ARTICLE

Transition Nuclear Proteins Are Required for NormalChromatin Condensation and Functional SpermDevelopmentMing Zhao,1 Cynthia R. Shirley,1 Shotaro Hayashi,2 Ludovic Marcon,3 Bhagyalaxmi Mohapatra,1Ryota Suganuma,2 Richard R. Behringer,4 Guylain Boissonneault,3 Ryuzo Yanagimachi,2 andMarvin L. Meistrich1*1Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center,Houston, Texas2Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii3Department of Biochemistry, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada4Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Received 31 October 2003; Accepted 21 January 2004

Summary: The histone-to-protamine transition is impor-tant in the formation of spermatozoa. In mammals thisinvolves two steps: replacement of histones by transi-tion nuclear proteins (TPs) and replacement of TPs byprotamines. To determine the functions of the TPs andtheir importance for sperm development, we generatedmice lacking both TPs, since mice lacking only TP1 orTP2 were fertile. Our results indicated that TP1 and TP2had partially complemented each other. In mice lackingboth TPs, nuclear shaping, transcriptional repression,histone displacement, and protamine deposition pro-ceeded relatively normally, but chromatin condensationwas irregular in all spermatids, many late spermatidsshowed DNA breaks, and protamine 2 was not post-translationally processed. Nevertheless, genomic integ-rity was maintained in mature spermatids, since efficientfertilization and production of offspring were achievedby intracytoplasmic sperm injection. However, manymature spermatids were retained in the testis, epididy-mal spermatozoa were drastically reduced in numberand were highly abnormal, and the mice were sterile.Most epididymal spermatozoa were incapable of fertili-zation even using intracytoplasmic sperm injection.Thus, in mammals TPs are required for normal chroma-tin condensation, for reducing the number of DNAbreaks, and for preventing the formation of secondarydefects in spermatozoa, eventual loss of genomic integ-rity, and sterility. genesis 38:200–213, 2004.© 2004 Wiley-Liss, Inc.

Key words: transition nuclear proteins; knockout mice;spermatogenesis; protamine; infertility; intracytoplasmicsperm injection

In many species, sperm formation involves replacementof histones by protamines (Ausio, 1999). Although re-placement occurs directly in fishes and birds (Oliva andDixon, 1991), in mammals and some marine vertebrates

(Wouters-Tyrou et al., 1998) histones are first replacedby the transition nuclear proteins, TP1 and TP2. Theseare subsequently replaced by protamine 1 and a precur-sor of protamine 2, which is then processed to its matureform by proteolysis (Chauviere et al., 1992). This chro-matin remodeling is accompanied by changes in nuclearshape, the conversion of negatively supercoiled nucleo-somal DNA into a nonsupercoiled state (Ward et al.,1989), induction of transient DNA breaks (McPhersonand Longo, 1993), and chromatin condensation.

TP1 is a 6.2-kDa, highly basic (about 20% each ofarginine and lysine) protein with evenly distributed basicresidues (Kistler et al., 1975; Kleene et al., 1988),whereas TP2 is a 13-kDa basic (10% each of arginine andlysine) protein with distinct structural domains (Grimes

M.Z., C.R.S., and S.H. contributed equally to this work.Present address for C.R. Shirley: University of New Orleans, Department

of Biological Sciences, Audubon Institute for Research of Endangered Spe-cies, New Orleans, LA 70131.

Present address for S. Hayashi: Department of Obstetrics and Gynecol-ogy, Fukushima Medical University, Fukushima, 960-1295, Japan.

Present address for B. Mohapatra: Department of Pediatrics-Cardiology,Baylor College of Medicine, Houston, TX 77030.

Present address for L. Marcon: Department of Pharmacology and Thera-peutics, McGill University, Montreal, Quebec Canada H3G1Y6

* Correspondence to: Marvin L. Meistrich, PhD, Department of Experi-mental Radiation Oncology, Unit 066, M. D. Anderson Cancer Center, 1515Holcombe Blvd., Houston, TX 77030.

E-mail: meistrich@mdanderson.orgContract grant sponsors: National Institutes of Health, Contract grant

number: HD-16843 (to M.L.M.), HD-30284 (to R.Y.), CA-16672 (CancerCenter Support Grant), Contract grant sponsors: Katherine and HaroldCastle Foundation (to R.Y.), Kosasa Family Foundation (to R.Y.), CanadianInstitutes for Health Research, Contract grant number: MOP-39771 (toG.B.).

DOI: 10.1002/gene.20019

© 2004 Wiley-Liss, Inc. genesis 38:200–213 (2004)

et al., 1977; Kleene and Flynn, 1987). The only similar-ities between the two are their high basicity, exon–intron genomic patterns (Schluter et al., 1996), anddevelopmental expression.

The transition nuclear proteins (TPs) are exclusivelylocalized to nuclei of elongating and condensing sper-matids (Meistrich, 1989). They are first detected in step10–11 spermatids (Alfonso and Kistler, 1993; Heidaranet al., 1988) (M. Zhao, M.L. Meistrich, ms. in prep.).They reach maximal levels during steps 12–13, duringwhich they constitute 90% of the chromatin basic pro-tein, with the levels of TP1 being about 2.5 times thoseof TP2 (Yu et al., 2000). They are not detected in thenucleus after the early part of step 15 (Alfonso andKistler, 1993; Heidaran et al., 1988) (M. Zhao, M.L.Meistrich, ms. in prep.).

Since the TPs constitute 90% of the basic chromatinproteins in condensing spermatids (Yu et al., 2000), theyshould be very important, but evidence has been limited toin vitro studies indicating possible roles for the TPs in someof these remodeling processes. First, TP1 can destabilizenucleosomes and prevent bending of the DNA, both ofwhich could contribute to displacement of histones (Baska-ran and Rao, 1990; Levesque et al., 1998). Second, the zincfingers of TP2 selectively bind to CpG sites and could beresponsible for the global repression of RNA synthesis(Kundu and Rao, 1996). Third, both TPs have been pro-posed to be alignment factors for DNA strands, and TP1 isinvolved in the repair of strand breaks (Boissonneault,2002; Caron et al., 2001). Fourth, both TPs can condenseDNA, TP2 being the more effective (Baskaran and Rao,1990; Brewer et al., 2002; Levesque et al., 1998).

