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Testis |
Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, sperm maturation, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TPs are removed from the condensing chromatin and replaced by two protamines, PRM1 and PRM2, which constitute the major nuclear proteins of condensed spermatids and mature spermatozoa [8]. In mice, Prm1 and Prm2 mRNAs are first detected in step 7 spermatids, and they are degraded in step 14 spermatids at stage II and step 15 spermatids at stage V, respectively [3]. Biochemical measurements showed that PRM1 and PRM2 first appear at step 12 and step 14, respectively, and then remain in sperm [9]. Whereas PRM1 is synthesized as the mature protein, PRM2 is synthesized as a precursor with 106 residues and is sequentially cleaved to intermediates to reach the mature form of 63 residues [10].
To investigate the roles of TPs during spermiogenesis, we generated Tnp1-, Tnp2-, and double-mutant mice [11– 13]. In these mice, histone displacement occurred normally and spermatid development progressed in the absence of TPs, but chromatin condensation was abnormal, and the few spermatozoa that were produced were not functional. The subtle phenotypes observed in Tnp single-mutant mice, compared with the major defects observed in the double-mutant mice, indicated that TP1 and TP2 might have redundant functions or that in the absence of one TP, the other TP may partially compensate to allow for efficient sperm production. Our previous results showed elevations of TP2 in total testicular homogenates from Tnp1 –/– mice and of TP1 levels from Tnp2 –/– mice [11, 12]. However, the elevation mechanism and whether the elevated remaining protein compensates for the function of deleted protein are still unclear.
Previous results also indicated a slightly increased level of PRM bound to the chromatin of step 12–13 spermatids of Tnp1-null mice and defects of PRM2 processing in both Tnp1- and Tnp2-null spermatids [11, 12]. However, no information was available as to whether the Tnp mutations affected the time of appearance or overall levels of PRM.
Furthermore, the previous studies showed that Tnp1 +/– mice possess about 50% of normal levels of TP1 protein and Tnp2 +/– mice about 50% of normal levels of TP2 protein, as expected from their heterozygous genotypes. However, the distribution of the proteins among the different spermatids is not known. Meiotic chromosome segregation followed by postmeiotic gene expression might give rise to gametes with different levels of proteins when the genomes of the meiotic segregants are not genetically equivalent. Whereas the levels of certain mRNAs and proteins appear to be equally distributed [14, 15] because spermatids are connected by cytoplasmic bridges, others are not [16].
To address these questions regarding protein levels and distributions in Tnp-mutant mice, in the present study we determined the presence and qualitative levels of TPs and PRM in spermatids at different steps of development in Tnp1 and Tnp2 heterozygotes and homozygotes using immunohistochemical methods.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult mice (8–25 wk) carrying a Tnp1-null mutation, 129S-Tnp1tm1Mlm [11], or a Tnp2-null mutation, 129S-Tnp2tm1Mzh [12], were used in this study. All of them were maintained on a 129Sv genetic background and caged in a controlled environment at M.D. Anderson Cancer Center (12L:12D). All procedures were approved by the institutional animal care and use committee.
Preparation and Analysis of Nuclear Proteins
For selective extraction of TP1, TP2, and histone H1, two testes from wild-type, Tnp1 +/–, and Tnp1 –/– mice were placed in 2 ml water containing a cocktail of protease inhibitors and homogenized and sonicated at 4°C as described previously for Tnp2 mutants [12]. Basic proteins were then extracted with 0.25 N HCl and precipitated with 3.5% trichloroacetic acid (TCA), and the supernatant was precipitated overnight with 25% TCA. The precipitates were washed with acidified acetone, followed by acetone, and dried [17].
Proteins were separated by electrophoresis in acid-urea 18% polyacrylamide gels and quantified by using IMAGEQUANT version 5.0 software (Amersham Biosciences, Piscataway, NJ) as described previously [12].
