INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The development of spermatids into spermatozoa, termed spermiogenesis, is characterized by striking morphological and molecular transformations (43). In elongating and condensing spermatids, major restructuring of the somatic chromatin takes place in which the histones are first replaced by a group of arginine- and lysine-rich proteins called transition proteins (TPs), which are in turn replaced by protamines (42). At that time, transcription ceases, the nucleosomal-type chromatin is transformed into a smooth fiber, and condensation begins (30).

TP1 and TP2 are the predominant TPs found in rodent spermatids (24). TP1 is a 6.2-kDa, highly basic chromosomal protein with evenly distributed basic residues (32, 34). TP2, by contrast, is a 13-kDa protein with distinct structural domains (39). The carboxyl third of the molecule is enriched in basic residues and is likely to be a major site of electrostatic DNA binding (14), whereas the amino-terminal region has two proposed zinc fingers (41). The preferential binding activity of TP2 to CpG sequences, which are often associated with promoter regions, is dependent on zinc (36).

TP2, in contrast to the highly conserved TP1 (34), is poorly conserved across mammals, showing only 75% nucleotide and 50% amino acid homologies (1, 29). Whereas TP1 is abundantly expressed in all mammals studied (27), representing 60% of the basic proteins of mouse step 12 to 13 spermatid nuclei (62), TP2 levels vary (1). For example, the mRNA in human testis is detectable only by reverse transcription-PCR (54), but in mice the protein constitutes about 30% of the basic proteins of step 12 to 13 spermatid nuclei (62). Based on these considerations, TP1 should have the more significant role in mouse sperm development, unless TP2 has a specific role that cannot be complemented by another protein.

The timing of the appearance of the TPs has been most extensively studied in the rat. Some studies indicated that both proteins are present at significant levels only in the condensing step 13 to 15 spermatids (1, 24, 27, 44). However, others suggested that TP2 first appears at step 10 or 11 during nuclear elongation, whereas TP1 appears slightly later at step 11 or 12 (31, 46). In the mouse, initial studies indicated that TP2 first appears at step 12, reaches a maximum at step 13, but disappears during step 14 (1), but a recent study reported the presence of TP2 as early as step 10 (60).

In the mouse, the TPs are replaced by two protamines, P1 and P2 (8). Whereas P1 is synthesized as the mature protein, P2 is synthesized as a precursor (pre-P2) with 106 residues (61) and is sequentially cleaved to produce six identifiable intermediates (int-P2) and the mature form with 63 residues (15). The genomic locus for TP2 is found in the same gene cluster as the protamines in all mammals examined (53), suggesting that they have an evolutionary relationship and therefore may share some common functions.

Different functions have been suggested for the TPs, including nuclear shaping, histone removal, transcriptional repression, chromatin condensation, and, most recently, repair of the DNA strand breaks that normally transiently occur during the removal of the nucleosomes (12). In vitro, both TP1 and TP2 stabilize DNA in a nonsupercoiled state and bring DNA molecules in close proximity (38). Other studies indicated that the two TPs have distinct roles. TP1 has been reported to decrease the melting temperature of DNA and decrease nucleosome compaction (55, 56), whereas TP2 does the opposite (7), leading to the suggestion that TP1 is involved in histone removal and TP2 is involved in chromatin condensation. Finally, the preferential binding of TP2 to CpG islands may indicate a role for it in global repression of transcription (41).

