Acknowledgments-We are grateful to Drs. Barbara M. Sanborn and Salvatore Gulizia ... S., Jr. (1982) in Genetic Expression in the Cell Cycle (Padilla,. G. M., and ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Societyof Biological Chemists, Inc.
Vol. 259, No.14. Issue of July 25, pp. 8840-8&p6,19&1 Printed in U.S.A.
Characterization of High Mobility Group Protein Levels during Spermatogenesis in the Rat* (Received for publication, March 1, 1984)
Luke R.BucciS, WilliamA. Brock, Ira L. Goldknopfi, and Marvin L.Meistrich From the Department of Experimental Radiotherapy, The University of Texas M . D.Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 andThe University of Texas at Houston, GraduateSchool of Biomedical Sciences, Houston, Texas 77030 and$Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
The distribution, quantitation, andsynthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic andtestis HMGB showed no differences and, therefore, failed to detect an HMGS variant. Testis levels ofHMG proteins, relative to DNA content, were equivalent to other tissues for H M G l ( l 3 gg/mg of DNA), HMG14(3 gg/mg of DNA), and HMG17 ( 5 pg/mg of DNA). The testis was distinguished in that itcontained a substantially higher level of HMG2 than any other rat tissue (32 pg/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing themajor stages of spermatogenesis; spermatogonia andearlyprimary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. Highlevels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 2 4 and 47 2 6 pg/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMGS (12 f 3 pg/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMGB (2 pg/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins weresynthesized in spermatogonia and primaryspermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A “slow”form comigrated with somatic cell HMG2, while the other “fast” band migrated ahead of the somatic form and appeared to be testis-specific. The “fast” form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very
high level ofHMGB in testis is not associated with proliferative activityas previously hypothesized.
In mammals, the four major HMG’ proteins can be divided into two categories: the high molecular weight proteins HMGl and -2 and the low molecular weight proteins HMG14 and 17 (1). HMGl4 and -17 are very similar proteins (2), and together with an analogous protein H6 in trout(3), they have been implicated in the maintenance of the chromatin configuration necessary for transcription (4). A function for HMGl and -2 has not been demonstrated yet, but a review of their properties suggests a role in arrangement of higher order chromatin structure (5). HMGl and -2 have closely related sequences, possessing basic NH2-terminal halves with clusters of hydrophobic amino acids and highly acidic COOH-terminal halves (2). In chromatin, HMGl and-2 reside on internucleosoma1 (linker) regions (6) and possess both relatively tight and loose populations bound to chromatin (7). Studies indicate that HMGl and -2 replace H1 on a subset of mononucleosomes (8) and are arranged nonrandomly, in clusters, on chromatin (8, 9). Nuclease digestion studies have associated HMG1, -2, and the analogous trout protein HMG-T (3) with transcriptionally active chromatin (10-12).However, these experiments require the preparation of nuclei, and other studies, incuding this one, show that HMGl and-2 are lost from cells during preparation of nuclei (7, 13-17), possibly because of an equilibrium with a cytoplasmic pool of HMGl and -2 (18, 19). The known properties of HMGl and -2 make it likely that theyare involved in manipulating the supranucleosomal structure of chromatin (5). By their sizes, charge domains, DNA-binding characteristics, and nucleosomal linker location, HMGl and-2 can be envisaged to separate nucleosomal strands, possibly providing access for enzymes or recognition proteins. If so, functions in DNA replication, transcription, or genetic recombination are likely. Although no direct proof of a role for HMGl and-2 exists, their levels have been shown to vary during cell differentia*This investigation was supported in part by Grants PCM-78- tion. In terminally differentiatedcells with reduced transcrip06097from the National Science Foundation and HD-16843,CA- tional activity, HMG protein levels are decreased or absent 17364,and CA-06294from the National Cancer Institute, Department (16, 20-22). Several reports suggest that in muscle (16, 23), of Health and Human Services. Animals used in this study were testis (16), and salivary gland tissues (24) the ratio of HMG2 maintained in facilities approved by the American Association for to HMGl increases in parallel with proliferative activity. Accreditation of Laboratory Animal Care and in accordance with However, other reports on regenerating rat liver (25) and current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services, Na- mouse neuroblastoma cells (22) do notnote an increased tional Institutes of Health. The costs of publication of this article HMG2/HMG1 ratio during cell proliferation. These studies
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. $ Recipient of the Rosalie B. Hite Memorial Fellowship.This paper is partial fulfillment for the Ph.D. requirements of this author.