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from mature mouse testes with the RNAgents Total RNA Isolation System (Promega, Madison, WI). Plasmids containing Tnp1 and Tnp2 cDNA were provided by Dr. Kenneth Kleene (University of Massachusetts, Boston, MA) [18, 19]. The 400-bp Tnp1 and 550-bp Tnp2 fragments were used as probes, which hybridized with bands centered on the blot at 600 and 700 bp, respectively. A 0.7-kb fragment from a plasmid containing rat cyclophilin cDNA provided by Dr. Miles Wilkinson (M.D. Anderson Cancer Center, Houston, TX) was used as a control probe and hybridized with a 720-bp band [20]. The levels of mRNA were quantified by using IMAGEQUANT software.
Seminiferous Tubule Squash Preparations
Seminiferous tubule segments at different stages of the seminiferous epithelial cycle were isolated in Dulbecco PBS (pH 7.2) under a stereomicroscope by transillumination-assisted microdissection [21]. For accurate identification of the stages, 0.5-mm segments of seminiferous tubules, preliminarily identified by transillumination, were placed on a microscope slide in 20 µl PBS and squashed between the slide and a coverslip; lens paper was used to withdraw excess fluid around the edges of the coverslip, carefully avoiding air bubbles [22]. The stages were identified based on acrosomal development, spermatid nuclear shape, and the presence of meiotic figures [23]. The selected stage-specific squash preparations were immediately frozen in liquid nitrogen, the coverslips were removed in the frozen condition with a scalpel, and the slides were immediately fixed in cold methanol for 15 min on dry ice, dried, and stored at –20°C.
Immunohistochemistry
For TP2 detection, testes and caput epididymis were fixed in Bouin solution for 12 h and paraffin embedded. Initially this method was also employed for TP1 detection, but some nonspecific staining was observed and squash preparations were used instead. Sections (4 µm) were cut, dewaxed, and rehydrated according to standard protocols. Endogenous peroxidase activity was quenched by incubating sections with 3% H2O2 for 10 min at room temperature. Sections were then subjected to antigen retrieval by bringing the slides just to the boiling point on a hot plate in 10 mM sodium citrate buffer, pH 6.0 (BioGenex, San Ramon, CA), and allowing them to gradually cool to room temperature. Slides were then incubated with PBS containing 0.3% Triton X-100 and 10% goat serum (PBS-TS) for 3 h at room temperature to block nonspecific binding sites; slides were then incubated with TP2 anti-serum diluted 1:1600 (courtesy of Dr. Stephen Kistler, University of South Carolina, Columbia, SC) in PBS-TS overnight at 4°C. Another section on the same slide was incubated only with PBS-TS as an immunohistochemical control. Following three washes in PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:200 in PBS-TS for 1 h at room temperature. Subsequently, the sections were incubated with VECTASTAIN ABC reagents (Vector Laboratories) for 30 min at room temperature, and TP2 was detected with 3,3'-diaminobenzidine substrate according to the manufacturer's protocol. The slides were then counterstained with periodic acid Schiff (PAS) and hematoxylin. The red PAS staining of the acrosome enables determination of the stage of the cycle of the seminiferous epithelium. The immunochemical specificity of the TP2 anti-sera has been previously demonstrated [6]. To further address their immunohistochemical specificity, sections from Tnp2 –/– mice were incubated with TP2 anti-serum.
