Expression of HSP70 in early pregnant mouse uteri and effect of estrogen on its synthesis

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1 49 (3) : , 2003 A cta Zoologica S inica (,, ) Western 70 ( Heat shock protein, HSP70) HSP70 : (1), HSP70, ( P < 0101) ; (2) (0128 g/ g ) (1110 g/ g ) HSP70 ( P < 0101) ; (3) Western HSP70 73 kd 1 68 kd 72 kd 73 kd 3 72 kd 73 kd 2 ; (4), HSP70, HSP70 HSP70 [ 49 (3) : , 2003 ] 70 Expression of HSP70 in early pregnant mouse uteri and effect of estrogen on its synthesis ZHAN G Shun2Li WEI Ji2Dong ZHAN G Wen2Xue XU Cun2Shuan ( College of L if e Sciences, Henan Normal U niversity, Henan Bioengi neeri ng Key L aboratory, Xi nxiang , Henan, Chi na) Abstract In order to investigate the expression, function and endocrine regulatory mechanism of heat shock protein 70 ( HSP70) in early pregnant (D1 - D6) mouse uteri, changes in immunoreactive positive cell count, content and protein bands of HSP70 and the ultrastructure localiztion of HSP70 in uteri stroma cells under different doses of dihydroxyestrin, were studied using immunohistochemistry, Western blotting, immuno2electron microscopy and image analysis techniques. The results indicate that : (1) positive HSP70 immunoreaction was localized in mouse uterine stroma and decidual cells. (2) HSP70 content increased markedly in pregnant mice and with the progress of gestation, increasing gradually from D1 to D5, peaking at D5 then decreasing from D6 ; (3) both small (0128 g/ g weight) and large doses (1110 g/ g weight) of dihydroxyestrin injected over three days can induce a marked increase in the HSP70 immunoreactionpositive stroma cell count in mouse uteri ( P < 0101, Control : / eyeshot, 1110 g/ g weight : / eyeshot) ; West2 ern blotting analysis showed that dihydroxyestrin also caused distinct changes in HSP70 protein bands ; there was just one 73 kd protein band in the control group, three protein bands (68 kd, 72 kd and 73 kd) in the small dose group, and two protein bands (72 kd and 73 kd) in the large dose group ( P < 0101) ; (4) electron microscopy revealed that positive HSP70 immunoreaction was localized in the cytoplasmic matrix and nucleolus of endometrial stroma cells ; aurosol granules were localized outside and inside the karyotheca of the same stroma cells and accumulated in a certain region of nucleolus. These results suggest that there is a significant relationship between HSP70 and the proliferation of uterine stroma cell during decidualization, and that dihydroxyestrin has a significant effect on the expression of HSP70 in mouse uteri that is , (No ) [ The research was funded by a grant from National Natural Science Foundation of China (No ) ] 33 (Corrresponding author). E2mail : com,, 38, : ν 2003 Acta Zoologica Sinica

2 3 : not dosage2dependent. One possible mechanism for this effect is that dihydroxyestrin influences the proliferation of uterine stroma cells by stimulating the synthesis of HSP70. There is possibly positive feedback between the regulation of dihy2 droxyestrin in the endometrium and the expression of HSP70 [ Acta Zoologica Sinica 49 (3) : , 2003 ]. Key words Mouse, Uteri, Decidualization, Heat shock protein 70, Dihydroxyestrin ( Heat shock protein, HSP) Ig G ( ) SDS Tween2 ( Brown et al., 1996 ; Piabowol et al., 1998 ;, 1999 ;, 2000 ) HSP70, Zymed, Tris2base BM, 113 SDS2PAGE Western ( Pechan et al., 1991), ( g, TGL216,, ) 10 min, Neuhoff et Smit h et al. (1979) ; Laemmli et al. ( 1993 ) Prapapanich et al. ( 1996 ) al. HSP V, 4 h) ; Towbin et al. (1979),, Western (200 ma, 16 h) HSP70, HSP70 ( NC) PBS/ Tween20 ( Koshiyama et al., 1995 ; Wu et al., 1996 ;, 2002) HSP70 ( ), HSP70 Ig G ( ) 37 1, HSP70 h, NB T2BCIP 114 HSP70,, 4 % 20 h,, 6 m , g, 10 min (1 600), 4 (, 20 ) (90 ) ( 2 1, 40 ) 1 200, min, DAB, 1 ( ) (D1), D6, (0128 g/ g ),, 2 % 8 : 00 9 : 00 1, 3 h, Epon812, 50 nm,, HSP70 ( ) Ig G (1 300) 37 Ig G ( ) ( 15 nm ) 20 Sigma Ig G ( ) - (1970) SDS2PA GE (110 V, 3 h ;, 2 %BSA (37, 1 h),, 30 m 1, 6, - ( St reptavidin2 proxidase, SP) 013 % H 2 O 2, 0101 mol/ L (p H 610), 24 h, Ig G SP, ( 1110 g/ g ), 20, 215 % % H 2 O 2 10 min, 20 % 30 min (1 500) 1 h, 5 % 012 % (p H 8119)

