Acid-soluble Nuclear Proteins of the Testis during Spermatogenesis in

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 255. No. 6 . Issue of March 25, pp 2533-2539, 1980 Prrnted m U.S.A.

Acid-soluble NuclearProteins of the Testis during Spermatogenesis in the Winter Flounder LOSS OF THE HIGH MOBILITY GROUP PROTEINS* (Received for publication,September 21, 1979)

Brian P. Kennedy and Peter L. Davies From The Growin Eukarvotic Molecular Biolom and Evolution,Department of Biochemistry, Queen’s University, Kingston, OntakoK7L 3N2 Canada I -

The basic proteins of the sperm nucleus from a variety of species have been catalogued by Bloch (1). Within the mammals and birds, protamines are utilized to package the sperm DNA whereas, in more primitive organisms, protamines or histones or proteins which have been called either histonelike or intermediate between histones and protamines f u l f i this role (1). Our studies on fishes’ suggest that within this class there is a dichotomy between those fishes which, like the rainbow trout, replace their histones with protamines (2) and those which, like the carp, retain histones in the sperm (3). Although basic nuclear proteins are found in most sperm * This work was supported by a grant from the Medical Research Council of Canada and by the award of a Medical Research Council Scholarship to P.L.D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. St. L. Daisley and P. L. Davies, unpublished work.



in amounts comparable to those found in somatic cells the non-histone chromosomal proteins are significantly depleted or possibly absent from the sperm nucleus after thehistone to protamine transition (4). However, in species which retain histones inthe sperm nucleus, there areno specific indications about the fate of the high mobility group (HMG) proteins during spermatogenesis. These proteins are closely associated with histones and are a significant component of chromatin, being present at the level of lo5 molecules/nucleus (5). More recently, these proteins in the trout have been associated by Dixon and co-workers (6) with that portion of chromatin which is most sensitive to endonuclease digestion and which is enriched in both transcribed sequences ( 7 ) and acetylated histones (8). However, HMG’ proteins have also been found in the highly condensed, transcriptionally inactive, nucleus of avian erythrocytes in quantities comparable to those foundin calf thymus nuclei (9, 10). In this paper, we report on the transitions observed in the acid-soluble nuclear proteins of the testes during spermatogenesis in the winter flounder, a species which retains histones in the sperm nucleus. EXPERIMENTALPROCEDURES

Materials-Phenylmethylsulfonyl fluoride, bovine thyroglobulin, iodoacetamide, and guanidine HCl were obtained from Sigma, St. Louis, Mo.; Sephacryl S200 from Pharmacia, Uppsala, Sweden; bacterialalkaline phosphatase(BAPc)from Worthington,Freehold, N. J.; and carboxymethyl cellulose (CM52) from Whatman, Maidstone, Kent, Great Britain. T h e protein molecular weight standards, myosin, P-galactosidase, phosphorylase B, bovine serum albumin, and ovalbumin were obtained asa kit from Bio-Rad, Richmond, Calif. Tissue-Testes were removed from the winter flounder during the months of July to January at the Marine Sciences Research Laboratory, St. Johns, Newfoundland. They were immediately frozen on dry ice and kept a t -6OOC until used. Freeze-dried flounder liver nuclei were supplied by Dr. C-L. Hew, Memorial University of Newfoundland, St. Johns, Newfoundland. Rainbow trout testes were collected in early October 1978 during the histone to protamine transition and were purchased from Dantrout, Brande, Denmark. Preparation of Nuclei-The procedure for the isolation of nuclei was based on that of Marushige and Bonner (11).Frozen testes were partially thawed and dispersed by scissor-mincing before being homogenized in an Omnimixer a t maximum speed for four periodsof 30 s each in 3 to 4 volumes of 75 mM NaCl containing 25 mM NaEDTA (pH 8.0) and 0.5 m M phenylmethylsulfonylfluoride (PMSF). The homogenate was filtered through four layers of cheesecloth and centrifuged a t 1,500 X g for 10 min. The nuclear pellet was washed by resuspension in the NaCI/EDTA/PMSF buffer described above and was recovered by centrifugation a t 3,000 X g for 10 min. This washing procedure was repeated once more after which the washed nuclei were used immediately for the extraction of proteins. The abbreviations used are: HMG, high mobility group; PMSF, phenylmethylsulfonyl fluoride; LMG, low mobility group; SDS, sodium dodecyl sulfate.