To test these predictions and to understand the exact role of TPs in spermiogenesis, we first generated Tnp1-null mice (62). Although we found that TP1 was not essential for histone displacement or chromatin condensation, probably because its absence may be partially compensated for by TP2, the absence of TP1 did result in an abnormal pattern of chromatin condensation and in reduced fertility. Subsequently, we generated mice lacking TP2 by targeted deletion of the Tnp2 gene. In this paper, we describe the effects of this mutation on the appearance and protein composition of various stages of spermatids and on chromatin structure and function of spermatozoa.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Construction of the Tnp2-targeting vector. A Stratagene 129 mouse genomic library in the -FixII vector was screened with a Tnp2 cDNA (33). The restriction sites of one of the positive clones overlapped with a sequenced protamine gene cluster clone (GenBank accession number Z47352) for 10.5 kb (53), which was used to select additional restriction sites used in constructing the replacement vector from our clone (Fig. 1A).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Targeted deletion of the Tnp2 gene. (A) Genomic Tnp2 locus, the replacement vector used, and the targeted locus. Restriction sites: A, AvaI; B, BamHI; Bs, BsmI; C, ClaI; E, EcoRI; H, HindIII; N, NotI. The positions of the probes used to distinguish the targeted locus from the endogenous locus are indicated. (B and C) Southern blot genotyping performed by probing BamHI digests with the 5' probe (B) or probing EcoRI digests with the 3' probe (C). (D) PCR genotyping using primers for Tnp2 and neo.

Generation of Tnp2 mutant mice. The NotI-linearized Tnp2-targeting vector was electroporated into AB-1 ES cells, which subsequently were cultured in the presence of G418 and 1-(2-deoxy-2-fluoro--D-arabinofuranosyl)-s-iodouracil (FIAU) on mitotically inactivated STO fibroblasts (28). Resistant ES cell clones were screened by Southern blotting. The 5' probe external to the region of vector homology was a 350-bp EcoRI-BamHI fragment from a P2 (Prm2) cDNA clone (26), and the 3' probe was a NotI-EcoRI fragment (nt 13031 to 13405) external to the 3' region of homology. Correctly targeted ES clones were identified by Southern blotting and were microinjected into C57BL/6J (B6) blastocysts, which were transferred to pseudopregnant foster mothers.

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Isolation of spermatogenic cells and preparation of nuclei. For isolation of sonication-resistant spermatid nuclei (SRS), which represent step 12 to 16 spermatids, our standard procedures (59, 62) were modified as follows. The concentration of phenylmethylsulfonyl fluoride (PMSF) was increased to 0.5 mM before homogenization and a protease inhibitor cocktail (standard cocktail. [1 mM phenylmethylsulfonyl fluoride, 1 µg p-aminobenzamidine per ml, 1 µg of leupeptin per ml, 1 µg of pepstatin A per ml] unless otherwise mentioned) was added to the buffer at each later step in the procedure. After sonication for 3 min at 50% of maximum power using a 3-mm-diameter probe (model 250 Sonifier, Branson Ultrasonics, Danbury, Conn.), each milliliter of spermatid nuclei was mixed with 9 ml of 83.5% (wt/vol) sucrose and centrifuged through a 7-ml cushion of 83.5% (wt/vol) sucrose at 100,000 × g in a Beckman SW 27 rotor for 1 h. The pellet was washed with MP buffer (5 mM MgCl₂, 5 mM sodium phosphate, pH 6.5).

Preparation and analysis of nuclear proteins. All extraction and preparation procedures were performed at 4°C. For selective extraction of TP1, TP2, and histone H1, two testes first were homogenized in water with protease inhibitors and sonicated for 1 min in a 51-mm-diameter cup-horn sonicator. Basic proteins were then extracted with 0.25 M HCl and precipitated overnight with 3.5% trichloroacetic acid (TCA), and the supernatant was retained (11). In all procedures, the proteins were finally precipitated with 25% TCA and the precipitates were washed with acidified acetone, followed by acetone, and dried (47).

