Interaction of Exogenous DNA With the NuclearMatrix of Live SpermatozoaSIOBHAN MCCARTHY1,2* AND W. STEVEN WARD1,2

1Program in Cell and Developmental Biology, Rutgers University, Division of Urology, Robert Wood Johnson MedicalSchool, New Brunswick, New Jersey2Cancer Institute of New Jersey, New Brunswick, New Jersey

ABSTRACT Sperm chromatin is a highly orga-nized array of protamines and DNA, with the protaminesserving to tightly condense the DNA into a compact,defined structure. We have previously demonstratedthat the sperm nucleus is an ordered library of DNAorganized into functional zones, such as the nuclearmatrix and nuclear annulus. Other laboratories havesuggested that mouse spermatozoa can interact withexogenous pSV2CAT plasmid DNA. In this work, weexplored this interaction and examined the subcellularlocalization of the exogenous DNA. We found a repeat-able association of exogenous DNA with a specificregion of the sperm nuclear matrix. This region of thenucleus correlates with the equatorial segment of thesperm head. This interaction requires only a definedfertilization media, transfection quality DNA, and incuba-tion with spermatozoa. Mol. Reprod. Dev. 56:235–237, 2000. r 2000 Wiley-Liss, Inc.

Key Words: sperm DNA; chromatin; nuclear matrix;exogenous DNA

INTRODUCTIONIn 1989, Lavatrino and colleagues published the first

report that live mouse spermatozoa interacted withexogenously added DNA. In subsequent publications,this group presented evidence that live spermatozoacould bind to radioactively labeled DNA and that it wasinternalized into the nuclei, as detected by electronmicroscopy (Francolini et al., 1993; Zani et al., 1995).More recently, they have suggested that the DNA is alsointegrated into the sperm chromatin (Zoraqi and Spada-fora, 1997). Several other laboratories have reportedthat live spermatozoa both from the mouse (Huguetand Esponda, 1998) and other species (Lavitrano et al.,1989; Gagne et al., 1991; Camaioni et al., 1992; Horanet al., 1992) have the ability to bind to exogenous DNA.

Our laboratory has long been interested in the inter-action between the endogenous, chromosomal DNA andsperm nuclear structures. We have demonstrated thatmammalian sperm DNA is organized into loop domainsof about 30–50 kb in length that are attached at theirbases to a proteinaceous structure, the nuclear matrix(Getzenberg et al., 1991; Ward and Coffey, 1991; Nadelet al., 1995). This organization is cell specific, with thesame genes having different structural relationships

with the nuclear matrix in the sperm and the liver cell(Nadel et al., 1995). Evidence for this type of organiza-tion in sperm DNA has been presented by other labora-tories (Kalandadze et al., 1990; Choudhary et al., 1995;Yaron et al., 1998). We have also demonstrated thatwithin the sperm nucleus, another structure, the nuclearannulus, organizes DNA in much larger units, possiblyat the level of whole chromosomes (Ward and Coffey,1989, 1991; Farrington et al., 1991). We have predictedthat these relationships between sperm DNA and thesperm nuclear matrix and nuclear annulus have func-tional consequences in spermiogenesis and embryogen-esis (Ward, 1994).

Given our interest in the structural and functionalinteractions of sperm nuclear structures with its endog-enous, chromosomal DNA, we were intrigued by thepossibility that these same proteinaceous scaffolds mightinteract with exogenous DNA. It was particularly signifi-cant that the several laboratories cited above hadshown that live spermatozoa might be capable of absorb-ing exogenous DNA and transporting it to the nucleus.In this report, we present our preliminary studies,which suggest that this exogenous DNA binds directlywith the sperm nuclear matrix, at a specific regionwithin that structure.

MATERIALS AND METHODSLive mouse spermatozoa were incubated with exog-

enous DNA as described by Lavitrano and colleagues(1989), with some modifications. Briefly, recently matedmale CD1 mice were sacrificed. Cauda epididymideswere surgically removed and placed on ice duringtransport. The epididymides were cut and placed infertilization media (FM) under silicone oil. The FM hadbeen equilibrated in 5% CO2, at 37°C for 15–24 hr. TheFM was prepared as described in detail by Lavitrano etal. (1989). It contained 119 mM NaCl, 25 mM NaHCO3,2.7 mM KCl, 1.8 mM CaCl2, 500 mM MgCl2, 155 mMNa2HPO4, 0.1% glucose, 0.5 mM Na Pyruvate, pH 7.5,supplemented with 4 mg/ml of Fraction V BovineSerum albumin (Sigma). After 1 hr, the sperm insuspension were withdrawn from under the silicone oil

*Correspondence to: Siobhan McCarthy, Cancer Institute of NewJersey, 195 Little Albany Street, Room 2034, New Brunswick, NJ08903.

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r 2000 WILEY-LISS, INC.

and were mixed with the pSV2CAT plasmid (a gift ofCorrado Spadafora) in microcentrifuge tubes, at aconcentration of 10–20 µg/ml. The capped tubes werethen placed back into the incubator for 15 min to 1 hr.

