Spermatogonia apoptosis induction as a possible mechanism of Toxoplasma gondii induced male infertility

Document Type : Original Article

Authors

1 Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Department of Parasitology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Virology Department, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

4 Department of Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

5 Physiology Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

6 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Objective(s): The protozoan Toxoplasma gondii as an intracellular protozoan is widely prevalent in humans and animals. Infection generally occurs through consuming food contaminated with oocysts and tissue cysts from undercooked meat. The parasite is carried in sexual fluids like semen but there is little information about the effect of T. gondii on the male reproductive system. In this study, we examined the effect of T. gondii tachyzoites on apoptosis induction in type B spermatogonia (GC-1) cells.
Materials and Methods: Fresh tachyzoites taken of infected BALB/c mice, GC-1 spg cells were infected with increasing concentrations of tachyzoites of T. gondii, then apoptotic cells were identified and quantified by flow cytometry. The genes associated with apoptosis were evaluated by RT2 Profiler PCR Array.
Results: PCR array analysis of 84 apoptosis-related genes demonstrated that 12 genes were up-regulated at least 4-fold and that one gene was down-regulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The number of genes whose expression had increased during the period of infection with T. gondii was significantly higher than those whose expressions had decreased (18 versus 1) and Tnfrsf11b had the highest rate of gene expression.
Conclusion: T. gondii induce in vitro apoptosis of GC-1 spg cells. This effect shows a trend of concentration-dependent increase so that with an increase in the ratio of parasite burden to spermatogonial cells, in addition to an increase in the number of genes whose expression has changed, the fold of these changes has increased as well.

