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Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men

Abstract

Purpose

To observe changes in semen parameters, sperm DNA integrity, chromatin condensation and cysteinyl aspartate-spicific proteinases (Caspase-3) in adult healthy men after scrotal heat stress (SHS).

Methods

The scrotums of 19 healthy male volunteers were exposed to the condition of 40–43 °C SHS belt warming 40 min each day for successive 2 days per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, eosin Y (EY) staining sperm HOS and chromatin dispersion (HOS/SCD) test, HOS and aniline blue (HOS/AB) staining test were carried out before, during and after SHS. The activated Caspase 3 levels of spermatozoa were determined with a microtiter plate reader.

Results

The mean parameters of sperm concentration, motility and normal morphological sperm were significantly decreased in groups with sperm being collected during SHS 1, 2 and 3 months when compared with those in groups of pre-SHS (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane and vitality, and Caspase-3 activity were observed between the groups of before SHS and after SHS 3 months and the groups of during SHS 1, 2 and 3 months (P < 0.001). Three months the SHS stopped, various parameters recovered to the level before SHS. Abnormal sperm with HOS/AB and HOS/SCD showed a negatively significant correlation with normal sperm by HOS/EY test, and WBC in semen showed a positively significant correlation with Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS/SCD showed a positively significant correlation with that of HOS/AB.

Conclusions

The continuously constant SHS can impact the semen quality, sperm DNA integrity, chromatin condensation and Caspase-3, and the combination of HOS plus AB test may simultaneously determine the integrity of membrane and chromatin condensation at the same spermatozoon.

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References

  1. Paul C, Murray AA, Spears N, Saunders PT. A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice. Reproduction. 2008;136:73–84.

    Article  CAS  PubMed  Google Scholar 

  2. Banks S, King SA, Irvine DS, Saunders PT. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction. 2005;129:505–14.

    Article  CAS  PubMed  Google Scholar 

  3. Liu YX. Control of spermatogenesis in primate and prospect of male contraception. Arch Androl. 2005;51:77–92.

    Article  CAS  PubMed  Google Scholar 

  4. Garolla A, Torino M, Sartini B, Cosci I, Patassini C, Carraro U, et al. Seminal and molecular evidence that sauna exposure affects human spermatogenesis. Hum Reprod. 2013;28:877–85.

    Article  CAS  PubMed  Google Scholar 

  5. Shrivastava V, Pekar M, Grosser E, Im J, Vigodner M. SUMO proteins are involved in the stress response during spermatogenesis and are localized to DNA double-strand breaks in germ cells. Reproduction. 2010;139:999–1010.

    Article  CAS  PubMed  Google Scholar 

  6. Li XX, Chen SR, Shen B, Yang JL, Ji SY, Wen Q, et al. The Heat-Induced Reversible Change in the blood-testis barrier (BTB) is regulated by the androgen receptor (AR) via the partitioning-defective protein (Par) polarity complex in the mouse. Biol Reprod. 2013;89:1–10.

    Article  Google Scholar 

  7. Sakkas D, Mariethoz E, St John JC. Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas mediated pathway. Exp Cell Res. 1999;251:350–5.

    Article  CAS  PubMed  Google Scholar 

  8. Shen HM, Dai J, Chia SE, Lim A, Ong CN. Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality. Hum Reprod. 2002;17:1266–73.

    Article  PubMed  Google Scholar 

  9. Love CC, Kenney RM. Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod. 1999;60(3):615–20.

    Article  CAS  PubMed  Google Scholar 

  10. Karabinus DS, Vogler CJ, Saacke RG, Evenson DP. Chromatin structural changes in sperm after scrotal insulation of Holstein bulls. J Androl. 1997;18(5):549–55.

    CAS  PubMed  Google Scholar 

  11. Wang C, Cui YG, Wang XH, Jia Y, et al. Transient scrotal hyperthermia and levonorgestrel enhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis. J Clin Endocrinol Metab. 2007;92(8):3292–304.

    Article  CAS  PubMed  Google Scholar 

  12. Qiu Y, Wang LG, Jia YF, Yang DT, Zhang MH, Zhang YP, et al. The effects of the extract of Chinese Polygala tennuidolia willd on human sperm in vitro. J Zhejiang Univ Sci B. 2011;12:448–54.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Qiu Y, Wang LG, Zhang LH, Li J, Zhang AD, Zhang MH. Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation. J Assist Reprod Genet. 2012;29:185–94.

