Vol. 10, No. 6MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 3239-32420270-7306/90/063239-04$02.00/0Copyright ©D 1990, American Society for Microbiology

Expression of HSP86 in Male Germ CellsSE-JIN LEE

Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway,Baltimore, Maryland 21210-3399

Received 27 October 1989/Accepted 16 February 1990

A comparison of HSP84 and HSP86 mRNA expression in adult mouse tissues revealed distinct expressionpatterns for these highly homologous genes. Particularly striking is the germ cell specificity of HSP86expression in the testis, suggesting distinct roles for HSP84 and HSP86 with respect to testicular function anddevelopment.

The period of mouse postimplantation development duringwhich early organogenesis takes place represents one of themost complex and important phases of mouse embryogene-sis with respect to cell differentiation. To begin to charac-terize some of the molecular events underlying these differ-entiation processes, I have been carrying out differentialscreening of embryonic cDNA libraries using stage-specificcDNA probes. Nucleotide sequence analysis of one cloneisolated during this screening process revealed an openreading frame potentially encoding a protein 85.8% homol-ogous to the gene for the murine 90-kilodalton heat shockprotein, HSP84. While this work was being completed,Moore et al. (14) reported isolating a virtually identicalclone, which they have designated HSP86. In order toinvestigate possible functional differences between HSP84and HSP86, a cDNA clone for HSP84 was isolated byrescreening the day 9.5 embryonic cDNA library at lowstringency by using the HSP86 clone as a probe. Portions ofthese clones (nucleotides 1321 to 2415 from HSP84 andnucleotides 1347 to 2571 from HSP86 [14]) were then used asprobes to compare the expression patterns of these twogenes.The 90-kilodalton heat shock protein is synthesized at high

levels in the midgestation mouse embryo (1, 3). To investi-gate whether the observed expression represents HSP84,HSP86, or both, Northern (RNA) blot analyses (7, 10) ofembryonic RNA were carried out with the two probes. Thetwo genes showed similar temporal patterns of expression;the highest levels were detected at the earliest time analyzed(day 8.5), and gradually decreasing levels were detected atlater times during embryogenesis (Fig. 1). Hence, bothHSP84 and HSP86 are expressed at high levels in themidgestation mouse embryo.To determine whether the spatial patterns of expression of

the two genes are also similar, RNA was isolated fromvarious adult tissues and probed with HSP84 and HSP86.HSP84 mRNA was detected in virtually every tissue; thehighest levels were detected in the adrenal gland and theovary, and many other tissues showed an almost comparablelevel of expression (Fig. 2). In contrast, although HSP86mRNA was also detected in many tissues, its levels in thetestis were strikingly higher than in any other tissue exam-ined. Among the adult tissues, the next highest level ofexpression was in the brain, but this was significantly lowerthan the level in the testis.The adult testis contains somatic cells, consisting predom-

inantly of Leydig cells and Sertoli cells, as well as germ cellsat various stages of differentiation. Within the seminiferous

tubules, the physical organization of the germ cells reflectsthe extent of differentiation, so that the least mature stemcells are located furthest outside, and the most maturespermatazoa are located inside the lumen (15, 16). Todetermine which cell types in the testis express HSP86, thedistribution of HSP86 mRNA was analyzed by in situhybridization (20) with antisense and control sense RNAstranscribed in vitro (8, 13). The antisense HSP86 RNAhybridized in a ringlike pattern to the outside portion of theseminiferous tubules, corresponding to the distribution ofimmature spermatogenic cells (Fig. 3). No hybridization wasobserved in the lumen of the seminiferous tubules, wheremature spermatazoa are found, or the interstitial region,where Leydig cells reside.To verify that HSP86 is expressed in the germ cells of the

testis, RNA was prepared from the testis of the mutantmouse strain atrichosis (at). The testis from an at/at ho-mozygous mutant male mouse has been shown to be com-pletely devoid of germ cells (K. P. Hummel, Mouse Newsl.34:31-32) but has all of the normal somatic components,

mmMGM us-a=.-

FIG. 1. Expression of HSP86 and HSP84 during embryogenesis.Total RNA (10 ,ug) prepared from mouse embryos isolated at theindicated days of gestation was electrophoresed, blotted, andprobed with either HSP86 or HSP84.

3239

MOL. CELL. BIOL.