3 min, J EM2100SX, Western PBS, (: 2, 3) HSP70 GIS21000 ( Tanon ) Western HSP SP,,, HSP70, HSP70, HSP Western ( 1, 2), ( P < 0101) HSP70, Western 73, D1 D5 kd1 68 kd 72 kd 73, D5, D6 ( P < 0101) (: 4) HSP70 ( gx S ) (: 5 8), kd 3, 72 KD 73 KD HSP70 Western Fig. 1 Expression of HSP70 in early pregnant mouse uterus as shown by the Western blot test 21112, 3 HSP70 Western, HSP70 Fig. 3 Effect of dihydroxyestrin on HSP70 expression in the mouse uterus as revealed by the Western blot test Western 3 HSP70, (Brown et al., 1996 ; Piabowol et al., 1998 ;, 2000) HSP70 ( Pechan, 2 HSP ) ( Hcikkila, 1993) Fig. 2 Expression of HSP70 in early pregnant ( Ruther2 mouse uterus as revealed by image analysis ford et al., 1994),, HSP70 D : (Day) 3 3 P < 0101

4 3 : HSP70 Western Fig. 4 Western blot intensity analysis of the effect of dihydroxyestrin on HSP70 expression in the mouse uterus 33 P < 0101 (Contrast with control) 5 HSP70 Western Fig. 5 Western blot area analysis of effect of dihydroxyestrin on HSP70 expression in the mouse uterus 3 3 P < 0101 (Contrast with control) 2001), HSP70, ( Helmbrecht et al., 2000 ), HSP70 HSP70, HSP70 Pechan ( 1991 ) Stoklosinski (1992),,,,, HSP70, HSP70, ( Prapapanich et al ; Chen et al., 1996), HSP70, HSP70 HSP70,, HSP70 HSP70,, ( ;,, 2002), Western,, HSP70 HSP70,, -, D5, D5 ( Prapapanich et al. 1996) ; D6, HSP70 ( P < 0101) Cdk4 prb p53 p27 HSP70 ( Koshiya2, ma et al., 1995), (Helmbrecht et al., 2000), HSP70 HSP70 ( Koshiyama et al., 1995 ; Wataba et al.,, HSP70 HSP70,

5 ,,, 73 kd, 1 72 kd HSP70,, 73 kd 72 kd, 68 kd 3 ;,, 70 kd HSP, HSP70 mrna (Wu et al., 1996), HSP70 ( References), HSP70 HSP90, HSP70, DNA (Smith et al., 1993),,, ( Read et al., 1988), HSP70 HSP90 mrna, ( Wu et al., 1996) HSP70,, HSP70,,, HSP70,, ; Western, HSP70 HSP70, HSP70 ( ) HSP70,,, (, 2000) HSP70 Western Angela S., H. Kruse, C. R. Landsberg and L. Rensing 1992 Effect of heat shock on neuroblastoma (NIE 115) cell proliferation and differentiation. Ex p. Cell. Res. 200 (1) : Brown, C. R., L. Q. H. Brown and S. J. Doxsey 1996 Molecu2 lar chaperones and the centrosome. J. Biol. Chem. 271 ( 2) : Chen, S. Y., V. Prapapanich, R. A. Rimerman, B. Honore and D. F. Smith Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins HSP90 and HSP70. Mol. Endocri nol. 10 : Dai, L., H. Shen, Y. Z. Yu, Y. D. Wang, W. L. Zuo and X. Kan 2000 Endometrial nuclear progesterone receptor in infertile wom2 en. Chi n. J. Pathol. 29 (3) [,,,,, (3) : ] Hcikkila, J. J Heat shock gene expression and development. Developmental Gentics 14 (1) : Helmbrecht, K., E. Zeise and L. Rensing 2000 Chaperones in cell cycle regulation and mitogenic signal transduction : a review. Prolif. 33 (6) : Cell Karl, T. R., L. A. Mizzen and J. W. William 1988 Heat shock is lethal to fibroblasts microinjected with antibodies against HSP70. Science 242 (21) : Koshiyama, M., I. Konishi, K. Nanbu, M. Mandai, T. Komatsu, S. Yamamoto, T. Mori and S. Fujii 1995 Immunohistoche2 mical location of heat shock protein HSP70 and HSP90 in the hu2 man endometrium : correlation with sex steroid receptors and ki267 antigen expression. J. Clin. Endocrinol. Metab. 80 ( 4 ) : Laemmli, U. K Cleavage of structural proteins in the assem2 bly of the head bacteriophue T 4. N at ure 227 : Neuhoff, V., K. Philipp and H. G. Zimmer 1979 A simple, versa2 tile, sensitive and volume2independent method for quantitative pro2 tein determination which is independent of other external influ2 ences. Physiol. Chem. 360 : Paul, M. P Heat shock proteins and cell proliferation. Fed2 eration of European Biochemical Societies 280 (1) : 1 4. Prapapanich, V., S. Y. Chen, S. C. Nair, R. A. Rimerman and D. F. Smith 1996 Molecular cloning of human p48, a transient component of progesterone receptor complexes and an HSP702bind2 ing protein. Mol. Endocri nol. 10 : Read, L. D., C. E. Snider, J. S. Miller, G. L. Greene and B. S. Katzenellenbogen 1988 Ligand modulated regulation of proges2