Immunoblot analysis. The nuclear proteins were electroblotted from the gels onto a Hybond polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, N.J.) in 0.7% acetic acid at 320 mA for 20 min. After staining with Ponceau S, strips were cut off based on the position of marker extracts and blocked with 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBS-T). The strips were incubated for 1 h at 25°C with anti-TP1 antiserum (1:6,000), anti-TP2 antiserum (1:1,500) (both courtesy of Stephen Kistler, University of South Carolina, Columbia), and anti-H1 antiserum raised against calf thymus H1 (1:1,000) (courtesy of Sylviane Muller, Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France) diluted in PBS-T. After being washed three times for 15 min each in PBS-T, the blots were incubated with anti-rabbit immunoglobulin G antibody linked to horseradish peroxidase. The binding to the proteins was detected with ECL-plus reagents (Amersham Pharmacia), imaged with a Molecular Dynamics Storm gel and blot system, and quantified using IMAGEQUANT software.

RNA isolation and Northern blot analysis. Total RNA was extracted from mature mouse testes with the RNAgents Total RNA Isolation System (Promega, Madison Wis.). Plasmids containing Tnp1 and Tnp2 cDNAs were provided by Kenneth Kleene (University of Massachusetts, Boston) (32, 33). Prm1 and Prm2 cDNA plasmids (61) were purchased from the American Type Culture Collection. Northern blotting and hybridization were performed as described elsewhere (52). The 400-bp Tnp1, 550-bp Tnp2, 480-bp Prm1, and 550-bp Prm2 fragments were used as probes, which hybridized with bands centered at 600, 700, 550, and 760 bp, respectively, on the blot. A 0.7-kb fragment from a plasmid containing rat cyclophilin cDNA, provided by Miles Wilkinson (M.D. Anderson Cancer Center, Houston, Tex.), was used as a control probe and hybridized with a 720-bp band (58). The levels of mRNA were quantified using IMAGEQUANT software.

Light and electron microscopy. For light microscopy, testes were fixed overnight in Bouin's solution and embedded in paraffin. Tissue sections, 4 µm thick, were stained with periodic acid-Schiff stain and counterstained with hematoxylin. For analysis of sperm retention, averages of 34, 7, 9, and 19 round or nearly round stage VIII, IX, X, and XI tubules, respectively, were counted in three mice of each genotype.

Testicular and epididymal sperm counts. Individual testes were homogenized in 1 ml of deionized water for 5 min using a Polytron homogenizer at setting 9 and sonicated in a 51-mm-diameter cup-horn sonicator for 3 min to remove sonication-sensitive cells. Each cauda epididymis was minced in 1 ml of PBS, and after 30 min the tissue pieces were separated from sperm by pipetting and then passing through an 80-µm-pore-size filter. All counts were performed using a hemacytometer.

Sperm morphology. Air-dried smears were prepared from sperm in PBS, stained with hematoxylin, and examined using light microscopy at a magnification of ×1,000. Head or tail morphology was determined independently for each mouse with separate counts of at least 100 cells each.

Preparation of nuclei for flow cytometry. The epididymides from each mouse were minced in 2 ml of TNE buffer (0.1 M Tris, 0.15 M NaCl, and 1 mM EDTA, pH 7.4). After ~30 min at room temperature, the suspension was pipetted several times and the debris was removed by filtering through 80-µm mesh. Sperm were diluted to 1 × 10⁶ to 2 × 10⁶ cells/ml. Because it proved necessary to use isolated nuclei, sperm heads were separated from tails in two ways, both yielding >90% separation.

Propidium iodide staining and flow cytometry. Aliqouts of sperm nuclei were stained with 25 µg of propidium iodide (Sigma) per ml in the presence of 0.3% Nonidet P-40 (Sigma) and RNase (40 µg/ml) (Boehringer Mannheim) for 1 h at room temperature (35). Samples were stored overnight at 4°C before being analyzed with an Epics 752 flow cytometer (Beckman-Coulter Corp., Hialeah, Fa.). The excitation wavelength was 488 nm, and the propidium iodide fluorescence signals (area under peak) were collected in list mode using a 610-nm long-pass filter. For normalization of the data, samples from a wild-type mouse were analyzed both before and after analysis of samples from all mutation-carrying mice.