A

Sam" a UN

B

.. r.'4 *4

FIG. 2. Expression of HSP84 and HSP86 in adult tissues. TotalRNA (10 ,g) prepared from various adult tissues and from day 10.5embryos and placenta was electrophoresed, blotted, and probedwith either HSP84 (A) or HSP86 (B).

including Leydig and Sertoli cells (6, 9). A male mouseheterozygous for the at mutation has normal testes and isfully fertile. No HSP86 expression was detected in the at/attestis, although a high level of expression was found in thetestis of a heterozygous littermate (Fig. 4). The levels ofHSP86 mRNA were equivalent in the brains of homozygousand heterozygous mice, making it unlikely that this mutantstrain has a primary defect in HSP86 expression. Hence,HSP86 expression in the testis appears to be restricted to thegerm cells. In contrast, there was no difference in the testesof homozygous and heterozygous mice in the expression ofHSP84. Thus, whereas HSP86 expression is restricted to thegerm cell lineage, HSP84 is expressed in the somatic cells ofthe testis.To further investigate the contrasting patterns of expres-

sion of these two genes in the testis, Northern blot analyseswere carried out by using RNAs isolated from testes atvarious stages of prepuberal development. Embryonic testesand neonatal testes (before day 9 of development) are knownto contain all of the somatic components present in adulttestes as well as germ cells at all mitotic stages but to lackgerm cells that have reached the meiotic stages of develop-ment (2). Although HSP86 RNA was detected in testes at allstages, its levels were significantly higher in day 21 testes

t~~~~~~~~~~~~~~~"MII WI7 -s

FIG. 3. In situ hybridization to adult testis. Adjacent sections of adult testis were hybridized to sense (A) and antisense (B) 35S-labeledHSP86 RNA transcribed in vitro, dipped in Kodak NTB-3 photographic emulsion, exposed, developed, and stained with hematoxylin andeosin.

3240 NOTES

I

NOTES 3241

Ps.B- HSP86ies,s O!"a: te t-as. s tL-0eS G

+ i_ rr ~ i

,iV V C. .T_ 7 -

o2! cvE

0~V V-

o

o~ o~L VI_l ra

HSP84

'E T5;~-C

, c cc

0 o 0 0iD a l:L a:ND D T; 'ar ,O_ O_ V V

;

'A aa

S01*all.

FIG. 5. Expression of HSP86 and HSP84 during testicular de-velopment. Total RNA (10 ,ug) prepared from day 17 embryonictestes or from testes of 7-, 14-, or 21-day-old mice was electro-phoresed, blotted, and probed with either HSP86 or HSP84.

FIG. 4. Expression of HSP86 and HSP84 in the mutant strainatrichosis. Total RNA (10 ,ug) prepared from the brain or testes of a

homozygous at/at mutant mouse or a heterozygous atl+ littermatewas electrophoresed, blotted, and probed with either HSP86 or

HSP84.

than in day 17 embryonic testes or day 7 neonatal testes (Fig.5). Hence, HSP86 expression appears to be enriched in germcells that have reached meiotic prophase. In contrast,HSP84 mRNA was expressed at high levels throughouttesticular development, with slightly higher levels beingdetected in immature fetal and neonatal testes than in day 21testes, which is consistent with its expression in the somaticcells.The 90-kilodalton heat shock protein (HSP90) is one of the

major proteins synthesized by cells subjected to a variety ofstressful stimuli (for a review, see reference 11). Althoughthe biological function of HSP90 has not yet been described,recent data have suggested that HSP90 forms a complex withsteroid hormone receptors (5, 18, 19) as well as with retro-viral tyrosine kinases (4, 12, 17). The identification of asecond HSP90 gene raises the possibility that distinct HSP90proteins may be responsible for these diverse interactions.The possibility that HSP84 and HSP86 perform differentfunctions is supported by the findings that they have distincttissue and cell type distributions. Contrasting expressionpatterns in the adult testis and the developing testis have alsorecently been observed by D. Wolgemuth and C. Gruppi(personal communication).Whatever functions the two HSP90 genes perform during

testicular development, it is likely that they play a role inother developmental events as well, since they are bothexpressed at high levels in the embryo at a time whentesticular development has not yet been initiated. A deter-mination of the specific functions performed by HSP84 andHSP86 in adult tissues as well as during embryonic devel-opment awaits further characterization of the expressionpatterns of these two genes and comparison of the biochem-ical properties of their encoded products.

I thank Daniel Nathans, in whose laboratory this work was

initiated, for his continued interest and guidance, and JoshuaFarber, Anthony Lanahan, Michael McLane, and Allan Spradlingfor helpful discussions.

This work was supported by the Carnegie Institution of Washing-ton and by Junior Faculty Research Award JFRA-254 from theAmerican Cancer Society.

LITERATURE CITED1. Barnier, J. V., 0. Bensaude, M. Morange, and C. Babinet. 1987.