Keywords


1. Dvorakova-Hortova K, Sidlova A, Ded L, Hladovcova D, Vieweg M, Weidner W, et al. Toxoplasma gondii decreases the reproductive fitness in mice. PLoS One 2014; 9:96770-96780.
2. Flegr J, Prandota J, Sovičková M, Israili ZH. Toxoplasmosis–a global threat. Correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. PLoS One 2014; 9:90203-90224.
3. Flegr J, Klapilová K, Kaňková Š. Toxoplasmosis can be a sexually transmitted infection with serious clinical consequences. Not all routes of infection are created equal. Med Hypotheses 2014; 83:286-289.
4. Su C, Evans D, Cole R, Kissinger J, Ajioka J, Sibley L. Recent expansion of Toxoplasma through enhanced oral transmission. Science 2003; 299:414-416.
5. Saeij JP, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol 2005; 21:476-481.
6. Shwab EK, Zhu X-Q, Majumdar D, Pena HF, Gennari SM, Dubey JP, et al. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology 2014; 141:453-461.
7. Jensen KD, Camejo A, Melo MB, Cordeiro C, Julien L, Grotenbreg GM, et al. Toxoplasma gondii superinfection and virulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination. mBio 2015; 6:02280-02214.
8. Zhao Y, Ferguson DJ, Wilson DC, Howard JC, Sibley LD, Yap GS. Virulent Toxoplasma gondii evade immunity-related GTPase-mediated parasite vacuole disruption within primed macrophages. J Immunol 2009; 182:3775-3781.
9. Khan W, Khan K. Congenital toxoplasmosis: An overview of the neurological and ocular manifestations. Parasitol Int 2018; 67:715-721.
10. Chaudhry SA, Gad N, Koren G. Toxoplasmosis and pregnancy. Can Fam Physician 2014; 60:334-336.
11. Gunes S, Al-Sadaan M, Agarwal A. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility. Reprod Biomed Online 2015; 31:309-319.
12. Kumar V, Abbas A, Aster J. Mechanisms of Cell Injury. Robbins Basic Pathology 9th ed Canada: Elvesier Saunders 2012:1-26.
13. Chen Z, Hauser R, Trbovich AM, Shifren JL, Dorer DJ, Godfrey‐Bailey L, et al. The relationship between human semen characteristics and sperm apoptosis: a pilot study. J Androl  2006; 27:112-120.
14. Inoue H, Tani K. Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments. Cell Death Differ 2014; 21:39-49.
15. Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl J Med 2009; 361:1570-1583.
16. Aitken RJ, Findlay JK, Hutt KJ, Kerr JB. Apoptosis in the germ line. Reproduction 2011; 141:139-150.
17. Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell 2011; 147:742-758.
18. Dong Y, Hou W, Li Y, Liu D, Hao G, Zhang H, et al. Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells. Cell Death Differ 2016; 23:787-800.
19. Murphy CJ, Richburg JH. Implications of Sertoli cell induced germ cell apoptosis to testicular pathology. Spermatogenesis 2014; 4:979110-979116.
20. Russell LD, Chiarini-Garcia H, Korsmeyer SJ, Knudson CM. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol 2002; 66:950-958.
21. Zheng S, Turner TT, Lysiak JJ. Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis. Biol 2006; 74:1026-1033.
22. Brugnon F, Van Assche E, Verheyen G, Sion B, Boucher D, Pouly J, et al. Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients. Hum Reprod 2005; 21:685-693.
23. Oishi K, Barchi M, Au AC, Gelb BD, Diaz GA. Male infertility due to germ cell apoptosis in mice lacking the thiamin carrier, Tht1. A new insight into the critical role of thiamin in spermatogenesis. Dev Biol 2004; 266:299-309.
24. Shaha C, Tripathi R, Mishra DP. Male germ cell apoptosis: regulation and biology. Philos Trans R Soc Lond B Biol Sci 2010; 365:1501-1515.
25. Tanaka H, Fujisawa M, Tanaka H, Okada H, Kamidono S. Apoptosis‐related proteins in the testes of infertile men with varicocele. BJU Int 2002; 89:905-909.
26. Jurisicova A, Lopes S, Meriano J, Oppedisano L, Casper RF, Varmuza S. DNA damage in round spermatids of mice with a targeted disruption of the Pp1cγ gene and in testicular biopsies of patients with non-obstructive azoospermia. Mol Hum Reprod 1999; 5:323-330.
27. Taylor S, Weng S, Fox P, Duran EH, Morshedi M, Oehninger S, et al. Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality. Mol Hum Reprod 2004; 10:825-834.
28. Zhou Y, Lu Y, Hu Y. Experimental study of influence of Toxoplasma tachyzoites on human sperm motility parameters in vitro. Chinese Journal of Zoonoses 2003; 19:47-49.
