48 (5) :648 653, 2002 A cta Zoologica S inica HSF2 mrna 11 3 33 (, 100080) (, 650000) HSF2 mrna, 11 ( TU), 3 ( TUN EL), HSF2 TUN EL, 5 30, HSF2 mrna ( ), Hsf2 Hsp7022, HSF2 Hsp7022, HSF2 HSF2 11 2 ( HSF2) HSF, HSF1 ( Rabindran et al., 1991 ), HSF2 ( Schuetz et al., 1991 ), HSF3 (Nakai et al., 1993 ) HSF4 ( Nakai et al., 1997), Hsp ( HSF HSP, Hsf Hsp ) ( HSE) HSF1 HSF3 111 ( Baler et al., 1993 ; Nakai et al., 1995 ) HSF2,, cdna E. K. Sulliyan HSF4 ; RNA Boehringer, HSF Mannheim ; TUN EL, T3 T7 RNA DNA ( Nakai et al., 1997), Xho EcoR Hind, HSF2 Hsp7022 HSE, HSF2 Hsp7012 ; Sigma ; (Sarge et al., 1994) APES TU HSP7012 Hsf2, HSF2 Hsp7012 HSF2 9 (5 7),,,, HSF2,,,, 2001204216, 2001210216 3 973 ( G1999055901) ( KSCX222SW2201) (No. 39870368) 33 Email : Liuyx @panda1ioz1ac1cn,, : Email : zhouxc @panda1ioz1ac1cn Hsf2 1, HSP 21 (5 7 ) HSF2, BCIP NB T Promega 112
5 : HSF2 mrna 11 649 1 3 Fig11 In situ 3 2end labeling of D NA fragmentation on testicular sections from unilateral cryptorchid monkeys A. (Normal testis) B D E 5 10 15 200 (B, D, and E represent cryptorchid testis on day 5, 10 and 15, respectively 200) C B 400 (C is the magnify cation of B 400) Gc ( Germ cell) ST (Seminiferous tubule), Bouinπs 4 24 h,,, Xho, T3 RNA (6 m), APES ; EcoR, T7 RNA, 40 200 bp 113 11 400 bp, - 20,, K 10 TU, (8 g/ ml) 37 10 min, 4 % 7 1 4, 5 min,, (2 SSC/ 50 %, 20 mg/ kg ) 2 h, D I G2HSP70122cRNA, Bouinπs DIG2HSF22cRNA (400 ng/ ml ISH ; 4 24 h ISH : 2 SSC/ 50 % / 10 mm Tris, p H 715/ 114 3 ( TUNEL) 400 g/ ml t RNA/ 015 % SDS/ 1 Denhardtπs) 48, 18 h 2 SSC, 1 SSC,, PBS 011 SSC, 42 15 min 2 DIG2cRNA 3 5 min ; 10 g/ ml K37 15 min, PBS 2 5 min ; ( TdT) Boehringer Mannheim Buffer 10 min, TdT DIG2112, 1 mm ) 2 du TP 37 1 h 4 h, 011 M Tris, p H 715/ 0115 M NaCl Buffer (011 M, 0115 M NaCl, p H 715) 3 5 min, Buffer ( 1 % ) 1 h ; 1 500 anti2dig2fab 2 h Buffer ( 013 %Tween 20) 3 5 min ; Buffer (011 M NaCl, 50 mm MgCl 2, 011 M Tris2Cl, p H 915) 5 min NB T/ BCIP, Buffer TUN EL ( 1 : A) 5, 115 DIG c RNA HSF2 crna : HSF2 cdna anti2dig2fab (,, 2 211 TUNEL,, TUN EL,
50 6 48 2 3 11 Fig12 In situ 3 2end labeling of D NA fragmentation in testicular cells of TU2treated monkey A. (Normal testis) B, C, D E 7 14 30 60 200 (B, C, D and E represent the testis on day 7, 14, 30 and 60 after TU injection, respectively 200) Gc : ( Germ cell) ST : (Seminiferous tubule) 3 HSF2 mrna Fig13 In situ hybridization of HSF2 mrna in monkey normal and cryptorchid testis A. (Normal testis) B H 5 10 15 20 30 45 60 100 (B2H represent cryptorchid testes on day 5, 10, 15, 20, 30, 45 and 60, respectively 100) Gc : ( Germ cell) ST : (Seminiferous tubule) ( 1 : B, C), 212 11 TUNEL ; 10 11 7,,, ;, 30 ( 1 : D) 15 ; ( 2) ( 1 : E)
5 : HSF2 mrna 11 651 4 HSF2 mrna TU Fig14 In situ hybridization of HSF2 mrna in monkey testes A. (Normal testis) B E 7 14 30 60 100 ( C2E represent TU2treated monkey testes on day 7, 14, 30 and 60 after injection, respectively 100) Gc : ( Germ cell) ST : (Seminiferous tubule) 213 HSF2 mrna (Brinkworth et al., 1995), L H ( Marthe et, HSF2 al., 1995) EDS ( Troiano et al., 1994 ; Hen2 ( 3 : A), riksen et al. ; 1995 ; Woolveridge et al., 1999) 5, HSF2 mrna, ( 3 : B) ; 10, HSF2 mrna ( 3 : C) ; 15 TUN EL HSF2 mrna ( 3 : D H), 214 HSF2 mrna 11 HSF2 mrna TU 7 TR2 ( Guo et al., 2000 ; Mu et 14 30, al., 2001) P16 (, 2002) Hsp7022 60 ( 4) ( Guo et al., 2001) HSF2 3,, HSF2 mrna ( 5 7 ( Kandell et al., ), 1988),, Hsp7012, HSF2 Hsp7012, (Chowdhury et al., 1964), ( HSF2 mrna Yin et al., 1997) 3, HSF2,, ( Patanelli, 1978 ; Knut h et al., mrna (Alastalo et al., 1985 ; Handelsman et al., 1992), 1998), HSF2, Hsf2, ( Wu, 1995), HSF2 mr2 ( Russel et al., 1977 ) GnRH