Previous studies showed that mice lacking either TP1or TP2 alone had normal numbers of sperm with onlyminor abnormalities and were fertile (Adham et al.,2001; Yu et al., 2000; Zhao et al., 2001), indicating eitherthat the TPs were not essential or that the individual TPscomplemented each other. To distinguish betweenthese possibilities and to test whether the roles sug-gested above for the TPs applied in vivo, we producedmice lacking both proteins and studied the effects onsperm development and function.

RESULTS

Specificity of Testicular Effects of Null Mutationsin Tnp GenesMale mice lacking both TPs did not appear to have anysystemic abnormalities. They had normal life-spans; bodyweights, serum testosterone levels, and seminal vesicleweights (which is also a measure of testosterone levels)showed no significant difference from wildtype mice(data not shown).

Testicular histology (Fig. 1A,B) in male mice lackingboth TPs appeared normal in the earlier stages of devel-opment (note tubules containing steps 9–14 sperma-tids), except there were fewer elongated spermatids inthe later stages of development (tubules containing step16 spermatids) and some had abnormalities. Through

step 13 of spermatid development, the numbers wereidentical to those in control testes, but by step 16 theydeclined to 34% of control (Fig. 1C). These histologicalresults were consistent with near normal testis weights(92% of control) but a reduction in numbers of step12–16 spermatids prepared by sonication (44% of con-trol) (Fig. 2A,B).

The release of spermatids from the seminiferous epi-thelium, which occurs after step 16 (stage VIII tubules)in normal mice, was also markedly defective in micelacking TPs. About half of the spermatids were stillvisible (in stage IX tubules) about 17 h after they shouldhave been released, and many remained there for about3 days (stage XII tubules) (Fig. 1C). Very few werereleased, as sperm counts in both the caput and caudaepididymides from mice lacking TPs were only 1% ofcontrol (Fig. 2C,D). This release process was age-depen-dent, as there was a 50% decline in late spermatids in thetestis and a 10-fold increase in epididymal sperm countin 52-week-old mice (Fig. 2C,D).

Defective Sperm Quality and Sterility in MiceLacking TPs

The quality and function of the epididymal spermatozoawere very poor. Over 80% were dead, and most of thelive ones were immotile (Table 1). Almost all had abnor-mal head morphology; many had blunted apical tips (Yuet al., 2000). Although no defects in the ultrastructure ofthe tail were apparent (C.R. Shirley, unpubl. results),light microscopy showed that most spermatozoa hadmissing mitochondria or abnormal configurations of thetail, and many were aggregated in clumps (Table 1). Theabnormal configurations and clumps were connected bycytoplasmic structures and sometimes an intact plasmamembrane could be seen enclosing the structures.

In contrast, late spermatids in the testis had fewerdefects that did the epididymal sperm. In smears, step12–13 spermatids from wildtype mice were identified byelongated or irregular nuclei that were somewhat con-densed, partial or full complements of cytoplasm, andabsence of a visible flagellum (Fig. 3A,B). Most step12–13 spermatids from mice lacking TPs had a normalnuclear shape (Fig. 3C,D), but about one-third had minor(Fig. 3E) or major head shape abnormalities (Fig. 3F,G),compared to 6% among cells from wildtype mice. Ma-ture step 16 testicular spermatids from wildtype micewere identified in phase contrast images of suspensionsby formation of a mitochondrial sheath along the mid-piece or, if mitochondria were absent, by a thickenedprincipal piece of the sperm tail, indicating a fibroussheath had formed (Fig. 3H). Similarly, about half ofthese mature spermatids from mice lacking TPs hadrelatively normal nuclear shapes (Fig. 3I,L,N), but manywere abnormal (Fig. 3J,K,M). In addition, many of thesemature spermatids had abnormalities in the midpieceand tail configurations (Fig. 3J–N) similar to, but not assevere as, those in epididymal sperm.

All five male mice lacking TPs tested by natural matingwere sterile. Cauda epididymal spermatozoa of male

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mice were unable to fertilize any oocytes by in vitrofertilization, whereas wildtype sperm fertilized 85% ofoocytes.

Specific Effect of TPs on Chromatin Remodeling:Functional Aspects

One of the first changes associated with chromatin remod-eling in spermatids is the global repression of transcription.

FIG. 2. Comparisons between wildtype mice and mice lacking TPsas young adult and as older mice. Results are mean � SEM;*significantly different from wildtype, P � 0.05. †Significantly differ-ent from value in young adult mice, P � 0.05. n � 5–25 mice perdata point. A: Testis weight was slightly lower in mice lacking TPsand this difference increased with age. B: Testicular sperm headcounts were moderately reduced in mice lacking TPs and declinedwith age, whereas (C,D) epididymal sperm counts were drasticallyreduced in mice lacking TPs but increased significantly with age.

FIG. 1. Relatively normal spermatogenesis in mice lacking TPs except for the decondensation and loss of late spermatids. Wildtype (A) andTnp1-/-Tnp2-/- (B) testes showing tubules with spermatids at specific steps of development. Scale bar � 50 �m. C: Spermatid:Sertoli-cellratio (log scale) in tubules at specific stages of the seminiferous epithelial cycle (Roman numerals) and the corresponding steps of spermatiddevelopment (Arabic numerals). At least 135 tubules from two mice per genotype were counted. Values are averages and SEM betweentubules. D: Percentage of mutant spermatids that were less condensed than normal or uncondensed at the light microscope level.

