Proc. Nat. Acad. Sci. USAVol. 72, No. 7, pp. 2714-2718, July 1975Cell Biology

New histones found in mature mammalian testes(spermiogenesis/nucleoproteins/protamines/histones)

ANN SHIRES*, MARY P. CARPENTER*, AND ROGER CHALKLEYtt*Oklahoma Medical Research Foundation and Department of Biochemistry, University of Oklahoma, Oklahoma City, Okla. 73104; and tDepartment ofBiochemistry, College of Medicine, University of Iowa, Iowa City, Ia. 52242

Communicated by James Bonner, April 18, 1975

ABSTRACT Two new histones have been found in sex-ually mature testes of several mammals. The new histoneshave been identified as a new F1 (TFI) and F2b (TF2b) on thebasis -of their behavior upon chemical fractionation proce-dures and electrophoresis at several urea concentrations. Thenew testis-specific histones are absent in very immature ani-mals and in somatic tissues. However, by day 20, in the rat,the histone pattern is that of the adult, displaying nearly afull complement of TF1 and TF2b. The new histone comple-ment evidently is characteristic of prespermatid germ cells.The degree of evolutionary variations in these histones andin other basic sperm proteins, as detected by change in elec-trophoretic mobility, appears to be much greater than thatseen in somatic histones.

The process of spermiogenesis involves the production of ahaploid genome, packaged in a highly organized and com-pact fashion. In echinoderms the compact nucleoproteincontains DNA in association with histones which are eitheridentical or similar to those found in somatic cells (1-4). Onthe other hand, in arthropods and teleosts somatic histonesare replaced in mature spermatozoa by a low molecularweight, arginine-rich class of proteins known as protamines(5, 6). Early studies of mammalian species indicated thatthere was a shift toward an arginine-rich nucleoprotein dur-ing spermiogenesis, and indeed several workers have isolatedsmall basic proteins from spermatozoa. Coelingh et al. isolat-ed and sequenced a protein of molecular weight about 6000from bull spermatozoa which contained large amounts of ar-ginine and cysteine (7, 8). Kistler et al. have described an-other nuclear protein (MW about 6200) isolated only frommature rat testes, but which is not present in rat spermato-zoa (9, 10).We initiated a program designed to compare the basic

proteins from spermatozoa of a range of mammals. As partof this approach we analyzed the testes tissue. We made theunexpected observation that sexually mature testes containtwo new testes-specific histones in substantial amount. Thesehave been characterized and are identified in this report asnew F1 and F2b histones. The testes-specific basic protein ofKistler et al. (9) was also observed in all varieties of maturetestes studied, and we have assayed for animal-specific vari-ation in its electrophoretic mobility.

MATERIALS AND METHODS

Histones were isolated from whole tissue by the method ofPanyim et al. (11) as modified by Balhorn et al. (12).

Protamines were obtained from the headpieces of maturespermatozoa isolated from the testis, intact epididymis, orsemen. The headpieces were separated from the tails of thespermatozoa by blending. During the acid extraction of thehistones from the nuclei, the sperm heads were precipitated

t To whom reprint requests should be made.

in the acid-insoluble fraction. The pelleted sperm headswere washed twice with deionized water, suspended in15-20 ml (1 ml/g of starting tissue) of 5 M guanidine-hydro-chloride, 0.5 M 2-mercaptoethanol, and homogenized. Theensuing gel was adjusted to pH 7 with NaOH and sheared ina Virtis model 45 homogenizer at 70 V for 45 sec. The ionicstrength was decreased by adding 2.5 volumes of deionizedwater. Protamines were extracted in 0.2 M H2SO4 at 40 for 2hr. After centrifugation at 27,000 X g for 20 min, the super-natant was dialyzed against 0.2 M H2SO4 to remove guani-dine-hydrochloride.The bull protamine was obtained from spermatozoa iso-

lated from bull semen, utilizing a slightly modified proce-dure of Marushige and Marushige (13).

Electrophoresis of histones was performed on 15% acryl-amide gels as described (14). Electrophoresis of protamineswas performed on 9-cm gels (20% acrylamide) containing 5M urea in 0.9 M acetic acid at 130 V for 2.5 hr.Whole histone of rat testis was chemically fractionated ac-

cording to the method of Oliver et al. (15).

