mechanisms of displacement of sperm basic …pate in the post-fertilization displacemen of thet...

J. Cell Sri. 53, 227-244 (1982) 227Printed in Great Britain © Company of Biologists Limited 1982

MECHANISMS OF DISPLACEMENT OF SPERM

BASIC NUCLEAR PROTEINS IN MAMMALS.

AN IN VITRO SIMULATION OF POST-

FERTILIZATION RESULTS

TOBY C. RODMAN*, FRED H. PRUSLIN ANDVINCENT G. ALLFREYCornell University Medical College and The Rockefeller University,New York, N.Y. 10021, U.S.A.

SUMMARY

A standardized cytological preparation of mature mouse sperm has been devised to serveas an in vitro system for probing the intra-ooplasmic molecular events of transformation ofthe fertilizing sperm. Two parameters of the early phase of transformation in vivo are definedat the resolution of the light microscope: deletion of sperm-unique nuclear proteins, detectableby immunofluorescence, and retention of homogeneity of the residual DNA complex, withintact chromatin boundaries detectable by ethidium bromide staining. These studies showthat both parameters are conserved when in vitro sperm preparations are treated with NaClunder reducing conditions. The deletion of 2 different classes of the unique basic proteinsof mouse sperm nuclei is specified by the NaCl concentration: 0-7 M-NaCl displaces thenon-protamine class but not the protamines, while 1 M-NaCl displaces both. On the otherhand, the effects of treatment with trypsin at various concentrations and intervals are lessconsistent with the in vivo parameters, indicating fragmentation and displacement, not onlyof the sperm-unique basic proteins, but also of structural proteins believed to maintain thefundamental cohesive organization of the DNA matrix. These observations suggest thatmechanisms other than proteolysis, e.g. localized changes in ionic concentrations, may partici-pate in the post-fertilization displacement of the sperm-unique nuclear proteins in vivo. Thisstudy also supports the validity of the in vitro simulation as a model with which to probethe progression of transformation of the sperm nucleus to the zygote pronucleus.

INTRODUCTION

The chromatin of the mammalian spermatozoon differs markedly from that ofsomatic cells, not only in its genetic inertness, but also in its organization andmolecular constituency. Therefore, the transformation of the fertilizing spermnucleus to the pronucleus of the zygote requires: first, disassembly of that uniquenucleoprotein complex; and secondly, reconstitution of transcriptionally competentsomatic-type chromatin. These studies are concerned with the first of those twophases of transformation. We report here the development and application of anin vitro system for examining possible mechanisms by which the disassembly processmay occur in vivo.

* Address correspondence to: Dr Toby C. Rodman, Department of Anatomy, CornellUniversity Medical College, 1300 York Avenue, New York, N.Y. 10021, U.S.A.

228 T. C. Rodman, F. H. Pruslin and V. G. Allfrey

The sperm chromatin is the end product of the nuclear events of spermiogenesis,which include deletion of histones, incorporation of low molecular weight basicproteins rich in cysteinyl and arginyl residues, extreme condensation throughformation of disulphide bonds and concomitant progressive decrease in nuclearvolume with assumption of the species-specific morphology of the sperm head(Fawcett, Anderson & Philips, 1971; Calvin & Bedford, 1971; Platz, Grimes,Meistrich & Hnilica, 1975; Rodman, Litwin, Romani & Vidali, 1979).

Cytological studies of fertilizing sperm in situ (McGaughey & Chang, 1969;Stefanini, Oura & Zamboni, 1969; Rodman, Pruslin, Hoffmann & Allfrey, 1981)have shown that, upon entry into the ooplasm the sperm nucleus increases in volume,the nuclear membrane is dispersed and decondensation of the chromatin is initiated;for a considerable period of the progressive nuclear swelling, the general contoursof the specific morphology of the sperm head are retained.

Although those morphological indications of the reversal of the events of spermio-genesis have been defined for several species, the underlying molecular mechanismsare not known. Logically, those would be expected to include reduction of thedisulphide bonds to allow chromatin decondensation and deletion of the uniquesperm basic nuclear proteins (SBNP) to free the DNA for complexing withhistones in nucleosomal assembly. Implicit in that expectation is the provision thatthe process of SBNP deletion does not impair the integrity of the DNA, and thuscompromise the transformation of the paternal template to the embryo genome.

