Adverse reproductive effects of S100A9 on bovine sperm and early embryonic development in vitro

Autoři: Natsumi Funeshima aff001;  Nao Tanikawa aff001;  Hikari Yaginuma aff002;  Hiroyuki Watanabe aff003;  Hisataka Iwata aff001;  Takehito Kuwayama aff001;  Seizo Hamano aff002;  Koumei Shirasuna aff001
Působiště autorů: Department of Animal Science, Tokyo University of Agriculture, Atsugi, Kanagawa, Japan aff001;  Animal Bio-Technology Center, Livestock Improvement Association of Japan Inc., Tokyo, Japan aff002;  Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan aff003;  Maebashi Institute of Animal Science, Livestock Improvement Association of Japan Inc., Gunma, Japan aff004
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227885


The phenomenon of aging arises from multiple, complex interactions causing dysfunction in cells and organs. In particular, fertility drastically decreases with age. Previously, we have demonstrated that the functional characteristics of the bovine oviduct and uterus change with the age-dependent upregulation of inflammation and noted that S100A9 triggers inflammatory responses in oviduct epithelial cells. In the present study, we investigated the hypothesis that S100A9 affects reproductive events to aspect such as sperm function, fertilization, and the development of the embryo in cows. To investigate the effect of S100A9 on bovine sperm, we incubated sperms in vitro with S100A9 for 5 h and observed significantly decreased sperm motility and viability. During in vitro fertilization, S100A9 treatment for 5 h did not affect the rate of fertilization, time of first division of embryos, or embryo development to blastocyst stage. Treatment of 2-cell stage embryos with S100A9 for 5 h significantly reduced the proportion of cells undergoing normal division (4–8 cell embryos) and embryo development to the blastocyst stage. In experiment involving 24 h treatment of 2-cell embryos, the development of all embryos stopped at the 2-cell stage in the S100A9-treated group. In blastocyst-stage embryos, S100A9 treatment significantly stimulated the expression of endoplasmic reticulum (ER) and the mRNA expression of ER stress markers, and activated caspase-3 with subsequent nuclear fragmentation. Pre-treatment with an ER stress inhibitor significantly suppressed caspase-3 activation by the S100A9 treatment, suggesting that S100A9 induces blastocyst dysfunction by apoptosis (via caspase-3 activation) depending on ER stress. These results indicate that direct exposure to S100A9 exerted adverse effects on sperm function and embryo development. These findings suggest that excessive dose of S100A9 may have an adverse effect to the reproductive machinery by inducing inflammation and tissue dysfunction.

Klíčová slova:

Blastocysts – Embryo development – Embryos – Endoplasmic reticulum – Fertilization – Fluorescence imaging – Inflammation – Sperm


1. Ulbrich SE, Zitta K, Hiendleder S, Wolf E. In vitro systems for intercepting early embryo-maternal cross-talk in the bovine oviduct. Theriogenology. 2010;73(6):802–16. doi: 10.1016/j.theriogenology.2009.09.036 19963260

2. Diskin MG, Murphy JJ, Sreenan JM. Embryo survival in dairy cows managed under pastoral conditions. Anim Reprod Sci. 2006;96(3–4):297–311. doi: 10.1016/j.anireprosci.2006.08.008 16963203

3. Tanaka H, Ohtsu A, Shiratsuki S, Kawahara-Miki R, Iwata H, Kuwayama T, et al. Age-dependent changes in inflammation and extracellular matrix in bovine oviduct epithelial cells during the post-ovulatory phase. Mol Reprod Dev. 2016;83(9):815–26. doi: 10.1002/mrd.22693 27580129

4. Malhi PS, Adams GP, Mapletoft RJ, Singh J. Oocyte developmental competence in a bovine model of reproductive aging. Reproduction. 2007;134(2):233–9. doi: 10.1530/REP-07-0021 17660233

5. Nelson SM, Telfer EE, Anderson RA. The ageing ovary and uterus: new biological insights. Hum Reprod Update. 2013;19(1):67–83. doi: 10.1093/humupd/dms043 23103636

6. Wilding M. Potential long-term risks associated with maternal aging (the role of the mitochondria). Fertil Steril. 2015;103(6):1397–401. doi: 10.1016/j.fertnstert.2015.03.034 25936236

7. Meldrum DR, Casper RF, Diez-Juan A, Simon C, Domar AD, Frydman R. Aging and the environment affect gamete and embryo potential: can we intervene? Fertil Steril. 2016;105(3):548–59. doi: 10.1016/j.fertnstert.2016.01.013 26812244

8. Tanikawa N, Ohtsu A, Kawahara-Miki R, Kimura K, Matsuyama S, Iwata H, et al. Age-associated mRNA expression changes in bovine endometrial cells in vitro. Reprod Biol Endocrinol. 2017;15(1):63. doi: 10.1186/s12958-017-0284-z 28806906