RESULTSPolyacrylamide gel electrophoretic analysis revealed thatcertain critical differences existed between the testis histoneand histone from other tissues, as is shown in Fig. la and b.Although there are variations among different creatures,several points distinguish the testis histone complement,namely, an additional band in the F1 region (denoted TF1)and a greater intensity of staining in the F2b-F3 region of thegel (denoted TF2b + F3). The rabbit and monkey testes areless unusual than the rodent tissues; nonetheless additionalstaining between the F2b and the F3 band is observed. Thetestis tissues also show the presence of an additional bandmigrating much more rapidly than F21, as shown in Fig. lb.This latter band appears to be identical to the "TP" proteinstudied by Kistler et al. (9). Using the higher molecularweight histones as an internal gel reference, it is possible toconclude that the monkey testis "TP" band migrates signifi-cantly faster than that obtained from rat or rabbit.The nature of the testis-specific pattern of the higher mo-

lecular weight histone becomes more apparent upon a high-er resolution analysis, as shown in Fig. 2. The gels were elec-trophoresed through 25-cm gels to resolve the new band inthe F3-F2b region. The new band migrates between the F3and the F2b histones. It appears to be totally absent in thehistone complement of somatic cells of other mammaliantissues. The new band varies slightly in mobility dependingupon its species of origin. Thus, the mouse and rat testisband is only fractionally faster than F3, whereas the monkeytestis band moves with a mobility almost directly in betweenthat of F3 and F2b and the rabbit band moves with a mobili-ty closer to that of F2b. The amount of the two new bands

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Proc. Nat. Acad. Sci. USA 72 (1975) 2715

cl TF2b

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FIG. 1. (a) Electrophoretic patterns of histones from monkey,rat, rabbit, and mouse testis, calf thymus, and monkey liver. His-tones were isolated as described in Materials and Methods. Poly-acrylamide gel electrophoresis was performed on 9-cm (15% acryl-amide) gels containing 2.5 M urea in 0.9 M acetic acid at 130 voltsfor 4.5 hr. (b) Electrophoretic separation of histones and the testis-specific protein (TP) (9) from adult monkey, rabbit, and rat testis.The testis-specific protein is not present in monkey or rat liver.Electrophoresis was performed on 17-cm gels using the same gelsystem as described in the legend of panel a at 40 and 200 V for 7hr.

relative to the normal histone fractions appears to vary as a

function of species. Thus, rat testes has a larger fraction ofthe two new bands relative to the other histones than othertestis tissue studied. The somatic histone fractions of the tes-tis appear to be present in abnormal relative proportionscompared to other tissues. Histone fractions F21 and F3 are

present to a much higher degree than F2a and the somaticF~b fraction. Finally, we note from Fig. 2 that there is little

:f

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FIG. 2. Microdensitometer scans of high resolution electro-phoretic analysis of histones from testis of adult rat, rabbit, mon-key, and mouse, from monkey liver and rat hepatoma tissue cul-ture cells (HTC). Electrophoresis was performed on 25-cm (15%acrylamide) gels containing 2.5 M urea in 0.9 M acetic acid at 40and 200 V for 48 hr.

microheterogeneity in the new band (TF1) in the F1 region,although it is clear that this band also appears to possess ani-

mal-specific mobility.The electrophoretic mobility of F2b is highly influenced

by the urea concentration (16). The faster of the two testis-specific histone fractions behaves identically to the normalhistone F2b, as is shown in Fig. 3. In 1 M urea gels, the fastermoving new band comigrates with the F3 band (see also thedensitometer traces); at intermediate urea concentration theband can be visualized as a discrete band; and at higher urea

concentration it migrates closer to the Fa band.Identification of histone fractions can also be achieved by

chemical fractionation, and the data of Fig. 4 indicate thatthe slower moving testis histone behaves like a characteristi-

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FIG. 3. High resolution gel electrophoretic patterns at 1 M, 2.5 M, and 6 M urea concentrations of histones from rat testis and rat hepa-toma tissue culture (HTC) cells. Electrophoresis was performed as described in the legend of Fig. 2.

cally lysine-rich histone in that it is fully soluble in 5% per-

chloric acid. The other testis-specific histone was separatedinto the F2b fraction, confirming the indications given bythe gels of different urea concentrations. No new fractionswere detected along with fraction F3 or Fad and F21. Asimilar conclusion for rat testis has recently been reported byBranson et al. (17).

