The Nuclear Form of Phospholipid Hydroperoxide Glutathione Peroxidase Is a Protein Thiol Peroxidase Contributing to Sperm Chromatin Stability

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase(PHGPx) is regarded as the major molecular target of selenodeficiencyin rodents, accounting for most of the histopathological andstructural abnormalities of testicular tissue and male germcells. PHGPx exists as a cytosolic form, mitochondrial form,and nuclear form (nPHGPx) predominantly expressed in late spermatidsand spermatozoa. Here, we demonstrate that mice with a targeteddeletion of the nPHGPx gene were, unlike mice with the fullknockout (KO) of PHGPx, not only viable but also, surprisingly,fully fertile. While both morphological analysis of testis andepididymis and sperm parameter measurements did not show anyapparent abnormality, toluidine blue and acridine orange stainingsof spermatozoa indicated defective chromatin condensation inthe KO sperm isolated from the caput epididymis. Furthermore,upon drying and hydrating, KO sperm exhibited a significantproportion of morphologically abnormal heads. Monobromobimanelabeling and protein-free thiol titration revealed significantlyless extensive oxidation in the cauda epididymis when comparedto that in the wild type. We conclude that nPHGPx, by actingas a protein thiol peroxidase in vivo, contributes to the structuralstability of sperm chromatin.

INTRODUCTION

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Introduction

Sperm chromatin condensation during the final steps of spermatogenesisin mammals is a multistep process that includes the sequentialreplacement of the majority of histones by transition proteinsand protamines in testis (6, 7). During epididymal transit ofspermatozoa, protamine thiol oxidation is completed and intra-and intermolecular cross-links are formed. Hence, a transcriptionallyinactive and tightly packed haploid genome is generated renderingsperm nuclei more resistant to mechanical and chemical insults(2). Recently, Cho and colleagues showed that chimeric micehemizygous for protamine 1 or 2 fail to transmit the targetedallele to the germ line (8).

Selenium depletion studies of rodents clearly demonstrated theimportance of this trace element in male fertility. Third generationselenium deficiency is associated with structural abnormalities,such as broken midpieces of sperm tails, giant heads, and reversibletesticular atrophy (5, 34). Due to its particular high expressionin mammalian testis (21) and its resistance to selenium deprivationin testis, the selenoenzyme phospholipid hydroperoxide glutathioneperoxidase (PHGPx) is thought to account for most of the defectsassociated with severe selenium deficiency.

PHGPx was initially characterized as a lipid peroxidation-inhibitingprotein (33) and was later shown to be an unusual member ofthe glutathione peroxidase family, in particular for its scarcespecificity for both the oxidizing and reducing substrates (32).Most relevant in this respect was the observation that, in thepresence of low glutathione (GSH) concentration, specific protein-SH groups may act as a reductant in the catalytic cycle witha stoichiometry of 2 equivalents of thiol per mole of hydroperoxide(13, 22, 28, 31). Apparently due to this reaction, during spermatogenesis,PHGPx behaves as a soluble peroxidase as long as GSH levelsare high; however, due to the drop of GSH levels (29), PHGPxbecomes enzymatically inactive and results in a cross-linked,insoluble protein representing the major structural constituentof the mitochondrial sheath of the midpiece of mature spermatozoa(31).

Three different transcripts of PHGPx exist, differing in their5' extension and coding for a cytosolic protein, mitochondrialprotein, and nuclear protein (24, 25). The latter was recentlyidentified as a sperm nucleus-specific 34-kDa selenoprotein(nPHGPx; also called snGPx, for sperm nucleus-specific glutathioneperoxidase) (24).

The nPHGPx gene shares all exons except the first with the cytosolicand mitochondrial forms of PHGPx. Alternative first exon (Ea)usage, and not alternative splicing, was shown to be responsiblefor the generation of the mRNA encoding nPHGPx (20, 23). ThisEa of nPHGPx encodes both a nuclear import signal and clustersrich in arginine and lysine residues, reminiscent of protamines(24). Circumstantial evidence suggested a crucial role for nPHGPxin sperm chromatin condensation: its nuclear localization, thecoincidence in the spatial and temporal expression with protamines,its structural similarity to protamines, its in vitro documentedthiol peroxidase activity, and the presence of improper condensedgiant cells in sperm specimens from selenium-depleted rodents.We hypothesized that nPHGPx may bind via its highly basic Nterminus to sperm chromatin and may introduce disulfide bridgesinto protamines in order to achieve a tight package of the malehaploid genome. To prove this hypothesis, we generated micespecifically lacking nPHGPx.