For PRM1 and PRM2 detection, testes were punctured in several places with an 18-gauge needle and fixed in cold Carnoy solution overnight. Carnoy fixation was used, instead of Bouin fixation, to improve penetration into more highly condensed late spermatid nuclei, which were expected to contain the PRMs. The loss of acrosomal staining with Carnoy fixation was not considered to be much of problem because the critical stages for the appearance of the PRMs were stages IX–XII, which could be identified on nuclear morphology alone. Sections were treated with 6 mM dithiothreitol in 50 mM Tris-HCl (pH 8.5) for 1 h at room temperature and then incubated with PBS-TS for 3 h at room temperature to block nonspecific binding sites. Monoclonal antibodies Hup1N (diluted 1:100, unless otherwise stated) and Hup 2B (diluted 1:400; both courtesy of Rod Balhorn, Lawrence Livermore National Laboratory, Livermore, CA) were used to detect PRM1 and PRM2, respectively [24, 25]. Sections incubated only with PBS-TS were used as a control. Antibody binding was detected with Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes, Eugene, OR). The sections were counterstained with VECTASHIELD mounting medium containing 1.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) for staging. Stages IX, X, and XI were identified by the shape of step 9, 10, and 11 spermatids, respectively. Stage XII was identified by the presence of meiotic cells [26]. Stage I–VIII tubules were identified by the presence of round spermatids. Stages VII and VIII tubules were distinguished by the presence of condensed spermatids lining the luminal surface of the seminiferous epithelium, whereas in stages I–VI the spermatids were not aligned but were embedded in the epithelium. Precise staging between stage I and VI tubules is difficult by DAPI staining. However, the percentages of tubules at each of the six stages—stages I, II, III, IV, V, and VI—are known to be 24%, 14%, 5%, 19%, 19%, and 19%, respectively, of the total tubules at stages I–VI [27]. If a change in staining occurs during this period and is maintained through stage VI, the percentage of stage I–VI tubules not showing this alteration in staining can be compared with the cumulative percentages of tubules at stages I, I–II, I–III, I–IV, and I–V, which are 24%, 38%, 43%, 62%, and 81% of stages I–VI tubules to determine the stage at which the change occurs. Because this calculation is dependent on the constancy of the relative durations of the stages of the cycle of the seminiferous epithelium, the percentage of a particular stage was determined by counting the cross-sectioned tubules with PAS-hematoxylin staining from wild-type, Tnp1 –/– and Tnp2 –/– mice. Approximately 450 total tubules in tissue sections from two different mice of each genotype were staged and counted. A chi-square analysis showed that neither Tnp mutation altered the frequency of occurrence, and hence relative timing, of each stage in the cycle of the seminiferous epithelium compared with wild type (data not shown).
Immunocytochemistry
To clearly visualize individual spermatid nuclei for the study of protein sharing in heterozygotes and to minimize interference by nonspecific staining when determining the stage specificity of TP1, immunocytochemistry was applied with squash preparations. The squash preparations were equilibrated to room temperature for 30 min, fixed again with a 1:250 dilution of saturated HgCl2 (made up in PBS) in PBS for 5 min, washed 3 times in PBS (5 min each), and incubated with PBS-TS for 2 h at room temperature. The slides were incubated with the primary antibodies used in the following dilutions: TP1 1:100 (courtesy of Dr. Stephen Kistler, University of South Carolina, Columbia, SC) and TP2 1:50 in PBS-TS overnight at 4°C. The slides were incubated with Alexa Fluor 488 goat anti-rabbit IgG (10 µg/ml; Molecular Probes) in PBS-TS for 2 h at room temperature and counterstained by VECTASHIELD mounting medium containing 1.5 µg/ml DAPI (Vector Laboratories). The immunochemical specificity of the TP1 anti-sera has been previously demonstrated [5].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether TP1 and TP2 were distributed among all condensing spermatids from Tnp1- and Tnp2-heterozygous mice, respectively, fluorescence immunocytochemistry studies were carried out. Since it was important for this analysis to distinguish individual spermatids, squash preparations of seminiferous tubule segments at stages XII and I, in which high levels of TP1 and TP2 were known to be present, were used instead of tissue sections. Elongated spermatids were identified by DAPI staining (Fig. 1, A and C). Immunocytochemistry with TP1 and TP2 antibodies (Fig. 1, B and D) showed that all of the step 13 spermatids from Tnp1 and Tnp2 heterozygous mice were immunopositive for TP1 or TP2 proteins, respectively. The same results were found with step 12 spermatids (data not shown). The levels of immunofluorescence appeared to be roughly equivalent in all cells. These data show that although the genomes of meiotic segregants were not genetically equivalent in Tnp1 and Tnp2 heterozygotes, both Tnp1 and Tnp2 gene expression products were shared among postmeiotic cells.