2533

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In December, at the end of spermatogenesis in the winter flounder (Pseudopleuronectes arnericanus) histones are themajor acid-soluble proteins of the sperm chromatin. Protamines are not detectable. These histones are identical with the histones present in the testes cell nuclei at the onset of spermatogenesis and in liver cell nuclei as judged by their mobilities in two polyacrylamide gel systems. Moreover, the ratioof the five histones does not differ significantly within these samples. During the early stages of testis development there isextensive covalent modification of the histones, in particular acetylation of H3 and H4, and phosphorylation of H2A. These modifications are not detectable at the end of spermatogenesis, and coincident with their disappearance is theloss of the two predominant high mobility group proteinswhich are theequivalent in the flounder of H6 and HMG-T of the trout. The loss of the high mobility group proteins is in accord with recent observations that H6 and HMG-T are associated with that portion of the chromatin which is transcriptionally competent, and theirloss from the sperm chromatin may reflect a need to erase the gamete’s previous history of gene activation. In addition to histones, the mature sperm of the flounder also contains a family of high molecular weight basic proteins ranging in molecular weight from 80,000 to 200,000 in which four amino acids make up 75% of the total residues. These are arginine, 23.7 mol %; serine, 22.8 mol 8;lysine, 14.9 mol 8;and proline, 13.7 mol %. Acidic residues account for only 4% of the total residues in these proteins and cysteine is not detectable.

2534

Changes in Nuclear Proteins during

:I

B. F. Bhullar and P. Candido, personal communication.

identical manner butwith the omission of bacterial alkaline phosphatase. Amino AcidAnalysis-Dried protein samples werehydrolyzed for 24 h a t 110°C in 6 N constant boiling HCI. The hydrolysatewas analyzed on a Beckman 120C amino acid analyzer modified for single column analysis. No corrections were made for losses during hydrolysis. RESULTS

From July to January, the average testis volume in the winter flounder increases more than 20-fold, particularly in the period September to November. During the same period, the yield of acid-soluble nuclear proteins per g wet weight of tissue increases by a similar amount as shown in Table I and reaches a maximum value around November or December. In TABLE I Yields of acid-soluble nuclear proteins obtained throughout the months of spermatogenesis Acid-soluble, nuclear proteins were extracted from 10- to 25-g lots of testes as described under “Experimental Procedures.” The testes used for these extractions were collected at the beginning of each month. Yield of acid-soluble nuclear protelns m g / g wet weight tissue

Month

July August September October November December Januarv

1

2

3

0.3

.o

1

1.6

6.2 14.4 18.4 14.2

4

5

6

7

8

9

1

0

-0

X3 nmG-r-

H3d

143 1 4 2 ~ ~

H2BY

144

H6

P

FIG. 1. Acid/urea gel electrophoresis of the acid-soluble nuclear proteins of flounder testes atdifferent stages throughout spermatogenesis. All samples wereisolated as described under “Experimental Procedures.” Sample I was a partially purified preparation of flounder HMG-T. Samples 2 to 7 were the acid-soluble nuclear proteins extracted from flounder testes collected during early August, September,October,November,December,andJanuary, respectively. Sample 8 contained flounder liver histones; Sample 9 contained theacid-soluble nuclear proteins from October trout testes; and Sample IO was a 5% trichloroaceticacidextract of October flounder testes. All samples except Sample 1 contained 100 pg of protein. Electrophoresis was donefor 22 h at 200 V as described under “Experimental Procedures.” P refers to the trout protamines, H3d to the H3 dimer,0 to the origin of the gel, and X to the high molecular weight proteins in Channels 6 and 7.