AO staining and flow cytometry. Other aliquots of sperm nuclei were stained with AO, a metachromatic dye that produces green fluorescence when bound to double-stranded DNA and red fluorescence when bound to single-stranded DNA. A 0.2-ml aliquot was mixed with 0.4 ml of acid denaturation solution (0.15 M NaCl, 0.08 N HCl, and 0.1% Triton X-100, pH 1.4) (18). After 30 s, the cells were stained with 1.2 ml of AO solution (6 µg/ml), (Polysciences, Warrington, Pa.) in a buffer consisting of 0.1 M citric acid, 0.2 M Na₂HPO₄, 1 mM EDTA, and 0.15 M NaCl (pH 6.0).

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Bivariate histograms of green fluorescence peak height versus peak area for acid-denatured, AO-stained preparations from wild-type mice. Regions containing intact sperm (S), free sperm heads (H), and free sperm tails (T) are marked. (A) Intact sperm. (B) Trypsin-treated sperm. (C) Sonicated sperm. (D) Histograms of green fluorescence peak height for the indicated gated areas from intact sperm (thin solid lines), trypsin-treated sperm (dashed lines), and sonicated sperm (thick solid line).

Statistical analysis. Student's t test was used to compare averages in different experimental groups.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Targeted deletion of Tnp2 in the mouse germ line. The Tnp2 gene was disrupted in ES cells by homologous recombination with a targeting vector in which the entire Tnp2 gene was replaced by a neo gene flanked by loxP sites (Fig. 1A). Germ line transmissions were obtained from chimeras of two independent clones. Clone 2C8 was selected for detailed analysis; clone 2E3 was used for comparison with 2C8. Heterozygous offspring produced by each chimera were intercrossed. Their offspring were genotyped by Southern blotting of tail DNA using external probes (Fig. 1B and C) or by PCR (Fig. 1D), which was in agreement with Southern analysis. Out of 206 offspring, there were 50 wild-type, 111 heterozygous, and 45 homozygous mice, a ratio consistent with 1:2:1 Mendelian inheritance.

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View larger version (70K): [in this window] [in a new window]   FIG. 3.   Analysis of 3.5% TCA-soluble proteins from testes of wild-type, heterozygous, and Tnp2 mutant mice by acid-urea gel electrophoresis. (A) Coomassie blue-stained gel. A total basic nuclear protein extract (BNP) from unfractionated wild-type mouse testis nuclei was used as a marker. (B) Immunoblots using antibodies to TP1, TP2, and H1.

Subtly abnormal spermatogenesis in the absence of the Tnp2 gene. When Tnp2 mutant mice were mated with individual 8-week-old B6 females, the percentage of mutant mice that were fertile was not significantly different from that of normal mice (Table 3). However, the average litter size produced by the Tnp2-null males (3.9 ± 0.7) was significantly reduced from that observed for the wild type (7.4 ± 0.4).

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View larger version (23K): [in this window] [in a new window]   FIG. 4.   Retention of mature spermatids in tubules at stages IX to XI in testes of wild-type (circles) and Tnp2 mutant (triangles) mice. The fractions of tubules with 3 to 15 sperm (A), 1 to 2 sperm (B), and no sperm (C) per cross-section are shown. Asterisks indicate values that are significantly different from those for the wild type (P < 0.05). Error bars indicate standard errors.

