
Comet assay
Cells were prepared and lysed as previously described (25). Briefly, testes from 12- to 16-wk-old mice were removed, decapsulated, and immediately placed in ice-cold PBS. Tissues were finely chopped with a scalpel in ice-cold PBS, filtered through a 100-µm cell strainer (Falcon no. 2360), and diluted to 3 x 104 cells/ml. Aliquots of 104 cells were mixed with 1% (wt/vol) low-melt agarose prepared in double-distilled water and equilibrated to 42 C. The mixture was pipetted onto a frosted microscope slide and allowed to gel. For alkaline comet assay, slides were submerged in alkaline-lysis solution (0.03 M NaOH, 1 M NaCl, and 1% N-lauroyl sarcosine) for 1 h and then rinsed twice, for 30 min each, in comet buffer (0.03 M NaOH and 2 mM EDTA). All manipulations were performed in darkened conditions at room temperature. After the rinse, slides were subjected to electrophoresis at 0.6 V/cm in freshly made comet buffer for 25 min, to allow extrusion of DNA from the cells. For neutral comet assays, slides were submerged in neutral-lysis solution (30 mM EDTA, 0.5% sodium dodecyl sulfate, pH 8.0) for 4 h at 50 C and then soaked in TBE buffer (90 mM Tris, 2 mM EDTA, 90 mM boric acid, pH 8.5) for 4 h at room temperature. Electrophoresis was performed at 0.6 V/cm in TBE for 25 min. After electrophoresis, slides for either the alkaline or neutral comet assay were rinsed for 15 min in distilled water, submerged in 2.5 µg/ml propidium iodide for 20 min, rinsed in distilled water, and placed in a humidified, air- and light-tight container until they were scored. Comets were scored using a Hamamatsu color chilled 3CCD camera (no. C5810) and a Zeiss Axioplan2 fluorescence microscope. Color TIFF images of the comets were captured using Optimas 6.5 on Windows NT and then converted to gray-scale GIF images using Photoshop 5.5 on MacOS 9.0.4. Images were then analyzed using comet analysis software (Euclid Analysis, St. Louis, MO) to determine (1) the comet tail moment (calculated using the values for comet tail length and percent of total DNA in the comet tail) and (2) total DNA content in both the cell and its tail. The former reflects the amount of broken DNA, whereas the latter reflects ploidy.
Spectrofluorimetric caspase-3 quantification
Testes from 12- to 16-wk-old mice were removed, decapsulated, and immediately placed in ice-cold PBS. Tissues were finely chopped with a scalpel in ice-cold PBS, filtered through a 100-µm cell strainer (Falcon no. 2360), and diluted to 1 x 106 cells/ml. Cells were lysed in 200 µl lysis buffer (100 mM HEPES, 10% sucrose, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, pH 7.5; 1 mM EDTA; 10 mM dithiothreitol; 2 mM phenylmethylsulfonylfluoride; 100 µM pepstatin; 10 µg/ml leupeptin). Lysates were incubated for 1 h on ice. Samples were divided for duplicate reactions; thus, 100 µl lysate was transferred to a fresh Eppindorf tube, and 900 µl reaction buffer (100 mM HEPES; 10% sucrose, pH 7.5; 1 mM EDTA) was added to each sample. Fluorogenic substrate specific for caspase-3 (Ac-Asp-Glu-Val-Asp-AMC ammonium salt; Bachem California, Inc., Torrance, CA) at a concentration of 25 nM (in dimethylsulfoxide) was added (4 µl) to each sample and incubated for 2 h at 37 C. Reaction buffer (1 ml) was added to each sample, and the sample was analyzed at an excitation wavelength of 380 nm and an emission wavelength of 460 nm on an RF-1501 spectrofluorimeter (Shimadzu Scientific Instruments, Columbia, MD). Relative fluorescence was calculated by subtracting the blank fluorescence (buffer plus substrate only) from the sample fluorescence value.