Mouse 89 kD heat shock protein. Exp. Cell Res. 170:186-194.2. Belive, A. R., J. C. Cavicchia, C. F. Miliette, D. A. O'Brien,

Y. M. Bhatnagar, and M. Dym. 1977. Spermatogenic cells of theprepuberal mouse. J. Cell Biol. 74:68-85.

3. Bensaude, O., and M. Morange. 1983. Spontaneous high expres-sion of heat-shock proteins in mouse embryonal carcinoma cellsand ectoderm from day 8 mouse embryo. EMBO J. 2:173-177.

4. Brugge, J. S., E. Erikson, and R. L. Erikson. 1981. The specificinteraction of the Rous sarcoma virus transforming protein,pp6src, with two cellular proteins. Cell 25:363-372.

5. Cateili, M. G., N. Binart, I. Jung-Testas, J. M. Renoir, E. E.Baulieu, J. R. Feramisco, and W. J. Welch. 1985. The common90-kd protein component of non-transformed '8S' steroid recep-tors is a heat-shock protein. EMBO J. 4:3131-3135.

6. Chubb, C., and C. Nolan. 1984. Genetic control of steroidogen-esis and spermatogenesis in inbred mice. Ann. N.Y. Acad. Sci.438:519-522.

7. Goldberg, D. A. 1980. Isolation and partial characterization ofthe Drosophila alcohol dehydrogenase gene. Proc. Natl. Acad.Sci. USA 77:5794-5798.

8. Golomb, M., and M. J. Chamberlin. 1977. T7- and T3-specificRNA polymerases: characterization and mapping of the in vitrotranscripts read from T3 DNA. J. Virol. 21:743-752.

9. Handel, M. A., and J. J. Eppig. 1979. Sertoli cell differentiationin the testes of mice genetically deficient in germ cells. Biol.Reprod. 20:1031-1038.

10. Lehrach, H., D. Diamond, J. M. Wozney, and H. Boedtker. 1977.RNA molecular weight determinations by gel electrophoresisunder denaturing conditions, a critical reexamination. Biochem-istry 16:4743-4751.

11. Lindquist, S. 1986. The heat-shock response. Annu. Rev. Bio-chem. 55:1151-1191.

12. Lipsich, L. A., J. R. Cutt, and J. S. Brugge. 1982. Association ofthe transforming proteins of Rous, Fujinami and Y73 aviansarcoma viruses with the same two cellular proteins. Mol. Cell.Biol. 2:875-880.

VOL. 10, 1990

MOL. CELL. BIOL.

13. McAllister, W. T., and A. D. Carter. 1980. Regulation ofpromoter selection by the bacteriophage T7 RNA polymerase invitro. Nucleic Acids Res. 8:4821-4837.

14. Moore, S. K., C. Kozak, E. A. Robinson, S. J. Ulirich, and E.Appella. 1989. Murine 86- and 84-kDa heat shock proteins,cDNA sequences, chromosomal assignments, and evolutionaryorigins. J. Biol. Chem. 264:5343-5351.

15. Oakberg, E. F. 1956. A description of spermiogenesis in themouse and its use in analysis of the cycle of the seminiferousepithelium and germ cell renewal. Am. J. Anat. 99:391-409.

16. Oakberg, E. F. 1956. Duration of spermatogenesis in the mouseand timing of stages of the cycle of the seminiferous epithelium.Am. J. Anat. 99:507-516.

17. Opperman, H., W. Levinson, and J. M. Bishop. 1981. A cellularprotein that associates with the transforming protein of Rous

sarcoma virus is also a heat-shock protein. Proc. Natl. Acad.Sci. USA 78:1067-1071.

18. Sanchez, E. R., D. 0. Toft, M. J. Schlesinger, and W. B. Pratt.1985. Evidence that the 90-kDa phosphoprotein associated withthe untransformed L-cell glucocorticoid receptor is a murineheat shock protein. J. Biol. Chem. 260:12398-12401.

19. Schuh, S., W. Yonemoto, J. Brugge, V. J. Bauer, R. M. Riehl,W. P. Sullivan, and D. 0. Toft. 1985. A 90,000-dalton bindingprotein common to both steroid receptors and the Rous sarcomavirus transforming protein, pp6VSvC. J. Biol. Chem. 260:14292-14296.

20. Wilkinson, D. G., J. A. Bailes, and A. P. McMahon. 1987.Expression of the proto-oncogene int-1 is restricted to specificneural cells in the developing mouse embryo. Cell 50:79-88.

3242 NOTES