Sperm nuclear chromatin transformations in somatic cell-free extracts

MOLECULAR REPRODUCTION AND DEVELOPMENT 37:305-317 (1994)

Sperm Nuclear Chromatin Transformations in Somatic Cell-Free Extracts SUBHASIS BANERJEE AND MAJ HULTEN LFS Research Unit, DNA Laboratory, Birmingham Heartlands Hospital, Yardley Green Road, Birmingham, United Kingdom

ABSTRACT HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was com- pleted within 10-20 min. The average enlargements of human and pig sperm nuclei were 15-fold and 3-fold, respectively. The structural organization of pig and human sperm chromatin was significantly different. Decondensa tion was differentially inhibited by Mg++ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleoso- ma1 structures within 60 min. The remodelled chromatin could be recondensed to chromosome-like structures, when incubated further in extracts from mitosis arrested HeLa cells. o 1994 Wiley-Liss, Inc.

Key Words: HeLa extracts, Chromatin decondensation, Remodelling, Recondensation

INTRODUCTION The earliest chromosomal event during fertilization

is the decondensation (Longo and Kunkle, 1978) of protamine and nucleosome-bound supercondensed sperm chromatin (Bedford and Calvin, 1974; Balhorn, 1982). Besides chromatin dispersion, the removal of sperm- specific basic proteins, remodelling of chromatin by assimilation of egg histones, and recondensation of chromatin are necessary steps towards the formation of a male pronudeus (Poccia, 1986 Longo, 1990). Many of these macromolecular events can be studied by insemi- nating eggs (Uehara and Yanagimachi, 1976; Per- reault et al., 1984; Clarke and Masui, 1986) or by incubating demembranated sperm nuclei in extracts from either Xenopus eggs (Lohka and Masui, 1983, 1984; Brown et al., 1987; Ohsumi et al., 1988; Philpott et al., 1991) or Drosophila embryos (Ulitzur and Gruenbaum, 1989; Berrios and Avilion, 1990). Extensive work on Xenopus extracts has revealed that nucleoplasmin, besides facilitating nucleosome assembly (Laskey et al., 1978; Dilworth et al., 1987; Kleinschmidt et al., 19901, decondenses frog sperm chromatin (Philpott et al., 1991; Ohsumi and Katagiri, 1991). On the other hand, 0 1994 WILEY-LISS, INC.

the maturation promoting factor (MPF), which is abundant in activated eggs, has been shown to be one of the components needed to trigger meiotic maturation and condensation of interphase nuclear chromatin (Masui and Clarke, 1979; Sunkara et al., 1979; Miake-Lye and Kirschner, 1985; Newport and Spann, 1987; Adachi et al., 1991).

Xenopus eggs contain stockpiles of proteins which can support thousands of rounds of cell division without measurable RNA synthesis (Graham and Morgan, 1966; Newport, 1987). On the contrary, the factors required for pronuclear and chromosome formation in mammalian eggs are very limited (Witkowska, 1981; Clarke and Masui, 1986). Cell-fusion studies have been unable to indicate clearly whether differentiated mammalian cells can induce decondensation of sperm chromatin in heterokaryons (Johnson et al., 1970; Sawicki and Koprowski, 1971; Gledhill et al., 1972). Biochemi- cal experiments to identify chromatin decondensation and remodelling factors using mammalian egg or oocyte extracts are impracticable, if not impossible. However, such issues would be amenable to experimental analysis if mammalian somatic cell extracts were able to mimic these activities. Here we have tested this possibility and report that HeLa cell extracts are able to decondense permeabilized sperm chromatin very rapidly and facilitate chromatin remodelling. Addition- ally, the decondensed sperm chromatin can be recondensed to chromosome-like structures in extracts derived from HeLa cells arrested at mitosis.

MATERIALS AND METHODS HeLa Cell Extracts

The HeLa interphase extracts (HIE) were prepared from cultures grown exponentially in suspension (6- 8 x lo6 cells per ml, S3 type, kindly provided by ICRF, London) as described previously (Banerjee and Cantor, 1990; Banerjee et al., 1991). One microliter of these extracts (18-22 pg of protein) could assemble 16-25 ng of DNA into nucleosomes as determined by DNA supercoiling and micrococcal nuclease (MNase) digestion as-

Received June 1,1993; accepted July 28,1993. Address reprint requests to Subhasis Banerjee, LFS Research Unit, DNA Laboratory, Birmingham Heartlands Hospital, Yardley Green Road, Birmingham B9 5PX, UK.

306

says. The HeLa mitotic extracts (HME) were prepared as follows: Cells were grown in suspension culture a t a concentration of 5 x lo6 cells per milliliter and arrested a t S-phase by adding an equal volume of fresh medium and thymidine at a final concentration of 2.5 mM. At 20 h, following thymidine addition, the cells were spun at 1,000 rpm at room temperature for 5 min. The cells were washed twice with suspension culture medium (S-MEM) without serum to remove traces of thymidine. The washed cells were resuspended in a separate spinner flask using two thirds of the original volume of complete medium. After 4-6 h of incubation, nocodazole, [5-(2-thienylcarbonyl)-lH-benzimidazole- 2-ylI carbamate, was added at a concentration of 10 pg/ml. At 10 h following nocodazole addition, 5 ml of the culture was aliquoted and the mitotic index was determined by staining methanol-acetic acid fixed cells with Giemsa and was observed using bright-field mi- croscopy.