Table 1Lack of Transition Nuclear Proteins Produces Severe Effects on

Epididymal Sperm Quality From Mice Lacking TPs

WildtypeTnp1�/�Tnp2�/�

Viability (%) 65 � 4 13 � 3*Total motility (%) 68 � 5 2 � 1*Progressive motility (%) 62 � 5 1 � 1*Heads with blunt tips (%) 2 � 0 41 � 5*Configurational defects involving

sperm tail (%)a10 � 2 69 � 2*

Defects involving midpiece (%)b 1 � 0 89 � 4*aConfigurational tail defects include sperm with the tail tightly

wrapped around the dorsal surface of the head, the midpiece bentback on itself, coiled tails, and multiple sperm clumped together.

bMidpiece defects include missing mitochondria and/or protrud-ing fibers.

*Significantly different from wildtype, P � 0.05, n � 3–5.

202 ZHAO ET AL.

The gross level of transcription, as measured by uridineincorporation, was maintained in round spermatids fromsteps 1–7, both in wildtype mice and in mice lacking TPs,with averages of 19 and 16 silver grains per nucleus, re-spectively (Fig. 4). Consistent with a previous report (Ki-erszenbaum and Tres, 1975), labeling of spermatids dramat-ically declined in wildtype mice during steps 8, 9, and 10.The decline in transcription in spermatids from micelacking TPs occurred during the exact same steps ofdevelopment. The levels of transcription were not sig-nificantly different from background (0.08 grains pernuclear area) by step 11 in either wildtype or micelacking TPs. The average grain counts in steps 11–14from wildtype and mice lacking TPs, after subtraction ofbackground, were less than 0.5% of the counts in steps1–7, indicating that transcription was reduced to verylow levels independently of the presence of TPs.

FIG. 4. Global transcriptional repression in mice lacking TPs. Graincounts over nuclei of spermatids at various steps of developmentfrom wildtype mice and mice lacking TPs. At least four tubules perstage (average 29 cells per tubule) were counted. Values are aver-ages and SEM between tubules.

FIG. 3. Morphology of late sper-matids in cell suspensions. Sper-matids prepared from testes ofwildtype mice (A,B,H) and micelacking TPs (C–G,I–N) show thatmice lacking TPs produce somenormal spermatids but have ahigher frequency of abnormalities.A–G: Step 12–13 spermatids, iden-tified by nuclear morphology, par-tial or full complement of cyto-plasm, and absence of a visibleflagellum. A,B: Spermatids from awildtype mouse, normal shape anddegree of condensation. Sperma-tids from mice lacking TPs with arange of nuclear shapes from (C,D)normal or (E) normal with blunt tip(arrowhead) to (F,G) very abnormal.H–N: Step 16 spermatids, identi-fied in phase contrast images insuspension by the formation of amitochondrial sheath along mid-piece (mp) or, if mitochondria wereabsent, by a thickened principalpiece (pp) of the sperm tail. H:Spermatid from wildtype mouse,normal morphology. Spermatidsfrom mice lacking TPs showing (I)essentially normal morphology,(J) abnormal nuclear shape, (K) anuncondensed nucleus, (J,K,L) mid-pieces missing some or all mito-chondria, and (K,M,N) configura-tional tail abnormalities such asbent tails and the head reflectedback on the midpiece. Scalebars � 10 �m.

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Specific Effects of TPs on Chromatin Remodeling:Morphological Aspects

Light microscopy indicated normal chromatin condensa-tion was initiated at step 12 in mice lacking TPs (Fig. 5A)and had progressed in most nuclei at later steps (Fig.5B,C). The histological studies confirmed observationsmade above on smears that some of the nuclei stilldeveloped a precise and specific shape (insets, Fig. 5A–C), which was indistinguishable from the nuclear mor-phology of wildtype spermatids.

In contrast to light microscopy, electron micros-copy revealed major abnormalities in condensation inall spermatid nuclei. In normal mice, chromatin con-densation began, as expected, during steps 12–13with a thickening of chromatin fibers and uniformcondensation in an anterior-to-posterior direction (Fig.5E). Between steps 13 and 16, the fibers aggregatedinto uniformly dense chromatin (not shown). How-ever, in mice lacking TPs the fibers never thickened;instead focal chromatin condensations appeared at

FIG. 5. Normal spermatid shaping but abnormal chromatin condensation in mice lacking TPs. A–C: Light microscopy of tubules withspermatids at different steps of development from mice lacking TPs showing normal nuclear shaping of some of the late spermatids (insets).D: Various nuclei appear normally condensed (solid arrow), less condensed (unfilled arrow), or uncondensed (arrowhead). Scale bars � 10�m. E–H: Electron micrographs of late spermatids showing (E) in wildtype mice, thickening and condensation of chromatin fibersproceeding in an anterior-to-posterior direction, but, in mice lacking TPs, (F) only focal condensation, (G) incomplete condensation, or (H)variable condensation. Scale bars � 1 �m.

204 ZHAO ET AL.

FIG. 6. Histone displacement in sper-matids from mice lacking TPs. A–C: Im-munohistochemical staining (brown) ofhistone TH2B and hematoxylin counter-staining (blue) showing histones in step12 spermatids (A) and a few step 13-15spermatids (B), but absent from moststep 13–15 spermatids (C) of mice lack-ing TPs. Z, zygotene spermatocyte; P,pachytene spermatocyte; M, meiotic fig-ure; T, round spermatid. Solid arrows,elongated spermatids with positivestaining for TH2B; unfilled arrows, weakor partial staining; arrowheads, un-stained nuclei. Scale bars � 10 �m. D:Chromatin basic proteins of step 12–16spermatids showing the absence of pro-cessed forms of protamine 2 (intP2 andmature P2, which would comigrate withP1) in mice lacking TPs. Percentage oftotal protein in each band is indicated.P1 and P2, protamine 1 and 2, respec-tively; preP2, precursor of protamine 2;intP2, partially processed protamine 2.