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Altered protein levels in Tnp2 mutant mice. TP1 and TP2 levels in the 3.5% TCA-soluble protein extracts of total testis were analyzed after separation by gel electrophoresis. Although quantification of Coomassie blue-stained bands was valid for measuring the levels of TP1 (62), there were some proteins in Tnp2-null mice, with a total intensity of 23% of that of TP2 from wild-type mice, that migrated in the TP2 region of the gel (Fig. 3A). However, immunoblotting confirmed the absence of TP2 in these Tnp2^/ mice (Fig. 3B). In heterozygotes, the level of TP2 was about 50% of that in wild-type mice. The level of TP1 was elevated by about 45% in Tnp2-null mice as shown by both the Coomassie blue-stained gels and immunoblots (Table 4). TP1 was also elevated in Tnp2 heterozygotes, but to an intermediate level.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   Basic nuclear proteins from sonication-resistant (step 12 to 16) spermatids (A) and epididymal sperm (B) from wild-type, Tnp2+/, and Tnp2/ mice. A total basic nuclear protein extract (BNP) from unfractionated wild-type mouse testis nuclei was used as a marker for the histone bands and TP1. The SRS marker represents sonication-resistant step 12 to 16 spermatids prepared from the testes of wild-type mice. In panel B, the heterozygote samples were overloaded on this gel so that the low level of P2 precursors would be clearly visible. Parts of the epididymal preparations were aminoethylated to separate P1 and the mature form of P2. pre-P2 is the primary translation product of the Prm2 gene; pre-P2/11, pre-P2/16, pre-P2/20, and pre-P2/32 result from proteolytic cleavage before the 11th, 16th, 20th, and 32nd amino acids, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Basic nuclear proteins in sonication-resistant spermatids (A) and epididymal sperm (B) as a percentage of total basic protein. Error bars indicate range (n = 2).

Altered chromatin condensation in Tnp2 mutant spermatids and sperm. The first abnormalities in chromatin condensation in Tnp2^/ mice were focal condensations, which appeared cylindrical, averaging 95 nm long and 65 nm across as measured in thin sections of randomly oriented units. They were in all late step 11 spermatids (Fig 7B), a stage in which there was no condensation in wild-type spermatids (Fig. 7A). These abnormal condensations appeared primarily in the anterior part of the nucleus. The remainder of the chromatin appeared as a fine needle-like network both in wild-type and Tnp2^/ mice. During steps 12 to 13, these focal condensations increased in number and, in some cases, were found throughout the entire spermatid nucleus (Fig. 7D). The differentially condensed core of chromatin representing the pericentromeric heterochromatin (25), which developed and increased in density during steps 11 to 12 in wild-type mice (Fig. 7C), was similarly observed in spermatids from Tnp2^/ mice (Fig. 7D). During steps 12 to 13 in both wild-type and Tnp2^/ mice, the chromatin fibrils similarly thickened and appeared more rope-like, and the fibrils also moved closer to each other in the process of condensing the chromatin and decreasing the nuclear volume (Fig. 7C and D), sometimes with a gradient from the anterior to the posterior end (not shown). In wild-type mice, the most dramatic condensation occurred as the spermatids progressed through step 13 and into step 14 (Fig. 7E). However, in step 14 spermatids from Tnp2^/ mice, the chromatin, consisting of fibrils and focal condensations, had not completely compacted (Fig. 7F), and occasionally the focal condensations and the differentially condensed pericentromeric heterochromatin remained distinguishable (Fig. 7F). In wild-type mice, the chromatin became uniformly dense when spermatids reached step 15 (not shown), and its appearance by electron microscopy did not change further during step 16 (Fig. 7G) or epididymal maturation (Fig. 7I). However, step 16 spermatids from Tnp2^/ mice still displayed incomplete condensation with lacunae, i.e., small spaces scattered between the dense chromatin, and occasionally a larger area that lacked condensed material (Fig. 7H). These areas of incompletely condensed chromatin were further reduced, but still present, in sperm from the cauda epididymis and vas deferens (Fig. 7J). The chromatin appeared to be arranged in randomly but closely packed spherical objects, with an 11-nm average diameter and an 18-nm average spacing, evenly distributed throughout the nucleus.