9. Nakamura Y, Iwata H, Kuwayama T, Shirasuna K. S100A8, which increases with age, induces cellular senescence-like changes in bovine oviduct epithelial cells. Am J Reprod Immunol. 2019:e13163. doi: 10.1111/aji.13163 31237976

10. Ibrahim M, Peter S, Gartner MA, Michel G, Jung M, Einspanier R, et al. Increased mRNA expression of selected antimicrobial peptides around ovulation and during inflammatory processes in the bovine endometrium postpartum. Theriogenology. 2016;86(8):2040–53. doi: 10.1016/j.theriogenology.2016.06.022 27531713

11. Swangchan-Uthai T, Lavender CR, Cheng Z, Fouladi-Nashta AA, Wathes DC. Time course of defense mechanisms in bovine endometrium in response to lipopolysaccharide. Biol Reprod. 2012;87(6):135. doi: 10.1095/biolreprod.112.102376 23077171

12. Swindell WR, Johnston A, Xing X, Little A, Robichaud P, Voorhees JJ, et al. Robust shifts in S100a9 expression with aging: a novel mechanism for chronic inflammation. Sci Rep. 2013;3:1215. doi: 10.1038/srep01215 23386971

13. Schiopu A, Cotoi OS. S100A8 and S100A9: DAMPs at the crossroads between innate immunity, traditional risk factors, and cardiovascular disease. Mediators Inflamm. 2013;2013:828354. doi: 10.1155/2013/828354 24453429

14. Wang S, Song R, Wang Z, Jing Z, Wang S, Ma J. S100A8/A9 in Inflammation. Front Immunol. 2018;9:1298. doi: 10.3389/fimmu.2018.01298 29942307

15. Tong L, Lan W, Lim RR, Chaurasia SS. S100A proteins as molecular targets in the ocular surface inflammatory diseases. Ocul Surf. 2014;12(1):23–31. doi: 10.1016/j.jtos.2013.10.001 24439044

16. Nair RR, Khanna A, Singh K. Role of inflammatory proteins S100A8 and S100A9 in pathophysiology of recurrent early pregnancy loss. Placenta. 2013;34(9):824–7. doi: 10.1016/j.placenta.2013.06.307 23850136

17. Nair RR, Khanna A, Singh K. Association of increased S100A8 serum protein with early pregnancy loss. Am J Reprod Immunol. 2015;73(2):91–4. doi: 10.1111/aji.12318 25252120

18. Shimada M, Mihara T, Kawashima I, Okazaki T. Anti-bacterial factors secreted from cumulus cells of ovulated COCs enhance sperm capacitation during in vitro fertilization. Am J Reprod Immunol. 2013;69(2):168–79. doi: 10.1111/aji.12024 23034013

19. Schelbergen RF, Blom AB, van den Bosch MH, Sloetjes A, Abdollahi-Roodsaz S, Schreurs BW, et al. Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4. Arthritis Rheum. 2012;64(5):1477–87. doi: 10.1002/art.33495 22127564

20. Koy M, Hambruch N, Hussen J, Pfarrer C, Seyfert HM, Schuberth HJ. Recombinant bovine S100A8 and A9 enhance IL-1beta secretion of interferon-gamma primed monocytes. Vet Immunol Immunopathol. 2013;155(3):162–70. doi: 10.1016/j.vetimm.2013.07.002 23890724

21. Simard JC, Cesaro A, Chapeton-Montes J, Tardif M, Antoine F, Girard D, et al. S100A8 and S100A9 induce cytokine expression and regulate the NLRP3 inflammasome via ROS-dependent activation of NF-kappaB(1.). PLoS One. 2013;8(8):e72138. doi: 10.1371/journal.pone.0072138 23977231

22. Hamano S, Kuwayama M. In vitro fertilization and development of bovine oocytes recovered from the ovaries of individual donors: A comparison between the cutting and aspiration method. Theriogenology. 1993;39(3):703–12. doi: 10.1016/0093-691x(93)90255-4 16727247

23. Takeo S, Kimura K, Shirasuna K, Kuwayama T, Iwata H. Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality. Reprod Fertil Dev. 2017;29(4):759–67. doi: 10.1071/RD15228 26829061

24. Yaginuma H, Funeshima N, Tanikawa N, Miyamura M, Tsuchiya H, Noguchi T, et al. Improvement of fertility in repeat breeder dairy cattle by embryo transfer following artificial insemination: possibility of interferon tau replenishment effect. J Reprod Dev. 2019;65(3):223–9. doi: 10.1262/jrd.2018-121 30745523