FIG. 7. Protamine levels duringspermatid development. Immu-nofluorescence of protamines(red fluorescence) in spermatidsfrom (A–C) wildtype mice and(D–F) mice lacking TPs. Blue flu-orescence indicates DNA. Solidarrows, nuclei with positive stain-ing for protamine; unfilled arrows,weak staining for protamine; ar-rowheads, spermatid nuclei notstained for protamine. A,D: Highlevels of protamine 1 are alreadypresent in step 12 spermatids inboth genotypes. B,E: Step 12spermatids from both genotypescontain low levels of protamine 2in a stippled pattern over the nu-cleus. The brightly stained nu-cleus in the mouse lacking TPscorresponds to a mature sperma-tid that was not released duringthe previous cycle. C,F: Prota-mine 2 reaches high levels in step14–15 spermatids from both ge-notypes. Some of the uncon-densed late spermatid nuclei inthe mutant mice lack protamine(arrowhead). Cells are labeled asin Figure 6; 2°, secondary sper-matocyte. Scale bar � 10 �m.

205TRANSITION NUCLEAR PROTEINS, SPERM DEVELOPMENT

step 13 (Fig. 5F). Subsequently, in many spermatidsthe focal condensation units enlarged and movedcloser together, but condensation remained incom-plete even at step 16 (Fig. 5G).

Even this limited condensation process went wrongin a subset of the late spermatids in mice lacking TPs(Fig. 5D,H). Some spermatid nuclei were consideredless condensed than the maximally condensed nucleiat these stages (open arrows) and others were com-pletely uncondensed (arrowheads), being even largerand having less heterochromatin than round sperma-tids. These were conclusively identified as late, ratherthan aberrant round spermatids, by the shape of thenucleus and the development of the acrosome. Duringsteps 13–16, the less-condensed nuclei appeared first,markedly uncondensed elongated spermatid nuclei ap-peared later, and finally both disappeared in the sameorder, coincident with the loss of spermatids (Fig.1C,D), suggesting that nuclei were decondensing andbeing lost.

Specific Effects of TPs on Chromatin Remodeling:Biochemical Aspects

In an attempt to explain the chromatin abnormalities,we next investigated whether histone removal and/orprotamine deposition were altered in mice lacking TPsby both immunohistochemistry and protein analysis. Im-munohistochemistry showed that the male-germ-cellspecific histones TH2B (Fig. 6A–C) and H1t (not shown)were present, both in wildtype mice (not shown) and inmice lacking TPs, in spermatid nuclei through step 12,but not at step 16, the last step of development beforerelease from the testis. Only a small fraction of the step13–15 elongated spermatids (8–23%) of both genotypescontained these histones. Since these histones werepresent at the start of step 13, but absent at the end ofstep 15, and the duration of step 13 is 24% of steps13–15 combined (Clermont and Trott, 1969), we calcu-lated that histone removal was completed during step13. Electrophoresis of proteins extracted from step12–16 spermatids (Fig. 6D) confirmed that the histoneswere almost completely displaced. The slightly higherhistone percentage (11 vs. 4%) in these cells from micelacking TPs than in wildtype mice can partly be ac-counted for by the reduction in the total protein result-ing from the absence of the TPs. Thus, TPs do not playa role in removal of histones.

Immunohistochemistry indicated that step 12 sperma-tids from both wildtype mice and mice lacking TPscontained high levels of protamine 1 (Fig. 7A,D), whichwere maintained throughout the remainder of spermatiddevelopment (not shown). However, step 12 spermatidsfrom both genotypes contained low levels of protamine2 (Fig. 7B,E), present in a stippled pattern over thenucleus. These levels markedly increased in the nuclei ofboth genotypes during steps 14–15 (Fig. 7C,F). Thus,the timing of the deposition of both protamines 1 and 2in the nuclei of the majority of spermatids was unaf-fected by the absence of TPs. However, some of the

uncondensed late spermatid nuclei in the mutant micelacked protamine 2 (Fig. 7F). In contrast, the maturespermatids that were retained in the tubules past theirnormal time of release contained high levels of prota-mine 2 (Fig. 7E).

Although the deposition of protamines was normal, asconfirmed by the high levels of protamines extractedfrom chromatin of step 12–16 spermatids (Fig. 6D), theabsence of TPs completely prevented the stepwise post-translational processing of protamine 2. Whereas in wild-type mice most of the protamine 2 was present as par-tially cleaved or mature forms, in mice lacking TPs nearlyall protamine 2 remained as uncleaved precursor.

Eventual Loss of Genomic Integrity

Breaks detected by terminal deoxynucleotide trans-ferase-mediated deoxy-UTP nick end labeling (TUNEL)normally occurred during the initial phase of chromatinremodeling in wildtype mice during steps 9–11 and incells that were not destined to undergo apoptosis (Mar-con and Boissonneault, 2004; Smith and Haaf, 1998).Similarly, more than 70% elongating spermatids in micelacking TPs were TUNEL-positive (Fig. 8A). However, inwildtype mice the breaks were transient; by step 12TUNEL staining was restricted to a narrow region of thespermatid nucleus (Fig. 8B), and at later stages no stain-ing was observed (not shown). But in mice lacking TPs,40% of step 12 spermatids remained TUNEL-positiveover the entire nucleus (Fig. 8C). At later steps it becameapparent that the uncondensed spermatids were TUNEL-positive, whereas most of the more condensed oneswere TUNEL-negative (Fig. 8D). Thus, even in the ab-sence of TPs, DNA nicks were repaired in some sperma-tids, but in others either the repair did not proceednormally or new DNA strand breaks were formed duringsteps 12–14.