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Results |
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). Three founder animals carrying this Pem transgene (Pem-121.2, Pem-121.5, and Pem-121.7) were identified and were used to establish transgenic lines. Using a probe specific to the bGH polyadenylation region of the Pem transgene for RPA, we detected transgene expression in the testis of all three transgenic mice but not in wild-type (WT) control mice (Fig. 1B). Northern blot analysis with a probe specific to the Pem coding region demonstrated that all three transgenic mouse lines expressed higher levels of Pem mRNA in the testis than did littermate controls (Fig. 1C). Pem-121.2 mice had approximately 3-fold higher Pem mRNA levels than did control mice, whereas Pem-121.5 and Pem–121.7 mice had approximately 7-fold increased Pem transcript levels.
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), consistent with the higher Pem mRNA levels in these mice (Fig. 1C
). Pem protein was limited to Sertoli cells (primarily in their nuclei) in both transgenic and control animals; it was not observable in any germ cell population. However, in contrast with control mice, the transgenic mice expressed Pem protein in a stage-independent manner such that all Sertoli cells in all stages of the seminiferous epithelial cycle expressed Pem. This is clearly demonstrated by comparing Fig. 2, E with F; the latter shows strong Pem expression in a stage IX tubule from a Pem-121 transgenic mouse, whereas the former shows lack of Pem expression in a stage IX tubule in a control littermate mouse. This unique expression pattern afforded us an opportunity to examine the functional consequences of Pem-121 expression in the correct cell type but during all stages of the seminiferous epithelial cycle.
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, A and B, and 4A), and elongating spermatids at step 9–10 (Figs. 3, E and F, and 4C). Single-factor ANOVA and Tukey tests, the latter of which compares differences between multiple comparisons, revealed a significant difference in the percentages of both positively stained preleptotene spermatocytes [F(3, 116) = 264.34, P < 0.001] and elongating spermatids [F(3, 56) = 27.45, P < 0.001] in the three Pem transgenic mice lines, as compared with control littermates. The increased frequency of TUNEL-positive spermatids was specific for steps 9 and 10, as there was a normal frequency of TUNEL-positive step 11 spermatids in the Pem transgenic mice (data not shown). Both control and transgenic mice had apoptotic TUNEL-positive pachytene spermatocytes and round spermatids; whereas spermatogonia, leptotene spermatocytes, and elongated spermatids were not detectably TUNEL-positive (data not shown).
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, C and D). Each transgenic line exhibited a different percentage of TUNEL-positive pachytene spermatocytes (Fig. 4B) that did not correlate with the levels of Pem mRNA expression (Fig. 1C). Statistical analysis by ANOVA and Tukey tests revealed a significant difference [F(3, 116) = 6.80, P < 0.01] in the percentages of TUNEL-positive pachytene spermatocytes in only one Pem transgenic mouse line (Pem-121.2), as compared with control littermates. We conclude that it is unlikely that Pem expression in Sertoli cells influences germ cell apoptosis.
Increased frequency of double-strand DNA breaks (DSBs) and single-strand DNA breaks (SSBs) in germ cells as a result of Pem expression in Sertoli cells
As an independent assay to assess whether ectopic Pem expression increases the frequency of DNA strand breaks, we used the alkaline comet assay. The advantage of the comet assay is that it allowed us to measure the amount of DNA strand breaks per cell and to identify and categorize cells based on DNA content. Analysis by the comet assay revealed that all three Pem transgenic mice lines had a significant increase in the average tail moment (the amount of DNA damage, as defined by the tail length and the percent of the total DNA in the tail) in both the haploid and tetraploid populations but not in the diploid cell population (Fig. 5, A–C). We do not believe that this increase in migrating DNA was the result of an increase in the number of apoptotic bodies, because these would have had very long tail moments, a high percentage of DNA in the tails, and a low percentage of DNA in the comet head (27). Rather, the increase in the average moment that we observed indicates a significant increase in the number of strand breaks in viable germ cells. The increase that we observed in the average tail moment in the tetraploid population is consistent with the increase in TUNEL-positive preleptotene spermatocytes in stage VIII tubules (Fig. 4A). The increase in average tail moment in the haploid population is consistent with the increase in TUNEL-positive elongating spermatids (Fig. 4C). The lack of a significant difference in average tail moments of the diploid cells between the Pem transgenic mice and control mice confirms our TUNEL assay results in which we found no increase in DNA strand breaks in diploid germ cells. Moreover, the lack of difference in tail moments in the diploid population suggests that Pem did not induce DNA strand breaks in Sertoli cells, the cells in which Pem is expressed. It should be noted that the comet assay detected a smaller difference in DNA strand breaks between the transgenic and WT mice, compared with the TUNEL assay. One explanation for this difference is that, unlike the TUNEL assay, the comet assay does not distinguish between cell types but instead distinguishes cells by ploidy. Thus, the comet assay would measure DNA breaks in all haploid cells, not just elongating spermatids, which would dampen effects observed.