MATERIALS AND METHODS

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Materials and Methods

Generation of nPHGPx KO mice. An AflII/NarI (NarI site blunt ended) fragment isolated fromvector pMC42, which carries an 18-kbp fragment including themurine PHGPx locus, was inserted in pBluescript SK+ (StratageneInc., La Jolla, CA) digested with AccI and XbaI (blunt ended)in order to obtain pMC69. By PCR cloning, a replacement cassettewas amplified containing a 5' NotI site, a characteristic EcoRVsite, the enhanced green fluorescent protein (EGFP) gene followedby a translational STOP element in every possible reading frame,and a characteristic AseI site (EaGFPkoforw1/EaGFPkorev1, 5'-TGCGGCCGCCGATATCGTGAGCAAGGGCGAGGAGCTGTTC-3'/5'-TAGTACTGGATTAATTAGTTATCTAGATCCGGTGGATCTG-3')(Fig. 1A). This cassette was inserted in pMC69 (NotI/ScaI),thereby replacing the main part of Ea (pMC70). A NotI fragmentincluding the in-frame EGFP cassette replaced parts of the 5'homologous arm of pPNT4.8 (unpublished), a targeting vectorderived from pPNT4 (9) that already harbored both homologousarms required for gene targeting (pPNT4.9). Gene targeting inembryonic stem (ES) cells was carried out as described previously(15). DNA of 500 ES cell clones digested with EcoRV was screenedfor homologous recombination by Southern blotting with a 3'external probe. Random integration was excluded with a probespecific for neomycin phosphotransferase. Positive ES cell clonesarose at a frequency of 1%. Three ES cell clones were microinjectedinto C57BL/6J blastocysts, and all three ES cell clones generatedchimera that finally transmitted the nPHGPx knockout (KO) alleleto the germ line. To facilitate genotyping of nPHGPx KO mice,two primer pairs were used: one specific for the wild-type (WT)(I1f2/Earev1, 5'-TCGGCGGCGCCTTGGCTACCGGCTC-3'/5'GGATCCGCCGCGCTGTCTGCAGCGTCCC-3')and one specific for the KO (I1f2/eGFPrev, 5'-TGAAGAAGTCGTGCTGCTTCATGTGG-3')allele.

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FIG. 1. Gene targeting of nPHGPx in mice. (A) Strategy for disruption of the nuclear form of PHGPx. WT PHGPx is shown in the upper line; localization of the selenocysteine codon UGA is marked with an asterisk. To specifically delete nPHGPx, most of the coding region of Ea was replaced by the EGFP gene. A translational STOP cassette was inserted 3' of EGFP. For negative selection of transfected ES cells, the thymidine kinase gene (TK) was placed downstream from the 3' arm. Underneath, expected DNA fragments, resulting from a characteristic restriction digest with EcoRV and detection with a 3' external probe (checkered rectangles), are depicted (neo, neomycin phosphotransferase gene; BS, pBluescript SK+ backbone). (B) Verification of homologous recombination in ES cell clones (lane 1) by Southern blotting. Below, germ line transmission of the targeted allele and genotyping of offspring obtained from heterozygous intercrosses by two PCRs, one specific for the WT and one specific for the KO allele. (C) Semiquantitative RT-PCR analyses of testis mRNA. (D and E) In situ hybridization of testis sections confirmed the absence of the targeted region in KO testis (E). Arrows point at nPHGPx expression around the luminal face of WT germinal epithelium (D). Stars indicate the lumen of tubuli seminiferi. (F) Western blotting of testis extracts revealed low levels of the longer truncated product of nPHGPx (25.9 kDa) in WT tissue (arrow), which is absent in KO tissue. Protein levels of cytosolic/mitochondrial forms of PHGPx (20 kDa) are equal in WT and KO tissue. Cytosolic extracts from pig heart served as a positive control. (G) No difference in litter sizes between WT (n = 30 litter) and KO (n = 20 litter) mice. Bars = 50 µm (D and E).