Microscopic examination revealed that immature sperm

heads are isolated with the testicular chromatin. The sperm

heads isolated intact from the acid-extracted testicular chro-

HTC

STONES

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FIG. 4. Rat testes F2b and F1 histone fractions obtained bychemical fractionation of rat testis whole histone. Chemical frac-tionation was performed by the method of Oliver et al. (15). Elec-trophoresis was performed as described in the legend of Fig. 2. Rathepatoma tissue culture cell (HTC) was used as a standard.

matin and from epididymal spermatozoa were dispersed in5 M guanidine-hydrochloride and 0.5 M 2-mercaptoethanoland extracted in 0.2 M H2SO4. The material so obtained isshown in Fig. 5A, in which the basic protein of testis sperm(protamine) is compared to that from epididymal sperm andalso to the testis-specific (TP) protein isolated from choma-tin. In agreement with the previous report of Kistler et al.(9), the testis (TP) protein can be distinguished electrophore-tically from sperm protamines. Further, the basic proteinfrom those spermatozoa which are still located in the testismoves slightly more slowly that that obtained from epididy-mal spermatozoa. This is consistent with recent reports thatboth fish and mammalian sperm basic proteins are phospho-rylated upon deposition onto sperm DNA and that they are

only slowly dephosphorylated (18-20). In Fig. 5B, we havecompared the basic proteins from the spermatozoa presentin the testis of several mammals. The protamines show vari-ations in mobility among the different creatures studied.Calf thymus histone was added as a marker to aid in assign-ment of the relative mobilities of the smaller protamines.

Clearly, the testis-specific F1 and F2b do not appear to bea reflection of the presence of mature spermatozoa withinthe testis. For a further analysis of these proteins, we haveexploited the earlier observations of Clermont and Perey(21), who showed that the nuclei fractions of spermatocytesand spermatids increase rapidly with age in the young rats.Accordingly, we have prepared histones from testes ob-tained from rats at various stages of sexual maturity. The re-

sults of such an analysis are shown in Fig. 6a and b. The tes-tis-specific histones are clearly absent in 5-day testis, but are

present (almost to the degree seen in adult tissue) by the20th day. During the period between the 20th and 36th daysthe amount of the somatic form of F2b declines slightly, andconcomitantly the TF2b increases. Sperm protamine was notisolated from testis by the methods described above until the45th day.

DISCUSSIONEvidence has been presented that two new proteins isolatedfrom testis of several mammals are new histone F1 and F2bfractions. This is based on the following evidence: (i) Theyare highly basic chromosomal proteins. (ii) Upon chemical

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A+ - S~ .xI...+

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Rat Testis + Monkey Testis

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Rot Epididymis

FIG. 5. (A) Variation in electrophoretic mobility of the rapidlymigrating basic proteins extracted from whole testis, testis sperm,

and epididymal sperm of adult rat. The basic proteins were isolat-ed as described in Materials and Methods. (a) Basic protein fromepididymal sperm, (b) basic protein from testicular sperm, (c) tes-tis-specific protein (TP) as isolated using the method of Kistler etal. (9), (d) testis-specific protein (TP) isolated with the testis his-tones by the procedure described in Materials and Methods, (e) band c combined. The basic protein from testis sperm migratesmore slowly than the testis-specific protein (TP). Electrophoresiswas performed on 9-cm (20% acrylamide) gels containing 5 M urea