Extracts were prepared only from cultures in which more than 90% cells were arrested a t metaphase. The cells were sedimented by centrifuging a t 1,200 rpm for 5 min at 4°C and washed three times in a buffer containing 20 mM HEPES, pH 7.9, 80 mM P-glycerophosphate, 15 mM MgClz, and 20 mM EGTA. The pellet was resuspended in 4 volumes of the same buffer containing 1 mM y-S ATP, 0.5 mM PMSF, I X protease inhibitor cocktail (leupeptin, chymostain, APMSF, pepstain A at 10 pglml; antipain and aproteinin at 5 pg/ml), and ho- mogenized on ice with 15 strokes in a Teflon-coated glass homogenizer. The homogenate was transferred to a flask containing 4 volumes of 50 mM Tris-HC1, pH 7.9,lO mM MgCl,, 0.75 M sucrose, 2 mM DTT, and 50% glycerol. The mixture was gently stirred on ice for 5 min. Cellular proteins were extracted with saturated ammonium sulphate, pH 7.6, at a final concentration of 0.5 M, for 30 min. The lysate was spun at 45,000 rpm for 3 h a t 4°C. The clear supernatant was transferred to a flask held on ice, and the proteins were precipitated with 0.33 g of ammonium sulphate per milliliter of the supernatant. The precipitated proteins were sedimented by centrifugation at 30,000 rpm for 30 min. The pellet was resuspended in a dialysis buffer (one fifth the original volume of the supernatant) containing 20 mM Hepes-KOH, pH 7.9, 27 mM P-glycerophosphate, 150 mM potassium glutamate, 15 mM MgCl,, 6.5 mM EGTA, 1 mM DTT, 0.1 mM y-S ATP, and 15% glycerol. Dialysis was carried out using one change of 30 volumes of the buffer for 8 h. The dialysed extract was clarified by centrifugation at 30,000 rpm for 30 min at 4°C. The clear supernatant was aliquoted, frozen in liquid nitrogen, and stored at -70°C.

Preparation of Sperm Nuclei Lysolecithin permeabilized Xenopus sperm nuclei

were kindly provided by Julian Blow, Clare Hall Labo- ratories (ICRF, England). The human and pig sperm nuclei were prepared as follows: 5 ml of human semen was liquified by incubating at 37°C for 45 min, and diluted to 50 ml by adding sperm wash buffer (SWB: 20

S. BANERJEE AND M. HULTEN

mM Hepes-KOH, pH 7.0, 100 mM potassium glutamate, 250 mM sucrose, 0.5 mM spermidine free base, and 0.2 mM spermine tetrahydrochloride). After filtra- tion through 8 layers of gauze, the diluted semen was spun at 2,000 rpm for 15 min. The pellet was washed once with 50 ml of SWB and resuspended in 2 ml of SWB. One milliliter of this suspension was gently lay- ered over 4 ml of 70% Percoll equilibrated with SWB and centrifuged at 3,000 rpm for 30 min. The sediment was carefully removed, washed twice with SWB, resuspended in 2 ml of SWB containing 0.05% lysolecithin (L-a-lysophosphotidylcholine) and incubated on ice for 10-12 min. The reaction was diluted by adding 3-4 volumes of cold SWB containing 3 mg/ml of BSA. The sperm were sedimented by centrifuging at 2,000 rpm for 15 min.

The pellet was washed twice with SWB containing BSA and finally with SWB alone. The permeabilized sperm were treated with 5 mM DTT for 15 min at room temperature. After pelleting again, the sperm were resuspended in SWB containing 1 x protease inhibitor and 20% glycerol, counted using a haemocytometer, aliquoted, frozen in liquid nitrogen, and stored a t -70°C. Fresh pig semen was diluted in HTB buffer (84 mM Hepes-KOH, pH 6.5, 40 mM potassium sodium tartrate, 14 mM NaHCO,, 7.5 mM EDTA, 35 mM KC1, 3.7% glucose, 600 pg/ml penicillin, 1 mg/ml streptomy- cin, and 32 pgiml gentamycin; osmolarity, 505 mOs/kg H,O) and stored at room temperature for up to 3 days. The sperm were permeabilized with lysolecithin as described above. In some preparations the SWB contained 2.5 mM MgCl, instead of spermine and spermidine.

Sperm Chromatin Decondensation Reaction Lysolecithin permeabilized sperm nuclei were incu-

bated at 35°C in a 25-50 pl reaction mixture containing 1,000-5,000 sperm per microliter of HIE (18-22 pg of protein), 20 mM Hepes-KOH, pH 7.9; 150 mM KC1,O.Ol mM EDTA, l x protease inhibitors, 1 mM DTT, 5 mM creatine phosphate (CP), and 1 mM ATP, either with or without 5 mM Mg++. To examine decondensation of the sperm nuclear chromatin, 4 pl of the reaction mixture was mixed with 4 pl of the buffer, containing 50% glycerol and 5 pg/ml of propidium iodide (PI), on a microscope slide and viewed using a fluorescence microscope (Nikon, Japan), equipped with phase rings and a photo- graphic camera (FX-35DX). The phase and fluorescent micrographs were taken on either Kodak technical pan film (TP-135-36) or Fujichrome 400 D. The nuclear membrane was examined by mixing an aliquot of decondensed sperm with 10 pg/ml of DHCC (3,3’-dihexyl- oxacarbocyanine iodide, Kodak), dissolved in DMSO, and subsequently in a buffer containing 50% glycerol, and was viewed using a fluorescein filter (Nikon, B1 A).

Measurement of Sperm Nuclear Chromatin Decondensation: A Time-Course Analysis

Lysolecithin permeabilized human and pig sperm nuclei were incubated in the extracts or in the reaction

SPERM CHROMATIN TRA .NSFORMATIONS IN HELA EXTRACTS 307

Sperm Nuclear Recondensation Sperm chromatin recondensation was carried out fol-

lowing two different protocols: (a) the sperm chromatin was decondensed in the presence of 1 mM ATP, 5 mM creatine phosphate, and 2.5 mM Mg++ in a 50 pl reaction for 1 h at 34°C. Twelve microliters of mitotic extract and 250400 pM y-S ATP were added and further incubated for 90 min; (b) the nuclei were first decondensed in the presence of ATP and creatine phosphate for 20 min, then 5 mM Mg" was added and further incubated for 40 min before adding mitotic extract and y-S ATP. Aliquots were stained with PI or DAPI (0.5 Fg/ml), examined, and photographed as described above.

buffer. Aliquots were taken onto microscope slides at different time points, stained with PI, and the nuclear fluorescence was visualized as described above. The images with the lowest fluorescence intensity were captured employing a calibrated Prismview software pro- gram on an Apple Macintosh I1 Ci. Images were automatically processed for maximum contrast and stored in a 44 megabyte (SQ 400) removable cartridge. The nuclear areas were measured and the data were transferred to a Microsoft Excel spreadsheet for further processing and statistical analysis. The areas of permeabilized human and pig sperm nuclei were determined by measuring 40 nuclei of each sample. The average areas of lysolecithin permeabilized human and pig sperm nuclei were determined to be 12 pm2 and 36 pm2, respectively.