After incubation, sperm nuclear matrices were pre-pared from the spermatozoa, as previously described(Ward et al., 1989). Briefly, spermatozoa were washedin sodium dodecyl sulfate (SDS) and subjected to su-crose gradient centrifugation. Isolated nuclei were thenextracted with 2 M NaCl, 25 mM Tris, pH 7.4, and 10mM dithiothreitol, to remove the protamines. A controlaliquot, not incubated with pSV2CAT, was reserved formixing with plasmid DNA just prior to the detergentwash. Aliquots of this preparation were prepared forfluorescent in situ hybridization (FISH) as previouslydescribed in detail (Nadel et al., 1995). The probe usedin these experiments was a directly labeled Cy3pSV2CAT probe. DNA was counterstained with DAPI.

RESULTSThe results reported in this manuscript are prelimi-

nary, and contain the data that was presented at theInternational Symposium on DNA Transfer in Sperma-tozoa (Siena, Italy, 1999 May 24–27). We wanted todetermine if the exogenous DNA that spermatozoaappear capable of absorbing interacted directly withthe nuclear structures that we have been studying. Todo this, we first incubated live mouse spermatozoa withexogenous pSV2CAT DNA. We then washed the sperma-tozoa with 0.5% SDS, a strong ionic detergent thatsolubilizes all the membranes and most of the cytoplas-mic proteins. We next purified the nuclei throughsucrose step gradients. Finally, the sperm nuclei wereextracted with 2 M NaCl and 10 mM DTT to extract theprotamines and other nonnuclear matrix proteins. Af-ter this treatment, the DNA forms a halo around thenucleus that consists of naked loops of DNA attached attheir bases to the nuclear matrix.

After all of these treatments, these nuclear haloswere then hybridized to CY3-labeled pSV2CAT. CY3 is afluorescent molecule that allowed us to detect hybrid-ized DNA directly, without having to induce the pos-sible artefact that comes from secondary antibodiesused in other methods of FISH detection. Our results

have so far demonstrated that the exogenous DNAbinds to a specific region in the sperm nuclear matrix ata position that corresponds to the point at which theacrosomal matrix (a cytoplasmic structure) ends (Fig.1B). We incubated the spermatozoa with DNA forvarious times, from 0 to 60 min, and found that withincreasing time more nuclei contained pSV2CAT DNA.Two types of control experiments were performed. Inthe first, DNA was mixed with live spermatozoa at thesame time as the addition of SDS, to ensure that thebinding we detected was not an artifact induced by thedisruption of the plasma membrane by the detergent.In the second, DNA was mixed with spermatozoa at thesame time as SDS, and incubated for up to 1 hr as onetest that the absorption of exogenous DNA onto thesperm nuclear matrix over time depended on the sper-matozoa being alive. In neither control were we able tofind a single nucleus that demonstrated the specificpattern of DNA binding shown in Figure 1B.

CONCLUSIONSOur data demonstrate that live mouse spermatozoa

have the ability to take up exogenous DNA and thatthis DNA binds very tightly to the sperm nuclearmatrix. This DNA cannot be removed by treatmentwith 0.5% SDS, by sucrose gradient centrifugation, orby subsequent treatment with 2 M NaCl. The bindingappears to require that the spermatozoa are alive, or atleast that the cells are structurally intact, since wash-ing with SDS simultaneously with the addition of theexogenous DNA prevents the association of the DNAwith the nuclear matrix.

The fact that live spermatozoa have this ability toabsorb exogenous DNA (Lavitrano et al., 1989; Spada-fora, 1998) and that it binds to a specific area of thenuclear matrix, as described in this report, seems tochallenge our models of both the function and structureof spermatozoa. We approached these experiments withskepticism, but are compelled by the data to accept theconclusions we have stated in this manuscript. Infuture experiments, we will begin to examine themechanism of this phenomenon in an effort to under-stand the reason that spermatozoa have this ability,which seems so detrimental to their primary function of

Fig. 1. Detection of binding of exog-enous DNA to the nuclear matrix oflive spermatozoa. Live spermatozoa wereincubated with exogenously addedpSV2CAT DNA, and the nuclear matri-ces were subsequently isolated andtested for binding, by FISH. A: Totalchromosomal DNA associated with thesperm nuclear matrix, as detected byDAPI staining. B: The same nucleus asin (A), shown in the red filter forpSV2CAT binding. The exogenouslyadded DNA binds to a specific region inthe sperm nuclear matrix. Bar 5 5 µm.

236 S. MCCARTHY AND W.S. WARD

transferring a pristine genome from the male to thefemale completely intact and uninfected. For now, wewould suggest that sperm DNA uptake indirectly re-sults from another mechanism that is necessary fornormal sperm function. Maione and colleagues (1997)have even suggested that spermatozoa contain a protec-tive mechanism against such inadvertent intrusions ofexogenous DNA, in which nucleases are activated todestroy them. Clearly, much work on the biology of thisfascinating mechanism remains.

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