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Elevated Levels of TP Proteins and Normal Levels of mRNA in Testes of Tnp-Mutant Mice
TP1 and TP2 levels in the 3.5% TCA-soluble protein extracts of the total testis of Tnp-mutant mice were analyzed after separation by gel electrophoresis. Histone 1, TP2, and TP1 were the major bands on the gel. In Tnp1-null mice, the level of TP2 was about 2.6 times that in wild-type mice, but it was not significantly elevated in heterozygotes (Table 1), similar to the results reported earlier for Tnp1-null mice on a hybrid genetic background [11]. In Tnp2-null mice, the level of TP1 was about 140%–145% the level in wild-type mice. In Tnp2 heterozygotes, TP1 was also elevated, but to an intermediate level.
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To investigate the mechanism of the elevation in Tnp-mutant mice, Tnp1 and Tnp2 mRNA levels were measured by Northern blot with RNA isolated from the total testis of Tnp-mutant mice. As expected, levels of Tnp1 and Tnp2 mRNA were reduced to about 50% of control in both Tnp1 +/– and Tnp2 +/– mice, respectively, and to zero in the homozygotes (Table 2). In both Tnp1 +/– and Tnp1 –/– mice, the Tnp2 mRNA levels were not significantly different from those in wild-type mice. Similarly, Tnp1 mRNA levels were not altered in Tnp2 +/– and Tnp2 –/– mice. Therefore, the elevated levels of the other TP protein in each of the Tnp-mutant mice are due to regulation at the posttranscriptional level.
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Abnormal Retention of TP2 in Tnp1 –/– Mice
In wild-type mice, immunohistochemistry showed that TP2 was absent in the nuclei of step 9 spermatids and first appeared in the nuclei of late step 10 spermatids (Fig. 2, A and B). Strong immunoreactions for TP2 occurred in step 13 spermatids at stage I (Fig. 2C) and step 14 spermatids at stage II–III (Fig. 2D). In step 15 spermatids at stage IV, TP2 staining significantly decreased in some nuclei and could not be detected in other nuclei (Fig. 2E). TP2 was completely absent from early stage V onward (Fig. 2F). In Tnp1 –/– mice, the appearance pattern of TP2 in step 10 spermatids was the same as that in wild-type mice, but TP2 staining remained strong in all step 15 spermatids at stage V (Fig. 2G), whereas TP2 stain was absent in wild-type mice at this stage. Furthermore, this strong staining was observed in the majority of step 16 spermatids at stage VII and VIII tubules (Fig. 2H). The results of counts of these tubules are summarized in Figure 3A. Despite the staining of most spermatids at step 16, nuclear staining was completely absent in caput epididymal sperm prepared in an identical manner and also after dithiothreitol treatment (data not shown).