Since biochemical studies of fertilizing sperm in situ are not feasible, the definitionof those mechanisms requires the construction of an in vitro system designed tosimulate the putative events of transformation. However, biochemical treatments ofa population of sperm, while providing significant information of the character of thenuclear proteins, may not allow for adequate consideration of the provision notedabove: that the process of SBNP displacement must be entirely benign with respectto the DNA. We have, therefore, devised a system in which the biochemical treatmentsmay be carried out in parallel, cytochemically, where the progressive displacement ofthe basic chromosomal proteins and maintenance of the DNA organization are moni-tored on the same spermatozoa. The system includes the immunofluorescent demonstra-tion of the basic chromosomal proteinsofmousespermaspreviouslydescribed(Rodmanet al. 1979), followed by staining with ethidium bromide or propidium iodide, thatare fluorochromes with high specificity for DNA (Le Pecq & Paoletti, 1967; Crissman& Steinkamp, 1973).

Utilizing that system, we have studied the effects and effectiveness of 2 mechanismsof SBNP displacement from mouse sperm in vitro: (1) elevated salt concentrationand (2) proteolysis, each in a reducing medium.

The first mechanism takes cognizance of the possibility that ooplasmic factorsmay mediate or participate in the displacement process and finds some support inobservations that a large influx of Ca2+ and Na+ occurs in invertebrate eggs immedi-ately following sperm penetration and preceding or concomitant with sperm chromatindecondensation (Hagiwara & Jaffe, 1979). Especially pertinent to the experimentsof this study, in which SBNP displacement was induced by NaCl treatment, are

Displacement of sperm basic nuclear proteins 229

Fig. 1. Types of immunofluorescence {if) patterns seen on mouse sperm after thevarious treatments listed in Table 1. A. Normal (pre-immune) rabbit serum; B, base-line fluorescence (i.e. that seen following treatment with buffer only); c, intra-headhaze; D, small extraction haloes with more brilliant fluorescence at chromatinboundaries; E, larger extraction haloes (B, C, D and E are unswollen); F, swollennuclei with brilliant intra-head fluorescence and large extraction haloes.

T. C. Rodman, F. H. Pruslin and V. G. Allfrey

Fig. 2. Sperm treated with 0-7 M-NaCl, 0-5 % 2-ME in 0-02 M-PO4 buffer (pH 7-2).A, 5 min; B, 15 min; c, 60 min. Left to right: phase-contrast prior to staining;//with SBNP antiserum, ethidium bromide fluorescence (eb) of DNA-protein complexresidual after treatment. The progression appears to be that of extraction tocompletion of an immunofluorescently reactive fraction of SBNP. None orminimal swelling of sperm heads is seen in this progression. The residual DNAcomplex appears homogeneous at this resolution and sharp definition of the chromatinboundaries is retained.

reports that metaphase chromosomes and interphase nuclei of somatic cells, havingbeen depleted of histones by high salt concentrations, retain both DNA organizationand their morphological contours (Adolph, Cheng, Paulson & Laemmli, 1977;Adolph, 1980). Similarly, the residual structures of the salt-extracted sperm nuclei,though swollen, retain their characteristic contours and DNA organization, as revealedby ethidium bromide staining and, in general, resemble intra-ooplasmic sperm inthe early stages of transformation (Rodman et al. 1981).

The possibility that displacement of SBNP in vivo occurs through proteolysis


by an enzyme residing in some organelle of the sperm has been suggested by studiesshowing that certain basic nuclear proteins may be extracted from bull (Marushige &Marushige, 1978) and rabbit (Zirkin, Chang & Heaps, 1980) sperm by treatmentin vitro with acrosin- or trypsin-like enzymes. However, examination of trypsin-treated sperm by the cytochemical methods of this study revealed no set of conditionsin which the SBNP were deleted without disruption of the residual DNA complex.