    Article  PubMed Central  PubMed  Google Scholar 

  14. Qiu Y, Wang LG, Zhang LH, Zhang AD. Quality of sperm obtained by penile vibratory bratory stimulation and percutaneous vasal sperm aspiration in men with spinal cord injury. J Androl. 2012;33:1036–46.

    Article  CAS  PubMed  Google Scholar 

  15. Zhang LH, Qiu Y, Wang KH, Wang Q, Tao G, Wang LG. Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labeling assay. Fertil Steril. 2010;94:102732.

    Google Scholar 

  16. Fariello RM, Del Giudice PT, Spaine DM, Fraietta R, Bertolla RP, Cedenho AP. Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity. J Assist Reprod Genet. 2009;26:151–7.

    Article  PubMed Central  PubMed  Google Scholar 

  17. Liu DY, Baker HW. Human sperm bound to the zona pellucida have normal nuclear chromatin as assessed by acridine orange fluorescence. Hum Reprod. 2007;22:1597–602.

    Article  CAS  PubMed  Google Scholar 

  18. Fernández JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl. 2003;24:59–66.

    PubMed  Google Scholar 

  19. Fernández JL, Muriel L, Goyanes V, Segrelles E, Gosálvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 2005;84:833–42.

    Article  PubMed  Google Scholar 

  20. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science. 1980;210:1131.

    Article  CAS  PubMed  Google Scholar 

  21. Paasch U, Grunewald S, Fitzl G, Glander HJ. Deterioration of plasma membrane is associated with activation of caspases in human spermatozoa. J Androl. 2003;24:246–52.

    CAS  PubMed  Google Scholar 

  22. Faleiro L, Lazebnik Y. Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol. 2000;151:951–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Beyret E, Lin H. Pinpointing the expression of piRNAs and function of the PIWI protein subfamily during spermatogenesis in the mouse. Dev Biol. 2011;14215–26.

  24. World Health Organisation. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. New York: Cambridge University Press; 1999.

    Google Scholar 

  25. World Health Organisation 2010 WHO laboratory manual for the Examination and processing of human semen. 5th ed. WHO Press, Prepublication version. p30–32.

  26. Sellami A, Chakroun N, Ben Zarrouk S, Sellami H, Kebaili S, Rebai T, et al. Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility. Adv Urol. 2013;578–631.

  27. Alkhayal A, San Gabriel M, Zeidan K, Alrabeeah K, Noel D, McGraw R, et al. Sperm DNA and chromatin integrity in semen samples used for intrauterine insemination. J Assist Reprod Genet. 2013;30:1519–24.

    Article  PubMed Central  PubMed  Google Scholar 

  28. Setchell BP. The Parkes Lecture. Heat and the testis. J Reprod Fertil. 1998;114:179–94.

    Article  CAS  PubMed  Google Scholar 

  29. Liu YX. Temperature control of spermatogenesis and prospect of male contraception. Front Biosci (Schol Ed). 2010;1:730–55.

    Article  Google Scholar 

  30. Lue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, et al. Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology. 1999;140:1709–17.

    CAS  PubMed  Google Scholar 

  31. Lue Y, Hikim AP, Wang C, Im M, Leung A, Swerdloff RS. Testicular heat exposure enhances the suppression of spermatogenesis by testosterone in rats: the “two-hit” approach to male contraceptive development. Endocrinology. 2000;141:1414–24.

    CAS  PubMed  Google Scholar 

  32. Lue Y, Wang C, Liu Y-X, Hikim AP, Zhang XS, Ng CM, et al. Transient testicular warming enhances the suppressive effect of testosterone on spermatogenesis in adult cynomolgus monkeys (Macaca fascicularis). J Clin Endocrinol Metab. 2006;91:539–45.

    Article  CAS  PubMed  Google Scholar 

  33. Guo J, Tao SX, Chen M, Shi YQ, Zhang ZQ, Li YC, et al. Heat treatment induces liver receptor homolog-1 expression in monkey and rat Sertoli cells. Endocrinology. 2007;131:1137–48.

    Google Scholar 

  34. Zhang XS, Lue YH, Guo SH, Yuan JX, Hu ZY, Han CS, et al. Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (MACACA FASCICULARIS). Front Biosci. 2005;10:3110–21.

    Article  CAS  PubMed  Google Scholar 

  35. Kandeel FR, Swerdloff RS. Role of temperature in regulation of spermatogenesis and the use of heating as a method for contraception. Fertil Steril. 1988;49:1–23.