29. Terpsidis KI, Papazahariadou MG, Taitzoglou IA, Papaioannou NG, Georgiadis MP, Theodoridis IT. Toxoplasma gondii: reproductive parameters in experimentally infected male rats. Exp Parasitol 2009; 121:238-241.
30. Yang R, Zhao J, Liu Z, Zhang Q, Rao H. Effect of Toxoplasma infection on the apoptosis of sperm in mice. J Trop Med 2006; 6:1153-1156.
31. Ryan AE, Lane S, Shanahan F, O’Connell J, Houston AM. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data. J Carcinog 2006; 5:5-12.
32. Hamouda M-A, Jacquel A, Robert G, Puissant A, Richez V, Cassel R, et al. BCL-B (BCL2L10) is overexpressed in patients suffering from multiple myeloma (MM) and drives an MM-like disease in transgenic mice. J Exp Med 2016; 213:1705-1722.
33. Vijayalingam S, Pillai SG, Rashmi R, Subramanian T, Sagartz JE, Chinnadurai G. Overexpression of BH3-only protein BNIP3 leads to enhanced tumor growth. Genes Cancer 2010; 1:964-971.
34. Tat SK, Pelletier JP, Lajeunesse D, Fahmi H, Lavigne M, Martel-Pelletier J. The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells. Clin Exp Rheumatol 2008; 26:295-304.
35. Kawamura T, Ogawa Y, Shimozato O, Ando T, Nakao A, Kobata T, et al. CD70 is selectively expressed on Th1 but not on Th2 cells and is required for Th1-type immune responses. J Invest Dermatol 2011; 131:1252-1261.
36. Dziarmaga A, Hueber P-A, Iglesias D, Hache N, Jeffs A, Gendron N, et al. Neuronal apoptosis inhibitory protein is expressed in developing kidney and is regulated by PAX2. Am J Physiol Renal Physiol 2006; 291:F913-F920.
37. Metais J-Y, Winkler T, Geyer JT, Calado RT, Aplan PD, Eckhaus MA, et al. BCL2A1a overexpression in murine hematopoietic stem and progenitor cells decreases apoptosis and results in hematopoietic transformation. PLoS One 2012; 7:48267-48278.
38. Tone M, Tone Y, Babik JM, Lin C-Y, Waldmann H. The role of Sp1 and NF-κB in regulating CD40 gene expression. J Biol Chem 2002; 277:8890-8897.
39. Tomasini R, Tsuchihara K, Wilhelm M, Fujitani M, Rufini A, Cheung CC, et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev 2008; 22:2677-2691.
40. Payne TM, Molestina RE, Sinai AP. Inhibition of caspase activation and a requirement for NF-κB function in the Toxoplasma gondii-mediated blockade of host apoptosis. J Cell Sci 2003; 116:4345-4358.
41. Nash PB, Purner MB, Leon RP, Clarke P, Duke RC, Curiel TJ. Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis. J Immunol 1998; 160:1824-1830.
42. Goebel S, Gross U, Lüder CG. Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly (ADP-ribose) polymerase expression. J Cell Sci 2001; 114:3495-3505.
43. Krishnamurthy S, Konstantinou EK, Young LH, Gold DA, Saeij JP. The human immune response to Toxoplasma: Autophagy versus cell death. PLoS Pathog 2017; 13:1006176-1006181.
44. Kim WH, Shin EH, Kim JL, Yu SY, Jung BK, Chai JY. Suppression of CD4+ T-cells in the spleen of mice Infected with Toxoplasma gondii KI-1 Tachyzoites. Korean J Parasitol 2010; 48:325-329.
45. Begum-Haque S, Haque A, Kasper LH. Apoptosis in Toxoplasma gondii activated T cells: The role of IFNγ in enhanced alteration of Bcl-2 expression and mitochondrial membrane potential. Microb Pathog 2009; 47:281-288.
46. Wang G, Gao M. Influence of Toxoplasma gondii on in vitro proliferation and apoptosis of hepatoma carcinoma H7402 cell. Asian Pac J Trop Dis 2016; 9:63-66.
47. Abbasi M, Kowalewska-Grochowska K, Bahar MA, Kilani RT, Winkler-Lowen B, Guilbert LJ. Infection of placental trophoblasts by Toxoplasma gondii. Int J Infect Dis 2003; 188:608-616.
48. Nishikawa Y, Makala L, Otsuka H, Mikami T, Nagasawa H. Mechanisms of apoptosis in murine fibroblasts by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum. Parasite Immunol 2002; 24:347-354.
49. Besteiro S. Toxoplasma control of host apoptosis: the art of not biting too hard the hand that feeds you. Microbial Cell 2015; 2:178-181.
50. Blader IJ, Koshy AA. Toxoplasma gondii development of its replicative niche: in its host cell and beyond. Eukaryot Cell 2014; 13:965-976.
51. Gavrilescu LC, Denkers EY. IFN-γ overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. J Immunol 2001; 167:902-909.