Certain subsets of nuclear proteins are believed to maintain the structural organi-zation of somatic chromatin (Agutter & Richardson, 1980; Kaufmann, Coffey &Shaper, 1981). It is likely that a comparable complement of proteins is present insperm chromatin and, to fulfil their role of maintenance of DNA organization,those proteins should be unimpaired by the mechanisms of transformation. Theobservations of this in vitro study imply that proteolysis by an acrosin-like enzymemay not meet that requirement, while displacement of the SBNP by elevation ofthe ionic concentration of the milieu of the fertilizing sperm may be entirely benignwith respect to the putative residual DNA/protein structure.

METHODS

Cytological preparations of sperm

Sperm were collected from the distal J of the vasa of 8-week-old ICR mice, treated for10 min with 1 % Triton Xioo in phosphate-buffered saline (PBS) (pH 7-2), centrifuged, re-auspended in PBS and washed 5 x to remove the detergent. The final pellet, suspended in0-02 M-PO4 buffer (pH 7-2), was distributed on well-cleaned slides and placed upright inCoplin jars containing the respective reaction solutions, all at pH 7-2.

At the specified time interval the slide was withdrawn, washed, and fixed by immersion inmethanol/acetic acid (3:1, v/v) for 1 min, rinsed well in PBS, and treated by the indirectimmunofluorescence method (IF), using rabbit antiserum to a basic nuclear protein fractionof mouse sperm, prepared and characterized as described (Rodman et al. 1979, 1981), andfluorescein isothiocyanate (FITC)-labelled goat anti-rabbit immunoglobulin G (IgG). Theslides were examined, and characteristic fields were photographed. The slides were thenrestained with ethidium bromide (io/tg/ml PBS) (EB) for 5 min and the same fields wererephotographed as described below.

Photomicrography

Display of the basic proteins by IF. The Zeiss photomicroscope was used with transmittedlight for all procedures. Phase-contrast, with tungsten lamp as light source, was used todefine sperm morphology. IF was photographed with a mercury burner as source, FITCexciter filter (band pass, 400-495 nm), 530 nm barrier filter, Kodak tri-X film and 30 sexposure.

Display of DNA by EB. In order to display the EB fluorescence with no input from theFITC, the 546 nm interference filter was used for excitation. That filter passes the narrowband of about 540 to 560 nm that includes the excitation maximum of DNA-bound EB, butexcludes absorption by conjugated FITC. Emission from the DNA/EB was filtered by thered 580 nm filter, which cuts off transmission below 570 nm, thereby restricting all FITCfluorescence. EB-stained sperm were photographed on tri-X film with 5 s exposures.

Immunochemical characterization of the treatment effects

The moieties of SBNP differentially displayed by the various treatments of the cytologicalpreparations were identified as follows: the total fraction of mouse SBNP was used to immunizea rabbit, as described (Rodman et al. 1979). A similar preparation of SBNP was alkylated andthe components separated into 2 classes of basic proteins, protamines and non-protamines, by


3A BFig. 3. A. SDS/polyacrylamide electrophoresis (PAGE), Coomassie blue-stained.Lane 1, calf thymus histone preparation (Vidali, Boffa, Mann & Allfrey, 1978); lane2, 07 M-NaCl extract; lane 3, non-protamine subset of SBNP (13 % to 18 % eluatefrom BioRex-70 chromatogTam of SBNP; Rodman et al. 1981); lane 4, molecularweight markers: alpha lactalbumin, 14400; soy bean trypsin inhibitor, 20000;carbonic anhydrase, 30000; ovalbumin, 43000; bovine serum albumin, 67000;phosphorylase b, 94000. B. Autoradiogram of immunological reactivity of the anti-serum with an unstained duplicate of the gel of A except that lane 4 is a repeat oflane 2. The principal components (e.g. mol. wt 15-18000) and immunological reactivityare present, although not in the same proportions, in the 0-7 M-NaCl extract andthe non-protamine subset of SBNP. The antiserum is not reactive with histones (ortrace amounts of other basic proteins) of calf thymus nuclei.

elution from a BioRex-70 chromatography column with ascending concentrations of guanidiniumchloride (GuCl) as described (Rodman et al. 1981). The antiserum used in this study has beenshown to contain antibodies to the 2 protamines obtained in the 20 % to 50 % GuCl eluateand to the main components of the non-protamine subset of SBNP, obtained in the 13 to18 % eluate (Rodman et al. 1981).