Assays for Studying Chromatin Structure and Chromatin Remodelling

To examine the structural organization of sperm chromatin, human and pig sperm nuclei, with or without preincubation in the presence of cofactors, were digested with MNase in reaction buffer containing 3 mM CaC1, a t 35°C. To study chromatin remodelling, the sperm nuclear chromatin was first decondensed in HIE in the presence of 1 mM ATP and 5 mM CP for 20 min and further incubated for 40 min in the presence of 5 mM Mg++. CaC1, and MNase were then added at concentrations of 3 mM and 300 U/ml, respectively, to digest the chromatin. Nuclease activity, of aliquots taken at different times, was stopped with a mixture of EDTA, EGTA, and N-lauroylsarcosine at a final concentration of 12.5 mM, 12.5 mM, and 62.5 mM, respectively. The mixtures were then treated with RNase A, SDS, and proteinase K, followed by phenol-chloroform extraction.

The DNA was ethanol precipitated and resolved in 1.5% agarose gels as described previously (Banerjee and Cantor, 1990). To analyse the protein composition of the remodelled chromatin, the decondensation reac- tions were diluted with 4 volumes of the buffer containing 5 mM Mg++ and 250 mM sucrose, and centrifuged at 6,500 rpm for 10 min. The pellet was washed twice with the same buffer and extracted with 0.25 N HC1 on ice for 1 h. The acid extracted proteins were precipitated with 20% trichloroacetic acid (TCA) for 1 h on ice. The precipitate was washed once with cold acidified acetone and twice with cold acetone. The proteins were separated in 18% discontinuous polyacrylamide-SDS gels as described previously (Banerjee et al., 1991). To examine the protein composition of the sperm nuclei, the acid soluble (0.25 N HCI) proteins extracted from the nuclei were precipitated with TCA and resolved in SDS gels as above or loaded on 15% polyacrylamide acid urea Triton (AUT) gels as described previously (Banerjee et al., 1991), except that the stacking gels did not contain potassium acetate. The gels were stained with Coomassie blue, destained, and photographed.

RESULTS HeLa Cell-Free Extracts Induce Sperm Nuclear

Chromatin Decondensation HeLa cell-free extracts, like those of the Xenopus

oocyte, are capable of assembling physiologically spaced nucleosomes (Banerjee and Cantor, 1990). Both systems require energy and acidic proteins to assemble nucleosomes. Phosphorylation of histone H2a corre- lates with the physiological spacing of nucleosomes assembled in both of these extracts (Kleinschmidt and Steinbiser, 1991; Banerjee et al., 1991). Moreover, nucleosome assembly and sperm chromatin decondensation are mediated by the same factor, nucleoplasmin, in Xenopus extracts (Philpott and Leno, 1992). These results led us to investigate whether HeLa cell extracts are also capable of sperm chromatin decondensation.

Initially we used lysolecithin permeabilized Xenopus sperm nuclei for these studies. As shown in Figure 1A (c,d), the sperm chromatin decondensed within 5-10 min of incubation in HeLa interphase extract. Decon- densation did not occur in reaction buffer without the extract (Fig. lA, a,b), suggesting that it was mediated by factors present in the extract. Prolonged incubation (60-90 min) did not alter the size of the decondensed nuclei; however, some rounded nuclei were observed (Fig. lA, e,f). The sperm chromatin dispersion was independent of exogenous ATP and Mg++. In fact, 5 mM 5' AMP-PNP, a nonhydrolyzable ATP analog, did not inhibit the decondensation (data not shown), indicating the apparent lack of energy requirement for chromatin decondensation. Like Xenopus sperm nuclei, both human and pig sperm chromatin decondensed within 10-20 min in HIE, as shown in Figure 1B (e,n and 1C (e,f). Once again, decondensation was not observed in buffer controls (Fig. 1B c,d; and 1C c,d). These results indicated that the decondensation factors present in HIE were not species specific.

Divalent and Polyvalent Cations Inhibit Sperm Nuclear Chromatin Decondensation

Experiments described above were carried out in HIE dialysed in Mg++-free buffers. These extracts contained very low levels of endogenous Mg++ and ATP, insuffi- cient to generate physiologically spaced nucleosomes

308 S. BANERJEE AND M. HULTEN

A. X E N O P U S B. P I G C H U M A N

Fig. 1. Decondensation of sperm chromatin in HeLa extracts. A: (a and b), nuclei incubated in buffer for 60 min at 35°C; c and d, same as a and b, except that extract was added and incubation was for 10 min; e and f, same as c and d, except incubation was for 60 min. B: (a and b), lysolecithin-treated pig sperm; c and d, sperm nuclei incubated in

the buffer for 30 min at 35°C; e and f, nuclei incubated in the extract for 15 min. C: (a and b), lysolecithin-treated human sperm nuclei; c and d, nuclei incubated in the buffer for 30 min at 35°C; e and f, nuclei incubated in the extract for 10 min. Bar: A, 58 km; B and C, 34 km.

on naked DNA (Banerjee et al., 1991). In these extracts, decondensation of Xenopus sperm chromatin occurred equally well in the presence or absence of exogenous ATP and Mg+ + (data not shown). However, human and pig sperm nuclear dispersion was inhibited when ATP and Mg++ were incorporated into the reaction. Subse- quent analyses showed that exogenous Mg+ + and polyvalent cations (spermine and spermidine) inhibited the decondensation of mammalian sperm chromatin (see below and data not shown).