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Extraction of Acid-soluble Nuclear Proteins-Freshly prepared testis nuclei or freeze-dried liver nuclei were extracted with 10 to 15 volumes of 0.2 M H&Or for 30 min. At the endof this period the solid residue was recovered by centrifugation a t 17,000 X g for 20 min and then re-extracted as above. The proteins in the combined supernatants were precipitated overnight a t -20°C by the addition of 3 to 4 volumes of 95% ethanol, and were recovered next day by centrifugation at 13,500 X g for 30 min. The pellet obtained was redissolved in water, neutralized with Tris base, and then reprecipitated with 3 to 4 volumes of 95% ethanol as describedabove. The final precipitate was dissolved in water, freeze-dried, and weighed before storage a t -20°C. Extraction of High Mobility Group Proteins-Flounder H6 was isolated along with HI, from October testis nuclei by the method of Wigle and Dixon (12) using 5% trichloroacetic acid. H6 contained in the 5% (w/v)trichloroacetic acid extract was precipitated by the addition of 100% (w/v) trichloroacetic acid to a final concentration of 20% (w/v). The precipitate was collected by centrifugation a t 12,000 X gfor 20 min and redissolved in water prior to overnight precipitation in 3 to 4 volumes of ethanol containing 2% HCI. The precipitate was recovered by centrifugation at 13,500 X g for 30 min, washed with 95% ethanol, dissolved in water, and neutralized with Tris base, and then freeze-dried. H6 was purified from the 5% trichloroacetic acid extract by cation exchange chromatography on a column (1.3 X 40 cm) of Whatman CM52 previously equilibrated with 0.1 M LiCl in 20 mM ammonium acetate (pH 5). The 5% trichloroacetic acid extract (50 mg) was loaded onto the column in this solution and was eluted into 5-ml fractions using a linear gradientof 500 ml each of 0.1 M LiCl in 20mM ammonium acetate (pH 5) and 1 M Licl in 20 mM ammonium acetate (pH 5). After reading the absorbance of the eluate at230 nm fractions were pooled, dialyzed in Spectrapor No. 3 membranes against water, and thenfreeze-dried. Flounder HMG-Twas partially purified from Octobertestes nuclei based on the procedure of Goodwin et al. (13) for the isolation of HMG proteins fromcalf thymus as modified by Bhullar and Candido” in which a 0.3 M NaCl extract of thetestischromatin was fmt prepared. Low mobility group (LMG) proteinswere then precipitated from the 0.3 M NaCl extract by the addition of 100% (w/v) trichloroacetic acid to a final concentration of 2% (w/v) and were removed by centrifugation a t 19,000 X g for15min. HMG-T was precipitated from the resulting supernatant by the addition of solid ammonium sulfate to 55% saturation followed by stirring on ice for 1 h and then centrifugation at 19,000 X g for 1 h. T h e pelletwasretained for analysis by gel electrophoresis. Carboxymethylation-Some preparations of acid-soluble nuclear proteins were carboxymethylated with iodoacetamide to prevent dimerization of H3 and HMG-T using the method of Konigsberg (14, 15). The 50-fold exce.ss of dithiothreitol was calculated on the basis of H3 and HMG-Tmaking up 20% of the protein samples. Purification of the High Molecular Weight Basic Proteins-The high molecular weight basicproteins presentin December testeswere separated from histonesby gel filtration through SephacrylS200. The purification was checked by gel electrophoresis. Gel Electrophoresis-Acid-soluble proteins were analyzed by gel electrophoresis on 15% polyacrylamide slabs (0.15 X 18 X 27 cm). The acetic acid/urea gels were run either by the method of Panyim and Chalkley (16) or by the modified version according toAlfageme et al. (17) which includes in the gel 0.22% of the nonionic detergent Triton X-100 to change the relative mobilitiesof the histones. The Panyim and Chalkley gelscontained 6.25 M urea and the Triton gels contained 4 M urea. All gels were pre-electrophoresed for at least 15 h a t 170 V. Samples were applied to gels in 0.9 N acetic acid containing 4 M urea. The electrophoresis buffer was 0.9 N acetic acid and methyl green was used as the tracking dye.After electrophoresis the gelswere stained in 7.5% acetic acid containing0.1%.Amido black for at least 2 h before destaining in 7.5% acetic acid. Molecular weight determinations on proteins were done by SDSslab gel electrophoresis according to the method of Laemmli (18). T h e S D Sgels werestained in7.5% acetic acid, 15%ethanol containing 0.1% Coomassie blue for a t least 2 h before destaining in 7.5% acetic acid, 15% ethanol. Alkaline Phosphatase Treatment-Acid-soluble nuclear prot.eins (2.5 mg/ml) were treated with bacterial alkaline phosphatase at a final concentration of0.1 mg/ml in 0.2 M sodium glycine (pH 9.2) containing 1 mM MgC12.After incubating the mixture for 1 h a t 37°C theproteins wererecovered byacetoneprecipitation a t 0°Cfor analysis by gel electrophoresis. A control sample was treated in an

Spermatogenesis

2535

Changes in Nuclear Proteins during Spermatogenesis 1

2

3

4

5

6

7

8

9

-

H3d

I-HMG-T cH1 H2A 4 H2B H4

*H3

-L

FIG. 2. Extended electrophoresis on an acid/urea gel of the acid-soluble nuclear proteins of flounder testes isolated at The description of the gel and of the samples is the sameas in the legend to Fig. 1 with the different stages throughout spermatogenesis. exceptions that the flounder liver histones were run in Channel 1 on this gel, the Samples 9 and 10 from Fig. 1 were run in Channels 8 and 9, respectively. The gel was electrophoresed for 36 h at 200 V.