View larger version (113K): [in this window] [in a new window]   FIG. 7.   Abnormalities in chromatin condensation in spermatids from Tnp2 mutant mice. (A and B) Step 11 spermatids from wild-type (A) and Tnp2/ (B) mice. (C and D) Step 13 spermatids from wild-type (C) and Tnp2/ (D) mice. (E and F) Step 14 spermatids in stage II from wild-type mice (E) and in stage III from Tnp2/ mice (F). (G and H) Step 16 spermatids in stage VIII from wild-type mice (G) and in stage VII from Tnp2/ mice (H). (I and J) Cauda epididymal sperm nuclei from wild-type mice (I) and sperm nuclei from the vas deferens from Tnp2/ mice (J). f, abnormal focal condensations; h, differentially condensed core of pericentromeric heterochromatin. Magnifications: A, ×25,000; B, ×16,000; C, ×31,000; D, ×10,000; E, ×12,000; F, ×13,000; G, ×11,000; H, ×16,000; I, ×12,000; J, ×12,000.

View larger version (34K): [in this window] [in a new window]   FIG. 8.   Flow cytometric analysis of sperm chromatin defects. (A) Distributions of propidium iodide fluorescence intensities for sperm heads prepared by sonication from Tnp2-null mice (solid line) and from wild-type mice (dashed line). (B) Distributions of the relative amount of red fluorescence (red/total fluorescence) of acid-denatured, AO-stained sperm heads prepared with trypsin from Tnp2-null mice (solid line) and from wild-type mice (dashed line). (C and D) Bivariate histograms of green versus red fluorescence for acid-denatured, AO-stained sperm heads prepared by sonication from wild-type (C) and Tnp2-null (D) mice.

View larger version (39K): [in this window] [in a new window]   FIG. 9.   Dye uptake and susceptibility to acid denaturation of DNA in cauda epididymal sperm nuclei from Tnp2 mutant mice on different genetic backgrounds and derived from different ES cell clones. All values were normalized to those for wild-type mice derived from the same matings as were the mutants. (A) Propidium iodide fluorescence intensity. (B) Denaturability as defined by the ratio of red fluorescence to total (red plus green) fluorescence of AO-stained sperm nuclei. For clone 2C8 on the 129 background, n = 3 to 5; for clone 2C8 and clone 2E3 on the hybrid (hyb) background, n = 2. When n = 2, the error bars represent the range of values; otherwise the error bars indicate standard errors. * and **, significantly different from 1 at P values of <0.05 and <0.005, respectively.

Susceptibility of sperm DNA from Tnp2 mutants to acid denaturation. The metachromatic fluorescence of sperm nuclei measured after AO staining and acid denaturation showed that elimination of the Tnp2 genes resulted in an increase of green fluorescence of only 16% but an increased of red fluorescence of 62% (Fig. 8C and D). The ratio of red to total fluorescence intensity was increased significantly in sperm from Tnp2-null mice over that observed in wild-type mice (Fig. 8B and 9B), indicating a greater susceptibility of the DNA to denaturation. The increase was homogenous, as there was no significant broadening of the distribution as measured by its coefficient of variation.

No qualitative differences with different genetic backgrounds, clones, and removal of neo. Since the effects of the null mutation for Tnp2 could be different on various genetic backgrounds, we examined whether the abnormal phenotypes observed in 129 mice were also present in F₂ hybrid (129/B6) mice. Tnp2-null hybrid mice showed the same trends towards reduced litter size and abnormal sperm tail morphology as did the ones on the 129 background (Table 3), but on the 129/B6 hybrid background, the differences from wild type were not statistically significant. In sperm from the Tnp2-null mice on the hybrid genetic background, the levels of P2 precursors were still elevated, at 17% of the total protamine. This value was above control levels (<1%) although not as high as the 33% value found in sperm from Tnp2^/ mice on the 129 background (Fig. 10).

View larger version (45K): [in this window] [in a new window]   FIG. 10.   Protamine 2 precursor levels (pre-P2 plus int-P2) expressed as percentages of total protamine in Tnp2 mutant mice produced from different ES cell clones, on different genetic backgrounds, and after the excision of the neo gene from the Tnp2 locus. (A) SRS nuclei (step 12 to 16 spermatids) from testes. (B) Epididymal sperm nuclei. Note that pre-P2 was not present in epididymal sperm of any genotype studied here and that there was <1% int-P2 in epididymal sperm from all wild-type mice. hyb, hybrid. Error bars indicate range (n = 2).