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Abnormal Retention of TP1 in Tnp2 –/– Mice
To determine the distribution of TP1 in Tnp2 –/– mice, several different immunohistochemistry procedures were tried with TP1 antiserum. Although the staining was good with sections fixed in Carnoy solution, we could not precisely stage the tubule cross-sections because the acrosome of early stage spermatids did not stain with PAS. With sections fixed in Bouin solution, slight nonspecific staining of the acrosome was observed and occluded the clear identification of the specific nuclear staining. The specificity of the nuclear staining and the nonspecificity of the acrosomal staining were verified by the immunohistochemical reaction of sections from Tnp1 –/– mice with TP1 anti-serum (data not shown). Therefore, stage-specific squash preparations and fluorescence immunocytochemistry were used to determine the distribution of TP1. In wild-type mice, TP1 stain was absent in step 10 spermatids (Fig. 4A). Staining was first detected in step 11 spermatids (Fig. 4B) and began from the anterior tip of the nucleus and spread backward. A strong immunoreaction was shown in step 12 and 13 spermatids (Fig. 4C) and persisted in step 14 spermatids from stage II–III tubules (data not shown). Staining covered the entire nucleus during these stages. In stage IV tubules, although some step 15 spermatids still showed a weak immunoreaction with TP1 antiserum in the central part of the nucleus (Fig. 4D), most of them were negative (data not shown). From stage V onward, TP1 could not be detected (Fig. 4E), although DAPI staining showed step 15 spermatids in the field. The other cells in the squash preparation also did not react with the TP1 antiserum, except for a weak cross-reaction with the acrosome of round spermatids (Fig. 4E). In Tnp2 –/– mice, TP1 initially was detected at the same stages as in wild-type mice and was present at high levels at step 12–13 spermatids (data not shown). However, compared with wild-type mice, the disappearance of TP1 was significantly delayed. The step 15 spermatids from stage V tubules still showed strong immunoreaction with TP1 antiserum (Fig. 4F), which gradually disappeared in later step 15 spermatids from stage VI tubules (Fig. 4, G and H). The percentages of tubules showing positive immunostaining for TP1 are summarized in Figure 3B.
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Increase in TP Levels Primarily Results from Lower Turnover
To estimate how the abnormal retention of TPs contributes to the elevated levels of TP proteins in testes of Tnp-mutant mice, we calculated the percentages of all tubules showing positive immunostaining for TPs by taking the percentage of positive immunostaining tubules at each stage of the cycle of the seminiferous epithelium and multiplying by the percentage of that stage in the cycle of the seminiferous epithelium [27]. The results of these calculations are shown in Table 3. Compared with wild-type mice, in which 46% of tubules in the cycle of the seminiferous epithelium showed positive TP2 immunostaining, the percentage of tubules showing positive TP2 immunostaining was increased to 86% in Tnp1 –/– mice. Similarly, the percentage of tubules showing positive TP1 immunostaining was increased from 42% in wild-type mice to 61% in Tnp2 –/– mice. We derived the increase in amount of TP proteins due to abnormal retention after first assuming that each positive tubule had the same amount of TP. Thus, TP2 retention resulted in a 1.9-fold elevation of TP2 protein in Tnp1 –/– mice, and TP1 retention resulted in a 45% elevation of TP1 protein in Tnp2 –/– mice.
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Since TP2 was elevated 2.6 times in Tnp1 –/– mice and TP1 was elevated by 40%–45% in Tnp2 –/– mice in the 3.5% TCA-soluble protein extracts of total testes, the increases in TP levels could be largely explained by their retention. From the present data, it is not possible to determine whether the 1.9-fold increase calculated by retention of TP2 in Tnp1 –/– mice was statistically significantly different from the 2.6-fold increase observed by protein measurements.
Distribution Pattern of PRMs in Tnp-Mutant Mice
Since TP levels were reduced in step 11–14 spermatids in mice lacking TPs and elevated in later stages, it is possible that Tnp mutations change the temporal expression pattern of the PRMs. In wild-type mice, PRM1 could not be detected in step 9 spermatids (Fig. 5, E and I). Faint evidence of PRM1 first appeared in nuclei of step 10 spermatids (Fig. 5, F and J), followed by strong and uniform nuclear staining in step 11 spermatids (Fig. 5, G and K) when the 1:100 dilution was used. The strong, uniform reaction persisted until stage I. In the rest of spermiogenesis (stages II–VIII), strong PRM1 stain was detected in the corresponding late spermatids (Fig. 5, H and L); however, in most late spermatid nuclei, staining was peripheral, rather than uniform. This change might be attributable to incomplete penetration of the antibody into the condensed nucleus. Because these high concentrations of primary antibody could lead to saturated fluorescence signal, the monoclonal antibody Hup 1N was further diluted up to 1: 8000. At 1:8000, PRM1 staining in step 10 spermatid nuclei could not be detected, and a progressive increase in PRM1 staining was observed in spermatid nuclei between stages XI and I (Fig. 6).