Despite the chromatin abnormalities, the genomic in-tegrity of the relatively condensed, viable, late sperma-tids was good. When the nuclei of mature late sperma-tids (testicular sperm) were injected into oocytes, thosefrom mice lacking TPs showed normal developmentalcapability (Table 2). Although there was a slight reduc-tion in the percentage of surviving oocytes that showedfertilization (92% vs. 98% for wildtype), this differencewas not significant (P � 0.07), and the appearance of thefertilized oocytes was normal (Fig. 9G–I). The overallproduction of live offspring per surviving oocyte wascalculated from the data in Table 2 to be 27% for wild-type and 29% for mature spermatids from mice lackingTPs. Furthermore, the offspring developed normally andthe ones tested were fertile.

Fertilization using spermatozoa that had left the testiswas much worse. The few feebly motile cauda epididy-mal spermatozoa from mice lacking TPs fertilized only31% of oocytes with intracytoplasmic sperm injection(ISCI), compared to more than 99% for motile caudaepididymal spermatozoa from wildtype mice (Table 2;Fig. 9A,B). Most unfertilized oocytes remained inacti-vated despite successful sperm injection (Fig. 9C,E). Of-

206 ZHAO ET AL.

ten the sperm nuclei transformed into loosely arrangedchromosomes (Fig. 9D) or abnormal pronuclei (Fig. 9F).Even when oocytes were artificially activated with Sr��,the fertilization rate was not improved, and only thefemale pronucleus could be observed. The failure ofnormal male pronuclear formation indicates that thesesperm nuclei had lost genomic integrity in addition tothe ability to activate oocytes. The few oocytes that werefertilized normally by mutant spermatozoan nucleishowed a slight deficiency in development to the 2-cellstage but were able to develop further, producing twolive offspring (Table 2).

DISCUSSION

This report is the first to show that TPs are essential forfunctional sperm development. The previously reportedfertility of mice lacking only one of the TPs (Yu et al.,2000; Zhao et al., 2001) must have been a result ofcomplementation of the functions of one protein by theother. The primary functions of the TPs were shownhere to be the uniform condensation of chromatin andthe repair or prevention of DNA breaks.

The elimination of TPs specifically affected eventsoccurring after step 11 of spermatid development,which was expected since the proteins are not presentin wildtype mice prior to step 10 and reached maximallevels at steps 12–13. Cell counts, morphology, and mo-lecular changes were identical in the spermatids fromwildtype and mutant mice up until step 11. In particular,

the repression of transcription and initial induction ofDNA strand breaks were independent of the presence ofTPs. These two events may have resulted from a majorremodeling of the chromatin related to the hyperacety-lation of H4 (Lahn et al., 2002) and possibly from otherhistone modifications or binding of additional proteinsto the chromatin (Alami-Ouahabi et al., 1996). The activeprocesses of chromatin remodeling even includes theremoval of histones, which likely started earlier but wascompleted during step 13 and was independent of thepresence of TPs. Histone chaperone proteins, such asNASP (O’Rand et al., 2000), are candidates for involve-ment in this process.

In normal mice, chromatin condensation begins dur-ing steps 12–13 with a thickening of chromatin fibersand uniform condensation in an anterior-to-posterior di-rection corresponding to the direction of deposition ofTPs (M. Zhao, M.L. Meistrich, ms. in prep.) (Oko et al.,1996). One of the first and most dramatic effects of theelimination of both TPs was the complete absence of thisthickening of fibers and the directionality of the conden-sation, which was not observed when only one TP waseliminated. Thus, we conclude that these aspects of thechromatin condensation of spermatids are the directresult of the action of both TP1 and TP2 on DNA.

In place of this normal pattern of chromatin conden-sation, we observed numerous focal condensations on abackground of uncondensed chromatin in mice lackingTPs. These were similar to ones observed prominently in

FIG. 8. DNA breaks in spermatids from mice lacking TPs. Green fluorescence indicates TUNEL staining and blue fluorescence indicatesDNA. The only other cells that appear to be TUNEL-positive are the zygotene spermatocytes, which may be associated with strandbreakage during genetic recombination. A: Tubule section showing that most of the step 10 spermatid nuclei from mice lacking TPs containbreaks. B,C: Tubule sections showing repair of breaks throughout most of the spermatid nucleus by step 12 in wildtype, but, in mice lackingTPs, many spermatid nuclei are TUNEL-positive over the entire nucleus. D: Squash preparation showing that at step 15, primarily theuncondensed spermatid nuclei, are TUNEL-positive. Cells are labeled as in Figure 6. Solid arrows, TUNEL-positive; unfilled arrow, only partof the nucleus is TUNEL-positive; arrowheads, TUNEL-negative. Scale bars � 10 �m.

207TRANSITION NUCLEAR PROTEINS, SPERM DEVELOPMENT

mice lacking TP1 (Yu et al., 2000) and less prominentlyin mice lacking TP2 (Zhao et al., 2001).

We propose the following explanation for the forma-tion of these focal condensations: Immunohistochemicalstudies indicated that the protamines (predominantlyprotamine 1) were already present in nuclei of step 12spermatids in both wildtype (Wu et al., 2000) and micelacking TPs (Fig. 7), but biochemical analyses showedthat very little protamine was bound to chromatin of thesonication-resistant step 12–13 spermatid nuclei fromwildtype mice (Yu et al., 2000). Thus, we suggest thatthe protamine is normally sequestered in the nucleo-plasm. But in mice lacking TPs, protamines are prema-turely deposited onto histone-free regions of chromatinnormally occupied by TPs, thus producing the focalcondensations. This explanation is consistent with theobservation that there was slightly more chromatin-bound protamine in these cells isolated from Tnp1-nullthan from wildtype mice (Yu et al., 2000). Although theimmunofluorescence images in Figure 7 do not providesupport for this concept, they are limited by low reso-lution and the inability to distinguish between protaminemolecules that are in the nucleoplasm and those boundto chromatin. We are further testing this proposal byconfocal laser scanning microscopy and immunoelec-tron microscopy. The focal chromatin condensation inspermatids of several species of bony fish (Saperas et al.,1993), in which there is a direct histone-to-protaminetransition, supports the proposed difference in conden-sation patterns when protamines directly replace thehistones in the absence of TPs.