‘The abbreviations used are: HMG, high mobility group; PAS, periodic acid-Schiff reagent; SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine; APS, ammonium persulfate; BIS, N,N‘-methylenebisac~ylamide.
8840
HMG Proteins in Rat Testis Cell Types employed the use of ratios, and not quantitative levels, in order to express protein levels, and thus true increases or decreases in individual protein levels may have been undetected. This study was initiated to determine the levels of HMG proteins throughout a differentiating system, spermatogenesis. Cell proliferation, mitosis, DNA replication without cell division, meiosis,transcription of genes under developmental control, and terminal differentiation to a transcriptionally inactive state all occur during spermatogenesis in different cell types (26). Most of these cell types can be isolated in high purity and yield, offeringthe opportunity to possibly correlate HMG protein levels to cellular activities. This report: 1) identifies and characterizes the HMG proteins of rat testis; 2) studies levels of HMG proteins in each major cell type of the testis (including the somatic cell elements); 3) localizes synthesis of HMG proteins with respect to cell type;4) shows that changes in HMG2/HMG1 ratios are due to quantitative increases of a testis-specific form of HMG2; and 5) shows that very high levels of HMGB in spermatogenesis are associated with nonreplicative spermatocytes and spermatids, indicating another cause for elevation of HMGZ levels besides replication. MATERIALS AND METHODS AND RESULTS~
HMG Protein Levels in Rat Testis Cells-Comparisonof HMG protein levels, relative to DNA, in whole tissue or cells to levels in nuclei revealed a substantial loss of HMGl and -2 from nuclei (Table IV). This finding was observed in both testis and somaic tissues and agrees with previousreports (7, 13-17). Thus, HMG protein extracts from whole tissues or cells were routinely used for quantitation of HMG protein levels. HMG protein levelswere determined for 15 adult tissues. Levels of HMG1, -14, and -17 were similar for all tissues, and thus thevalues forsomatic tissues were averaged for comparison to testis levels (Table IV). The quantitative level of HMGS per unit of DNA varied among tissues according to proliferative status. Proliferating tissues contained a 2to &fold higherHMGS level than did nonproliferatingtissues. Testis tissue was unique, in that it contained a 3-foid higher HMGB level than did proliferating tissues (Table IV). In addition, on long acid-urea gels, testis HMGZ was resolved into two bands (Fig. 1).The “slow” testis band comigrated with HMGB from somatic tissues; however, the “fast” band was not seen in any somatic tissue and thus appeared to be testis-specific. Since all tissues, especially testis, are composed of a mixture of cell types, it is possible that whole tissue HMG levels reflect average values from populations of cells varyingwidely in their HMG contents. Thus, in order to determine why the testis contains high levels of HMG2, discrete cell types must be analyzed to ascertain the possible significanceof variations in HMG protein levels. In male rats, the only tissue that contains developing germ cells the is testis, and thepossibility that the uniquely elevated levels of HMG2 in the testis are due to germ cellswas examined. Germ cellscan be completely eliminated from rat testesby treatment with y irradiation (x-
* Portions of this paper (including “Materials and Methods,” part of “Results,” andTables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifyingglass. Full size photocopies are available fromthe Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. RequestDocument NO. “-657, cite the authors,andincludea checkormoneyorderfor $4.00 per set of photocopies. Fullsize photocopies arealso included in the microfilm edition of the Journal that is available from Waverly Press.