6 3 : terone receptor messenger ribonucleicacid and protein in human breast cancer cell lines. Mol. Endocri nol. 2 (3) : Rutherford, S. L. and C. S. Zuker 1994 Protein folding and the regulation of signaling pathways. Cell 79 (7) : Smith, D. F. and D. O. Toft 1993 Steroid receptors and their as2 sociated proteins. Mol. Endocri nol. 7 : Towbin, H., T. Staehelin and J. Gordon 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure ane some applications. Pro. Natl. Acad. Sci. 76 : Wataba, K, T. Satio, K. Fukunaka, K. Ashihara, M. Nishihimura and R. Kudo 2001 Over2expression of heat shock protein in carcinogenic endometrium. Int. J. Cancer 91 (4) : Wu, W. X., J. B. Derks, Q. Zhang and P. W. Nathanielsz 1996 Changes in heat shock protein290 and 270 messenger ribonu2 cleic acid in uterine tissues of the ewe in relation to parturition and regulation by estradiol and progesterone. Endocrinol. 137 (12) : Xu, C. S., M. Xia, A. L. Lu, X. Y. Li, Y. H. Li, X. Y. Zhao and Y. H. Hu 1999 Changes in the content and activity of HSP70/ HSC68, proteinase and phosphatase during liver regenera2 tion. Acta Physiol. Si n. 51 (5) : [,,,,,, (51) : ] Zhang, S. L., M. Guo, X. S. Zhang, W. X. Zhang, X. Y. Li and C. S. Xu 2000 Study on the expression of HSP70 induced by heat shock in mouse hepatocytes. J. Anat. 23 (4) : [,,,,, (4) : ] Zhang, S. L., J. D. Wei, J. T. Li, W. X. Zhang, L. S. Yang and C. S. Xu 2002 The immunohistochemical study of heat shock protein 70 in mouse endometrium during early pregnancy. J. A nat. 25 (1) : [,,,,, (1) : ] ( Explanation of Plate) ( Plate ) 11 [ Negeative control of stroma cells in mouse endometrium by immuno2electron microscope ] ( ) [ Collaurum granules ( ) distribute of stroma cells in normal mouse endometrium] D5 ( ) [ Collaurum granules ( ) distribute in cytoplasm matrix and specific area of the nucleolus] (N) ( ) [ Collaurum granules ( ) in nucleolus (N) of the endometrial stroma cells in small dose dihydroxyestrin injecting group mouse ] HSP70 ( ) [ Showing HSP70 immunoreaction ( IR) positive cells ( ) in normal mouse endometrium] HSP70 ( ) [ HSP70 IR positive cells ( ) increase in large dose dihydroxyestrin inject2 ing group mouse endometrium] HSP70 ( ) [ HSP70 IR positive cells ( ) increased remarkably in small dose dihydroxyestrin injecting group mouse endometrium] HSP70 ( ), ( g ) [ Endometrium stroma cells appear HSP70 IR positive ( ), glandular epithelium cells appears negative ( g ) in small dose dihydroxyestrin injecting group mouse ] 400

7 : 70 ZHAN G Shun2Li et al. : Expression of HSP70 in early pregnant mouse uteri and effect of estrogen on its synthesis Plate ( Explanation at the end of the text)

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