The other immediate consequence of the absence ofTPs was the presence of DNA strand breaks in many step12 spermatids. This result provides support for the pro-posal that TPs promote the repair and ligation of DNA(Caron et al., 2001; Levesque et al., 1998). However,they are not essential for this process because DNA nickswere repaired in some spermatids even in the absence ofTPs. This repair must be sufficient to allow normal de-velopment, since the condensed late spermatids werecapable of fertilizing oocytes by ICSI, embryos devel-oped normally, and offspring were produced. It is likelythat the TUNEL-positive spermatids at step 12 in micelacking TPs were a result of the failure to repair thebreaks that occurred naturally in earlier stages, but it ispossible that new DNA strand breaks were formed dur-ing step 12. The deficiency in basic nuclear proteinscould allow endonucleases or reactive oxygen speciesthe opportunity to attack the DNA, which would other-wise be protected by the TPs at this step. Despite thepresence of increased strand breakage at steps 12–13,spermatid number was maintained (Fig. 1C), indicatingthat a period of time and subsequent events are neces-sary for cell loss.

There were numerous consequences of the deficiencyof TPs at steps 12–13 that became apparent in the nucleiduring the later steps of spermatid development. Thesemust be considered as secondary, rather than direct,consequences of the absence of TPs, since in normal

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208 ZHAO ET AL.

mice the protamines have already replaced the TPs.Although the focal condensation units became larger andmoved closer together, the chromatin never becamecompletely condensed in any of the spermatids. Theenlargement of the focal condensation units during steps14–16 was most likely related to the increase in chro-matin-bound protamine, since at these stages the overalllevels of protamine 2 within the nucleus increased (Fig.7F) and both protamines became bound to the chroma-tin (Fig. 6D). Although both protamines were present ina normal ratio, the posttranslational processing of prota-mine 2 did not occur, which might also contribute to theincomplete condensation. This failure of protamine 2processing appears to be related to the lack of TPs. Thelevels of incompletely processed protamine 2 increased

from negligible levels in wildtype mouse sperm to sig-nificant levels in Tnp2-null mice, which lack the lessabundant TP (Zhao et al., 2001), still higher levels inTnp1-null mice (Yu et al., 2000), and to nearly 100% inmice lacking both TPs. Defective processing of prota-mine 2 also occurs in mice carrying mutations that affectthe timing or levels of protamine synthesis (Cho et al.,2001; Giorgini et al., 2002; Lee et al., 1995), and some ofthese mutants are known to have reductions in TP levels(Giorgini et al., 2002; Lee et al., 1995). Thus, we pro-pose that TPs may be required for recruiting or activat-ing a protease that processes protamine 2 in chromatin.

In addition, other nuclei underwent extreme decon-densation characterized by the complete absence of het-erochromatin (arrowhead, Fig. 5H). We believe that this

FIG. 9. Micrographs of oocytes after fertilization by ICSI using (A,B) epididymal sperm heads from wildtype mice, (C–F) epididymal spermheads from mice lacking TPs, and (G–I) mature spermatid (testicular sperm) heads from mice lacking TPs. A: Activated oocyte 1 h afterinjection of wildtype sperm head showing telophase II oocyte chromosomes and a swollen sperm head (arrow). B: Activated oocyte 6 h afterinjection showing normal female, F, and male, M, pronuclei. Inactivated oocytes 1 h after injection of mutant epididymal sperm showing (C)metaphase II oocyte chromosomes and (D) sperm head transformed into irregularly arranged chromosomes (arrow). E: Inactivated oocyte6 h after injection of mutant epididymal sperm. F: Activated oocyte 6 h after injection with mutant epididymal sperm with an abnormallysmall pronucleus, presumably of male origin (arrow). Activated oocytes injected with testicular sperm heads from mice lacking TPs showing(G) telophase chromosomes and (H) a swollen sperm head at 1 h after injection and (I) two pronuclei at 6 h after injection. Scale bars �10 �m.

209TRANSITION NUCLEAR PROTEINS, SPERM DEVELOPMENT

is related to the presence and persistence of DNA strandbreaks in these cells (Fig. 8D). Strand breaks have thecapability of inducing apoptosis in cells. It is not possibleto identify the usual morphological hallmarks of apopto-sis in these cells, since the late spermatids normally havenuclei with condensed chromatin and cytoplasm with anincreased density, compaction of organelles, and vacuo-lation in preparation for phagocytosis of the residualcytoplasm by neighboring somatic cells (Blanco-Rodri-guez and Martinez-Garcia, 1999). These cells with decon-densed nuclei are likely in the process of being lostbecause of their transient appearance during steps 14–16, which is when spermatid numbers dramatically de-cline (Fig. 1C,D).

In addition to the secondary defects in the nucleus,there are major indirect cytoplasmic abnormalitiescaused by the prior absence of TPs, including a weak-ened alignment and attachment of mitochondria alongthe midpiece (Fig. 3J–L), failure of release from the testis(Fig. 1C), and midpiece and tail configuration defects(Table 1). To explain this sequence of developmentalaberrations, a mechanism by which a primary nucleardefect signals a cytoplasmic response must be identified.The DNA strand breakage even persists in some rela-tively condensed spermatid nuclei at step 15 (lowerarrow, Fig. 8D) and can initiate such a signaling cascadeto induce the cytoplasmic events of apoptosis, which areknown to significantly involve the mitochondria in othercell types. The changes in the mitochondria could altertheir ability to attach to the midpiece of the late sper-matid, thus affecting tail morphogenesis. This and othercytoplasmic or membrane changes in the spermatidmust alter its interaction with the Sertoli cell in a waythat acts as a checkpoint to block the normal release atstep 16. The failure of spermiation observed here issimilar to that reported in several other mutations thataffect TP and protamine expression specifically in sper-matids (Giorgini et al., 2002; Zhao et al., 2001; Zhong etal., 1999). In the mice lacking TPs, this block appears tobe a result of the failure to achieve cytoplasmic removaland individualization of the spermatids, since most of thefew sperm that were released into the epididymis hadabnormal configurations or were in clumps that resultedfrom attachment through cytoplasmic structures. Spermwith similar abnormalities were observed in other micewith mutations in a nuclear matrix protein or in levels,translation, or posttranslational modification of prota-mine (Cho et al., 2001, 2003; Giorgini et al., 2002;Kimura et al., 2003; Wu et al., 2000; Zhong et al., 1999),further supporting the concept that these cytoplasmicdefects are likely to be secondary effects of the disrup-tion of the orderly process of nucleoprotein replacementduring spermatid development and not a direct effect ofthe absence of TPs.