8841
ray-treated testes), leaving onlythe somatic cells ofthe testisSertoli cells, Leydig cells, tubule myoid cells, blood vessel endothelial cells, blood cells, interstitial macrophages, and fibroblasts. Examination of HMG protein levelsin x-raytreated testes showed a lowlevelof HMG2 (2.0 pg/mg of DNA), similar to nonproliferatingtissues. These results agree with the previously reported relative decreaseof HMGB in rat testes-rendered cryptorchid, a process that greatly reducesthe number of germcells (16).Furthermore, examination of HMG protein levels from two types of isolated somatic cells of rat testes (Sertoli and Leydig cells) also showed a lowlevel of HMG2, similar to nonproliferating tissues (Table IV). Thus, HMG protein levelsfrom somatic cell types of the testis indicated 1) that increased levels of HMG2 in whole testis were present in germ cells;and 2) testicular somatic cells are typified by low levels of HMG2. In order to determine which germinal cell type(s) contained elevated HMGP levels, single cell suspensions of rat testis tissue were elutriated and subjected to equilibrium density centrifugation as described under “Materials and Methods.” Five separate populations of cells were isolated that encompassed nearly all germinal cell types of the testis (Table 11). HMG proteins from purified testis cells are shown in Fig. 1. The first population ofgerm cells, representing the initial stages of spermatogenesis,was enriched in spermatogonia and early primary spermatocytes (see Table 11).HMG1, -14, and -17 levels in this population were similar to both whole testis and other rat tissues (Table IV). On the other hand, HMG2 levels were significantly lessthan whole testis and similar to proliferating tissues. The next stage of spermatogenesis,pachytene spermatocytes, was isolated in high purity (Table 11) and showed levelsof HMG14 and -17 similar to those of whole testis (Table IV). Levels of HMGS in pachytene spermatocytes were increased 5-fold over the level in spermatogonia and early primary spermatocytes (Table IV). The subsequent stage of spermatogenesis, early spermatids, retained the same pattern of HMG protein levels as pachytene spermatocytes. There appeared to be a developmentally related decrease in HMGl levels in germinal cells.In the final stages of spermatogenesis, late spermatids (steps 9-19) are characterized by nuclear condensation and elongation caused by replacement of histones and nonhistones with late spermatidal basic nuclear proteins (46). Owing to the rigors of cell suspension preparations, some late spermatids separate into cytoplasts and nuclei (30). Both portions were isolated (Table 11), and each was analyzed forHMG protein content; both were found to be devoid of HMG proteins. In late spermatid nuclei, TP2 and TP4 obscured the region in gels whereHMGl4 migrated, as previously noted, and its absence, though extremely likely, cannot be unequivocally proved by this study. These results from isolated testis cells illustrated that the unique elevated levels of HMGB in testis tissue are due to specific cell types that contain greatly increased HMGZ levels, pachytenespermatocytes through early spermatids. All other cell types of the testis either possess no HMG proteins (late spermatids) or possess levelswithin the ranges of somatic tissues, Synthesis of HMG Proteins in Germinal Cells-hcorporation of tritiated lysine into cellular HMG proteins was examined. In whole testis or testis cell suspensions, radioactive lysine was incorporated into HMG1, -2, and -17 (Fig. 3A). HMG1.Qwas obscured by the late spermatidal proteins TP2 and TP4. Lysine was actively incorporated into HMG1, -2, and -17 in spermatogonia and early primary spermatocytes (Fig. 3B). Pachytene spermatocytes showed similar incorporation into HMGl as did spermatogonia and early primary spermatocytes (normalized to total DNA), but incorporation
HMG Proteins in Rat Testis Cell Types
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TABLE IV Quantitative levels of HMC proteins and HI histones in rat tissues and testis cells H1 histones are included for comparative purposes. A rat diploid nucleus (6 pg of DNA) should contain 160 pg of Hl/mg of DNA, assumingone H1 histone (31,000 daltons)per nucleosome, 165 basepairs of DNA per nucleosome, and equal amounts of DNA bases. HMGl H1
Sample
HMGP HMGl7
pg
Somatic tissuesb
12.9 f 0.8
2.9 1-5' 5-13 31.8k 2.0 12.3 1.6 f 2.4 40.5 f 2.3 1 1 . 5 f 3.1 56.4 f 4.0 46.6f 5.6 ND ND 1.5 f 0.1 2.1 f 0.1
H1'
n
15-45'
5od
HMGl4 proteinfmg DNA k S.E."
4.8 f 0.3
182 f 0.4
f6
0-7
Whole testis 13.5f 1.1 1.6 f 0.3 5.9 f 0.7 149k 9 3.9 f 0.6 Testis nuclei 2.4 f 1.03.2 3.7 177 f 39 4.6 f 0.2 2.4 4.1 k 0.3 163 f 10 9.2 f 0.6 Testis cell suspension 7.5 f 2.7 4.8 f 0.9 169 k 27 1.4 f 0.1 10.4 f 2.0 G/EPS' 4.1 f 1.0 162 k 11 4.2 f 0.6 Pachytene 8.0 f 0.9 1.8 128f 15 13 f 1.s' 2 . 8 f 0.3 1.0 f 0.4 spermatids Early 6.5 f 1.0 11 ND ND N P Late spermatids 106 k 4 5.3 f 1.3 1.9 4.2 f 2.3 10.6 f 2.2 Sertoli cells 114 f 10 8.3 1.9 9.5 f 1.5 15.6 f 4.4 Leydig cells "Values without standard errors represent one determination. HMG14 values for whole testis and G/EPS samples are theaverage of two determinations. * HMG protein levels determined from brain, bone marrow, cecum, epididymis, heart, kidney, leukocytes, liver, lung, pancreas, seminal vesicle, small intestine, spleen, and thymus tissueswere averaged. Nonproliferating tissues (brain,epididymis, heart, kidney, liver, lung, pancreas, and seminalvesicle) contained HMGZ levels between 1 and 5 pg/mg of DNA, and H1" levels between 15 and 45 pg/mg of DNA. Proliferating tissues (bone marrow, cecum, leukocytes, small intestine, spleen, and thymus) exhibited HMGZ levels between 5 and 13 pg/mg of DNA, and H1' levels between 0 and 7 pg/mg of DNA. HMG14 and HMG17levels are from 32 and 40 determinations, respectively. e G/EPS, spermatogonia and early primary spermatocytes (preleptotene, leptotene, and zygotene stages). 'The value of HI" also includes a cytoplasmic protein that migrates very close to H1' on acid-urea gels. Thus the true value of H1" in early spermatids islower than the statedvalue. ND, not detected.