Treatment with 07 M-NaCl. Biochemical extraction of sperm, paralleling the cytochemicaltreatment, was carried out by removing membranes with Triton Xioo (protease inhibitoradded), sonicating to separate heads from tails, collecting the heads by centrifugation througha sucrose gradient, and incubating the heads in the reaction solution used for the cytologicalpreparation (07 M-NaCl, 0-5 % 2-mercaptoethanol (2-ME) in 0-02 M-PO4 buffer, pH 7-2)for 2 h with stirring. Following centrifugation, the proteins in the supernatant were collectedby precipitation with 20 % trichloroacetic acid, resuspended in PBS and designated the07 M-NaCl extract. An aliquot of the 07 M-NaCl extract was co-electrophoresed on a sodium


1 2 3 4 5

Fig. 4. Acetic acid/urea PAGE, Coomassie blue-stained. Lane 1, protamine I (earlyfractions of the 20 % to 50 % eluate from BioRex-70 chromatogram of SBNP);lane 2, protamine II (late fractions); lane 3, 0-7 M-NaCl extract; lane 4, extract ofsperm heads residual after 0-7 M-NaCl treatment; lane 5, calf thymus histonepreparation. The 07 M-NaCl extract contains neither of the protamines, while theresidual sperm heads contain both.

dodecyl sulphate (SDS)/polyacrylamide gel with the 13% to 18% eluate obtained in theBioRex-70 chromatography of SBNP, and a solution of calf thymus histones. That gel wasstained with Coomassie blue (Fig. 3 A) and a duplicate gel was transferred to nitrocellulosepaper (Towbin, Staehelin & Gordon, 1979), treated with the antiserum and the immunologicalreactivity detected by luI-labelled protein A (Fig. 3B). Another aliquot of the 0-7 M-NaClextract was co-electrophoresed on an acetic acid urea gel with an early fraction (representingprotamine I), a late fraction (representing protamine II) of the 20% to 50 % GuCl eluate,and an extract of the sperm heads residual after the extraction with 0-7 M-NaCl (Fig. 4). Theresidual extract was prepared by the standard procedure used to obtain SBNP from spermheads (Rodman et al. 1979).

Treatment with 1 M-NaCl. A preparation of heads from Triton Xioo-rreated sperm wasincubated for 2 h, with stirring, in a solution of 1 M-NaCl, 0-5 % 2-ME in 0-02 M-PO4 buffer.The supernatant and the residual sperm heads were treated as described above and co-electrophoresed on an acetic acid/urea gel with an early fraction and a late fraction of the20 % to 50 % GuCl eluate (Fig. 6).

RESULTS

Removal of acrosomes and sperm head membranes

The outer acrosomal membrane and the acrosomal contents of the sperm aredispersed prior to its entry into the oocyte. In addition, EM studies have shownthat the intra-ooplasmic mouse sperm is devoid of plasma membrane and nuclearenvelope (Stefanini et al. 1969). In order, therefore, to simulate those in vivo


conditions, the sperm suspension used for the cytologic preparation (Figs, i, 2, 5, 7, 8)was treated with Triton Xioo. Examination of the preparations showed that fewerthan 2% of the sperm retained osmiophilic perinuclear structures.

Significance of the IF procedure

The rabbit antiserum to mouse SBNP used in this study has been characterizedpreviously by definition of its immunological reactivity with electrophoreticallyseparated components of the SBNP fraction used for immunization (Rodman et al.


Fig. 5. Sperm treated with 1 M-NaCl, 0-5 % j-ME in 0-02 M-PO4 buffer (pH 72).A, s min; B, 15 min; c, 30 min; D, 60 min; E, 120 min. Swelling and some proteinextraction occurs within 5 min. Brilliant intrahead fluorescence appears first in theposterior head region. The if reactive SBNP are progressively extracted to completion,while the residual DNA complex retains homogeneity and sharp chromatin bound-aries, eb, ethidium bromide.

1981). That characterization showed that the antiserum recognizes the 2 protaminesand the major components of the non-protamine subset of mouse SBNP and isdevoid of immunological reactivity with other components of mouse sperm headsor with calf thymus histones.

We conclude, therefore, that the IF procedure of this study may be used as aspecific probe for the cytological localization of the major components of SBNP andfor detection of their differential displacement.