RNA isolation and semiquantitative RT-PCR. Isolation of testis RNA, DNase treatment, and reverse transcription(RT) were carried out as described previously (23). Primer pairsspecific for the aldolase gene (Aldolase1/2, 5'AGCTGTCTGACATCGCTCACCG-3'/5'-CACATACTGGCAGCGCTTCAAG-3'),the GAPDH gene (GAPDH1/2, 5'-CTCACTCAAGATTGTCAGCAATG-3'/5'-GAGGGAGATGCTCAGTGTTGG-3'),and the mitochondrial form of PHGPx (P32/E2rev1, 5'-GCAAGCCATACTCGGCCTCGCGCGTCC-3'/5'-CCAGGCAGACCATGTGCCCGTCGATGTCC-3'),as well as one for the deleted region of the alternative exon(Eaforw2, 5'-CTCGCCGGATGGAGCCCATTCC-3'/E2rev1) were used.

In situ hybridization. The corresponding region of the deleted part of Ea of the PHGPxgene was amplified by PCR using pMC42 as a template (forward/reverseprimer, 5'-GGGACGCTGCAGACAGCGCGGCGGATCC-3'/5'-ACTGGGGAGGCTCTTGCAGCAGAGG-3')and cloned in pDrive (QIAGEN, Hilden, Germany). Generation ofdigoxigenin-labeled RNA probes and in situ hybridization wereperformed as described previously (23).

Biological samples. Spermatozoa, obtained from approximately 12-week-old mice, wereconsistently prepared from caput and cauda epididymis and vasdeferens by gentle squeezing in cold phosphate-buffered saline(PBS). After passing through a 100-µm mesh filter, thecell suspension was centrifuged at 600 x g for 10 min and washedtwice with 10 ml of the same buffer. Sonication-resistant nucleifrom testis were prepared according to reference 24.

Western blotting. Pelleted sperm were dissolved in 0.1 M Tris-HCl, pH 7.8, buffercontaining 0.1% (vol/vol) Triton X-100, 0.1 M 2-mercaptoethanol,and 6 mM guanidine-HCl, which was replaced by Laemmli bufferusing Micro Biospin P6 cartridges (Bio-Rad, München, Germany).Gel electrophoresis was performed according to reference 18.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis-separatedproteins (10 µg each) were blotted onto a nitrocellulosemembrane in 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS)-NaOH(0.01 M), 10% methanol, pH 11, and probed with a PHGPx-specificantibody at a dilution of 1:1,000 (27). Detection was achievedby incubation with biotinylated anti-rabbit immunoglobulin Gand streptavidin-alkaline phosphatase complex.

Hematoxylin-eosin histology of testis and epididymis. Freshly dissected tissues were immediately fixed in Bouin'ssolution or in 4% (wt/vol) paraformaldehyde in PBS at 4°Covernight, embedded in paraffin, and cut in 5-µm-thicksections. Sections were deparaffinized in xylol, hydrated ingraded ethanol series, stained with Mayer's hematoxylin (Sigma,Taufkirchen, Germany) for 5 min, washed in water, briefly treatedwith 1% acid ethanol, washed again, and stained with 1% eosinY (Merck, Darmstadt, Germany) for 3 min. Then the sections werewashed again in water, dehydrated in graded ethanol series,treated with xylol, and mounted.

Ultrastructural analysis. (i) SEM. Sperm were washed twice in Soerensen buffer, pH 7.4 (1:5 solutionof 0.07 M KH₂PO₄ and 0.07 M Na₂HPO₄-2H₂O), fixed in 1% glutaraldehydein Soerensen buffer at 4°C for 24 h, washed again, and centrifugedonto slides coated with 3-aminopropylene-ethoxysilane. Thensperm were dehydrated in an ascending series of acetone (10%,20%, 30%, 40%, 50%, and 60% twice for 5 min each; 70%, 80%,and 90% for 1 h each; 100% for 12 h) and dried in a Union Pointdryer CPD 030 (Bal-Tec, Walluf, Germany) using liquid CO₂ asthe transitional fluid. Dried sperm were coated with 12-nm gold-palladiumby a Union SCD 040 sputtering device (Bal-Tec). Scanning electronmicroscopy (SEM) observations were made with the Zeiss scanningelectron microscope DSM 950 using magnifications of x200 tox10,000 (Carl Zeiss, Jena, Germany).

(ii) Transmission electron microscopy. Transmission electron microscopy for ultrastructural analysesof testis and epididymis was principally carried out as describedin reference 10.