In order to investigate this phenomenon in detail, the quantitative increase in area of the sperm nuclei, in the presence and absence of 5 mM Mg++, was measured as a function of time. The results as shown in Figure 2A and 2B revealed that both human and pig sperm chromatin decondensation began almost immediately on incubation in HIE in the absence of exogenous Mg++. However, human sperm chromatin decondensed by an average of 15-fold compared to an average %fold enlargement of pig sperm nuclei. Pig sperm chromatin decondensation was inhibited more effectively by exogenous Mg++ compared to its human counterpart. In the presence of 5 mM Mg++, human sperm nuclei were enlarged 5-fold compared to a 30% increase in area of the pig sperm nuclei (Fig. 2A,B). Since identical experimental conditions were used for both sperm nuclei, we concluded that the extent of decondensation and Mg++-

mediated inhibition in HIE were species dependent. Surprisingly, the cationic inhibition had an all-or-none effect, that is the nuclei enlarged 3- to 10-fold or failed to disperse a t all.

The differential cationic inhibition of mammalian chromatin decondensation led us to ask further if such an inhibition, which remained unidentified in our earlier experiments with Xenopus sperm, could be seen after chelating the endogenous Mg+' in the extract. To test this, we preincubated HIE with 15 mM EDTA prior to adding the Xenopus sperm nuclei and further incubation. This resulted in a dramatic enlargement of the nuclei (Fig. 2C, c-0, indicating that such cationic inhibition of decondensation did occur for Xenopus sperm nuclei. EDTA alone failed to induce chromatin dispersion (Fig. 2C, a,b), further suggesting that the cationic block was due t o the endogenous Mg++ of the extract. We concluded that Mg++ differentially inhibited the decondensation of Xenopus, human, and pig sperm nuclei in HIE; inhibition was least for Xenopus and most for pig sperm chromatin.

Chromatin Organization of Human and Pig Sperm Is Different

In the absence of exogenous Mg++, the average enlargement of the human sperm nuclei in HIE was about 5-fold greater than that of pig sperm, even after an

A. HUMAN

SPERM CHROMATIN TRANSFORMATIONS IN HELA EXTRACTS 309

C. B* P IG XENOPUS

' 1 , T . . ..._ c.______

n 111 20 30 411 so 60

lncuhation Time (min)

Fig. 2. Cationic inhibition and time-course analysis of sperm chromatin decondensation. A and B Each time point represents the average area of 25-44 nuclear images captured randomly; closed circle (*), nuclei and extract; open circle (01, nuclei, extract plus 5 mM Mg++; closed triangle (A), nuclei and buffer; open triangle (A), nuclei in buffer plus 5 mM Mg' + . Right-hand panels of each graph are exam-

incubation of 2 h. We hypothesized that the difference could be due to intrinsic variation in biochemical composition and structural organization of the sperm chromatin. This possibility was tested by two complemen- tary experiments. Firstly, we examined the primary organization of chromatin by limited digestion of the human and pig sperm nuclei with micrococcal nuclease (MNase). Secondly, the abundance of basic proteins (protamines and histones) in these two nuclei was compared.

A striking difference in sensitivity of human and pig sperm chromatin to MNase was observed. The bulk of human chromatin DNA digested to give products of between 100 bp and 1 kb, whereas pig chromatin digestion generated mostly fragments of more than 1 kb ( Fig. 3A). Even when the concentration of the nuclease and the length of digestion of pig nuclei were increased, a similar resistance to MNase was evident (Fig. 3B). These results suggested that the bulk of human sperm chromatin was loosely packed and folded into domains with repeating structures, whereas pig sperm nuclei were more compactly organized and therefore less ac- cessible to MNase. Notably, no regularly spaced nucleosomal structures of either human or pig sperm chromatin could be detected under these nuclease digestion conditions. However, human sperm nuclei, incubated for 1 h at 35°C in the presence of ATP and Mg++ before MNase digestion, produced distinct mononucleosome length fragments (145-155 bp). Comparing the nucleo- soma1 DNA content of human and pig sperm revealed that human sperm had significantly greater amounts

ples of nuclear images processed for each condition. C: (a and b), Xenopus nuclei incubated in buffer and 15 mM EDTA for 30 min; c-f, nuclei incubated for 15 min in extract preincubated with 15 mM EDTA for 15 min. a, c, and e, phase; b, d, and f, fluorescence. Bar: 75 Wn.

of nucleosome-bound chromatin (Fig. 3 0 . Accordingly, histones extracted from the sperm chromatin showed that human sperm contained at least 3-fold more histones than pig sperm (Fig. 3D,E). Also, in agreement with previous reports (Gatewood et al., 1987, 1990a), the human sperm contained all the somatic histones, including the heteromorphous H2a (H2a.X), except H1. H4 and H3 were highly acetylated (Fig. 3E).

Sperm Nuclear Envelope Disintegrates Following Extensive Chromatin Decondensation

Lysolecithin increased the permeability of sperm nuclei without disrupting the general architecture of the nuclear envelope. This was verified by staining the permeabilized sperm nuclei with DHCC, a fluorescent stain specifically highlighting the membrane (Fig. 4a,b). The nuclear envelopes of decondensed nuclei were completely disintegrated, with remnants of the fluorescing particles loosely attached to the periphery of the nuclei (Fig. 4c,d). This observation prompted us t o examine the membranes of nuclei which failed to decondense in HIE in the presence of 5 mM Mg++. These nuclei exhibited more discrete envelopes compared to their untreated counterparts (Fig. 4; compare a with e, and b with 0. Moreover, pig sperm treated with 0.05% lysolecithin in sperm wash buffer, containing 2.5 mM Mg++ instead of spermine and spermidine, failed to decondense in HIE, even in the absence of exogenous Mg++ (data not shown). These results suggested that the presence of exogenous Mgt+ in HIE altered the


A B

SDS Fig. 3. Structural organization of pig and human sperm chromatin.