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PRM2 first faintly appeared in nuclei of elongated spermatids in some of the stage XI and all of the stage XII tubules of wild-type mice (Fig. 7, E and I). The weak stain persisted in 40% of stage I–VI tubules (Fig. 7, F and J), whereas 60% showed a strong immunoreaction (Fig. 7, G and K). The strong stain persisted in nuclei of step 16 spermatids in stage VII and VIII tubules (Fig. 7, H and L). Like PRM1 staining, most PRM2 staining in late spermatid nuclei was also peripheral. Since stage XII tubules showed weak staining and stage VII tubules showed strong staining, we assume that the weakly stained stage I–VI tubules were in the early part of these stages and the strongly stained ones were in the later part. Because the duration of stage I–II is 38% of that of stages I–VI and I–III is 43%, we calculated that the 40% of stage I–VI tubules with the weak PRM2 stain corresponded to those in stages I–II and half of stage III. Thus, the increase in PRM2 levels occurred in late step 14 spermatids.
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The staining patterns for PRM1 and PRM2 are displayed graphically in Figure 8. In both Tnp1 –/– mice and Tnp2 –/– mice, PRM1 and PRM2 showed the same timing and staining intensity as those in wild-type mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Quantitative biochemical analysis of purified rat and mouse spermatids suggested that nucleoprotein replacement was largely sequential, with little overlap of histones and TPs and minimal overlap of the TPs and PRMs during spermiogenesis. Histones were the major basic nuclear proteins in purified rat step 11–12 spermatids (corresponding to mouse step 10–11 spermatids), and only 4% of the proteins were TPs [28]. However, TPs constituted the major nuclear proteins in mouse step 12–13 spermatids purified based on their sonication-resistant character; only 1% of the basic nuclear protein was histone, and <10% was PRM [11]. Radioactive labeling studies of mouse sonication-resistant spermatids and epididymal sperm indicated that PRM1 synthesis starts in step 12 and PRM2 in step 13 spermatids [9], whereas the comparison of the protein content of purified step 12–13 spermatids with that of all of the sonication-resistant nuclei (step 12–16) indicated that the displacement of TPs did not occur until just after step 13 [11]. Similarly, studies on microdissected tubules from rat testes showed that PRM1 first appeared in step 15 spermatids (corresponding to mouse step 13 spermatids), whereas TP2 was last detected in stage I tubules (rat step 15, corresponding to mouse step 13), and TP1 was last observed in stage II– III tubules (rat step 16, corresponding to mouse step 14) [29].
In this study, we applied sensitive immunohistochemical methods to determine the distributions of histones, TP1, TP2, PRM1, and PRM2 in wild-type and Tnp-mutant mice. The distributions of these proteins in the nucleus (Fig. 9) showed more overlap than expected from the biochemical studies. Although histones were not completely removed until step 13 spermatids [13], TP2 and TP1 were first detected in steps 10 and 11 spermatid nuclei, respectively, and both of the TPs showed strong immunostaining in steps 12 and 13 spermatid nuclei. This suggested that histone displacement by TPs is a gradual process. PRM1 distribution also overlapped that of TPs and even the histones. It was first detected immunohistochemically in the spermatid nucleus as early as step 10, progressively increased from step 11 through steps 13 or 14, and then persisted through the rest of spermiogenesis. TPs also persisted in the nuclei later than expected, through early step 15 spermatids during stage IV, which contributed to more overlap with PRM1. PRM2 was also first detected in step 12 spermatid nuclei, although it remained at low levels until late step 14 spermatids in stage III. Thus the TPs showed appreciable overlap with the low levels of PRM2, but their disappearance rather quickly followed, and may be coordinated with, the major increase in PRM2.