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). In contrast, we found no significant difference in either the haploid [F(3, 224) = 2.19, P < 0.09] or diploid [F(3, 85) = 1.54, P < 0.25] populations for all three transgenic lines, compared with control mice (Fig. 5D). These observations, coupled with our alkaline comet results, suggest that the increase in DNA strand breaks that we observed in elongating spermatids were SSBs, whereas the breaks we observed in preleptotene spermatocytes were DSBs. Although our analysis does not rule out the possibility that preleptotene spermatocytes also had some SSBs (because the alkaline comet assay does not distinguish between SSBs and DSBs), the combined results from the neutral and alkaline comet assays strongly support our contention that Pem overexpression in Sertoli cells results in an increase in specific types of DNA strand breaks in distinct germ cell populations.
Effect of Pem overexpression in Sertoli cells on fertility and germ cell subsets
Despite the dramatically increased frequency of preleptotene spermatocytes and elongating spermatids possessing increased DNA strand breaks in Pem transgenic mice, these mice had no obvious disruption of spermatogenesis or any overt histological anomalies in the seminiferous tubules, as judged by histological examination of hematoxylin-andeosin- and periodic acid/Schiff-stained sections (data not shown). Pem transgenic mice also exhibited normal fertility, given that there was no significant difference in litter sizes born to WT (6.40 ± 0.29) and transgenic parents (Pem-121.2, 6.34 ± 0.31; Pem-121.5, 6.92 ± 0.22; and Pem-121.7, 7.03 ± 0.22).
However, we obtained evidence that the Pem transgenic mice had modest alterations in specific germ cell subsets. Statistical analysis, using ANOVA, revealed that there was a statistically significant increase in the mean number of steps 9–10 elongating spermatids per tubule in the three transgenic lines, as compared with the control mice [F(3, 56) = 4.97, P < 0.005] (Fig. 6C). The Tukey test demonstrated that the three Pem transgenic lines did not have statistically significant differences between them, indicating that the effect was attributable to Pem (Fig. 6C). The mean number of preleptotene spermatocytes per tubule was also slightly greater in all three transgenic lines, compared with control littermates (Fig. 6A). However, ANOVA and Tukey analysis demonstrated that only one transgenic line, Pem-121.7, exhibited a significant difference, compared with control littermates [F(3, 116) = 7.78, P < 0.001]. Statistical analysis of pachytene spermatocytes in stages XII–VI of the seminiferous epithelial cycle demonstrated a statistically significant difference between two of the transgenic lines, compared with control littermates [F(3, 116) = 21.13, P < 0.001] (Fig. 6B). However, these two transgenic lines displayed a different response (Pem-121.2 showed an increase and Pem-121.7 showed a decrease), indicating that the changes in pachytene spermatocytes were unlikely to be attributable to Pem overexpression.