A: 4.8 X lofi sperm nuclei in reaction buffer were digested with 330 U per milliliter of MNase up to 24 min at 35°C and aliquots were taken at 4,8 ,16 , and 24 min. DNA extracted from aliquots was resolved in 1.5% agarose gel; pig, lanes %5; human, lanes 7-10, B same as A, except that the nuclei were digested with 600 U per milliliter of MNase for up to 32 min and aliquots were taken at 8, 16, 24, and 32 min (lanes 2-5). C: same as A, except that the nuclei were incubated at 35°C for 60 min in the presence of 1 mM ATP, 5 mM creatine phosphate (CP), and 5 mM Mg++ which was added after first 20 min of incubation; human, lanes 14; pig, lanes 8-11. A (lanes 1 and Il), B

C

AUT (lanes 1 and 6) and C (lanes 5 and 7) are DNA size standards; A (lane 6), B (lane 7), and C (lane 6) are MNase digested products from HeLa nuclei. D: Acid (0.25 N HCl) extracted proteins from pig (3 x lo7 nuclei) and human (1.4 x lo7 nuclei) sperm nuclei were resolved in 18% polyacrylamide-SDS gel. E Same as D except that twice the number of each nuclei were extracted and proteins were resolved in 15% polyacrylamide acid urea Triton (AUT) gel. A faint band comi- grating with protamine (lanes 1 and 4) is pyronin Y dye front. HeLa nuclei: Proteins acid extracted from nuclei; HeLa histones and HeLa HI are chromatographically purified; the recovery of H3 and H4, lane 1 (E), was substoichiometric.


S P E R M NUCLEI SPERM NUCLEI SPERM NIJCLEI, &NO EXTRACT 8z EXTRACT EXTRACT & ~ g + 2

Fig. 4. Sperm nuclear membrane disintegrates on chromatin decondensation or becomes distinct when incubated in extract in the presence of Mg++. a and b, permeahilized sperm nuclei stained with 10 pg/ml of DHCC; c and d, nuclei incubated in the extract for 15 min; e and f, nuclei incubated in extract in the presence of 5 mM Mg' for 30 min. Bar: 21.4 pm.

structure of the permeabilized nuclear envelope such that chromatin decondensation was inhibited.

Chromatin Decondensing Factors Are Thermostable

Nucleoplasmin, the sperm chromatin decondensing factor in Xenopus egg extracts, is heat resistant (Mills et al., 1980). We asked whether the decondensation factors in HIE had catalytic activities or were structural proteins. Any enzymatic activity of putative decondensing factors in HIE should be abolished by incubating the extract at nonphysiological temperature. Prior to incubation with sperm nuclei a t 35"C, the extract was incubated at 65°C for 15 min and cooled to room temperature. Both Xenopus and human sperm nuclei decondensed in heat-treated extracts (Fig. 5).

Thus the putative decondensing factors in HIE were thermostable.

Decondensed Chromatin Is Remodelled in the Extracts

Maximum decondensation of human sperm chromatin occurred in HIE in the absence of exogenous Mg++ (Fig. 2). Moreover, once decondensed, exogenous Mgtt had no effect on size of the nuclei (data not shown). Previous studies have shown that exogenous Mg' + and ATP are essential to increase the spacing of close- packed nucleosomes formed in the presence of endogenous Mg++ in HIE (Banerjee et al., 1991). Therefore, we examined changes in the primary organization of decondensed sperm chromatin upon further incubation with exogenous Mg++.


PHASE FLUORESCENCE

0 z w x

Fig. 5. Chromatin decondensation activity is thermostable. a and b, nuclei were incubated for 5 min in extracts preheated a t 65°C for 15 min; c and d, same as a and b, except that incubation was for 10 min. Bar: 20 pm for a and b and 75 pm for c and d.

To do this, human sperm nuclei were incubated in HIE with ATP and creatine phosphate for 20 rnin at 35"C, and further incubated for 40 min in the presence of 5 mM Mg++. The first incubation ensured dispersion of nuclear chromatin, whilst the second allowed for physiological spacing of the assembled nucleosomes. As a control, an equal number of nuclei were incubated under identical conditions but without extract. Subse- quent MNase digestion of nuclei revealed a distinct nucleosome ladder, similar to that of HeLa cells (Fig. 6A,B), suggesting that the decondensed sperm chromatin had remodelled to a somatic-type nucleosomal structure. Analysis of the basic protein content of decondensed sperm provided further evidence of chromatin remodelling. Appearance of somatic H1, homomor- phous H2a, and loss of protamines indicated the assimilation of somatic histones from HIE by decondensed sperm chromatin (Fig. 6C,D). The incomplete loss of protamines (Fig. 6D, lane 3) indicated that the decondensation factors were limiting under these conditions.

Decondensed Chromatin Can Be Recondensed

Having achieved chromatin dispersion and remodelling of the chromatin to somatic nucleosomal structures, we asked if the remodelled sperm chromatin could be recondensed using HeLa mitotic extracts. Prior to adding mitotic extracts and y-S ATP, the nuclei were incubated in HIE for 1 h in the presence of ATP and creatine phosphate. Mg++ was added at the beginning or after 20 min of incubation.

After 10-15 rnin of incubation in HME the nuclear periphery became uneven and discrete fluorescent chromatin domains appeared (Fig. 7a). Chromosome- like structures became visible after 30-60 min of incubation. When 2.5 mM Mgt+ was incorporated at the beginning of incubation, 20-30% of the decondensed nuclei were recondensed to well-separated chromosome-like structures (Fig. 7A, MI. When Mg+ + addition was delayed to achieve maximum dispersion of nuclei, more than 90% of the nuclear chromatin was


Fig. 6. Remodelling of human sperm chromatin in HIE. A 6 x lo6 nuclei were incubated in buffer (lanes 1 4 ) or in extract (5,000 nuclei per microliter of extract) for 1 h in the presence of 1 mM ATP, 5 mM CP, and 5 mM Mg' ' , which was added after the first 20 min of incubation (lanes 6-9). MNase digested DNA was separated in agarose gel. B same as A, except that the extract and the source of sperm were different (lanes 4-7) and the buffer control lanes are not shown.