ISEL and immunohistochemistry. The in situ end-labeling (ISEL) method and immunohistochemistrytests were performed as follows. Detection of apoptotic cellsand proliferating cellular nuclear antigen (PCNA) staining werecarried out as described previously (10). The latter protocolwas also applied for 8-hydroxyguanosine staining of paraffin-embeddedsections after boiling sections twice in 10 mM sodium citratebuffer for antigen retrieval (30). Anti-8-hydroxyguanosine wasthe primary antibody (QED Bioscience Inc., San Diego, CA); thesecondary antibody was an anti-mouse biotinylated antibody,BA-2000 (Vector Laboratories Inc.).

Sperm parameter analysis. Spermatozoa were collected from the cauda epididymis. Theirconcentration, motility, and progressivity were estimated byusing the Integrated Visual Optical System (IVOS) for spermanalysis according to the manufacturer's recommendation (HamiltonThorne Research, Beverly, MA).

TB and AO staining (modified Rigler-Roschlau method). Both the toluidine blue (TB) and acrid orange (AO) methods wereperformed with fresh sperm smears as reported recently (12).In brief, sperm were fixed in 96% ethanol-acetone (1:1) for30 min at 4°C, denatured in 0.1 N HCl at 4°C for 5 min,washed in distilled water, and stained with 0.05% TB in McIlvainbuffer (200 mM Na₂HPO₄, 100 mM citric acid, pH 3.5) for 10 minat room temperature. Slides were dehydrated in butanol at 37°Cand embedded in Roth medium (Roth GmbH, Karlsruhe, Germany).For AO staining, sperm were fixed in 96% ethanol-acetone (1:1)at 4°C for 30 min, rehydrated in a graded ethanol seriesat room temperature, washed in PBS, treated with 1 N HCl for1 min at 60°C, and rinsed three times in distilled water.Slides were dipped into McIlvain buffer for 5 min, stained withfreshly prepared AO (0.1 mM), and subsequently rinsed threetimes for 5 min each with AO (0.001 mM) in the same buffer.Sperm were immediately evaluated by fluorescence microscopy.

Sperm chromatin decondensation assay with SDS. Prior to decondensation, reactive thiols were blocked by incubatingisolated sperm in 10 mM N-ethylmaleimide (Sigma, Taufkirchen,Germany) in PBS for 30 min at 37°C with gentle shaking.Subsequently, sperm were washed in PBS, incubated in 0.5% SDS,50 mM Tris-HCl, pH 7.5, for 20 min at 37°C with gentle shaking,stained with ethidium bromide, and immediately evaluated byfluorescence microscopy (Axioplan; Carl Zeiss, Oberkochen, Germany).

Protein thiol measurement. A stock solution of 50 mM monobromobimane (mBrB [Thiolyte];Calbiochem, Schwalbach, Germany) was prepared in 100% acetonitrileand stored at 4°C in the dark for up to 1 month. This wasdiluted to 1 mM with PBS and added to spermatozoa resuspendedin PBS at a final concentration of 20 x 10⁶ cells/ml. Afterlabeling at room temperature for 30 min in the dark, spermatozoawere twice washed with PBS. To solubilize labeled proteins,spermatozoa were dissolved in 0.1 M Tris-HCl, pH 7.8, containing0.1% (vol/vol) Triton X-100, 0.1 M 2-mercaptoethanol, and 6mM guanidine-HCl. Proteins were precipitated in 4% (wt/vol)trichloroacetic acid and washed with cold ethanol. Final solubilizationwas achieved in 10 mM NH₄HCO₃, pH 8.3, containing 1% (wt/vol)SDS. Fluorescence emission was measured by a RF-5000 spectrofluorophotometer(Shimadzu) with excitation and emission wavelengths of 380 and470 nm, respectively. Total thiol content was measured by repeatingthe labeling procedure after the solubilization and reductionstep.

Ex vivo labeling of spermatozoa. Spermatozoa were resuspended in 0.5 ml PBS, labeled with a solutionof 1 mM mBrB prepared as described above, washed twice withPBS, and resuspended in a minimal volume of PBS prior to fluorescencemicroscopy evaluation (Axioplan; Carl Zeiss) (excitation andemission wavelengths of 365 to 395 and 420 nm, respectively).