± b

ifI 11

11

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11 0HI111111111

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20-day-old Rat Testis

36-day-old Rat Testis

60-day-old Rat Tes t i s

I2F20 TP

FIG. 6 (a) Electrophoretic separation of histones isolatedfrom the testis of 5-day-old, 20-day-old, and 60-day-old rats. Thetestis-specific histones are absent in the 5-day-old rat but are pres-ent (almost to the degree seen in the adult testis) by the 20th day.Electrophoresis was performed as described for Fig. la. (b) Ap-pearance of the testis-specific protein (TP). Electrophoretic pat-terns of testis basic proteins isolated from 20-day, 36-day, and 60-day-old rats. The "TP" protein appears after the 36th day. Thesomatic forms of F1 and F2b are declining as the TF1 and TF2b in-crease during sexual maturation. Electrophoresis was performedusing 17-cm gels as described for Fig. lb.

extraction procedures the proteins behave in a manner char-acteristic of histone F1, on the one hand, and histone F2b, on

the other. (ii{) Behavior of testis-specific F2b upon electro-phoresis in gels of various urea concentrations is typical ofnon-testicular tissue F2b. These conclusions are in agreementwith those obtained by Branson et al. and reported in a re-

cent abstract (17).The contribution of the testis-specific fractions to the total

histone complement varies with the source of the testiculartissue; in rat testis about 25% of the total histone comple-ment is made up of the two new fractions. Analysis of-theamounts of the various histone fractions in mature testis re-

veals (i) Fw and F2b are present in less than 50% of thatnormally seen in somatic cells, (ii) F3 is present to a greaterdegree than in any other tissue, (iii) there is slightly more

TF2b than somatic F2b, (iv) there is as much TF1 as somaticF1, and (v) F2.1 is present in normal amounts. In the mature

in 0.9 M acetic acid run at 370 at 130 V for 2.5 hr. (B) A compari-son of the electrophoretic mobilities of the protamines from thespermatozoa present in the testis of rabbit, monkey, and rat, theepididymis of rat, and the ejaculate of bull. Calf thymus histonewas added to each gel as a marker. Electrophoresis was performedas described for panel A.

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1 111

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testis about 30% of the cells are somatic (21), and if we makethe reasonable assumption that testis somatic cells containFU2, F2b, and F1 in normal amounts, then it is necessary toconclude that the F&2, F2b, and F1 of whole testis histone iscoming entirely from the somatic supporting cells, andtherefore, that the germ line cells contain appreciableamounts of neither FW nor somatic F2b or F1. The histonecomplement of the germ line cells can then be calculated bysimple subtraction, based on Clermont's estimates of thefraction of somatic cells in the testis (21). We conclude thatthe mean germ line histone composition is TF1, F3, TF2b,and F2.1 in the ratio 13:21:20:15, respectively. The bulk ofthe prespermatid cells are spermatocytes, although type Bspermatogonia do contribute about 20% to the total numberof testes cells and the data are not sufficiently precise to in-dicate whether the new histone pattern is characteristic ofall germ cells (including spermatogonia and spermatocytes).Clermont and Perey have shown that the 5-day-old rat testiscontains only 4% of germ line cells and 96% of somatic sup-porting cells. We have shown the histones of such testes aresomatic in nature. On the other hand, by day 20 the testescontain about 30% support cells, 40% spermatocytes, and ap-proximately 30% spermatogonia (primarily of type B) (21).At this stage the histone pattern is essentially that of theadult, showing a full complement of TF1 and TF2b and dras-tically reduced yields of the somatic F2b and FU2. Sperma-tids are not present at day 20, and so clearly the new histonecomplement is characteristic of prespermatid germ cells.The TP protein first described by Kistler et al. (9) is absentat this stage. By day 36 spermatids appear in the tubuleswith a development up to stage 7 (21). In the strain of ratswe are using, neither TP protein nor protamines are presentat day 36 (see Fig. 6b), and we may conclude that sperma-tids in the earlier stages of maturation contain "T" histones.