    CAS  PubMed  Google Scholar 

  36. McNitt JL, First NL. Effects of 72-hour heat stress on semen quality in boars. Int J Biometeorol. 1970;14:373–80.

    Article  CAS  PubMed  Google Scholar 

  37. Wettemann RP, Wells ME, Johnson RK. Reproductive characteristics of boars during and after exposure to increased ambient temperature. J Anim Sci. 1979;49:1501–5.

    Google Scholar 

  38. Stone BA. Thermal characteristics of the testis and epididymis of the boar. J Reprod Fertil. 1981;63:551–7.

    Article  CAS  PubMed  Google Scholar 

  39. Larsson K, Einarsson S. Seminal changes in boars after heat stress. Acta Vet Scand. 1984;25:57–66.

    CAS  PubMed  Google Scholar 

  40. Malmgren L, Larsson K. Semen quality and fertility after heat stress in boars. Acta Vet Scand. 1984;25:425–35.

    CAS  PubMed  Google Scholar 

  41. Chihara M, Nakamura T, Sakakibara N, Otsuka S, Ichii O, Kon Y. The onset of heat-induced testicular calcification in mice: involvement of the telomeric locus on chromosome 1. Am J Pathol. 2014;184:2480–92.

    Article  CAS  PubMed  Google Scholar 

  42. Paul C, Teng S, Saunders PT. A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death. Biol Reprod. 2009;80:913–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Hjollund NH, Storgaard L, Ernst E, Bonde JP, Olsen J. Impact of diurnal scrotal temperature on semen quality. Reprod Toxicol. 2002;16:215–21.

    Article  CAS  PubMed  Google Scholar 

  44. Zhang ZH, Hu ZY, Song XX, Xiao LJ, Zou RJ, Han CS, et al. Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism. Int J Androl. 2004;27:234–9.

    Article  PubMed  Google Scholar 

  45. Chen M, Cai H, Yang JL, Lu CL, Liu T, Yang W, et al. Effect of heat stress on expression of junction-associated molecules and upstream factors androgen receptor and Wilms’ tumor 1 in monkey sertoli cells. Endocrinology. 2008;149:4871–82.

    Article  CAS  PubMed  Google Scholar 

  46. Chen M, Yuan JX, Shi YQ, Zhang XS, Hu ZY, Gao F, et al. Effect of 43 degrees treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells. Asian J Androl. 2008;10:474–85.

    Article  CAS  PubMed  Google Scholar 

  47. Wang DH, Hu JR, Wang LY, Hu YJ, Tan FQ, Zhou H, et al. The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis. PLoS One. 2012;7e39920.

  48. Simon L, Liu L, Murphy K, Ge S, Hotaling J, Aston KI, et al. Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment. Hum Reprod. 2014;29:904–17.

    Article  CAS  PubMed  Google Scholar 

  49. Dadoune JP. The nuclear status of human sperm cells. Micron. 1995;26:323–45.

    Article  CAS  PubMed  Google Scholar 

  50. Morel F, Mercier S, Roux C, Elmrini T, Clavequin MC, Bresson JL. Interindividual variations in the disomy frequencies of human spermatozoa and their correlation with nuclear maturity as evaluated by aniline blue staining. Fertil Steril. 1998;69:1122–7.

    Article  CAS  PubMed  Google Scholar 

  51. Morel F, Roux C, Bresson JL. Disomy frequency estimated by multicolour fluorescence in situ hybridization, degree of nuclear maturity and teratozoospermia in human spermatozoa. Reproduction. 2001;121:783–9.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank doctor Feng Chen (Shandong Provincial Xintai Family Planning Service Station) and Hua-Qiang Liu (Shandong Provincial Pingyin Family Planning Service Station) for their help in semen processing and their technical assistance.

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Correspondence to Yi Qiu.

Additional information

The work was supported by the National “China’s 12th 5-Year Plan (2011–2015)” of Science and Technology (No: 2012BAI31B08)

Capsule The continuously constant SHS can impact the semen quality, sperm DNA integrity, chromatin condensation and Caspase-3, and the combination of HOS plus AB test may simultaneously determine the integrity of membrane and chromatin condensation at the same spermatozoon.

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Zhang, MH., Shi, ZD., Yu, JC. et al. Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men. J Assist Reprod Genet 32, 747–755 (2015). https://doi.org/10.1007/s10815-015-0451-0

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  • DOI: https://doi.org/10.1007/s10815-015-0451-0

Keywords

  • Sperm
  • Scrotal heating
  • Sperm chromatin condensation
  • Caspase-3
  • Aniline blue staining