Swelling of sperm head and displacement of the basic proteins. Separate roles of reducingagent and NaCl

We have previously shown that SBNP may be detected in situ by immuno-fluorescence of sperm heads that have been made to swell and the antigenic sitesof the proteins revealed, and, conversely, that those sites are not displayed in

236 T. C. Rodman, F. H. Pruslin and V. G. AUfrey

Fig. 6. Acetic acid/urea PAGE, Coomassie blue-stained. Lane 1, 1 M-NaCl extract;lane 2, extract of sperm heads residual after 1 M-NaCl extraction; lane 3, protamineII; lane 4, protamine I of SBNP (see legend to Fig. 4). Both protamines are presentin the 1 M-NaCl extract, while neither is present in the residual sperm heads. Thus,the 1 M-NaCl treatment extracts SBNP to completion, confirming the cytologicaldata of Fig. 5.

unswollen sperm heads (Rodman et al. 1979; Pruslin, Romani & Rodman, 1980).The swelling and 'unmasking' of the antigenic sites were attributed to the reductionof the disulphide bonds between cysteinyl residues of the protamines. Since swellingof the sperm head is an early event in transformation in vivo, we considered it ofinterest to determine whether any of the components of SBNP could be displacedprior to, or in the absence of, swelling. Using the 2 probes, changes in sperm headmorphology and immunofluorescent display of SBNP, we investigated the separateeffects of treatment of sperm with reducing agent and salt (Table 1). The morpho-logical and cytochemical indications of the effect are illustrated in Fig. 1. Thosedata indicate that in the absence of reducing agent NaCl does not extract SBNP,whereas, in the presence of reducing agent extraction of SBNP does occur, apparentlyprogressively with increasing concentrations of NaCl. 'Baseline fluorescence' (Fig.1 B), in which some peripheral regions of the demembraned sperm head are stained,is similar to, although consistently more pronounced than, that seen with pre-immuneserum (Fig. 1 A). It may, therefore, represent non-specific background binding ofthe anti-rabbit goat serum, increased because of higher immunoglobulin titres in theimmune rabbit serum. The difference in response to 2-ME with and without the


protease inhibitor, phenylmethane sulphonyl fluoride (PMSF) (Table 1), indicated thepresence and activity of an endogenous protease, confirming previous observations(Marushige & Marushige, 1978). All experiments probing the action of NaCl, there-fore, included PMSF.

'Intrahead haze' (Fig. ic) may represent a minor component of the SBNP thatis more readily reactive with the reducing reagent. The fluorescent haloes (Fig. 1 D,E, F) clearly represent some components that are extracted and, as they are highlyinsoluble in the reaction solution (pH 7-2), are deposited on the slide and areadherent. An ancillary point of interest that may be noted is the fact that SBNP areimmunologically reactive both in the chromatin complex and free in the extractionhaloes. Swelling represents a stage in decondensation of the chromatin where volumechanges are perceptible (Fig. 1 F). The volume change is presumably related to stericchanges in SBNP (Rodman et al. 1979), which follow reduction of the disulphidelinkages and result in the display of antigenic sites, since swelling is accompaniedby display of far more brilliant intrahead fluorescence (Fig. 1 F) than that seen inunswollen sperm (Fig. 1 B, C, D, E).

The data of Table 1 show that both protein extraction and swelling require theaction of a reducing agent, but that the 2 progressions are not entirely interdependent:some extraction takes place in the absence of swelling. Since those observationssuggested that different components of SBNP are extracted at specific salt con-centrations, we carried out a series of treatments with 0-7 M- (Fig. 2) and I-M-(Fig. 5) NaCl, taking samples over a 2-h period. We also monitored the retentionof chromatin organization in the extraction and swelling progressions by EB stainingof the DNA complex.

Treatment with 07 M-NaCl

The micrographs in Fig. 2 show that some components of the sperm nucleus areextracted by 0-7 M-NaCl with no alteration of sperm head morphology or dispersionof the DNA complex. The progression appears to be that of continued extractionwithout considerable accumulation within the nucleus, since the intrahead fluorescenceis never as intense as might be expected from the size and density of the subsequentlyproduced extraction haloes. At any given interval, the diameters of the haloes areremarkably constant. After 2 h of treatment (Fig. 2c) the haloes appear to reachtheir maximum size and density and the DNA/protein complex remaining in thenucleus appears to be intact. Even after that extended treatment with 0-7 M-NaClthere is no swelling, other alteration of nuclear morphology or dispersion of anyDNA-complexed moiety.

