Advertisement

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

  • Mei-Hua Zhang
  • Zhi-Da Shi
  • Jian-Chun Yu
  • Yan-Ping Zhang
  • Lei-Guang Wang
  • Yi Qiu
Reproductive Physiology and Disease

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.

Keywords

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

Notes

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.

References

  1. 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.CrossRefPubMedGoogle Scholar
  2. 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.CrossRefPubMedGoogle Scholar
  3. 3.
    Liu YX. Control of spermatogenesis in primate and prospect of male contraception. Arch Androl. 2005;51:77–92.CrossRefPubMedGoogle Scholar
  4. 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.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefGoogle Scholar
  7. 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.CrossRefPubMedGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  13. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  14. 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.CrossRefPubMedGoogle Scholar
  15. 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. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 20.
    Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science. 1980;210:1131.CrossRefPubMedGoogle Scholar
  21. 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.PubMedGoogle Scholar
  22. 22.
    Faleiro L, Lazebnik Y. Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol. 2000;151:951–9.CrossRefPubMedCentralPubMedGoogle Scholar
  23. 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.Google Scholar
  24. 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. 25.
    World Health Organisation 2010 WHO laboratory manual for the Examination and processing of human semen. 5th ed. WHO Press, Prepublication version. p30–32.Google Scholar
  26. 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.Google Scholar
  27. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Setchell BP. The Parkes Lecture. Heat and the testis. J Reprod Fertil. 1998;114:179–94.CrossRefPubMedGoogle Scholar
  29. 29.
    Liu YX. Temperature control of spermatogenesis and prospect of male contraception. Front Biosci (Schol Ed). 2010;1:730–55.CrossRefGoogle Scholar
  30. 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.PubMedGoogle Scholar
  31. 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.PubMedGoogle Scholar
  32. 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.CrossRefPubMedGoogle Scholar
  33. 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. 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.CrossRefPubMedGoogle Scholar
  35. 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.PubMedGoogle Scholar
  36. 36.
    McNitt JL, First NL. Effects of 72-hour heat stress on semen quality in boars. Int J Biometeorol. 1970;14:373–80.CrossRefPubMedGoogle Scholar
  37. 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. 38.
    Stone BA. Thermal characteristics of the testis and epididymis of the boar. J Reprod Fertil. 1981;63:551–7.CrossRefPubMedGoogle Scholar
  39. 39.
    Larsson K, Einarsson S. Seminal changes in boars after heat stress. Acta Vet Scand. 1984;25:57–66.PubMedGoogle Scholar
  40. 40.
    Malmgren L, Larsson K. Semen quality and fertility after heat stress in boars. Acta Vet Scand. 1984;25:425–35.PubMedGoogle Scholar
  41. 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.CrossRefPubMedGoogle Scholar
  42. 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.CrossRefPubMedCentralPubMedGoogle Scholar
  43. 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.CrossRefPubMedGoogle Scholar
  44. 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.CrossRefPubMedGoogle Scholar
  45. 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.CrossRefPubMedGoogle Scholar
  46. 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.CrossRefPubMedGoogle Scholar
  47. 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.Google Scholar
  48. 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.CrossRefPubMedGoogle Scholar
  49. 49.
    Dadoune JP. The nuclear status of human sperm cells. Micron. 1995;26:323–45.CrossRefPubMedGoogle Scholar
  50. 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.CrossRefPubMedGoogle Scholar
  51. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Mei-Hua Zhang
    • 1
  • Zhi-Da Shi
    • 1
  • Jian-Chun Yu
    • 1
  • Yan-Ping Zhang
    • 1
  • Lei-Guang Wang
    • 1
  • Yi Qiu
    • 1
  1. 1.Key Laboratory of Birth Regulation and Control Technology of National Health and Family Planning Commission of China, Key Laboratory for Improving Birth Outcome TechniqueShandong Provincial Family Planning Institute of Science and TechnologyShandongPeople’s Republic of China

Personalised recommendations