16 7 14 3 7 5 1 3 2
HMGl +MG2 "HMG 1 "slow HMGP &-fast HMGP
-HI at Ql)l)-Uw-dWWW-lilbcde -HO
-Ubiq.
A
B
C
-TP2 -HMG 14 -TP4
FIG.1. Acid-urea gel of rat HMC protein extracts from: ( A ) spleen, ( B )testis, and (C) testis cell suspension. Note the presence of the "fast" band in testis samples only.
into HMG2 wasmuchhigher(Fig. 3C). Earlyspermatids showed only a trace of radioactivity corresponding toHMGl and -2 peaks and noradioactivity with HMGl4 and -17 peaks (Fig. 30). We concluded that HMGl and -2 are synthesized in spermatogonia, early primary spermatocytes, and pachytene spermatocytes, withnegligible levels of synthesis in early spermatids3
-HMG 17
A B C D E F G H I
FIG.2. Acid-urea gel of HMG protein extracts from rat testis cell types. Samples were rat HMG protein extracts from: A, testis nuclei; E , whole testis tissue; C, testis cell suspension; D, spermatogonia and early primary spermatocytes;E , pachytene spermatocytes; F, early spermatids; C , Sertoli cells; H , Leydig cells; and I , whole liver tissue. Migration is from top (+) to bottom (-1.
DISCUSSION
This communication hasidentified HMG1, -2, -14, and -17 in the rat testis by their extraction and solubility character-
istics, electrophoretic mobility, ion exchange chromatography, NH2-terminalamino acids, andamino acidanalysis. The ~results show that rat testis HMG proteins are almost identical Although we cannot be assured that incorporation of [3H]lysine HMG14, to their calf thymus counterparts, except for rat reflects quantitative measurement of HMG protein synthesis due to lack of knowledge about specific activities of amino acid pools in the which possesses twoamino acids, histidine andisoleucine, not different cell types, relative levels of HMG protein synthesis can be present incalf HMG14. The determinationof absolute HMG compared. The incorporation of [3H]lysine into acid-insoluble chro- protein levels in various rat tissue extends the previous obmosomal proteins (47) andubiquitin(thispaper)aresimilarin pachytene spermatocytes and early spermatids, indicating that dif- servations of HMGl and -2 ratios reported by Seyedin and ferent testis cell types exhibit little variation in specific activities of Kistler (16). The advantage of determining absolute levels, rather than ratios only, is that actual increases or decreases lysine pools.
HMG Proteins in Rat Testis Cell Types WHOLE TESTIS Hlal I Hl bcde
.o Ubiguitin
i
M
-.
i
TP2.4
1
”
HMG17 ”
PRIMARY SPERMATOCYTES
TP2.4
mat
C
‘
HMGZ 1
1‘ lIlbCde
I .o
3.5
PACHYTENE SPERMATOCYTES
1 U
4.OU 0
z 4
m
M
8m
2.04 Ubiquilin
....^ HMQU
HMQ17
EARLYSPERMATIDS
2.0 1.o
HMGU
HMQ17
FIG. 3. Protein (-) and radioactivity (- - -) profiles of testis cell type HMG protein extracts. Incorporation of tritiated lysine into ( A ) whole testis, ( B ) spermatogonia and early primary spermatocytes, (C) pachytene spermatocytes, and (D)early spermatids electrophoresed on acid-urea cylindrical gels. Migration is from left (+) to right (-). Absorbance was measured at 525 nm.