Figs. 3 and 4 present biochemical data paralleling the cytological data of Fig. 2.The 0-7 M-NaCl treatment extracts the major components of the non-protaminesubset of SBNP, but extracts neither of the 2 protamines. The autoradiogram of theimmunological reactivity of the antiserum (Fig. 3B) shows no recognition of histones,while previous characterization of the antiserum (Rodman et al. 1981) showed thatit does not contain antibodies to other moieties of mouse sperm that might be presentin trace amounts in the SBNP fraction. The IF of Fig. 2, therefore, may be interpreted


B

D


to represent one or more of the components of the non-protamine subset of SBNP.The data also imply that displacement of the non-protamine subset occurs withoutconcomitant swelling of the sperm nucleus.

Treatment with 1 M-NaCl

Treatment of the sperm preparations with 1 M-NaCl results in a progression ofeffects (Fig. 5) quite different from those induced by the 07 M-NaCl (Fig. 2). The1 M-NaCl progression includes swelling and change in conformation of the nuclearproteins that result in display of a large number of antigenic sites of those proteinswithin the sperm nucleus. The latter occurs in a specific regional sequence: theposterior regions of the sperm nuclei light up first (Fig. 5B), then the entire spermhead displays brilliant fluorescence (Fig. 5 c). With continued incubation in the1 M-NaCl solution, the intrahead fluorescence is progressively depleted as thoseproteins are extracted (Figs. 5 D, E). At the end of the 2-h interval, the nuclei areentirely depleted of the immunofluorescently reactive proteins (Fig. 5E). However,even though the nuclei are greatly swollen, the general contours of the sperm mor-phology are conserved and the distribution of the DNA complex, as revealed bystaining with EB, appears to be homogeneous and contained within sharply definedchromatin boundaries. Again, as in the 0-7 M-NaCl treatment (Fig. 2) the 1 M-NaClextraction of SBNP (Fig. 5) appears not to disrupt the general organization of theDNA in its residual complex. Biochemical analysis of the effects of the 1 M-NaCltreatment showed that SBNP is extracted to completion (Fig. 6), confirming thatthe non-fluorescent residual sperm heads of Fig. 5E are depleted of SBNP.

Treatment with trypsin

Since the possibility has been considered that the biological method of displacementof SBNP might be that of proteolysis (Marushige & Marushige, 1978; Zirkin et al.1980), we utilized the double-staining system to determine whether orderly extractionof the proteins without disruption of DNA organization could be induced by mildtreatment with trypsin (Figs. 7, 8). One of the striking differences between the saltand the trypsin series is the heterogeneity of effects observed in the latter. In theNaCl treatment series (Figs. 2, 5), homogeneity of reaction on a single slide withrespect to morphological changes, halo size and density was characteristic. In thetrypsin series (Figs. 7, 8), although the general level of response was reproduciblein repeated experiments and the degree of response was progressively greater withincrease in both trypsin concentration and reaction time, in any given cytologicalpreparation there was a spectrum of morphological changes and immunofluorescencepatterns. Some sperm in each preparation resembled, to some extent, those of the1 M-NaCl treatment: swollen, with the general contours of the mouse sperm

Fig. 7. Sperm preparations treated with 1 /Jg/ml trypsin, 0-5 % 2-ME in 0-02 M-PO«buffer (pH 7-2). A, 5 min; B, 15 min; c, 30 min; D, 60 min. From left to right: phasecontrast, if, eb. With increased swelling, the antigenic sites of the SBNP are revealed,and those proteins are deleted. However, the progression appears to be randomand the residual DNA complex is not conserved in the SBNP-depleted structures.