52. Liesenfeld O, Kosek JC, Suzuki Y. Gamma interferon induces Fas-dependent apoptosis of Peyer’s patch T cells in mice following peroral infection with Toxoplasma gondii. Infect Immun 1997; 65:4682-4689.
53. Kim JY, Ahn MH, Jun HS, Jung JW, Ryu JS, Min DY. Toxoplasma gondii inhibits apoptosis in infected cells by caspase inactivation and NF-κB activation. Yonsei Med J 2006; 47:862-869.
54. Vutova P, Wirth M, Hippe D, Gross U, Schulze‐Osthoff K, Schmitz I, et al. Toxoplasma gondii inhibits Fas/CD95‐triggered cell death by inducing aberrant processing and degradation of caspase 8. Cell Microbiol 2007; 9:1556-1570.
55. Goebel S, Lüder C, Gross U. Invasion by Toxoplasma gondii protects human-derived HL-60 cells from actinomycin D-induced apoptosis. Med Microbiol Immunol 1999; 187:221-226.
56. Angeloni M, Guirelli P, Franco P, Barbosa B, Gomes A, Castro A, et al. Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression. Placenta 2013; 34:973-982.
57. Keller P, Schaumburg F, Fischer SF, Häcker G, Groß U, Lüder CG. Direct inhibition of cytochrome c-induced caspase activation in vitro by Toxoplasma gondii reveals novel mechanisms of interference with host cell apoptosis. FEMS Microbiol Lett 2006; 258:312-319.
58. Villena I, Ancelle T, Delmas C, Garcia P, Brezin A, Thulliez P, et al. Congenital toxoplasmosis in France in 2007: first results from a national surveillance system. Euro Surveill 2010; 15:19600-19605.
59. El-Tantawy N, Taman A, Shalaby H. Toxoplasmosis and female infertility: is there a co-relation? American Journal of Epidemiology and Infectious Disease 2014; 2:29-32.
60. Kankova S, Flegr J, Calda P. The influence of latent toxoplasmosis on women’s reproductive function: four cross-sectional studies. Folia Parasitol 2015; 62:041-046.
61.  Stahl W, Dias JA, Turek G. Hypothalamic-adenohypophyseal origin of reproductive failure in mice following chronic infection with Toxoplasma gondii. Exp Biol Med 1985; 178:246-249.
62. Abdoli A, Dalimi A, Movahedin M. Impaired reproductive function of male rats infected with Toxoplasma gondii. Andrologia 2012; 44:679-687.
63. Aitken RJ, Baker MA. Oxidative stress, spermatozoa and leukocytic infiltration: relationships forged by the opposing forces of microbial invasion and the search for perfection. J Reprod Immunol 2013; 100:11-19.
64. Howe DK, Summers BC, Sibley LD. Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii. Infect Immun 1996; 64:5193-5198.
65. Harba NM, Afifi AF. Evaluation of DNA damage by DNA fragmentation and comet assays in experimental toxoplasmosis with virulent strain. PUJ 2012; 5:189-198.
66. Nishikawa Y, Kawase O, Vielemeyer O, Suzuki H, Joiner K, Xuan X, et al. Toxoplasma gondii infection induces apoptosis in noninfected macrophages: role of nitric oxide and other soluble factors. Parasite Immunol 2007; 29:375-385.
67. Channon JY, Kasper LH. Toxoplasma gondii-induced immune suppression by human peripheral blood monocytes: role of gamma interferon. Infect Immun 1996; 64:1181-1189.
68. Candolfi E, Hunter CA, Remington JS. Roles of gamma interferon and other cytokines in suppression of the spleen cell proliferative response to concanavalin A and toxoplasma antigen during acute toxoplasmosis. Infect Immun 1995; 63:751-756.
69. Xu X, Liu T, Zhang A, Huo X, Luo Q, Chen Z, et al. Reactive oxygen species-triggered trophoblast apoptosis is initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the JNK pathway in Toxoplasma gondii infection in mice. Infect Immun 2012; 80:2121-2132.
70. Contreras-Ochoa CO, Lagunas-Martínez A, Belkind-Gerson J, Díaz-Chávez J, Correa D. Toxoplasma gondii invasion and replication within neonate mouse astrocytes and changes in apoptosis related molecules. Exp Parasitol 2013; 134:256-265.
71. Hwang I-Y, Quan JH, Ahn M-H, Ahmed HAH, Cha G-H, Shin D-W, et al. Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70. Parasitol Res 2010; 107:1313-1321.
72. Nagata S. Apoptosis by death factor. cell 1997; 88:355-365.
73. Lenardo M, Chan FK-M, Hornung F, McFarland H, Siegel R, Wang J, et al. Mature T lymphocyte apoptosis—immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immunol 1999; 17:221-253.
74. Desagher S, Martinou J-C. Mitochondria as the central control point of apoptosis. Trends Cell Biol 2000; 10:369-377.