25. Funeshima N, Noguchi T, Onizawa Y, Yaginuma H, Miyamura M, Tsuchiya H, et al. The transfer of parthenogenetic embryos following artificial insemination in cows can enhance pregnancy recognition via the secretion of interferon tau. J Reprod Dev. 2019;65(5):443–50. doi: 10.1262/jrd.2019-026 31378757

26. Bakri NM, Ibrahim SF, Osman NA, Hasan N, Jaffar FH, Rahman ZA, et al. Embryo apoptosis identification: Oocyte grade or cleavage stage? Saudi J Biol Sci. 2016;23(1):S50–5. doi: 10.1016/j.sjbs.2015.10.023 26858565

27. Kim JK, Kang KA, Ryu YS, Piao MJ, Han X, Oh MC, et al. Induction of Endoplasmic Reticulum Stress via Reactive Oxygen Species Mediated by Luteolin in Melanoma Cells. Anticancer Res. 2016;36(5):2281–9. 27127134

28. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. doi: 10.1006/meth.2001.1262 11846609

29. Ibrahim S, Salilew-Wondim D, Rings F, Hoelker M, Neuhoff C, Tholen E, et al. Expression pattern of inflammatory response genes and their regulatory micrornas in bovine oviductal cells in response to lipopolysaccharide: implication for early embryonic development. PLoS One. 2015;10(3):e0119388. doi: 10.1371/journal.pone.0119388 25764515

30. Swangchan-Uthai T, Chen Q, Kirton SE, Fenwick MA, Cheng Z, Patton J, et al. Influence of energy balance on the antimicrobial peptides S100A8 and S100A9 in the endometrium of the post-partum dairy cow. Reproduction. 2013;145(5):527–39. doi: 10.1530/REP-12-0513 23533291

31. Lee AS. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods. 2005;35(4):373–81. doi: 10.1016/j.ymeth.2004.10.010 15804610

32. Hu H, Tian M, Ding C, Yu S. The C/EBP Homologous Protein (CHOP) Transcription Factor Functions in Endoplasmic Reticulum Stress-Induced Apoptosis and Microbial Infection. Front Immunol. 2018;9:3083. doi: 10.3389/fimmu.2018.03083 30662442

33. Basar M, Bozkurt I, Guzeloglu-Kayisli O, Sozen B, Tekmen I, Schatz F, et al. Unfolded protein response prevents blastocyst formation during preimplantation embryo development in vitro. Fertil Steril. 2014;102(6):1777–84. doi: 10.1016/j.fertnstert.2014.09.004 25305729

34. Lin T, Diao YF, Kang JW, Lee JE, Kim DK, Jin DI. Tauroursodeoxycholic acid improves the implantation and live-birth rates of mouse embryos. Reprod Biol. 2015;15(2):101–5. doi: 10.1016/j.repbio.2015.01.004 26051458

35. Gianaroli L, Cristina Magli M, Ferraretti AP, Fiorentino A, Tosti E, Panzella S, et al. Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate. Hum Reprod. 1996;11(1):166–71. doi: 10.1093/oxfordjournals.humrep.a019011 8671180

36. Bungum M, Bungum L, Humaidan P. A prospective study, using sibling oocytes, examining the effect of 30 seconds versus 90 minutes gamete co-incubation in IVF. Hum Reprod. 2006;21(2):518–23. doi: 10.1093/humrep/dei350 16239314

37. Zhao LL, Ru YF, Liu M, Tang JN, Zheng JF, Wu B, et al. Reproductive effects of cadmium on sperm function and early embryonic development in vitro. PLoS One. 2017;12(11):e0186727. doi: 10.1371/journal.pone.0186727 29095856

38. Yoisungnern T, Das J, Choi YJ, Parnpai R, Kim JH. Effect of hexavalent chromium-treated sperm on in vitro fertilization and embryo development. Toxicol Ind Health. 2016;32(9):1700–10. doi: 10.1177/0748233715579805 25903088

39. Gu YH, Li Y, Huang XF, Zheng JF, Yang J, Diao H, et al. Reproductive effects of two neonicotinoid insecticides on mouse sperm function and early embryonic development in vitro. PLoS One. 2013;8(7):e70112. doi: 10.1371/journal.pone.0070112 23922925

40. Kostakis ID, Cholidou KG, Kallianidis K, Perrea D, Antsaklis A. The role of calprotectin in obstetrics and gynecology. Eur J Obstet Gynecol Reprod Biol. 2010;151(1):3–9. doi: 10.1016/j.ejogrb.2010.03.006 20378239

41. Barabé F, Laouedj M, Tessier P. Myeloid-Related Protein S100A9 Induces Cellular Differentiation in Acute Myeloid Leukemia through TLR2 and TLR4 Receptors. Blood. 2015;126:3858.

Článek vyšel v časopise


2020 Číslo 1