Fig. 8. Sperm preparations treated with 10 /tg/ml trypsin, 0-5 % 2-ME, in 002 M-PO4buffer (pH 7-2). Neither immunofluorescence nor chromatin decondensation pat-terns appear to be non-random or consistent in any preparation. With swelling anddisplacement of SBNP the residual DNA complex is irregularly dispersed andchromatin boundaries are eroded.

preserved and with brilliant intrahead fluorescence (e.g. Figs. 7, 8 A). Most, however,even in the low trypsin concentration short-interval preparations, developed bizarreshapes that bore little resemblance to the specific shape of the mouse sperm. Parti-cularly characteristic was the ' dumb-bell' shape in which the anterior and posteriorregions were swollen, while the mid-region remained condensed (e.g. Figs. 7B, c, 8 A).EB staining of those sperm indicated that the DNA of the swollen regions wasirregularly dispersed and the chromatin boundaries were eroded. The distributionof basic proteins, as displayed by the immunofluorescence, was inhomogeneous andgreatly variable from sperm to sperm. In the advanced stages of swelling (Figs. 7c, n,


Table 1. Imrnunofluorescence (IF) patterns produced on mouse sperm with antiserum toSBNP, following treatment with a series of NaCl concentrations with and withoutreducing agent (2-ME)

Treatment solution

Buffer onlyo-i M-NaCl0-3 M-NaCl0-5 M-NaClo-6 M-NaCl07 M-NaClo-8 M-NaCl1 M-NaCl2-ME2-ME, PMSF2-ME, PMSF, 01 M-NaCl2-ME, PMSF, 0-3 M-NaCl2-ME, PMSF, 05 M-NaCl2-ME, PMSF, 06 M-NaCl2-ME, PMSF, 0-7 M-NaCl2-ME, PMSF, o-8 M-NaCl2-ME, PMSF, 1 M-NaCl

Duration

5

BB

BBBBBBB

B

B

C

C

CDD

F

of treatmentA

30

BB

BBBBB

B

CB

B

CC

CE

B

F

(min)

60

BB

BBB

B

B

B

C

B

C

C

C

C

E

E

F

All solutions were in 0-02 M-PO4 buffer, adjusted to pH 7-2.The letters refer to the micrographs of Fig. 1. In the absence of reducing agent, no change

in sperm head morphology or IF is detected. In the presence of 2-ME, NaCl solutions inducechanges in SBNP that result in availability of antigenic sites. The effect is greater withincreasing concentrations of NaCl. Detectable increase in nuclear volume (swelling) occurswhen the NaCl concentration of the treatment solution exceeds o-8 M. With PMSF omitted,treatment with 2-ME results in some display of IF that may be attributable to proteolysis byacrosomal enzymes that have not been removed in the preparative procedure.

8 A, B) the basic proteins, as well as the DNA complex, appeared to be randomlydispersed. Immunofluorescently positive extraction haloes were seen infrequently,and when seen were not uniform in diameter, suggesting that the diffusing proteinfragments were of variable size.

DISCUSSION

Two requirements for the transformation of sperm chromatin to zygote pronucleusare recognized, a priori: (1) the sperm-unique basic nuclear proteins must be dis-placed ; and (2) the DNA of the paternal genome must be conserved. In this study,we have described an in vitro system in which the early events of transformationmay be simulated and in which those 2 conditions may be monitored. The displace-ment of the SBNP of mouse sperm is monitored by immunochemical methodsutilizing rabbit antiserum raised to the total fraction of mouse SBNP; that antiserumis an especially suitable probe since it contains antibodies to the principal componentsof both classes of SBNP identified in mouse sperm nuclei. Thus, a single immuno-


chemical reagent is used to display the differential displacement of each, the protamineand the non-protamine classes, and the relationship of those progressions to de-condensation of sperm chromatin.

Analysis of the data yielded by that probe suggests that the DNA-protein relation-ships of the sperm chromatin are not destabilized by treatment with 0-02 M-PO4