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The differences between immunoreactivity and electrophoretic detection could be due to several factors. The most likely contribution is that the immunohistochemical assay is more sensitive than the gel assay. However, it is difficult to quantify protein levels from immunohistochemical assays. Although large differences in protein levels were reproducibly detected, small differences could not be detected visually, and in some cases the staining was saturated so that increases were not detected. The biochemical assays are more quantitative; however, they suffer from several shortcomings. One shortcoming is the possible loss of nuclear proteins during preparation. Some nuclear isolation procedures, such as sonication for sonication-resistant spermatid purification, could result in the loss of proteins not bound to chromatin or bound to sonication-sensitive chromatin, such as that in nuclei containing only histones. This might cause the absence of histones in step 12–13 spermatids in the biochemical assays. Some TPs were also found in the sonication-sensitive fraction in one of two studies, but no PRMs were found in this fraction in either study [30, 31]. Even washing in salt solutions could dissociate basic nuclear proteins that are not bound to the chromatin. This could explain why only 4% of basic nuclear proteins were TPs in rat step 11–12 spermatid nuclei prepared by a procedure involving washing nuclei in 0.22 M NaCl [28], whereas high levels of TP2 were present in basic protein extracts from whole microdissected tubules containing rat step 12 spermatids [29]. Although the lack of purity of the cell fractions or tubule dissections could result in the presence of additional proteins from other cell types, this is not an issue for our discussion, since data from biochemical assays showed less overlap than those from immunohistochemical assays.
The presence of TP2, PRM1, and TP1 in mouse step 10–11 spermatid nuclei was shown by immunochemical methods. However, there were no ultrastructural changes or condensation of the chromatin at these stages [26] as would be expected by binding of these proteins, which have a high capacity to condense DNA [2, 32]. Although transcriptional repression occurred in step 10–11 spermatids, this cannot be taken as evidence of chromatin binding of TPs in these cells because it occurred similarly in mice lacking both TPs [13]. It is possible that the lack of chromatin condensation may be a result of the levels of the proteins that were detected by immunohistochemistry being very low at these steps. Alternatively these proteins may initially not be chromatin bound, but perhaps associated with some chaperone proteins within the nucleus.
In steps 12–13, biochemical studies have shown that TPs are the major chromatin proteins. However, immunohistochemistry showed that both PRMs are also present in the nucleus. Since TPs are capable of condensing DNA [2] and are essential for chromatin condensation at these stages [13], the role of PRMs in these early stages is not apparent. Because PRM binding to chromatin would render it sonication resistant and PRMs are not readily extracted from chromatin, it is possible that the PRMs are not chromatin bound. The delay in chromatin binding may involve the presence of chaperone proteins or possibly a contribution from PRM phosphorylation. The concept that the PRMs are present but not normally chromatin bound in step 12–13 spermatids with a full complement of TPs is supported by observations of a higher level of PRM in sonication-resistant step 12–13 spermatid nuclei from mice lacking TP1 than in those from wild-type mice [11].
The absence of TP1 or TP2 had remarkably little effect on many of the events of nucleoprotein replacement in spermatids, including transcription, translation, and nuclear transport. In a separate study, we showed that the absence of both TP1 and TP2 did not affect the displacement of histones [33], and therefore it is most likely that this is also the case in mice lacking one of the TPs. The present data further showed that the absence of one TP does not affect the appearance of PRMs. PRM1 first appeared in step 10– 11 spermatids in both wild-type and Tnp-mutant mice. In the absence of one TP, although the normal posttranslational processing of PRM2 was disrupted [11, 12], the temporal expression of PRM2 was not altered. Also, in mice lacking one of the TPs, the mRNA level for remaining TP was not altered. Furthermore, the apparent elevation in the other TP did not result from higher levels of translation, but rather was largely due to prolonged retention of the protein during spermiogenesis.