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Discussion |
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, A and C); 2) haploid and tetraploid germ cells in transgenic Pem mice demonstrated an increase in average tail length and percentage of DNA in the tail when assayed by the alkaline-comet assay (Fig. 5, A–C); and 3) there was a significant increase in free-DNA ends in tetraploid germ cells in Pem transgenic mice, as assayed by the neutral-comet assay (Fig. 5D). Though this data implicates Pem as a potent regulator of germ cell events, it should be stressed that our results were obtained from transgenic mice that ectopically express Pem; and thus, we cannot be sure that Pem’s normal physiological role is to control DNA strand breaks in germ cells. Many other transcription factors are expressed in Sertoli cells, including GATA-1, GATA-4, WT-1, Sox9, SF-1, and Spz-1 (28, 29, 30, 31, 32), but Pem is the first to be suggested to play a role in the adult testis. Our observations suggest a model in which Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that signal germ cells to alter DNA replication, DNA repair, or chromatin remodeling. The discovery that Pem expression in Sertoli cells alters the number of preleptotene spermatocytes and spermatids with DNA modifications is significant, because little is known about how Sertoli cells influence germ cell maturation events (1, 3).
Although there are many differences between preleptotene spermatocytes and elongating spermatids, two features they share in common are their high degree of chromatin remodeling and their positions at critical points of germ cell differentiation. Preleptotene spermatocytes are at the important juncture in which mitosis has ceased and meiosis has begun. During this stage, their chromatin undergoes reorganization as meiotic replication begins. Steps 9–10 haploid spermatids are also at a critical point of differentiation because they are undergoing cellular elongation and repackaging of their chromatin into a more condensed state. At step 10 in the mouse, histones become acetylated and begin to disassociate from DNA as they are replaced by the transition proteins (33).
Spermatocytes normally begin to accumulate DSBs during premeiotic S-phase (34). Why does Pem expression in Sertoli cells increase the proportion of preleptotene spermatocytes with these DSBs? One possible explanation is that Pem affects meiotic DNA replication by increasing topoisomerase activity, which would result in increased DNA nicking (SSBs) and a subsequent increase in DSBs caused by replication-fork stalling (35, 36). To date, the presence of topoisomerases in preleptotene spermatocytes has not been investigated. However, topoisomerase II
has been found to be present in meiotic prophase spermatocytes (37), and recent work in the rat has demonstrated that its expression is androgen-dependent (38), just as Pem is (5, 17). Furthermore, topoisomerase II has been localized to sites of DNA replication in chicken embryonic fibroblasts, suggesting that it plays a role in DNA unwinding during replication (39). Interestingly, in the mouse testis, topoisomerase II expression is initiated at d 8 (37), the same time that Pem expression is initiated (5). An alternative explanation is that Pem may promote a delay in the repair of DSBs by inhibiting either the production or activity of DNA-repair molecules. Targets for such regulation are the several ligases that repair DSBs resulting from DNA replication and recombination (40, 41, 42). Other potential targets are the many molecules localized at DSBs and arrested replication forks that collaborate to direct cell-cycle checkpoints that act during premeiotic S-phase, including H2AX-
, ATM, BRCA1, PCNA, Mre11, Rad50, and 53BP1 (34, 43, 44, 45, 46, 47). Regardless of the exact mechanism, the lack of detectable strand breaks in leptotene spermatocytes from Pem-121 transgenic mice suggests that the breaks we observed in preleptotene spermatocytes are repaired and do not hinder gametogenesis.
During steps 9–10 of spermiogenesis, spermatids repackage and remodel their chromatin into a more condensed state (33), which results in the formation of transient SSBs, probably as a result of the mechanical manipulation of the DNA to eliminate the presence of supercoiled DNA (48, 49, 50). The increase in the number of spermatids with SSBs caused by Pem expression in Sertoli cells suggests that this chromatin remodeling process may be directed by a Pem-induced Sertoli-cell signal. Though no proteins have yet been identified as responsible for causing these SSBs in spermatids, topoisomerase II is a likely candidate, because it has been localized to the chromosomes in nuclei of elongating spermatids in the rat (33). This enzyme is believed to serve as a loop fastener that organizes chromatin into loop domains, based on studies that show that topoisomerase II binds to chromosomal scaffolds in the nuclear matrices of somatic cells (33, 51, 52). Alternatively, Pem may not actually increase the frequency of SSB induction; but rather, it may increase the persistence of SSBs at a given point in time by delaying or inhibiting DNA repair. Regardless, it is clear that SSBs are necessary to relieve the torsional stress in DNA to better suit the conditions needed for the major structural rearrangement of chromatin in germ cells in later spermiogenesis (33), and our data suggests the possibility that Pem directs this activity via the Sertoli cell. It is likely that these strand breaks are repaired between steps 10 and 11, because we were unable to detect DNA strand breaks in step 11 spermatids. Because SSBs in spermatids are repaired as part of the normal program of spermiogenesis (33, 48), this underscores the likelihood that Pem impinges on a normal developmental pathway rather than directing an aberrant process. This is further supported by our finding that our Pem-121 transgenic mice were fertile and had no gross level alterations in seminiferous epithelial morphology.