Since the TP protein is present shortly after the 36th dayof t~estes development, at which time the first wave of mat-uring spermatids is progressing into the intermediate stagesof development, it seems reasonable to argue that the shift tomore arginine-rich histones is a result of the addition of theTP protein to the nucleus of the developing spermatids. Cy-tological observations indicate that the earlier stage "lysine-rich" histones are removed at this time (22, 23), and sincecalculations on the total amount of the TP protein indicatethat it contributes 5% to the mass of total testis histones, it islikely that if the TP protein represents the total histone com-plement of the developing germ line, it does so for a rela-tively short time period. Certainly the mature spermatidsand the immature testicular spermatozoa contain no TP pro-tein, and presumably the bulk of their protein is protamine(see Fig. 5A). Thus, we may conclude that the TP protein isassociated with the spermatid nucleus only transiently. Fi-nally, we note that the protamine extracted from late stage

spermatids and immature spermatozoa migrates slightlymore slowly than that associated with mature epididymalsperm (see Fig. 5A). Recent observations reported by Mar-ushige and Marushige (20) indicate that the reduction inelectrophoretic mobility is due to phosphorylation of the pa-rental protamine, which is found in the unphosphorylatedform in mature sperm.We thank Drs. Vaughn Jackson and Rod Balhorn for their advice

and assistance, and Connie Hunter, Bob Neer, and Patricia Cain fortheir valuable technical assistance. This work was supported by theOklahoma Medical Research Foundation and by the USPHS GrantsCA-10871 and HD-05641.

1. Hultin, T. & Herne, R. (1948) Ark. Kemi Mineral. Geol. A 26,20-26.

2. Subirana, J. A. & Palau, J. (1968) Exp. Cell Res. 53,471-477.3. Easton, D. & Chalkley, R. (1972) Exp. Cell Res. 72,502-506.4. Subirana, J. A. (1970) Exp. Cell Res. 63,253-258.5. Kaye, J. S. & McMaster-Kaye, R. (1973) Arch. Biochem. Bio-

phys. 158, 426-432.6. Sung, M. T. & Dixon, G. H. (1970) Proc. Nat. Acad. Sci. USA

67, 1616-1623.7. Coelingh, J. P., Rozijn, T. H. & Monfoort, C. H. (1969) Bio-

chim. Biophys. Acta 188,353-356.8. Coelingh, J. P., Monfoort, C. H., Rozijn, T. H., Leuverv, J. A.

G., Schiphof, R., Steyn-Parve, E. P., Braunitzer, G., Schrank,B. & Ruhfus, A. (1972) Biochim. Biophys. Acta 285, 1-14.

9. Kistler, W. S., Geroch, M. E. & Williams-Ashman, H. G.(1973) J. Biol. Chem. 248,4532-4543.

10. Kistler, W. S., Noyes, C. & Heinrikson, R. L. (1974) Biochem.Biophys. Res. Commun. 57,341-347.

11. Panyim, S., Bilek, D. & Chalkley, R. (19/1) J. Biol. Chem.246,4206-4215.

12. Balhorn, R., Balhorn, M., Morris, H. P. & Chalkley, Pt. (1972)Cancer Res. 32, 1775-1784.

13. Marushige, Y. & Marushige, K. (1974) Biochim. Biophys. Acta340,498-508.

14. Balhorn, R., Rieke, W. 0. & Chalkley, R. (1971) Biochemistry10,3952-3959.

15. Oliver, D., Sommer, K. R., Panyim, S., Spiker, S. & Chalkley,R. (1972) Biochem. J. 129,349-35.

16. Panyim, S., (1971) Ph.D. Dissertation, University of Iowa,Iowa City, Ia. 52242.

17. Branson, R. E., Grimes, S. R., Yonushot, G. & Irvin, J. L.(1973) Fed. Proc. 32,588 (abstr.).

18. Ingles, C. J. & Dixon, G. H. (1967) Proc. Nat. Acad. Sci. USA58, 1011-1018.

19. Louie, A. J. & Dixon, G. H. (1972) J. Biol. Chem. 247, 7962-7968.

20. Marushige, Y. & Marushige, K. (1975) J. Biol. Chem. 250,39-45.

21. Clermont, Y. & Perey, B. (1957) Am. J. Anat. 100, 241-268.22. Davis, J. R. & Langford, G. A. (1970) in The Testis, II, eds.

Johnson, A. D., Gromes, W. R. & Vandemark, N. L. (Academ-ic Press, New York), pp. 259-306.

23. Vaughn, J. C. (1966) J. Cell Biol. 31, 257-278.

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