RESULTS

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Results

Generation of nPHGPx KO mice. The PHGPx gene is composed of 8 exons spanning a region of approximately3 kbp. The mRNAs encoding the mitochondrial (mPHGPx) and cytosolic(cPHGPx) forms of PHGPx are initiated at alternative transcriptioninitiation sites (16, 25) and share exons 1 to 7 but excludeEa localized between exons 1 and 2 (Fig. 1A). In contrast, nPHGPxlacks the first exon and consists of exons Ea and 2 to 7 (23,24). The Ea encodes both a nuclear localization signal and clustersrich in arginine and lysine, most likely required for bindingof nPHGPx to DNA. Since loss of the whole PHGPx gene or of exonsEa to 7 is associated with early embryonic lethality in mice(14, 36), we replaced large parts of the coding region of Eawith EGFP, generating a fusion gene consisting of the six N-terminalamino acids of nPHGPx (MGRAAA) and EGFP. Its functionality wascontrolled by transient overexpression of the fusion proteinin HeLa and NIH 3T3 cells under the control of the human cytomegaloviruspromoter. Strong cytoplasmic staining of the cells could beobserved (data not shown); however, unfortunately this fusionprotein was not functional in vivo, meaning we could detectneither green spermatozoa nor green fluorescing testicular tissue.The EGFP gene was flanked by characteristic restriction sitesfor confirmation of homologous recombination of the targetingconstruct in ES cells. Additionally, a translational STOP cassettewas inserted in every possible reading frame 5' of the fusiongene. The remaining 3' coding region of Ea, corresponding to11 amino acids, did not contain any translational START codon.Hence, our strategy aimed at removing the coding region of Ea,thereby abolishing nPHGPx expression without affecting mPHGPxand cPHGPx expression.

The construct for targeting nPHGPx in ES cells, a Southern blotdemonstrating germ line transmission of the targeted allele,and genotyping of tail DNA are shown in Fig. 1B. Intercrossof nPHGPx^+/^– mice produced viable nPHGPx^–^/^–mice. Of 191 offspring from heterozygous breeding pairs, 22.5%were WT and 52.4% were heterozygous and 25.1% homozygous KOfor nPHGPx. Thus, we conclude that nPHGPx is not essential forembryonic development and survival. Semiquantitative RT-PCRanalysis with testis mRNA was used to verify the absence ofthe coding region of Ea. Primer pair P32/E2rev1 was used todetect the mitochondrial form of PHGPx. No differences in mPHGPxmRNA levels were noticeable in mice of the different genotypes(Fig. 1C). However, when the primer Eaforw2, specific for thedeleted part of Ea, was used in combination with E2rev1, a clearreduction of nPHGPx mRNA levels in nPHGPx^+/^– and absencein nPHGPx KO mice could be demonstrated. We confirmed our RT-PCRdata by in situ hybridization experiments on testis sectionsusing an antisense probe specific for the deleted region ofEa (Fig. 1D and E). No specific signal could be detected insections from nPHGPx^–^/^– mice (Fig. 1E). In contrast,a clear signal especially around the luminal face of tubuliseminiferi was noticed in WT mice (Fig. 1D), which is in accordancewith previous findings (20, 23). Furthermore, the absence ofthe 25.9-kDa immunoreactive band, representing the principalform of nPHGPx in late spermatids and spermatozoon heads (19),confirmed that nPHGPx is not expressed in the testes (Fig. 1F)and spermatozoa (data not shown) of KO mice.

Fertility of nPHGPx KO mice. We assumed a crucial role for nPHGPx in sperm chromatin condensation,and to test this hypothesis, breedings of male nPHGPx^–^/^–mice with C57BL/6 mice were performed. To our surprise, malenPHGPx^–^/^– mice were fully fertile and litter sizes(Fig. 1G) (6.4 ± 2.0 pups [n = 30] versus 7.3 ±2.7 pups [n = 20], control versus KO, respectively) and whelpingintervals (30.4 ± 13.0 days [n = 19] versus 30.5 ±8.3 days [n = 11], control versus KO, respectively) were indistinguishablefrom those of their WT counterparts.

Morphological analyses of germinal epithelium and germ cells. Histological examinations of testis and epididymal tissue didnot show any differences between nPHGPx^+/+, nPHGPx^+/^–,and nPHGPx^–^/^– siblings (Fig. 2, A to D). Likewise,SEM of isolated epididymal spermatozoa and ultrastructural analysisof testis and epididymis did not reveal any obvious differencesbetween WT and KO mice (Fig. 2E to K) (data not shown). Stainingwith PCNA and ISEL staining was performed to test whether proliferationof spermatogonia or apoptosis of spermatogenic cells was affectedby loss of nPHGPx. We could not detect any differences in WTand heterozygous and homozygous KO mice (data not shown). SincePHGPx is known to have antioxidant properties, we stained testissections with an antibody recognizing 8-hydroxy-deoxyguanine.Again, no signs of increased DNA damage of germ cells couldbe observed (data not shown).