-

early December, a morphological examination of the tissue > H3 > H1 to H4 > H2B H1 > H3 > H2A by the inclusion showed that greater than 95% of the cells were mature sper- of Triton X-100 in the acid/ureapolyacrylamide gels as in Fig. 3 the similarity of the sperm histones to somatic histones is matozoa although spawning does not take place until late further confirmed by virtue of their identical migrations in spring. The acid-soluble nuclear proteins from the flounder testes this second gel electrophoretic system. were examined by acid/urea polyacrylamide gel electrophoSome changes can be observed, however, in the pattern of resis a t intervals during spermatogenesis.Fig. 1 shows clearly acid-soluble nuclear proteins during spermatogenesis. Firstly, that histones are retained throughout this process and are there not are extensive covalentmodifications of the histones in replaced by protamines. Standards for comparison are the the early stages of testes development, particularly in the acid-soluble nuclear proteins extracted fromrainbow trout months September to November. In Fig. 2 there are multiple testes collectedin Octoberduringthetransitionbetween components which ran slightly slower than H4 and H3, rehistonesandprotamines in this species. Protaminesfrom spectively, which are suggestive of the acetylated forms of diverse species of fish have been found to migrate very close these histones. There is also a prominent band, particularly in to the trout protamines when electrophoresed on acid/urea the November preparations, which runs betweenH3 and H2A. polyacrylamide gels4 This protein component is more easily seen in November The mobilities of the histonesfrom mature spermwere the preparations whichwere not reductively alkylatedand in same as thoseobserved for the histones isolated from imma- which much of H3 was oxidized to the dimerform (Fig. 4). In ture testes in August or from flounder liver cell nuclei. This these preparations the intensity of the stained H2A band is latter sample and the trout testis acid-soluble nuclear proteins considerablyless than that of the H2B band,whereas in were not carboxymethylated and therefore much of their H3 December or January preparations theyof equal are intensity. exists as a dimer. This result seen in Fig. 1 was better demTreatment of the November acid-soluble nuclearproteins onstrated in gels whichwere electrophoresed for a longer with bacterial alkaline phosphatase completely eliminated the period of time, as shownin Fig. 2 where rainbow trout prota- band between H2A and H3, and restored the intensityof the mine has been run off the bottomof the gel. When the relative H2A band to equal that of the H2B band (Fig. 4). By these mobilities of the histones are altered from H4 > H2B > H2A criteria, the unknown component was identified as phosphorylated H2A. There is an indication fromFig. 2 that acetylation of H3 and H4 precedes phosphorylation of H2A, since St. L. Daisley and P. L. Davies, unpublished observations.

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* H6

Changes in Nuclear Proteins during

2536

Spermatogenesis

1

2

3

4

5

6

7

8

-

9

0

---

- m . .-

,

+H3d +H2A



*H3

- 0 -

+Hl+H2B *H4

HMG-T+ H1 +

1

n

L

H6+

FIG. 3. Electrophoresis on a Triton gel of the acid-soluble, purified flounder HMG-T (Sample 1 ) was run in Channel 2.Channels nuclear proteinsof flounder testes isolated at different stages 3 to 8 inclusive were loaded with the acid-soluble nuclear proteins throughout spermatogenesis.The preparation of the Triton gel is from flounder testes for the months August to January, respectively. described under “Experimental Procedures.” Otherwise, the description of the gel and the samples is again similar to that contained in the legend to Fig. 1 with the following exceptions. The 5% trichloroacetic acid extract (Sample IO) was run in Channel 1. The partially

Channel 9 received the flounder liver histones (Sample 8 in Fig. 1) and Channel 10 received the trout proteins (Sample9 in Fig. 1). This gel was electrophoresed at 200 V for 22 h and for a subsequent 5 h at 250 V.