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

To investigate the role of TP2 in mammalian spermiogenesis, we generated a null mutation in the Tnp2 gene using homologous recombination. Mice homozygous for the mutation completely lacked TP2 in their testes. However, the Tnp2 mutation did not severely impair spermiogenesis; many aspects were normal, but others showed minor defects as discussed below. These mice had relatively normal testis histology, normal numbers of mature sperm, and were still fertile. This suggests that TP2 is not essential for spermiogenesis, likely because the negative consequences caused by the absence of TP2 are minimized by compensation initially by TP1 and later by the protamines.

We did not detect any defects in histone displacement in Tnp2 mutant mice. This result supports our suggestion, based on similar observations with Tnp1-null mice, that histone displacement may be an active process and may start before the initiation of TP accumulation in the nucleus (62).

The transcription of the genes for the other basic nuclear proteins (Tnp1, Prm1, and Prm2) was not affected by the Tnp2 mutation, and the levels of Tnp2 mRNA in Tnp2^+/ mice were reduced to about half of that found in the wild type. These results indicate that there is no feedback at the mRNA level. Furthermore, these mRNAs are among the last to be transcribed in step 7 to 9 spermatids (40) before the global repression of transcription at step 10 (30). Therefore, the absence of increased levels of these mRNAs in Tnp2-null mice suggests that TP2 is not responsible for the global repression of transcription.

In contrast to the lack of effect on mRNA levels, TP1 protein levels were elevated by 22 and 45% in Tnp2^+/ and Tnp2-null mice, respectively, relative to the wild type. This regulation of TP1 must therefore occur at the posttranscriptional level. These results could be explained by the Tnp1 mRNA being capable of acting as a template for more protein than is produced in wild-type mice and translating more protein because of the reduced amount of TP2. An alternative explanation for the elevated level of TP1 could be its prolonged retention in spermatids beyond step 14. Precedence for this comes from observations of prolonged retention of TP2 in Tarbp2-null mice, which fail to translationally activate the protamines in some cells (63), and in Camk4-null mice, in which P2 is lost from the nucleus soon after deposition (60). However, since protamine levels are not reduced in the Tnp2-null mice, this alternative would appear not to apply.

The Tnp2 mutation also resulted in a deficiency in P2 processing by a mechanism that is not yet clear. Failure of P2 processing has also been observed in Tnp1^/ mice (62), mice that prematurely express P1 at step 8 (37), and mice with haploinsufficiency in either protamine gene (13), suggesting that the normal arrangement of both TPs and protamines on the chromatin is a prerequisite for complete P2 processing. The failure of TP2 removal in spermatids with failure of P2 synthesis (63) or P2 retention and maturation (60) indicates that there may be some interaction between TP2 and P2 by which abnormalities in TP2 levels can affect P2 processing.

Although some aspects of chromatin condensation, such as the thickening of the chromatin fibrils and their condensation during steps 12 and 13, were normal in spermatids from Tnp2-null mice, there were significant abnormalities both in the initiation of condensation and in the final outcome. First was the abnormal focal condensations in step 11 spermatids of Tnp2^/ mice, which were also found in spermatids from Tnp1-null mice, in which they were originally called rod-shaped units and were even more evident (62). The cause of this abnormality might be a consequence of the abnormal deposition of other nuclear proteins present in excess levels and/or an unstable chromatin structure caused by lack of TP2.