A (lanes 5 and 11) and B (lanes 1 and 3) are DNA size standards; A (lane 10) and B (lane 2) are MNase digested DNA from HeLa nuclei. C 1.4 x lo7 nuclei were incubated in buffer or extract exactly as described in A, the proteins were acid extracted from the nuclei and separated in 18% polyacrylamide-SDS gel. D same as C except that 2.8 x lo7 nuclei were incubated (8,000 nuclei per microliter of extract) and proteins were resolved on 15% polyacrylamide-AUT gel.


Fig. 7. Recondensation of sperm chromatin in HME. A: Human sperm chromatin was dispersed in HME in the presence of 2.5 mM Mg++ (a), 1 mM ATP (b), and 5 mM CP (c) for 20 min at 35°C; subsequently Mg' + was added to a final concentration of 5 mM (d) before further incubation for 40 min. Incubation was continued for

another 90 min in the presence of HME and y-S ATP at 34°C; B Same as A except no Mg ' ' was added during first 20 min of incubation; the data represent four different experiments in each set of conditions. Bar: A, 11.6 wm; B, 13.3 pm.

recondensed (Fig. 7B). However, these structures were rarely converted to thicker and well-separated chromosome-like structures, even after longer incubation. Such distinct differences in the extent and pattern of condensation of the nuclei, dispersed in the presence and absence of exogenous Mg++, led us to conclude that a Mg++-dependent activity of interphase extract was necessary for effi- cient recondensation of the sperm nuclear chromatin.

Several control experiments were run: (a) nuclei decondensed in HIE were incubated for further 2 h alone or (b) in the presence of fresh HIE. Generally, nuclei were seen to reduce in size and to have smooth periphery and uniform fluorescence. Moreover, 250400 ~J.M y-S ATP was essential to achieve condensation in HME. Also different degrees of chromatin condensation (Johnson et al., 1970) were observed when HeLa interphase nuclei were incubated in HME in the presence of 400 pM y-S ATP at 32°C for 60-90 min (data not shown), suggesting that HME contained all the components necessary for HeLa interphase chromatin condensation.

DISCUSSION The decondensation of Xenopus, human, and pig

sperm nuclear chromatin in HeLa extracts was surpris- ing in view of previous experiments. Sperm chromatin decondensation has so far been shown to occur only in lysates from oocyte, egg, or embryo (Lohka and Masui, 1983, 1984; Ohsumi et al., 1988; Philpott et al., 1991) and, in particular, postneurola stage embryo extracts of Xenopus have failed to decondense sperm chromatin, even in homologous studies (Ohsumi and Katagiri, 1991). The question then arises whether the components in HeLa extracts inducing chromatin dispersion correspond to the physiological factors in oocyte, egg, and embryo. The identification of any such putative factors would have important implication for future research in this area, especially because of the limitation in obtaining adequate amounts of mammalian oocyte or egg extracts.

There are precedents that many catalytic or structural proteins, such as MPF (Masui and Clarke, 1979;


Ford, 1985; Nurse, 1990) and histone H1 (Multigner et al., 19921, have essential yet diverse cellular functions. Notably, -450 Mb DNA of highly condensed human sperm chromatin retain somatic nucleosomal structure (Tanphaichitr et al., 1982; Gatewood et al., 1987). Therefore, the decondensing activity in HIE might have physiological relevance to the chromatin dynamics of somatic cells.

Mg’+ and polyamines might inhibit sperm chromatin decondensation fa) by neutralizing the negative charge of unbound phosphodiester groups of DNA and N, atoms of chromatin proteins (Barry and Merrium, 1972); (b) by interacting with the decondensation factors; or (c) indirectly by stabilizing the nuclear envelope, therefore making it less permeable to decondensation factors. We have no clear experimental evidence which can eliminate any of these possibilities. How- ever, Mg‘ + differentially inhibited Xenopus, human, and pig sperm chromatin dispersion (Fig. 2).

Maximum cationic inhibition correlated with the appearance of a distinct nuclear envelope compared to that of lysolecithin-treated control nuclei (Fig. 4). Addi- tionally, natural polyamines of seminal plasma regu- late the energy-dependent sugar transport by directly binding to the sperm membrane (Pulkkinen et al., 1977). Therefore, we reconcile that the cationic block is most likely at the level of sperm membrane.

The distinct differences in sensitivity of the human and pig sperm nuclei to MNase, in size of nuclease digestion products of the chromatin and in basic protein composition, suggest that the structural organization of chromatin in these two sperm nuclei is significantly different. It appears that the extent of decondensation of chromatin is predetermined by the packaging infor- mation in the sperm nuclei. Our analysis of the basic protein composition of pig and human sperm nuclei indicates that absolute protamine content (Balhorn, 1982) might not alone determine the size of the sperm head. Additional parameters, like spatial distribution of nucleosome bound chromatin and the degree of cova- lent (disulphide) or noncovalent (Zn++-coordination) crosslinking of protamines (Bedford and Calvin, 1974; Kvist et al., 1987; Gatewood et a]., 1990b), may also be important.

The formation of mononucleosome-length DNA fragments following MNase digestion of human sperm nuclei has significant implications, because nucleosome formation in sperm chromatin is sequence specific (Gatewood et al., 1987). Moreover, extensive acetyla- tion of histones H3 and H4 in sperm chromatin led to the speculation that nucleosomal chromatin in sperm represents transcriptionally active genes (Gatewood et al., 1987,1990a). Therefore, the isolation of pure mononucleosome-length DNA fragments from sperm chromatin would facilitate identification of these genes.