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the former modification is most extensive in October while flounder testis chromatin by 5% trichloroacetic acid as dethe latter is predominant in November. However, 1 month scribed under “Experimental Procedures.” The proteins relater, in December, all visible traces of histone modification covered from the 5% trichloroacetic acid extract by ethanol have disappeared. This transition can also be seen in the precipitation were primarily H1 witha lesser amount of flounTriton gel (Fig. 3) as a sharpening of the H3 and H4 bands der H6 as shown in Figs. 1 to 3. Flounder H6 was purified between November and December. Triton gels which have from the 5% trichloroacetic acid extract byion exchange been electrophoresed for up to 70 h show the changesin H2A chromatography on carboxymethylcellulose and eluted from phosphorylation particularly well. Phosphorylated H2A the column ahead of H1 a t a LiCl concentration of approximately 0.28 M (Fig. 5) in agreement with the findings for H6 which can alsobe seen in the liver histonepreparations migrates slightly slowerthan unmodified H2A. It is a t i t s most from trout (12). Final confirmation of its identity came from abundant in November preparations but is completely absentamino acid analysis donein quadruplicate which showed that, by January (not shown). In the Triton gel shown in Fig. 3, the as in the trout H6, lysine and alanine each made up almost one quarter of the amino acid residues, that there were not H2A band is in fact a doublet of H2A and phosphorylated H2A which were incompletely resolved during the shortelec- sulfur-containing or aromatic amino acids present, and that histidine was also absent (Table 11). One qualitativedifference trophoresis time. The second change in the basic nuclear proteins that was was the presence of isoleucine (1.7 mole 55) which is notfound observed during spermatogenesis was the coordinate loss of in trout H6 orcalf thymus HMG-17. Flounder H6 is richer in two major proteins shown in Fig. 1 that occurred between threonine and glutamicacid than its trout counterpart. The other HMG protein has been identified as the flounder November and December. These have been identified as high mobility group (HMG) proteins. Thelower of the two which equivalent of the trout protein HMG-T by virtue of their gels migrates faster thanH4 and marginally slower than H6 from similar migration on regular acid/urea polyacrylamide trout testes (Figs. 1 and 2) has been identified as the flounder (Fig. 1) in which both proteins migrate between the H3 dimer equivalent of H6 from the trout which in turn is similar to and H1. Moreover, an extraction procedure used to isolate HMG-17 of calf thymus (6).This protein was extractedfrom HMG-T from trout testis yielded three prominent proteins

Nuclear Proteins Changes Spermatogenesis during in 2

1

2537

3

from flounder testis, one of which co-migrated with the putative HMG-T (Fig. 1). The band immediately below this one might be the intramolecular disulfide-bonded monomer of HMG-T which has been observed by Watson et al. (21) to migrate slightly faster on starch gels than the reduced form. The presence of dimers, trimers, and tetramers of HMG-T was also reported by these workers. However, the mobilities of trout HMG-T and flounder HMG-T do differ on Triton gels. Flounder HMG-T migrates between H3 and H1 (Fig. 3) whereas trout HMG-T migrates between H2A and H1 (8). The third change in the acid-soluble nuclear proteins observed during spermatogenesis in the winter flounder is the sudden appearance after early November of a set of acid-

+O

+

H 3 dimer

TABLE I1 The amino acid composition of flounder H6 compared to H6 from the trout and HMG- I7 from calf thymus

I H1

P-H2A

The compositions for the latter two proteins were derived from Refs. 19 and 20, respectively. Flounder H6 was purified as described under “Experimental Procedures,” and a sample from Peak Y in Fig. 5 was used for the determination of i t s amino acid composition. T h e values displayed are the averageof four determinations. ..

cH2A ‘H2B

* H4

~~

FIG. 4. Dephosphorylation of phosphorylated H2A. Channel 3 of this acid/urea gel was loaded with acid-soluble nuclear proteins ( 6 O p g ) obtained from flounder testes collected in mid-November. The sample loaded in Channel 2 was recovered from treating 62.5 p g of the above material with bacterial alkaline phosphatase as described under “Experimental Procedures.” The sample in Channel I was the control for this digestion that was notexposed to theenzyme. 0 refers to the origin of the gel and P-H2A refers to the phosphorylatedform of H2A. This gel was run for 23% h at 170 V and then 20 h at 200 V.

. .