Later there was a failure of completion of chromatin compaction in Tnp2 mutant sperm. This was shown by the lacunae in step 15 to 16 spermatids, the granular appearance of the chromatin in epididymal sperm, and the increased uptake of intercalating dyes by the sperm nuclei. The level of intercalating dye fluorescence appears to be primarily related not to the protein composition of the nucleus (17) but rather to the degree of disulfide cross-linking of the protamine (10, 19, 48). Although propidium iodide dye uptake appears to measure a different aspect of chromatin condensation than does electron microscopy, the changes observed when Tnp2 is mutated vary in parallel. Thus, although TP2 is not essential for chromatin condensation, there are defects in the ultimate degree of condensation in Tnp2-null mice.

In addition to the incomplete condensation of the chromatin, there was an increase in DNA denaturation as indicated by the higher relative red fluorescence of AO-stained sperm from Tnp2-null mice. An increase in the relative red fluorescence was also observed in sperm from mice with haploinsufficiency for either of the protamine genes (13). The increased denaturability of the DNA is believed to result from DNA strand breakage, based on correlations between the numbers of cells with high red fluorescence and other measures of strand breakage (3, 23, 51) and the lack of correlation with protamine disulfide cross-linking (50) or phosphorylation (22).

Despite these defects in chromatin condensation, no significant alterations in sperm nuclear shape were observed in Tnp2^/ mice. It was surprising that the absence of TP2 affected tail structures of epididymal sperm to a greater extent than it did sperm heads, given that TP2 is a nuclear protein. The most common tail defect, a sharp bend in the midpiece, resulting in the flagellum being bent back on itself, was also observed in sperm from Tnp1-null mice and mice with haploinsufficiency for one of the protamines (13). Thus, it appears more likely that the tail defects are due to an indirect secondary effect caused by the absence of TP2 or the presence of incompletely processed P2, rather than a direct cytoplasmic role for TP2.

It is interesting that similar phenotypes were observed in both Tnp2 and Tnp1 mutant mice and in mice with haploinsufficiency for the protamines. Some of the abnormalities, such as the abnormal focal chromatin condensations, deficiency of P2 processing, sperm head abnormalities, and loss of fertility, were less marked in Tnp2-null than in Tnp1-null mice (62). The lower levels of defects observed in the Tnp2-null mice are consistent with TP2 being present in spermatids in smaller amounts than TP1 and being a less conserved protein than TP1 (29, 34). In contrast, the loss of even one of the protamine genes appeared to have greater impact, resulting in a complete failure to produce fertile sperm (13).

The role of TP2 in normal spermiogenesis and the exact mechanism by which a mutation in the Tnp2 gene results in defective spermiogenesis are not known, but the present study provides some information. The similarities of the phenotypes of the Tnp1^/ and Tnp2^/ mutants suggest that cooperative participation of both TP1 and TP2 may be required for completely normal spermiogenesis. Alternatively, the severity of the defect may be primarily dependent just on the dosage of the four basic protein genes, with loss of a protamine gene being most severe and loss of the Tnp2 genes being least severe.

The common mechanism underlying the chromatin abnormalities and sperm tail defects in Tnp1-null, in Tnp2-null, and apparently in protamine-haploinsufficient mice is not known. However, all are deficient in P2 processing, indicating that it may have a central role in the sperm abnormalities and infertility. This result is consistent with several studies that have implicated reduced levels of mature P2 in sperm, sometimes accompanied by elevation of the levels of P2 precursors, in human infertility (5, 9, 16), but the murine models will allow more rigorous study of a role for maturation of P2 in fertility. Based on sperm abnormalities and reduced sperm motility in Tnp mutant mice, it is probable that the reduced fertility is a result of failure to fertilize the ova, but the possibility remains that there is some postfertilization developmental defect of sperm nuclei containing incompletely processed P2.

The generation of single Tnp1 and Tnp2 mutations has been helpful in understanding the respective roles of TP1 and TP2 in spermiogenesis. However, due to the compensation between proteins, analysis of the single mutations is not sufficient to completely appreciate the roles of the TPs during mammalian spermiogenesis. Therefore, studies of Tnp1 Tnp2 double mutants are needed and are in progress.