Because the elevation of TP levels begins at step 15, obviously, the "elevated" TP protein does not compensate for the function of deleted protein in step 12–13 spermatids. This suggests that there may be a shortage of basic nuclear protein in the chromatin of step 12–13 spermatids, and the excess negative charge on DNA would need to be neutralized by divalent ions or polyamines if the amount of PRM binding is not sufficient to neutralize it. The abnormal presence of TPs in step 15 (and step 16 in the case of Tnp1 –/–) spermatids in mice lacking one TP was considered in the search for a cause of the failure of PRM2 processing and complete chromatin condensation in the mutants. Both of these possibilities can be ruled out because complete condensation and PRM2 processing also fail in mice lacking both TPs [13].
The mechanism of displacement of TP1 and TP2 and the cause of its disruption in Tnp-mutant mice are not known. In one line of male Camk4 –/– mice, observations of failure of PRM2 phosphorylation, specific loss of the mature form of PRM2, and prolonged retention of TP2 in step 15 spermatids led to the proposal that successful interaction of mature PRM2 with chromatin is required for displacement of TP2 [7]. In Tarbp2 –/– mice, failure to express PRM2 at the appropriate time also resulted in prolonged retention of TP2 in later-stage elongated spermatids [34]. In the present study, although the levels and distribution of PRM1 and PRM2 were normal in both Tnp1 –/– and Tnp2 –/– mice, the processing of PRM2 was disrupted in both of them. Thus, our suggestion above that the displacement of TPs at early step 15 was a consequence of the major increase of PRM2 at late step 14 might need to be modified to only apply to an increased mature form of PRM2. Delayed and incomplete processing of PRM2 might affect its interaction with chromatin and make it less effective at displacing the TPs than the mature form of PRM2.
That immunohistochemical studies showed more overlap during the nucleoprotein replacements in spermiogenesis than did the biochemical studies suggested that at certain stages these proteins may be in the nucleus but not chromatin bound. Further studies are needed to examine the chromatin binding, particularly of TP2 in step 10–11 spermatids and of histones and both PRM in step 12–13 spermatids. The mechanisms of displacement of the histones and deposition of the TPs and then of the displacement of the TPs and the deposition of the PRMs are not known. Chaperone proteins may be involved in keeping these proteins within the nucleus either before binding to chromatin or as part of their removal from chromatin. It will be important to identify such proteins in future research.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Ming Zhao, Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030. FAX: 713 794 5369; mzhao{at}mdanderson.org
Received: 5 February 2004.
First decision: 25 February 2004.
Accepted: 19 May 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 H. Zheng, C. J. Stratton, K. Morozumi, J. Jin, R. Yanagimachi, and W. Yan Lack of Spem1 causes aberrant cytoplasm removal, sperm deformation, and male infertility PNAS, April 17, 2007; 104(16): 6852 - 6857. [Abstract] [Full Text] [PDF]
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R. Oliva Protamines and male infertility Hum. Reprod. Update, July 1, 2006; 12(4): 417 - 435. [Abstract] [Full Text] [PDF]
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 R. Suganuma, R. Yanagimachi, and M. L. Meistrich Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI Hum. Reprod., November 1, 2005; 20(11): 3101 - 3108. [Abstract] [Full Text] [PDF]
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R.-M. Laberge and G. Boissonneault On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids Biol Reprod, August 1, 2005; 73(2): 289 - 296. [Abstract] [Full Text] [PDF]
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C. R. Shirley, S. Hayashi, S. Mounsey, R. Yanagimachi, and M. L. Meistrich Abnormalities and Reduced Reproductive Potential of Sperm from Tnp1- and Tnp2-Null Double Mutant Mice Biol Reprod, October 1, 2004; 71(4): 1220 - 1229. [Abstract] [Full Text] [PDF]
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