Human sperm chromatin decondensed in HIE and further incubated in the presence of Mg++ was remodelled to somatic chromatin structures containing physiologically spaced nucleosomes. However, we failed to obtain quantitative remodelling of the sperm chro-

matin in HIE (Fig. 6). This is possibly due to limiting amounts of nucleosome assembly factors in the extracts. Recently we observed that 70% saturated ammonium sulphate (used in the penultimate step during the preparation of HIE) failed to precipitate quantita- tively a group of proteins which were enriched in the salt supernatants. Proteins fractionated from these supernatants, when added to HIE, facilitated quantitative assembly of nucleosomes on naked DNA. The effect of these fractions on chromatin decondensation and remodelling is currently under investi- gation.

The factors in HIE facilitating sperm chromatin decondensation are thermostable, suggesting they are structural proteins; this is reminiscent of the heat-stable nature of nucleoplasmin (Mills et al., 1980). Phil- pott and Len0 (1992) proposed that a nucleoplasmin- like acidic protein, which was referred to as “Factor F1” (Longo, 1990), might exist in mammalian eggs. The mammalian somatic cells do contain a number of well- characterised acidic proteins whose functions still re- main elusive (Earnshaw, 1987). Given the distinct differences in growth and RNA metabolism during early cleavages between Xenopus (Graham and Morgan, 1966; Newport, 1987) and mammalian embryos (Saw- icki et al., 1981; McGrath and Solter, 1984; Cattanach and Kirk, 1985; Surani et al., 1986), it is tempting to speculate that sperm chromatin decondensing factors in mammalian eggs might be acidic proteins also repre- sented in somatic cells.

Recondensation of remodelled human sperm chromatin in HME confirms earlier reports (Burke and Gerace, 1986; Nakagawa et al., 1989) that many of the mitotic events can be successfully reproduced in lysates ob- tained from cultured mitotic cells. The absolute requirement of y-S ATP for chromatin condensation sug- gests a kinase-phophatase network has been activated during the incubation of the extracts. A Mg++-dependent activity in HIE was vital for generating well-separated chromosome-like structures in HME. This activity may either have a catalytic or structural function (Dunphy and Newport, 1988). Topoisomerase I1 has been proposed to have both catalytic (chromatin loop fastener) and structural (scaffold) roles in chromosome condensation (Adachi et al., 1989). We observed that topoisomerase 11-mediated DNA catenation (Cantor et al., 1988; Banerjee and Spector, 1992) and RNA pol 11-dependent transcription (S. Banerjee, unpublished data) in HIE dialysed in the presence of 10-12 mM Mg++ were much higher than that seen in Mg++-free extracts described in this work. Preincubation of HIE in the absence of Mg++ might have further inhibited these activities. Purified topoisomerase I1 could be used to verify this possibility. It cannot be ruled out that the selective loss of structural proteins, important for higher order folding of chromatin, is due to the precipi- tation of salt-extracted proteins. The cell-free extracts described here provide an opportunity to identify these proteins involved in sperm chromatin condensation and decondensation.


ACKNOWLEDGMENTS We thank Julian Blow for kindly providing sperm

nuclei to initiate the work on Xenopus, Madan Tamuli for the generous gift of pig semen, and various individ- uals for donating human sperm. We also thank Martin Lawrie for kind help with computer programs; Martin King for photography; and Alastair Goldman, Yvonne Wallis, Mike Stacey, Andrea Shepherd, Clare Pullar, Alan Smallwood, and Fiona Macdonald for reading the manuscript. We are grateful to Chris Ford and Azim Surani for comments on the manuscript. Finally, we thank the reviewers for suggestions which improved the Discussion. The work on Xenopus began with a grant from the SmithKline Foundation. S.B. is a Letten F. Saugstad Fellow.

REFERENCES Adachi Y, Kas E, Laemmli UK (1989): Preferential, cooperative bind-

ing of DNA topoisomerase I1 to scaffold-associated regions. EMBO J 839974006.

Adachi Y, Luke M, Laemmli UK (1991): Chromosome assembly in vitro: Topoisomerase I1 is required for condensation. Cell 64:137- 148.

Balhorn R (1982): A model for the structure of chromatin in mammalian sperm. J Cell Biol93:29&305.

Banerjee S, Cantor, CR (1990): Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract. Mol Cell Biol 10:2863- 2873.

Banerjee S, Spector, DJ (1992): Differential effect of DNA supercoiling on transcription of adenovirus genes in vitro. J Gen Virol73:2631- 2638.

Banerjee S, Bennion GR, Goldberg MW, Allen TD (1991): ATP dependent histone phosphorylation and nucleosome assembly in a human cell-free extract. Nucleic Acids Res 195999-6006.

Barry JM, Merrium RW (1972): Swelling of hen erythrocyte nuclei in cytoplasm from Xenopus eggs. Exp Cell Res 71:90-96.

Bedford JM, Calvin HI (1974): The occurrence and possible functional significance of -S-S- crosslinks in sperm heads, with particular ref- erence to eutherian mammals. J Exp Zoo1 188:137-156.

Berrios M, Avilion AA (1990): Nuclear formation in a Drosophzlla cell-free system. Exp Cell Res 191:64-70.

Brown DB, Blake EJ, Wolgemuth DJ, Gordon K, Ruddle FH (1987): Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus Laevis egg extracts. J Exp Zoo1 242:215-231.

Burke B, Gerace L (1986): A cell-free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 44:639-652.

Cantor CR, Banerjee S, Bramachari SK, Hui C-F, McCleland M, Morse R, Smith CL (1988): Torsional stress, unusual DNA structures, and eucaryotic gene expression. In RD Wells, SC Harvey (eds): “Unusual DNA Structures.” New York: Springer-Verlag, pp 73-78.

Cattanach BM, Kirk M (1985): Differential activity of maternally and paternally derived chromosome regions in mice. Nature 315:496 498.

Clark HJ, Masui Y (1986): Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse. J Cell Biol 102:1039-1046.

Dilworth SM, Black SJ, Laskey RA (1987): Two complexes that contain histones are required for nucleosome assembly in vitro: Role of nucleoplasmin and N1 in Xenopus egg extracts. Cell 51:1009-1018.