in protein levels can affect ratios and possibly lead to erroneous conclusions.For example, an increased HMGZ/HMGl ratio may be caused by a decrease in HMGl levels and not a true increase in HMGZ levels. Determination of absolute levels eliminates these sources of error and allows for valid comparisons among samples. Unlike HMG1, -14, or -17, the absolute levels of HMG2 from various tissues showed dramatic differences. Three categories of tissue HMGZ levels werefound low, intermediate, and high levels of HMG2. Nonproliferating tissues, with low levels of HMG2 (less than 6 pg/mg of DNA), invariably possessed much greater H1” levels (15-45 pg/mg of DNA) than tissues with intermediate or high HMGZ levels, in agreement with previous reports of a reciprocal relationship between HMG2 and H1” levels in tissues and cells (16, 22). Tissues with intermediate levels of HMGZ were either proliferating (bone marrow, cecum,small intestine, and thymus) or
8843
possessed a high proportion of lymphoid cells (bone marrow, leukocytes, spleen, and thymus). These tissues also possessed very low levels of H1” (0-7 pg/mg of DNA). These findings agree with previousreports that link elevated levelsof HMGZ with proliferation of rat tissues (16, 24). The testis, also a proliferating tissue, is the only organthat contains high levels of HMG2. This observation was also made in an amphibian (Xenopus laeuis), a marsupial (opposum),and other mammals (mouse, dog, and rhesus monkey): The testis is unique in that it contains germ cells. The association of high levels of HMG2 with germ cell-specific phenomenawas confirmed by characterization of HMG protein levels in testicular somatic and germ cell types.Testicular somatic cells donot contribute to high HMG2 levels inthe testisbut rather are overwhelmed by the greater number of germ cells which do contain high levels of HMG2. Interestingly, the only proliferating cells of the testis, spermatogonia, and preleptotene spermatocytes showed only intermediate levels of HMGS consistent with proliferating tissues and not the large testis-specific increase found in pachytene spermatocytes and early spermatids. By a method to be published elsewhere, populations of type A and type B spermatogonia as well as early, middle, and late stage pachytene spermatocytes were isolated, and HMG protein levels characterized. Levels of HMG proteins from spermatogonia and early pachytene spermatocytes4were not different from levels found in the mixtures of spermatogonia and early primary spermatocytes used in this study. This indicates that spermatogonia and early primary spermatocytes contain equivalent amounts of HMG proteins and that the particular cell type which starts thetremendous increase in HMGZ levels must be after the early pachytene stage. The high levelsof testis HMGS are attributable to the“fast” form of HMGZ in pachytene spermatocytes and early spermatids (data not shown), since late spermatids contain no HMG proteins. This is substantiated by calculationsconsideringthe percentage of each cell type in testis (29), the DNA contribution of each cell type, and the observed HMG2 levels of each cell type (Table IV). The calculated value of 28 pg of HMGZ/mg of DNA agrees well with the observed valueof 32 pg of HMG2/mg of DNA. Secondary spermatocytes, which occur betweenpachytene spermatocytesand early spermatids, comprise lessthan 1% of testis cells (29), and no method yet exists to isolate or enrich them. The HMG protein levels of these cells are unknown, but we see no reason for the levels to differ substantially from pachytenes or early spermatids. Thus, the fate of HMGZ during spermatogenesis canbe traced (Fig. 4). The appearance of a “fast” form of HMG2 during the pachytene spermatocyte stage, with high synthesis rates, and maintenance of these levels without synthesis in early spermatids, closely resembles the case of testis-specific histones in rat testis cells. Synthesis and accumulation of germ cellspecific variants of H1, H2A, and H2B occur in pachytene spermatocytes only, while early spermatids maintain the histone composition of pachytene spermatocytes with no detectable synthesis (32, 44, 46, 47). Although the “fast” form of HMGZ was not shown to be a sequence variant of HMG2, the mobility increase on acid-urea gels still leaves postsynthetic modification as a source of heterogeneity. Testis tissue from X. laeuis toads, mice, or dogs also exhibited a testisspecific or -enriched “fast” form of HMGP,’ indicating that heterogeneity of HMG2 may bea common phenomenon.Also, like the histones, HMG proteins were absent from late spermatids, presumably replacedby late spermatidal basic nuclear L. Bucci, unpublished results.