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FIG. 2. Histological analyses of testis and epididymis. (A to D) Hematoxylin-and-eosin staining of testis (A and B) and epididymis sections (C and D) did not show any gross abnormalities in KO (n = 8) (B and D) as compared to WT (n = 8) (A and C) tissue. Shown are scanning electron microscopy of spermatozoa isolated from the cauda epididymis (E and F) (n = 2 each) and ultrastructural analysis of sperm derived from the caput (G and H) (n = 2 each) or cauda (I and K) (n = 2 each) epididymis. Bars = 10 µm.

Sperm parameters. Sperm parameters such as concentration, motility, and progressivityof spermatozoa isolated from the cauda epididymis were determinedby using the IVOS. Similar to the histological findings describedabove, we could not find any difference in the analyzed spermparameters of mice of any genotype, supporting the notion thatmale nPHGPx KO mice are fully fertile (Fig. 3A) (data not shown).

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FIG. 3. Sperm parameters and TB and AO staining of spermatozoa. (A) No difference in motility and progressivity of spermatozoa of either genotype could be observed. (B to F) More than 50% of KO spermatozoa isolated from the caput epididymis showed violet-blue TB staining (black arrows in panel C) as compared to less than 10% in WT spermatozoa (B). No such difference was noticeable in spermatozoa from the caudal part of epididymis (D and E). Somatic cells are marked with an asterisk, and light blue spermatozoa are marked with open arrows. (F) Quantification of TB staining. The data are representative of two independent experiments and represent the mean ± standard deviation obtained from three control and three KO animals (n = 200 sperm each). (G to L) AO staining confirmed the results obtained from TB staining. Panels G and H show spermatozoa derived from the caput, and panels I and K show spermatazoa from the cauda epididymis. (L) Quantification of AO staining in caput. The data are representative of three independent experiments and represent the mean ± standard deviation obtained from three control and three KO animals (n = 200 sperm each). (M to O) Aberrant morphology of spermatozoa after drying and hydrating was observed in samples from caput epididymis of KO mice (arrows in panel N). (O) Quantification of these findings. The data are representative of four independent experiments and represent the mean ± standard deviation obtained from three control and three KO animals (n = 300 sperm each). (P to T) Treatment of sperm with 0.5% SDS revealed a large portion of decondensed spermatozoa (arrows in panel Q) derived from the caput epididymis of KO (Q) in contrast to WT (P) mice. (R and S) No difference was detectable in spermatozoa from the caudal part. (T) Quantification of the results obtained in panels P and Q. The data are representative of three independent experiments and represent the mean ± standard deviation obtained from four control and four KO animals (n = 200 sperm each). Bars = 10 µm.

Sperm chromatin condensation. We used TB and AO staining to study the integrity of sperm chromatin(12). Protamine cross-linking by formation of intra- and intermoleculardisulfide bonds is considered to render sperm chromatin resistantto mechanical and chemical insults. Thus, susceptibility todenaturation of chromatin by heat or acid can be used to monitorprotamine cross-linking. Upon acidic or heat treatment of spermDNA, green/yellow fluorescent AO staining indicates fully condensed,nondenatured double-stranded DNA, whereas red fluorescence indicatesnoncondensed partially single-stranded DNA. TB staining followsthe same principle, where pale blue staining and violet/darkblue staining visualize normal and incomplete sperm chromatincondensation, respectively.

Spermatozoa were consistently isolated from the caput and fromthe cauda epididymis and subjected to both staining techniques(Fig. 3B to L). A clear difference in TB staining was observedonly in spermatozoa derived from the caput epididymis. Approximatelyhalf of KO spermatozoa showed dark blue TB staining as comparedto 8% in WT spermatozoa (Fig. 3B, C, and F). No such differencecould be observed in spermatozoa isolated from the cauda epididymis(Fig. 3D to F). AO staining confirmed the results obtained byTB staining (Fig. 3G to L). Of note, when we prepared smearsof spermatozoa, let them dry without fixation, and hydratedthem with phosphate-buffered saline, around one-fourth of KOspermatozoa displayed morphologically abnormal sperm heads (Fig.3M to O). Again, this was only apparent in spermatozoa isolatedfrom the caput epididymis of KO mice. Additionally, spermatozoawere exposed to 0.5% SDS in order to assess the structural stabilityof sperm nuclei (Fig. 3P to T). Similarly to the TB and AO methods,more than half of KO sperm collected from the caput epididymisdisplayed strongly decondensed nuclei (Fig. 3Q). This differencewas again restricted to the caput epididymis. This strengthensthe observation that KO sperm derived from the caput epididymisare structurally less stable than WT sperm.