The genomic integrity of these epididymal spermato-zoa was much worse than that of the mature testicularspermatids, as demonstrated by their low frequency toproduce fertilization (31%) using ICSI (Table 2). This isconsistent with observations that many of the cauda

epididymal spermatozoa exhibited dim nuclear fluores-cence with DAPI, indicating that the DNA was degraded(C.R. Shirley, M.L. Meistrich, ms. in prep.). But the fewoocytes that were fertilized normally by these mutantepididymal spermatozoan nuclei showed only a slightdeficiency in development to the two-cell stage andwere able to develop further (75% of two-cell embryosdeveloped to morula-blastocyst), producing two live off-spring. These results differ from those obtained withICSI using spermatozoan heads from mice lacking onecopy of the protamine 2 gene, in which 79% of eggswere fertilized and developed to at least the two-cellstage (Cho et al., 2003), which is significantly higherthan the value of 26% from the mice lacking TPs (P �0.001) (Table 2). However, the numbers of survivingoocytes developing to the morula-blastocyst stage, 14%using sperm from germ cells with protamine haploinsuf-ficiency versus 20% with sperm from mice lacking TPs,were similar (P � 0.6). Thus, both mutations result insevere embryonic loss, but it appears that the damage tothe DNA in epididymal spermatozoa from sperm frommice lacking TPs may be more severe than those fromgerm cells with protamine haploinsufficiency (Cho et al.,2001, 2003) because the block in the former occurs at anearlier stage.

Finally, this work has direct clinical implications. Theresults show that, although epididymal sperm may bepresent, some infertile patients could benefit from ISCIusing their late spermatids (testicular sperm) instead.The ability of late spermatids that contain unprocessedprotamine 2 to initiate normal development proves thatprotamine 2 processing defects do not inhibit postfertil-ization processes, supporting limited clinical experience(Carrell and Liu, 2001). Thus, the well-known correla-tion between defective protamine 2 processing and in-fertility in humans (de Yebra et al., 1998) and mousemutants (Yu et al., 2000; Zhao et al., 2001) could be duesolely to the secondary cytoplasmic effects on spermdevelopment, resulting in a reduced ability to penetratethe egg. Therefore, failure of protamine 2 processingshould not be considered a contraindication to ICSI.

MATERIALS AND METHODS

AnimalsDouble heterozygotes carrying null alleles in the genes(Tnp1 and Tnp2) for transition nuclear protein 1 (TP1)and transition nuclear protein 2 (TP2), were producedfrom 129S-Tnp1tm1Mlm (Yu et al., 2000) and 129S-Tnp2tm1Mzh (Zhao et al., 2001) mice, respectively.Tnp1-/-, Tnp2-/- mice were produced by mating maledouble heterozygotes with either female double het-erozygotes or Tnp1-/-, Tnp2-/- females, which are alsofertile. Some mice (about 14% of wildtype, 25% of dou-ble Tnp null) had gross testicular abnormalities, mostcommonly a small left testis; these mice were excludedfrom other analyses. Except where noted, all data wereobtained with mice younger than 23 weeks of age. Micewere housed in accordance with current regulations and

210 ZHAO ET AL.

standards of the USDA and the DHHS, NIH under aprotocol approved by the Institutional Animal Care andUse Committee.

Cell Counts and Sperm Analysis

Counts of step 12–16 spermatids from the testis, pre-pared based on their resistance to sonication (Meistrichet al., 1976), were performed as described previously(Zhao et al., 2001). Cauda epididymal spermatozoa weresuspended in a modified Krebs-Ringer solution at 37°C.Sperm viability was determined using a dual fluores-cence assay (LIVE/DEAD sperm viability kit, MolecularProbes, Eugene, OR), and the percentage sperm thatwere motile was determined visually. Air-dried smears ofspermatozoa were hematoxylin stained for morphologi-cal analysis.

Hormone Measurements

Serum testosterone was measured using a DSL-4000-coated tube radioimmunoassay kit (Diagnostic SystemsLaboratory, Webster, TX) as described previously (Shettyet al., 2001).

Microscopic Techniques

For histological studies, testes were fixed in Bouin’ssolution, embedded in methacrylate, cut in 4-�m sec-tions, and stained with PAS-hematoxylin. The step ofdevelopment of elongated spermatids was identifiedfrom the stage of the cycle of the seminiferous epithe-lium of each tubule cross-section based on the develop-ment of the acrosome of round spermatids, nuclearshape of elongating spermatids, presence of meiotic fig-ures, and other criteria (Russell et al., 1990).

Cell suspensions were prepared either mechanicallyor with trypsin. Cells were viewed in air-dried, stainedsmears fixed in Bouin’s solution and stained with PAS-hematoxylin. They were identified according to previ-ously described criteria (Meistrich et al., 1973).

Electron microscopy was performed as described pre-viously (Zhao et al., 2001). The sections were stainedwith uranyl acetate-lead citrate.