HMG Proteins in Ra.t Testis Cell Types
8844
DAYS
0
REFERENCES
x ) 2 0 3 0 4 0 5 0 6 0
1. Johns, E. W. (1982) in The HMG Chromosomal Proteins (Johns, E. W., ed), pp. 1-8, Academic Press, New York CELL TYP 2. Walker, J. M. (1982) in The HMGChromosomal Proteins(Johns, E. W., ed) pp. 69-88, Academic Press, New York 3. Dixon, G. H. (1982) in The HMG Chromosomal Proteins (Johns, E. W., ed) pp. 149-192, Academic Press, New York 4. McCarty, K. S., Sr., Kellner, D. N., Wilke, K., and McCarty, K. S., Jr. (1982) in Genetic Expression in the Cell Cycle (Padilla, G.M., and McCarty, K. S., Sr., eds), pp. 55-102,Academic Press, New York 5. Goodwin, G. H., and Mathew, C. G. P. (1982) in TheHMG Chromosomal Proteins (Johns, E. W., ed) pp. 193-221,AcaHMGl.2 demic Press, New York PROTEIN 6. Peters, E. H., Levy-Wilson, B., and Dixon, G. H. (1979) J. Biol. LEVELS Chem. 254,3358-3361 7. Mathew, C. G. P., Goodwin, G. H., andJohns, E. W. (1979) Nucleic Acid Res. 6, 167-179 8. Jackson, J. B., Pollock, J. M., Jr., and Rill, R. L. (1979) Biochemistry 18,3739-3748 9. Itkes, A.V., Glotov, B. O., Nikolaev, L. G., and Severin, E. S. HMGP SYNl‘HESIS (1980) FEBS Lett. 1 1 8 , 63-66 HMGl 10. Goodwin, G. J., Mathew, C. G. P., Wright, C. A., Venkov, C. D., and Johns, E. W. (1979) Nucleic Acids Res. 7, 1815-1835 11. Levy W., B., Connor, W., and Dixon, G. H. (1979) J. Biol. Chem. FIG. 4. Spermatogenesis in the rat andaccompanying 254,609-620 changes in H M G l and -2 protein levels and synthesis. Germ 12. Vidali, G., Boffa, L. C., and Allfrey, V. G. (1977) Cell 12, 409cell figures (49) are not drawn to scale. Synthesis of HMGl and -2 415 depicts relative levels. Data for type A spermatogonia to zygotene 13. Gordon, J. S., Bruno, J., and Lucas, J. L. (1981) J. Cell Biol. 88, spermatocytes were obtained from the populations of spermatogonia 373-379 and early primary spermatocytes. 14. Albright, S. C., Wiseman, J. M., Lange, R. A., and Garrard, W. T. (1980) J. Biol. Chem. 255, 3673-3684 proteins (46). Since the DNA of late spermatids is highly 15. Wu, L., Reichsteiner, M., and Kuehl, L. (1981) J. Cell Biol. 91, 488-496 condensed to a transcriptionally inactive state (26), results 16. Seyedin, S. M., and Kistler, W. S. (1979) J. Biol. Chem. 254, suggest that HMG proteins are associated with functional 11264-11271 nucleosomal chromatin. 17. Isackson, P. J., Bidney, D. L., Reeck, G. R., Neihart, N. K., and The enormous increase of testis HMG2 is associated with Bustin, M. (1980) Biochemistry 19,4466-4471 meiosis and not proliferation. Possible functions for high 18. Bustin, M., and Neihart, N. K. (1979) Cell 16, 181-189 HMGB levels may include roles in testis-specific events such 19. Rechsteiner, M., and Kuehl, L. (1979) Cell 16,901-908 as genetic recombination, meiotic reductive divisions, or re- 20. Mezquita, C., Chiva, M., and Vidal-Sivilla, S. (1980) J. Cell Biol. 87, 153a (abstr.) placement of histones by late spermatidal basic proteins dur21. Kennedy, B. P., and Davies, P. L. (1980) J. Biol. Chem. 255, ing nuclear condensation. Conversely, high levels of HMG2 2533-2539 in germ cells may only reflect an increased need of germ cells 22. Seyedin, S. M., Pehrson, J. R., and Cole, R.D. (1981) Proc. Natl. for a function common to all cells, such as modulation of Acad. Sci. U. S. A. 78, 5988-5992 developmentally controlled gene expression, since pachytene 23. Gordon, J. S., Kaufman, R., and Rosenfeld, V. I. (1981) Arch. Biochem. Biophys. 