Thiol content of isolated spermatozoa. With the nPHGPx KO model at hand, we were able to test the long-standinghypothesis that PHGPx can act as a protein thiol peroxidasein vivo. We quantified the protein thiol content of isolatedspermatozoa by using the mBrB labeling technique (17) (Fig.4). In testis and caput epididymis, no difference in the ratioof free thiols versus total thiols could be observed in spermatidsand spermatozoa, respectively (Fig. 4A, C, and E). In WT spermatozoa,a strong decrease of free thiols was found along the epididymis,from more than 80% free thiols in testis to around 50% and 10%free thiols in caput and cauda, respectively (Fig. 4E), similarto the findings described for rat spermatozoa (29). However,this did not apply for KO spermatozoa, where significantly higherlevels of free protein thiols were observed in KO spermatozoaobtained from the cauda epididymis. Microscopic evaluation revealedthat the difference in fluorescence intensity was largely restrictedto the head region in sperm from cauda epididymis and was notevident in the midpieces and tails of spermatozoa (Fig. 4A to D).

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FIG. 4. Thiol-disulfide status of spermatozoa. (A to D) Microscopic evaluation of the fluorescence of spermatozoa from the caput (A and C) and cauda epididymis (B and D). No difference in fluorescence intensity could be observed in WT and KO spermatozoa from the caput epididymis (A and C), whereas less intensity was noticeable in WT spermatozoa from the cauda epididymis (B) as compared to KO spermatozoa. This difference was restricted to the head region. (E) Protein thiol status in testis sonication-resistant nuclei and spermatozoa from WT and KO mice as assessed by fluorescence photometric quantification. Significantly higher levels of free thiols were evident in KO spermatozoa. Results are expressed as a ratio between reactive -SH before and after reduction with 0.1 M ß-mercaptoethanol and are representative of the mean ± standard deviation of three independent experiments (*, P < 0.001 versus WT).

DISCUSSION

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Discussion

To investigate the physiological role of nPHGPx, in particularduring male gametogenesis, mice with a specific deletion ofthe nPHGPx were established. Furthermore, with this model athand, we aimed at providing an answer to the question whethernPHGPx is able to accept electrons from thiol groups other thanthat of glutathione in vivo. Due to early embryonic lethalityof PHGPx KO mice (14, 36), we specifically deleted the alternativeexon. Since nPHGPx is independently expressed from the cytosolicand mitochondrial forms of PHGPx (20, 23), we did not expectany adverse impact of the KO on expression of the cytosolicand mitochondrial forms of PHGPx. This was indeed the case,thus confirming the previously reported mechanism of PHGPx expression.Most surprisingly, nPHGPx KO mice are fertile and litter sizesare indistinguishable between WT and heterozygous and homozygousKO mice. This is also in line with histological and ultrastructuralexamination of testis and spermatozoa, which did not revealany morphological abnormalities (see Fig. 2).

Though nPHGPx KO mice are fully fertile, we were still interestedin whether nPHGPx is involved in protamine cross-linking. Duringepididymal transit, mammalian sperm protamines are extensivelycross-linked by inter- and intramolecular disulfide bridgesup to a variable extent, which is generally species specific(1, 3, 26, 29). (n)PHGPx has been proposed as the prime catalystinvolved in this oxidation process, due to its predominancein mammalian male germ cells and its peculiar reducing substratespecificity (21, 28, 32). Thus, we applied the AO and TB stainingmethods to assess chromatin susceptibility to denaturation inspermatozoa isolated from caput or cauda epididymis (11, 12).Our results show that while chromatin of the WT spermatozoaentering the epididymis is already resistant to denaturation,chromatin of KO spermatozoa is more labile, reaching stabilitysimilar to that of the WT only at a later stage in the cauda(Fig. 3). This indicates that there is a phase of transientchromatin instability in caput-derived KO spermatozoa that canbe unraveled by exposure to physical or chemical stress (Fig.3). From these experiments, we conclude that chromatin condensation,albeit notably delayed, does occur in nPHGPx KO mice.