~

Y

Z

~~

~

mol

Asp Thr Ser Glu Pro Gly Ala Val Leu Lys Arg Ile

5.1 6.6 4.5 9.7 9.5 5.1 23.3 4.8 1.4 22.1 6.3 1.7

I ,

12.4 1.1 2.2 10.1 12.4 11.2 18.0 2.2 1.1 24.7 4.5

7.2 1.5 4.4 5.8 10.1 8.7 27.5 4.4 1.5 21.7 7.2 ~.

~

~

Calf thymus HMG-17

Trout H6

~~~

T

Std

l x

2x

3x

-0 I

c

c

H6

0 0 N

a+

T I

b-r -

P

40

Fraction

+*

d-c

Y

0

C-r

80

e+

No.

FIG. 5. Purification of flounder H6 by CM-cellulose ion exchange chromatography. The 5% trichloroacetic acid extract (namely Sample 10 in Fig. 1) was prepared and chromatographed as described under “Experimental Procedures.” The flow rate through the column (1.3 X 40 cm) of Whatman CM52 was 0.5 ml/min and 5ml fractions were collected. The optical densityof individual fractions was measured a t 230 nm (A)and the molarity of the LiCl gradient was determined from conductivity measurements (- - -). The acid/ urea polyacrylamide gel electrophoresis profile included identifiesand shows the purity of Peahs Y (H6) and Z (HI). T refers to the acidsoluble, nuclearproteins isolatedfrom Octobertrouttestes.The loading on the gel was Y (40 p g ) , Z (30 p g ) , and T (60 p g ) . P refers to trout protamines, 0 to the origin of the gel, and X to an unidentified component eluted from the CM53column.

FIG. 6 . SDS-gel electrophoresis of the acid-soluble, nuclear proteins from January flounder testes prepared as described under “Experimental Procedures.” The amountof sample applied to the gel was 4 0 pg ( I X ) , 80 p g ( 2 X ) ,and 120 p g ( 3 X ) .Standards ( S t d ) , withmolecularweights in parentheses, were a. myosin (200,000); h, /I-galactosidase (130,000); c, phosphorylase B (94,000); d. bovine serum albumin (68.000); and e, ovalbumin (43,000). each a t 2 pg. 0 refers to the point of application of the sample and thestar to the major component (103,000) in the high molecular weight basic proteins. The gel was run withcoolingonaBio-Hadmodel 22 apparatus for 1 h a t 20 mA and then 2% h a t 45 mA.

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H6

Amino Flounder acid

Changes in Nuclear Proteins during Spermatogenesis

2538

-4

H4

Fraction No.

TABLE111 The amino acid composition of the high molecular weight basic proteins isolated from December testes

The acid-soluble,nuclearproteinsfromDecembertesteswere fractionated as in Fig. 7. The high molecular weight proteins eluted in Fraction A and were subiected to amino acid analysisin duplicate. Amino acid

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine

High M , basic nuclear proteins mol 7

1.1 5.1 22.8 3.0 13.7 1.1 2.8

Not detectable

phosphate,respectively. Samples A, B , and C wererecovered as described under “Experimental Procedures” and wereanalyzed by acid/urea polyacrylamide gel electrophoresis as shown on the righthand side. The samples applied to the gel were as follows. S , starting material as applied to the Sephacryl column (100 pg);A , B , C. 80 pg each from the respective pooled regions of the column. 0 refers to the origin of the gel and X refersto the high molecular weight components. The gel was electrophoresed at 200 V for 30%h. 7). Fraction A consisted of the higher molecular weight proteins witha trace of H1. Fraction C was made up of the nucleosomalcore histones together with some H1 and the intermediate Zone B contained predominantly H1. The amino acid composition of Fraction A from the Sephacryl column comprising the high molecular weight proteins seen in Fig. 6 is given in Table 111. This composition is unusual in that four amino acids, arginine, serine, lysine, and proline, acids. Cysteine together make up75.3 mol R of the total amino is absent. The imbalance in the composition indicates that the components of Fraction A can justifiably be considered as a distinct group of related proteins. DISCUSSION