21 1, 709-721 spermatocytes initiate synthesis of many germ cell-specific proteins, including enzymes and surface antigens (26). Gene 24. Pipkin, J. L., Hinson, W. G., Hudson, J. L., Anson, J., and Pack, L. D. (1981) Biochim. Biophys. Acta 655,421-431 expression of proteins necessary for cell replication may be 25. Kuehl, L. (1979) J. Biol. Chem. 254, 7276-7281 another example of how HMGB levels affect chromatin, since 26. Bellve, A.R. (1979) in Oxford Reuiews of Reproductive Biology Seyedin and Kistler also noticed that increased levels of (Finn, C. A., ed) Vol. I, pp. 159-261, Clarendon Press, Oxford, England HMG2 were present in proliferating tissues (16). This could account for the intermediate levels of HMG2 found in the 27. Leblond, C. P., and Clermont, Y. (1952) Ann. N. Y. Acad. Sci. 55,548-573 group of proliferating tissues. Although no direct evidence 28. Tack, B. F., Dean, J., Eilat, D., Lorenz, P. E., and Schechter, A. exists for such a hypothesis, the biochemical properties and N. (1980) J. Biol. Chem. 255,8842-8847 tissue levels of HMGl and -2 can lead one to speculate that 29. Meistrich, M. L. (1977) Methods Cell Biol. 15, 15-54 both HMGl andHMGB are involved in gene expression, with 30. Meistrich, M. L., Longtin, J. L., Brock, W. A., Grimes, S. R., and Mace, M. L. (1981) Biol. Reprod. 25, 1065-1077 HMGl being associated with chromatin regions necessary for cell survival common to all cells and HMG2 being associated 31. Bucci, L. R., Brock, W. A., and Meistrich, M. L. (1982) Exp. Cell Res. 140,111-118 with genes that only need to be expressed at certain times of 32. Steinberger, A., Heindel, J., Lindsey, J., Elkington, J., Sanborn, development. However, further studies on HMG proteins are B., and Steinberger, E. (1975) Endocr. Res. Commun. 2, 261necessary before a role of HMGl and-2 can be demonstrated. 272 33. Galdieri, M., Ziparo, E., Palombi, F., Russo, M. A., and Stefanini, Acknowledgments-We are grateful t o Drs. Barbara M. Sanborn M. (1981) J. Androl. 5, 249-254 and Salvatore Gulizia for advice on the preparation of Leydig cells, 34. Browning, J. Y., D’Agata, R., and Grotjan,H. E., Jr. (1981) Dr. Anna Steinberger and her laboratory for supplying Sertoli cell Endocrinology 109, 667-669 cultures, Barbara Martin and Drs. Juan Guevarra and Lewis Rodri- 35. Gulizia, S., D’Agata, R., Sanborn, B., and Steinberger, E. (1983) guez for silver staining of polyacrylamide gels, Patricia Trostle-Weige J. Androl. 4,248-252 for technical help and assistance with peptide mapping, and Ann 36. Platz, R. D., Meistrich, M. L., and Grimes, S. R., Jr. (1977) McCarver for typingthis manuscript. The histologic preparation and Methods Cell Biol. 16,297-316 37. Nicolas, R. H., and Goodwin, G. H. (1982) in The HMG Chrotechnical assistance were provided by the Histopathology Laboratories of The University of Texas, M. D. Anderson Hospital and Tumor mosomal Proteins (Johns,E. W., ed), pp. 41-68, Academic Institute at Houston. Press, New York I
I
u l
1
1
1
1
1
I