Immunohistochemistry and Strand-BreakDetection

Immunohistochemistry for histones was performed onsections prepared from Carnoy’s-fixed, paraffin-embed-ded tissue. A monoclonal antibody recognizing TH2B(Unni et al., 1995) (anti-tyrosine hydroxylase, Chemicon,Temecula, CA) at 0.6 �g/ml or a polyclonal guinea pigantiserum prepared against H1t (from M.A. Handel, Uni-versity of Tennessee) at 1:800 dilution was used. Sec-tions were microwaved for antigen retrieval. Antibodybinding was detected with the peroxidase reaction withdiaminobenzidine substrate (Vectastain Elite ABC kit,Vector Laboratories, Burlingame, CA). Slides were coun-terstained with hematoxylin.

Immunohistochemistry for protamines was done asabove except that sections were treated with 6 mMdithiothreitol for 1 h. Monoclonal antibodies Hup1N

(1:100) and Hup2B (1:400) were used to detect prota-mines 1 and 2, respectively (Stanker et al., 1987). AlexaFluor 594 goat antimouse IgG (Molecular Probes) wasused for detection of antibody binding. DNA was coun-terstained with DAPI.

TUNEL was performed using either Carnoy’s-fixed tis-sue or squash preparations of defined stage tubules. Thelatter were prepared by microdissection, flash-frozen inliquid nitrogen, and fixed in cold methanol (Parvinenand Hecht, 1981). The slides were incubated with theTUNEL reagents, including biotinylated-dUTP, at 37°Cfor 60 min, then with FITC-avidin at room temperatureas described elsewhere (Marcon and Boissonneault,2004). The slides were counterstained with DAPI. Sup-porting evidence that the TUNEL staining representsendogenous strand breaks is provided by the same-stagedependence observed with many different fixation andprocessing methods (this study; Marcon and Boisson-neault, 2004; Smith and Haaf, 1998), as well as withunfixed permeabilized cells and nuclei (McPherson andLongo, 1992), and the coincidence of TUNEL positivityin normal mice with only those cell types in whichstrand breaks are expected because of either geneticrecombination or changes in supercoiling.

Photomicrographs were taken with a digital cameraand processed with Adobe PhotoShop (v. 6.0; San Jose,CA) so that color differences would be clear when theimages were printed.

Cell Labeling and Autoradiography

One testis from a wildtype and one from a mouse lackingTPs were labeled with [3H]uridine in vitro by modifica-tions of previous procedures (Soderstrom and Parvinen,1976). Pieces (1–2 mm) were incubated with agitation inHEPES-buffered DMEM/F12 medium containing 200 �Ciof [5-3H]uridine (28 Ci/mmole) per ml and 10 �g/mleach of adenosine, cytidine, and guanosine at 32.5°C in5% CO2/air for 2 h. Additional medium containing 10�g/ml uridine was added for 1 h, and further incubationwas done in six changes of medium for 30 min each.Tissue was fixed in 2.5% glutaraldehyde and processedfor autoradiography as described previously (Kierszen-baum, 1974) except that Spurr resin, Kodak NTB2 emul-sion, a 30-day exposure time, and 0.025% Toluidine bluewere used. Tubules in which the spermatogonia or sper-matocytes were heavily labeled were selected and thenumbers of grains over the spermatid nuclei werecounted.

Analysis of Spermatid Proteins

Step 12–16 spermatid nuclei were prepared by sonica-tion and basic proteins were extracted and separated byPAGE in acid-urea gels (Zhao et al., 2001).

Fertility

For in vivo fertility testing, each male was mated, at 8–9weeks of age, with one known-fertile C57BL/6 female for8 weeks and was considered fertile if any pups wereborn. In vitro fertilization (Toyoda et al., 1971) and ICSI

211TRANSITION NUCLEAR PROTEINS, SPERM DEVELOPMENT

(Kimura and Yanagimachi, 1995a,b) were performedusing protocols described previously. For ICSI, motilespermatozoa were selected and the head was separatedfrom the tail by applying one or a few Piezo pulses to thehead–tail junction. Only the head was injected into eachoocyte obtained from adult B6D2F1 females. Fertilizationwas considered successful if two pronuclei developedand the second polar body appeared 5–6 h after insem-ination or ICSI. In some experiments, unfertilized oo-cytes were scored to determine whether activation did(female pronucleus present) or did not (arrest at meta-phase II) occur and what the status of the sperm chro-matin was. In additional experiments, oocytes were ar-tificially activated with 5 mM SrCl2 (Shamanski et al.,1999). Fertilized eggs were allowed to develop to themorula-blastocyst stage (except where noted), at whichtime they were transferred to the oviducts of pseudo-pregnant CD-1 females. When available, �10 embryoswere transferred per recipient; there were fewer em-bryos when epididymal sperm from mice lacking TPswere used, resulting in a lower percentage of pregnantfemales. Females were euthanized at 19.5 days postco-itum to count all implantation sites. Live fetuses weredelivered by cesarean section and raised by lactatingCD-1 foster mothers.

Statistical Analysis

Results are expressed as mean values � SEM. For somevalues, such as serum testosterone, sperm counts, andspermatids per Sertoli cell, the mean and SEM werecalculated from the log-transformed data points. A Stu-dent’s t-test was performed to determine the significanceof differences. Significance of differences in fertilizationrates and development of embryos resulting from ICSIwere determined using Chi-square analysis or Fisher’sexact test. A Bonferroni correction was used to adjustthe level of significance to P � 0.01 due to multiplecomparisons (n � 4, 0.05/4 � 0.0125). A computer-assisted statistics program (SPSS, Chicago, IL) was used.

ACKNOWLEDGMENTS

We thank Suzanne Mounsey for technical assistance,Lonnie Russell, Kenneth Dunner, and Angela Raymer forassistance with electron microscopy, Connie Weng fortestosterone measurements, Walter Pagel for editorialassistance, Miles Wilkinson and Jill Schumacher for crit-ical reading of the manuscript, and Mary Ann Handel, W.Stephen Kistler, and Rod Balhorn for antibodies.

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213TRANSITION NUCLEAR PROTEINS, SPERM DEVELOPMENT