By December spermatogenesis is essentially complete in the winter flounder. The size of the testes and the yield of acid-soluble nuclear proteins perg of tissue have both reached 1.1 plateau values (Table I). Acytological examination of the 1.1 Tyrosine 0.5 tissue reveals only mature sperm. Theincreased yield of acidPhenylalanine 0.5 soluble nuclear proteins per g of tissue that takes place be14.9 Lysine tween September and November can be accounted for both Histidine 0.5 by the loss of cytoplasmand by thecondensation of the Arginine 23.7 nucleus. Analysis on acid/urea polyacrylamide gels of the acid-solsoluble proteins which migrate more slowly than the histones uble nuclear proteinsisolated a t monthly intervals throughout on acid/urea polyacryalmide gels (Figs. 1 to 3). When these spermatogenesis shows that in this species of fish there is no is, for example, proteins from January testes wereexamined on SDS-7.5% replacement of histones by protamines as there in the polyacrylamide gels they migrated as discrete bands ranging in the salmonidfish (2). The histonesthatremain in molecular weight from 80,000 to 150,000 with minor com- sperm nucleus are indistinguishable from both the somatic in immature testis. ponents as large as 200,000 (Fig. 6). The predominant com- cell histones andfrom the histones present ponent had a molecular weight of 103,000. In this gel system No testis-specific histones are observedin this system as they have been in the rat (22) or sea urchin (23). Moreover, the the histones migrated with the dye front. In view of the marked size difference between this set of fact that the relative proportions of the five histones do not proteins and the histones, they were separated from each change noticeably during spermatogenesis suggests that the other by gel filtration on SephacrylS200 (Fig. 7). The optical nucleosome remains as the basic structural unit of the chrodensity of the eluant was monitored at 230 nm and showed matin in the sperm nucleus in this species. Extensive histone acetylation has been noted here in the that the sample hasbeen resolved into two main Fractions A and C and an intermediate Zone B. Fractions were pooled as flounder testis histones collected in September and October indicated by the bars in Fig. 7 and the sets of proteins from but not in the histones from the transcriptionally inactive, in these regions were recoveredseparately by dialysis and freeze- maturesperm.Thisobservationparallelsthesituation drying for analysis on an acid/urea polyacrylamide gel (Fig. Tetrahymena whereacetylatedhistonesare foundin the 3.0 4.9

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FIG. 7. Isolation of the high molecular weight components from the acid-soluble nuclear proteins of December flounder testes. The sample (20 mg) was chromatographed on a column (2.5 X 40 cm) of Sephacryl S2(M at a flow rate of 1 ml/min. The sample was applied in 4 ml of 10 mM Tris-HCI (pH 7.5) containing 0.75 M NaCl and was eluted with the same solutioninto 2-ml fractions. The void volume (V,,)and total volume ( V , ) of the column were determined by the elution positions of bovine thyroglobulin and inorganic

Changes in Nuclear Proteins during Spermatogenesis

Acknowledgments-We would like to thank Dr. Choy-L. Hew of the Marine Sciences Lab, St. John's, Newfoundland, for the collection

of tissue used in the work described here and Dr. Peter Candido of the University of British Columbia, Vancouver, BritishColumbia, for communicating to us his procedure for the partial purification of HMG-T from trout testis. REFERENCES 1. Bloch, D. P. (1969) Genet. Suppl. 61, Suppl. 1,93-111 2. Dixon, G. H., and Smith, M. (1968) Prog. Nucleic Acid Res.Mol. Biol. 8.9-34 3. Subirana, J. A,, Puigjaner, L. C., Roca, J., Llopis, R., and Suau, P. (1975) Ciba Found. Symp. 28 4. Kaye, J. A,, and McMaster-Kaye, R. (1966) J. Cell Biol. 31:1, 159-179 5. Johns, E. W., Goodwin, G. H., Walker, J. M., and Sanders, C. (1975) Ciba Found. Symp. 28 6. Levy-W., B., Wong, N. C. W., Watson, D. C., Peters, E. H., and Dixon, G. H. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 793-801 7. Levy-W., B., Connor, W., and Dixon, G. H. (1979) J . Biol. Chem. 254,609-620 8. Levy-W., B., Watson, D. C., and Dixon, G. H. (1979) Nucleic Acids Res. 6,259-274 9. Vidali, G . , Boffa, L., and Allfrey, V. G. (1977) Cell 12,408-415 10. Rabbani, A., Goodwin, G. H., and Johns, E. W. (1978) Biochem. Biophys. Res. Commun. 81,351-358 11. Marushige, K., and Bonner, J. (1966) J . Mol. Biol. 15, 160-174 12. Wigle, D. T., and Dixon, G. H. (1971) J . Biol. Chem. 246, 56365644 13. Goodwin, G. H., Nicolas, R. H., and Johns,E. W. (1975) Biochim. Biophys. Acta 405,280-291 14. Konigsberg, W. (1972) Methods Enzymol. 25, 185-188 15. Waxdal, M. J., Konigsberg, W. H., Henley, W. L., and Edelman, G. M. (1968) Biochemistry 7, 1959-1966 16. Panyim, S., and Chalkley, R. (1969) Arch. Biochem.Biophys. 