Testis HMG Rat Proteins in 38. Laemmli, U. K. (1970) Nature (Lord.)227, 680-685 39. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130,337-346 40. Mardian, J. K. W., and Isenberg, I. (1978) Anal. Biochem. 92,l12 41. Guevara, J., Johnston, D. A., Ramagli, L. S., Martin, B. A., Capetillo, S., andRodriguez, L. V. (1982) Electrophoresis 3 , 197-205 42. Gray, W. R. (1972) Methods Enzymol. 25, 121-138 43. Burton, K. (1956) Biochem. J. 62,315-323 44. Trostle-Weige, P. K., Meistrich, M. L., Brock, W. A., Nishioka,
Cell Types
8845
K., and Bremer, J. W. (1982) J. Biol. Chem. 2 5 7 , 5560-5567 45. Sterner, R.,Boffa, L. C., andVidali, G. (1978) J. Bwl. Chem. 283,3830-3836 46. Meistrich, M. L., Trostle, P. K., and Brock, W. A. (1981) in Bioreguhtors of Reproduction (Jagiello, A., ed) pp.151-166, Academic Press, New York 47. Brock, W. A., Trostle, P. K., and Meistrich, M. L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,371-375 48. Huckins, C. (1971) Cell Tissue Kin& 4, 139-154 49. Perey, B., Clermont, Y., and Leblond, C. P. (1961) Am. J. Anat. 108.47-77
Table 1 Modifled elutriator conditions
f o r separatim o f spermatogonia L
e a r l y primary spermatocytes f m a d u l t r a t t e s t i s Rotor Speed
(rp)
Flow Rate (nllmin)
3003
12.0
2'
3wO
3
2003
qb
2000
Fractim 1
cell rurpenrionr
Volm
.u
Predrmrnant Cell Types
I80
spermatid Late cytoplasts, spermatids. late residualbodler
25.0
150
Early rpennatidr. cytoplasts, spemtogmia, early p r i m l y spematocytes.leukocytes
25.0
250
Early Spenatlds, rpenatacyter. nucleatedIpematidr
38.0
150
Pachytene IpenMtacyteS. multi-nucleated r p e m t i d r .S e r t o l lc e l l s
multi-
HMG Proteins in Rat Testis Cell Types
8846 Purities
b. Cell t y p Sertoli
lmlated Testis c e l l m p u l a t i m s used for Ms Protein Level Deteminatims
Of
c e l FV!ty
cpeulrl i t y
Prep5.
3
99*1
97.8
Of
rMgt
Leydig
2
67 f 12
55.0
S p m t o g a i a hb early primry IpMtOCytel
3
39
+ 11
m.4
Pachytme rpnatocyter
7
93fl
Early s p m t i d s d
5
93 f 2
3
97*2
3
*+2
18.1
- 50.2
- 97.5 88.7 - 97.7 95.0
Late S P n a t i d cytop1asts
~
99.7
88.0
Late rpmtiff nuclei
~
91.4
- 98.7 - 97.4
D** C o n t r i b u t i m ~ from c e l l typ
99
Calf R4t Rat Calf testis thwr testis thws u61 Hlfil M32 M32
+I
71 2 14 78
*
3c
96
*
1
Asp Glu Lyr Arq His Ala
5;
93 f 1
Leu kt
IOf M9
Gly
Sly
11.0 17.3
18.0 3.3
M s 1 7 Hc.17
8.0 13.4 21.0 5.0 0.0
10.0
10.4
8.4
8.6
16.9 19.4
20.7 19.1 4.0
13.4 21.6 4.1 1.2 14.3
20.1
3.5 1.2
7.4
9.7
4.0 2.1 7.1
1.6
8.0
16.0
!1.5 :
7%
:1.2:
9”:;::!0.0
1.9
2.7
2.0
2.6
1.9 4.2
1.3
2.0
3.0 6.9 1.9
3.6
0.3 0.6
6.8
6.9
7.0
7.6 2.0 3.2 1.6
8.9
3.6
9.0 4.0
tr
0.0
3.0
1.9
phe
Pm
3.1 8.4
Ser
6.7
Thr ~ y r Val
2.3 4.0 2.2 2.1 3.5
2.9
1.9
2.9
lbtem Gly
Gly
3.0
Rat Calf testis thws
W14
20.1
2.0
6% 2.3 2.9 1.3
Rat Calf testis thws
a14
5.4 5.8
1.5
2.0 0.0 0.0
9.4
10.4
10.1 24.7
16.7 17.9
11.6
9.5 18.1
20.9
19.3
4.1 0.0 18.8
4.5
4.5
4.2
3.9
0.0 18.0
1.5 7.9
1.9 7.1
7.6
i:i
6:;
1;: 0.0 1.3
1:
0.0
1.1
? t’ tr
0.0 0.0
11.8 2.5 1.1
12.4
i:; 1.6 2.8 1.5
1.1
4.0
tr 2.5 0.0 2.9 2.2
2.4
pro
pro
Values fnm ref 16; 24-harr n y d r n l y r i s tie.
values f n n
12.4
22.2
3.2 7.6 6.9 2.6
a ValUeI fmn r e f 1.
c
13.7 10.0
Ratb RatC RatC testis liver salivavy Ms2 Hc.2 p l r n d rp62
ref 24: 22-hour hYdm1y*ir t n ly .
2.2
2.1 2.0 1.9 3.8
1.9
1.0 2.0 1.6
4.0
7.7
1.6
6.9 2.8 2.6
2.G 2.8
2.3
2.1
7.5