Dunphy WG, Newport JW (1988): Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo. J Cell Biol 106:2047-2056.

Earnshaw WC (1987): Anionic regions in nuclear proteins. J Cell Biol 105:1479-1482.

Ford, CC (1985): Maturation promoting factor and cell-cycle regula- tion. J Embryo1 Exp Morphol89(Suppl):271-284.

Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW (1987).

Sequence-specific packaging of DNA in human sperm chromatin. Science 236:962-964.

Gatewood JM, Cook GR, Balhorn R, Schmid CW, Bradbury EM (1990a): Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol Chem 26520662-20666.

Gatewood JM, Schroth GP, Schmid CW, Bradbury EM (1990b): Zinc- induced secondary structure transitions in human sperm protamines. J Biol Chem 265:20667-20672.

Gledhill BL, Sawicki W, Croce CM, Koprowski H (1972): DNA synthesis in rabbit spermatozoa after treatment with lysolecithin and fusion with somatic cells. Exp Cell Res 73:3340.

Graham CF, Morgan RW (1966): Changes in the cell cycle during early amphibian development. Dev Biol 14:439-460.

Johnson RT, Rao PN, Hughes HD (1970): Mammalian cell fusion 11. A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species. J Cell Physic1 76151-158.

Kleinschmidt J , Steinbeisser H (1991): DNA dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus Zue- uis oocytes: Involvement of protein phosphorylation in nucleosome spacing. EMBO J 10:30433050.

Kleinschmidt JA, Seiter A, Zentgraf H (1990): Nucleosome assembly in vitro: Separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laeuis oocytes. EMBO J 9:1309-1318.

Kvist U, Bjorndahl L, Kjellberg S (1987): Sperm nuclear zinc, chromatin stability, and male fertility. Scanning Microsc 1:1241-1247.

Laskey RA, Honda BM, Mills AD Finch J T (1978): Nucleosomes are assembled by an acidic protein which binds histones and transfer them to DNA. Nature 275:416420.

Lohka MJ, Masui Y (1983): Formation in vitro, of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220:719-721.

Lohka MJ, Masui Y (1984): Roles of the cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol98:1222- 1230.

Longo FJ (1990): Dynamics of sperm nuclear transformations at fertilization. In B Bavister, J Cummins, ERS Roldan (eds): “Fertilization in Mammals,” Norwell, MA: Serono Symposia, pp 297-307.

Longo FJ, Kunkle M (1978): Transformation of sperm nuclei upon insemination. Curr Topics Dev Biol12:149-184.

Masui Y, Clarke HJ (1979): Oocyte maturation. Int Rev Cytol57:185- 282.

McGrath J, Solter D (1984): Completion of mouse embryogenesis re- quires both maternal and paternal genomes. Cell 37:179-183.

Miake-Lye R, Kirschner MW (1985): Induction of early mitotic events in a cell-free system. Cell 41:165-175.

Mills AD, Laskey RA, Black P, De Robertis EM (1980). An acidic protein which assembles nucleosomes in vitro is the most abundant protein in Xenopus oocyte nuclei. J Mol Biol 139:561-568.

Multigner L, Gagnon J, Dorsselaer A, Job, D (1992): Stabilization of sea urchin flagellar microtubules by histone H1. Nature 360:33-39.

Nakagawa J , Kitten GT, Nigg EA (1989): A somatic cell-derived system for studying early and late mitotic events in vitro. J Cell Sci 94:449-462.

Newport J (1987): Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA. Cell 48:205-217.

Newport J , Spann T (1987): Disassembly of the nucleus in mitotic extracts: Membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell 48:219- 230.

Nurse P (1990): Universal control mechanism regulating onset of M-phase. Nature 344:503-508.

Ohsumi K, Katagiri C (1991): Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: Involvement of nucleoplasmin. Dev Biol148:295-305.

Ohsumi K, Katagiri CH, Yanagimachi R (1988): Human sperm nuclei can transform into condensed chromosomes in Xenopus egg extracts. Gamete Res 2O:l-9.

Perreault SD, Wolff RA, Zirikin BR (1984): The role of disulphide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol 101:160-167.


Philpott A, Len0 GH (1992): Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts. Cell 69759-767.

Philpott A, Len0 GH, Laskey RA (1991): Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. Cell 65:569- 578.

Poccia DL (1986): Remodelling of nucleoproteins during gametogene- sis, fertilization and early development. Int Rev Cytol 105:1-65.

Pulkkinen P, Sinervirta R, Janne J (1977): Mechanism of action of oxidized polyamines on the metabolism of human spermatozoa. J Reprod Fertil51:39%404.

Sawicki W, Koprowski H (1971): Fusion of rabbit spermatozoa with somatic cells cultivated in vitro. Exp Cell Res 66:145-151.

Sawicki JA, Magnuson T, Epstein CJ (1981): Evidence for expression of paternal genome in the two-cell embryo. Nature 294:45&451.

Sunkara PS, Wright DA, Rao PN (1979): Mitotic factors from mammalian cells: A preliminary characterization. J Supramol Struct 11: 18%195.

Surani MA, Barton SC, Norris ML (1986): Nuclear transplantation in mouse: Heritable differences between parental genomes after acti- vation of the embryonic genome. Cell 45:127-136.

Tanphaichitr N, Sobhon P, Chalermisarachai P, Chutatape C. (1982). Biochemical and ultrastructural characterizations of nucleoprota- mine in human sperm heads treated with micrococcal nuclease and salt. Gamete Res 6235-255.

Uehara T, Yanagimachi R (1976): Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 15467-470.

Ulitzer N, Gruenbaum Y (1989): Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos. FEBS Lett 259:113-116.

Witkowska A (1981): Pronuclear development and the first cleavage division in polyspermic mouse eggs. J Reprod Fertil62:493498.

Sperm nuclear chromatin transformations in somatic cell-free extracts

Documents

Transcript of Sperm nuclear chromatin transformations in somatic cell-free extracts