130,337-346 17. Alfageme, C. R., Zweidler, A,, Mahowald, A,, and Cohen, L. H. (1974) J. Biol. Chem. 249, 3729-3736 18. Laemmli, U. K. (1970) Nature 227,680-685 19. Watson, D. C., Wong, N. C. W., and Dixon, G. H. (1979) Eur. J. Biochem. 95, 193-202 20. Walker, J . M., Hastings, J. R. B., and Johns, E.W. (1977) Eur. J . Biochem. 76,461-468 21. Watson, D. C., Peters, E. H., and Dixon, G. H. (1977) Eur. J. Biochem. 74,53-60 22. Meistrich, M. L., Brock, W. A,, Crimes, S. R., Platz, R. D., and Hnilica, L. S . (1978) Fed. Proc. 37, 2522-2525 23. Subirana, J. A,, and Palau, J. (1968) Exp. Cell. Res. 53,471-477 24. Gorovsky, M. A., Pleger, G. L., Keevert, J . B., and Johman, C. A. (1973) J . Cell. Biol. 57, 773-781 25. Kierszenbaum, A. L., and Tres, L. L. (1975) J.Cell. Biol. 65, 258270 26. Soderstrom, K-O., and Parvinen, M. (1976) Mol. Cell.Endocrinol. 5, 181-199 27. Louie, A. J., and Dixon, G. H. (1972) J. Biol. Chem. 247, 54905497

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transcriptionally active macronucleus but not in the genetically repressed micronucleus (24), thereby strengthening the association between histone acetylation and transcription. Although the sperm in the winter flounder has retained the somatic type histones as the major structural element in its chromatin the high mobility group proteins as represented by the flounder equivalents of HMG-T andH6 are not kept.This finding is consistent with the postulated role of HMG proteins as structural elements attached to that portion of the chromatin which is transcriptionally competent (6, 7), since the production of RNA gradually diminishes and then ceases during spermatogenesis (25-27). However, it is a significant departure from the situation in the avian erythrocyte nucleus where it hasbeen reported that HMG proteins are present (9, lo), although the nucleus is relatively condensed and shows little transcriptional activity. Presumably, the HMG proteins remain associated with that portion of the chromatin which was earlier expressed. One can surmise, therefore, that in the developing testis cell HMG proteins are removed or phased out in order to erase the sperm cell's previous history of gene expression, thereby allowing the gamete DNA to begin a new program of gene activation within the egg. Finally, at thetime that both the HMG proteins and histone modifications are finally disappearing from the testis nuclei there begins the appearance of a set of high molecular weight (80,000 to 200,000), basic (lysine + arginine = 38.6 mol 5%; aspartic acid + glutamic acid = 4.1 mol %) proteins. Aside from the imbalance between basic and acidic residues serine + threonine amount to 27.9 mol % and proline is 13.7 mol %. These figures, taken together with the low values determined for histidine andthe aromatic residues, are suggestive of proteins intermediate between histones and protamines. The absence of cysteine from the composition means that the 15 or more bands thatcan be detected in thisfamily of proteins inFig. 6 have not arisen by the linkage through disulfide bridges of smaller molecules. The function of these proteins can only be conjectured a t the present time. On the basis of the data in Fig. 7 these proteins amount to between 25% and 30% of the total acidsoluble protein in the sperm nucleus. It is conceivable that they replace a portion of the histones of the sperm nucleus in the manner of the nucleohistone to nucleoprotamine transition in salmonid fish. However, we suggest that they more likely function in an auxiliary role with histones in making the sperm chromatincondensed and quiescent during the long storage period prior to spawning.

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Acid-soluble nuclear proteins of the testis during spermatogenesis in the winter flounder. Loss of the high mobility group proteins. B P Kennedy and P L Davies J. Biol. Chem. 1980, 255:2533-2539.

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Acid-soluble Nuclear Proteins of the Testis during Spermatogenesis in

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 255. No. 6 . Issue of March 25, pp 2533-2539, 1980 Prrnted m U.S.A. Acid-soluble NuclearProteins of the Testi...

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