52 6 48 NA, Hsf2, Alastalo, T., M. Lonnstrom, S. Leppa and J. D. ( References) forms in the rat seminiferous epithelium. Ex p. Cell Research 240 : 16 27. Baler, R., G. Dahl and R. Kirby 1998 Stage2specific expression and cellular localization of the heat shock factor 2 iso2 Voellmy 1993 Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1. Mol. Cell Biol. 13 : 331 333. Brinkworth, M. H., G. F. Weinbauer, S. Schlatt and E. Nieschlag 1995 Identification of male germ cells undergoing apoptosis in adult rats. J. Reprod. Fertil. 105 : 25 33. Chowdhury, A. K. and E. Steinberger 1964 A quantitative study of heat on germinal epithelium of rat testes. A m. J. A nat. 115 : 509 524. Guo, C. X., Z. Y. Hu, R. J. Zou and Y. X. Liu 2000 Expression and regulation of orphan receptor TR2 mrna in germ cells of cryp2 torchid testis in rat and rhesus monkey. Chi nese Sci. B ull. 45 : 720 725. [,,, 2001 TR2 mrna. 45 : 720 725. ] Guo, C. X., J. Ma, X. Ch. Zhou and Y. X. Liu 2001 Expression of Hsp7022 gene during germ cell apoptosis in rat unilateral cryptorchid testes. A rch. A ndrol. 46 (2) : 109 115. Handelsman, D. J., A. J. Conway and L. M. Boylan 1992 Suppression of human spermatogenesis by testosterone implants in man. J. Cli n. Endocri nol. Metab. 75 : 1 326 1 332. Henriksen, K., H. Hakovirta and M. Parvinen 1995 Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage2specific manner : i n sit u quantification in squash preparations after administration of ethane dimethane sulfonate. Endocri nol. 136 : 3 285 3 291. Kandell, F. R. and R. S. Swerdloff 1988 Role of temperature in regulation of spermatogenesis and the use of heating as a method for contracep2 tion. Fertil. Steril. 49 : 1 23. Knuth, U. A., H. Behre, L. Belkien, H. Bents and E. Nieschlag 1985 Clinical trial of 192nortestosterone hexoxyphenylpropionate (Anadur) for male fertility regulation. Fertil. Steril. 44 : 814 821. Marthe, G. K., J. Shetty and R. R. Dighe 1995 Selective immunoneutralization of luteinizing hormone results in the apoptotic cell death of pachytene spermatocytes and spermids in the rat testis. Endocri ne. 3 : 705 709. Mu, X. M., Y. X. Liu, L. L. Collins and C. Chang 2001 The p53/ retinoblastoma2mediated repression of testicular orphan receptor22 in the rhesus monkey with cryptorchidism. J. Bio. Chem. 275 : 23 877 23 883. Nakai, A., Y. Kawazoe, M. Tanabe, K. Nagata and R. I. Morimoto 1995 The DNA2binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6. Mol. Cell. Biol. 15 : 5 268 5 278. Nakai, A. and R. I. Morimoto 1993 Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway. Mol. Cell. Biol. 13 : 1 983 1 997. Nakai, A., M. Tanabe, Y. Kawazoe, J. Inazawa, R. I. Morimoto and K. Nagata 1997 HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol. Cell. Biol. 17 : 469 481. Patanelli, D. J. 1978 Hormonal control of male fertility. Washington (DC) : US Department of Health Education and Welfare. Publication No. (NIH) 78 : 1 097. Rabindran, S. K., G. Giorgi, J. Clos and C. Wu 1991 Molecular cloning and expression of a human heat shock factor, HSF1. Proc. N atl. Acad. Sci. USA 88 : 6 906 6 910. Russel, L. D. and Y. A nat. Rec. 187 : 347 366. Clermont 1977 Degeneration of germ cells in normal hypophysectomized and hormone treated hypophysectomized rats. Sarge, K. D., O. K. Park2Sarge, J. D. Kirby and S. Leppa 1994 Expression of heat shock factor 2 in mouse testis : potential role as a regu2 lator of heat2shock protein gene expression during spermatogenesis. Biol. Reprod. 