The increase in DNA strand breaks caused by Pem expression in Sertoli cells was limited to germ cells in stages VII–X, despite the presence of Pem, in all stages of the seminiferous epithelial cycle in our transgenic mice (Figs. 2 and 3). This selectivity correlates with Pem’s normal expression pattern in the mouse: Pem is dramatically induced in Sertoli cells when the adjacent germ cells are at the preleptotene stage (at d 8 post partum during the first wave of spermatogenesis), and its expression is extinguished just before elongating spermatids reach step 9 (in the adult seminiferous epithelial cycle) (4, 5). It will be interesting to determine whether this selectivity occurs at the level of the Sertoli cell or the germ cell. If at the level of the Sertoli cell, it may be attributable to a stage-specific inhibitor of Pem function or stage-specific alterations in Pem’s downstream genes that make them impervious to Pem regulation at stages other than VII–X. If at the level of the germ cell, perhaps only specific germ cell subsets are competent to receive a Pem-induced Sertoli-cell signal. The differentiation program of germ cells may dictate this receptivity. For example, the initiation of meiotic replication may induce receptivity to the Pem-induced signal, thus explaining why preleptotene spermatocytes respond but not mitotic spermatogonia. In yeast, meiotic DNA replication is distinguished from mitotic replication by promitotic cyclins and the promeiotic molecule Ime I (53); such molecules could also dictate receptivity to Pem-induced signals in mammalian germ cells. It is less clear what molecules might suppress receptivity in meiotic spermatocytes in prophase I, reinitiate receptivity in step 9 spermatids, and again suppress it at a later stage of spermatid differentiation.
Our findings suggest that the role of the Sertoli cell extends beyond the support role of providing nutrients to germ cells. In the future, it will be important to identify the Pem-induced molecules secreted or expressed on the surface of Sertoli cells that mediate this communication between Sertoli cells and germ cells. That Pem functions in activating signaling between somatic and germ cells is consistent with the fact that, in the ovary, Pem is expressed by granulosa cells, the somatic cells that surround the oocyte (6). Pem is also aberrantly expressed in tumor cells from several different lineages and tissues (18); and thus, it is possible that it also plays a role in tumor-cell progression by allowing tumor cells to circumvent normal checkpoint mechanisms that survey DNA strand breaks and control cell-cycle progression (54).
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Acknowledgments |
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Footnotes |
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Abbreviations: bGH, Bovine GH; DSB, double-strand DNA break; nt, nucleotide; RNase, ribonuclease; RPA, ribonuclease protection analysis; SSB, single-strand DNA break; tet, tetracycline; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick end labeling; WT, wild-type.
Received May 28, 2002.
Accepted for publication August 22, 2002.
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, but not ß, with sites of DNA replication and possible involvement of a C-terminal region of through its binding to PCNA. Chromosoma 110:102–114[Medline]
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35 nt) was included as a loading control. C, Northern blot analysis of adult testis RNA (10 µg) from WT and three transgenic founder lines with a Pem probe that recognizes both endogenous and transgene transcripts. Placenta RNA (10 µg) from a WT mouse was used as a positive control for Pem expression. Cyclophilin was used as a loading and normalization control.
, P < 0.35 for 2N. B, Pem-121.5 compared with littermate control; *, P < 0.01 for 1N and 4N; 
, P < 0.02 for 4N. D, Increase in average tail moments for all three transgenic lines in the tetraploid population, but not in the haploid or diploid population, as assessed by the neutral comet assay. Statistical analysis was performed as described for Fig. 4