buffer alone, but that the addition of 2-ME induces some configurational changethat results in rendering a small portion of the antigenic sites of those proteinsavailable for immunological reactivity (Table i). Presumably that change is relatedto reduction of disulphide bonds. In the absence of extraction haloes (Fig. i c), weinfer that no displacement of the proteins takes place with reducing agent alone.With an increase in ionic concentration by the addition of 0-7 M-NaCl, however,some or all of the non-protamine class of basic proteins (but neither of the 2 protamines)are dissociated and displaced from the chromatin. Whatever the mechanism of thatdisplacement, it appears not to be dependent upon nor the cause of sufficientdecondensation of the chromatin to be detectable as an increase in nuclear volume.Moderate elevation of the concentration of the NaCl treatment solution to 1 Minduces progressive swelling, regionally sequential display of antigenic sites ofproteins in situ in the chromatin and culminates in complete displacement of bothclasses of basic proteins of the SBNP fraction. In summary, the data suggest thatmere reduction of S-S bonds does not result in dissociation from the sperm chromatinof either class of the components of SBNP, and that decondensation (swelling)results only when dissociation of the protamines, which constitute 98% of theSBNP (Rodman et al. 1981), does occur.

A particularly significant index of the specificity of the 1 M-NaCl-induced deletionof the basic proteins is the observation that the extraction goes to apparent completionwithout disruption of the residual DNA complex or erosion of the chromatin bound-aries (Fig. 5 E). Those observations parallel an extensive body of reports on somaticcell interphase and metaphase chromatin: treatment with a spectrum of salt con-centrations results in selective extraction of histones (e.g. Ohlenbusch, Olivera,Tuen & Davidson, 1967), leaving a de-histoned structure in which the residualDNA complex is highly organized and the nuclear or chromosomal morphology isretained (Adolph, 1980).

Since proteolysis has been invoked as the primary in vivo mechanism of displacementof SBNP (Marushige & Marushige, 1978; Zirkin et al. 1980), we used the in vitrosystem to study the effectiveness of a protease in displacing the SBNP withoutdisrupting the DNA-residual complex. We selected trypsin as the protease since theenzyme postulated to participate in sperm transformation, acrosin, is very similarto trypsin with respect to active site conformation, splitting specificity and affinityfor natural and synthetic inhibitors (Schleuning & Fritz, 1976). However, althoughthe enzyme induced decondensation, display of antigenic sites and displacement ofthe basic proteins, no order or homogeneity of those effects could be defined. Further,we could find no set of conditions in which both complete extraction of the basicproteins and retention of DNA-complex organization could be achieved by treatmentof the sperm with trypsin in a reducing medium. Once again, we note the parallel


with histone-depleted somatic chromatin: the residual structure is dispersed bymild treatment with protease (Anderson, 1953; Adolph et al. 1977).

Extrapolation of the data obtained by the in vitro model to the fertilizing spermin vivo must as yet be considered speculative. Those data, however, indicate that it ismechanistically possible for an early and mandatory step in transformation, dis-placement of the SBNP, to be induced by the action of NaCl in reducing solution.The reducing potential of the fertilizable ooplasm has been confirmed by demon-stration of a high content of SH groups (Rodman et al. 1981), while the possibilityfor intra-ooplasmic elevation of cation concentration is suggested by observationsof post-fertilization cation influx into invertebrate eggs (Hagiwara & Jaffe, 1979).It is particularly relevant to note that the specific immunofluorescent display ofSBNP in the posterior region of the sperm head after a short interval of treatmentwith 1 M-NaCl (Fig. 5B) is similar to that seen in intra-ooplasmic sperm at a veryearly stage of transformation (Rodman et al. 1981). Clearly, the intra-ooplasmicelevation of cation concentration would not be expected to be of the magnitude ofthe 1 M-NaCl added to the 0-02 M-PO4 buffer of the in vitro reaction solution. Farless elevation of the complex ionic content of the ooplasm or a transient increase inthe microenvironment of the fertilizing sperm could reasonably be postulated.Further, it is logical to assume that the kinetics of transformation are modulatedby multiple factors. Although the data obtained in this study suggest that proteolysisalone is not the mechanism of SBNP displacement in vivo, we do not rule out thepossibility that a highly specific protease originating from sperm chromatin orooplasm may be activated at fertilization and may participate interactively with ionfluxes. The cytological model of this study provides an in vitro system in which suchinteractions may be studied.

This work was supported by grants from the Harry Winston Research Foundation Inc.and the National Institutes of Health (HD13664). We thank Ms Alinda Barth for excellenttechnical assistance, particularly in the preparation of the micrographs.

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{Received 12 May 1981)

mechanisms of displacement of sperm basic …pate in the post-fertilization displacemen of thet...

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