50 : 1 334 1 343. Schuetz, T. J., G. J. Gallo, L. Sheldon, P. Tempst and R. E. Kingston 1991 Isolation of a cdna for HSF2 : evidence for two heat shock factor genes in humans. Proc. N atl. Acad. Sci. USA 88 : 6 911 6 915. Troiano, L., M. F. Fustini, E. Lovato, A. Frasoldati, W. Malorni, M. Capri, E. Grassilli, P. Marrama and C. Franceschi 1994 Apoptosis and spermatogenesis : evidence from an in vivo model of testosterone withdrawal in the adult rat. Biochem. Biophys. Res. Com2 m un. 202 : 1 315 1 321. Woolveridge, I., M. D. Boer2Brouwer, M. F. Taylor, K. J. Teerds and F. C. Wu 1999 Apoptosis in the rat spermatogenic epithelium following androgen withdrawal : changes in apoptosis2related genes. Biol. Reprod. 60 : 461 470. Wu, C. 1995 Heat shock transcription factors : structure and regulation. A nnu. Rev. Cell. Dev. Biol. 11 : 441 469.
5 : HSF2 mrna 11 653 Yin, Y., K. L. Hawkins, W. C. Dewolf and A. Morgentaler 1997 Heat stress causes testicular germ cell apoptosis in adult mice. J. A n2 drol. 18 : 159 165. Zhou, X. C., Y. C. Li, Z. Y. Hu, R. J. Zou and Y. X. Liu 2002 Role of P16 protein and germ cell apoptosis in rhesus monkey a2 zoospermia or oligozoospermia induced by heat stress or testosterone undecanoate. Acta. Zool. Si n. 48 (3) : 363 367. [,,,, 2002 P16 11. 48 (3) : 363 367. ] ( Abstract) CHANGES OF HSF mrna EXPRESSION IN RHESUS MONKEY TESTES D URING GERM CELL APOPTOSIS IND UCED BY HEAT STRESS OR HIGH DOSAGE OF TESTOSTERONE UNDECANOATE 3 ZHOU Xin2Chang HU Zhao2Yuan ZUO Ru2Jin L IU Yi2Xun 33 ( State Key L aboratory of Reproductive Biology, Instit ute of Zoology, Chi nese Academy of Sciences, Beiji ng 100080, Chi na) ( Kunmi ng Instit ute of Zoology, Chi nese Academy of Sciences, Kunmi ng 650200, Chi na) In order to investigate the changes of HSF2 mrna expression and its role in germ cell apoptosis induced by heat st ress or a high of testosterone undecanoate, we established t he operation2induced unilateral cryptorchid and TU2injected monkey models, and examined i n sit u analysis of testicular cell DNA f ragmentation and t he expre2 ssion level of HSF2 mrna in germ cells after treatment. The TUN EL results showed that the apoptotic signals after treatment increased and the strongest apoptotic signals appeared in the germ cells of the cryptorchid testes on day 5 or the TU2treated monkey testes on day 30, suggesting that the failure of spermatogenesis in the testes of monkeys after treatment may be due to the germ cell apoptosis induced by heat stress or high TU. The HSF2 mrna decreased slightly at the early phase of the cell apoptosis just prior to the strongest signals of apoptosis, followed by dramatic decrease. This change was closely time2correlated to t hat of Hsp7022 mrna ( unpublished observation). It, t herefore, suggest s t hat HSF2 may regulate t he expression of Hsp7022. Moreover, HSF2 may control the spermatogenesis and the germ cell apoptosis by other signal pathways. Key words Rhesus monkey, HSF2, Germ cell, Apoptosis, Heat stress, Testosterone undecnoate 3 This work was supported by 973, Chuang2Xin and National Natural Science Foundation of China (No. 39870368) 33 Corresponding author. Liuyx @panda. ioz. ac. cn