Since chromatin resistance to acid or heat treatment is believedto rely on the presence of disulfides in protamines, we directlyaddressed the issue of protein thiol redox status in spermatozoaduring maturation by measuring the fluorescence of mBrB adducts.Our results with WT spermatozoa showed, in agreement with previousobservations (29), that protein thiol oxidation is indeed acontinuous process, becoming almost complete in the cauda epididymis,when approximately 90% of cysteine thiols are oxidized (Fig.4). Surprisingly, in spermatozoa isolated from the caput epididymis,where KO chromatin was shown to be less condensed and KO spermheads were more susceptible to dehydration-rehydration and SDStreatment, protein thiol redox status was identical to thatof the WT. Significantly higher levels of free sulfhydryl groupswere, however, detectable in KO spermatozoa isolated from thecauda epididymis. This indicates that (i) nPHGPx unquestionablyharbors protein thiol peroxidase activity in vivo; (ii) proteinthiol oxidation in sperm heads can take place also in the absenceof nPHGPx, albeit with less efficiency; and (iii) at an earlystage of sperm maturation when about half of the thiol groupsof protamines are oxidized, the stability of sperm chromatinunder denaturing conditions does not exclusively rely on theprotein thiol content. In this context, one should bear in mindthat oxidation is restricted not only to the head but also tothe tail region of spermatozoa, although bulk oxidation occursin the head of spermatozoa (29) (Fig. 4A and B).

The apparent discrepancy between the stability of the spermnuclei and head with thiol redox status, occurring in the caputof epididymis, was surprising and striking. It implies thatnPHGPx did already play a role in stabilizing nuclear structuresin spermatozoa in the caput before and independently of proteinthiol oxidation. The notion of a role for nPHGPx in structuralstability is in accordance with the proposed function of PHGPxin the spermatozoon midpiece (22, 28, 31). It is conceivablethat nPHGPx, already linked to DNA via its basic N terminus,becomes cross-linked to protamines via the -Se-S dead-end intermediateof the catalytic cycle, when accessible protamine thiols arelimiting (22). Even very small amounts of nPHGPx covalentlylinked to protamines and DNA may significantly contribute tostructural stability of caput sperm nuclei. Apparently, thisis no longer relevant at higher oxidation levels.

The general features of nPHGPx function highlighted in thisstudy imply that an enzyme(s) other than nPHGPx may be involvedin thiol oxidation or, alternatively, that the thiol oxidationprocess takes place spontaneously (4). The observation thatthe phenotype in the head region of nPHGPx KO spermatozoa ismuch less severe than that observed under selenium deficiencystrongly argues against a nonenzymatic process and for the contributionof another selenoenzyme (34). Cytoplasmic PHGPx (cPHGPx) issmall enough to enter the nuclear pore by diffusion (35) andhas been reported to be present in the head of spermatozoa (19).This strongly suggests that it is cPHGPx which fulfills thisimportant function in our nPHGPx KO mice.

Since nPHGPx KO mice exhibit abnormalities at the male germcell level not affecting fertility, we must conclude that thenuclear form of PHGPx is definitely not an indispensable playerin spermatozoon physiology. Far beyond this observation, themild phenotype, as observed in nPHGPx KO mice, revealed an outstandingtool to unmask the peculiar functions of PHGPx in vivo. Moreover,due to transient nuclear instability and delay in chromatincondensation, nPHGPx KO mice might prove an ideal model to studythe susceptibility of the male genome to chemically inducedmutagenesis (28a, 29a).

ACKNOWLEDGMENTS

We are most grateful to M. Krienke and A. Boersma for fruitfuldiscussions; N. Mayr, S. Lippl, and C. Neumüller for excellenttechnical assistance; and P. Braghetta for advice and collaborationin fluorescence microscopy experiments.

This work was supported by the DFG-Priority Programs SPP1087(to G.W.B., M.B., and M.C.), a fellowship from Alexander vonHumboldt-Stiftung to S. Moreno, and Fonds der Chemischen Industrieand the Italian Ministry of University and Scientific Research(grants PRIN 2002052331 to F.U. and 2003038920_002 to M.M.).

FOOTNOTES

* Corresponding author. Mailing address: Institute of Clinical Molecular Biology and Tumour Genetics, GSF Research Centre for Environment and Health, Marchioninistr. 25, D-81377 Munich, Germany. Phone: 49-89-7099525. Fax: 49-89-7099500. E-mail: marcus.conrad{at}gsf.de.

REFERENCES

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