Constitutive expression of toll-like receptor genes and signaling pathways in stallion testis and epididymis
Keywords:
Horse, testis, epididymis, toll-like receptor, innate immunity, transcriptome
Abstract
Toll-like receptors (TLRs) expression pattern and their associated molecules have not been described in stallions. Understanding the TLR expression and their responses to activation is key in interpreting the interactions between immune system and reproductive system in healthy and ill animals. Objective of this study was to describe the constitutive expression pattern of TLRs and their signaling pathways in stallion’s testis and epididymis. It was hypothesized that all TLRs and downstream signaling molecules are constitutively expressed in these tissues in reproductively normal stallions. Transcriptome analysis was performed in testicular and epididymal samples from reproductively normal stallions (n = 8). A detectable constitutive expression of all TLRs, costimulatory molecules, and most downstream adaptors and effectors was identified in stallion reproductive tissues. Most significant pathways were associated with TLR3, TLR2/TLR1, TLR2/TLR6, and the effector molecules C-C motif ligand 5 (CCL5) and CD40. Most genes were overexpressed in the epididymis compared to testis. Widespread and abundant expression of these genes supports an important function of the innate immunity in testicular and epididymal defense. These TLRs may also have a physiological role in maintaining the tolerogenic environment, supporting posttesticular maturation, or removing defective sperm from the tubular system.
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References
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24. Heckenbichler S, Deichsel K, Peters P, et al: Quality, and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology 2011;75:849-856. doi: 10.1016/j.theriogenology.2010.10.027
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26. Pickett BW, Voss JL, Bowen RA, et al: Seminal characteristics and total scrotal width (T.S.W.) of normal and abnormal stallions. Proc Am Assoc Equine Pract 1987;33:487-518.
27. Kenney RM, Kingston RS, Rajamannon AH, et al: Stallion semen characteristics for predicting fertility. Proc Am Assoc Equine Pract 1971;17:53-67.
28. Bielanski W: The evaluation of stallion semen in aspects of fertility control and its use for artificial insemination. J Reprod Fertil Suppl 1975;23:19-24.
29. Dott HM: Morphology of stallion spermatozoa. J Reprod Fertil Suppl 1975;23:19-24.
30. Stout TAE, Colenbrander B: Reproductive parameters of Draft Horse, Friesian, and Warmblood stallions. In: McKinnon AO, Squires EL, Vaala WE, et al: editors. Equine Reproduction. 2nd edition, Ames, IA; Wiley Blackwell: 2011. p. 1362-1366.
31. Trapnell C, Williams BA, Pertea G, et al: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotech 2010;28:511-515. doi: 10.1038/nbt.1621
32. Anders S, Huber W: Differential expression analysis for sequence count data. Genome Biol 2010. doi: 10.1186/gb-2010-11-10-r106
33. Wu H, Wang H, Xiong W, et al: Expression patterns and functions of toll-like receptors in mouse Sertoli cells. Endocrinology 2008;149:4402-4412. doi: 10.1210/en.2007-1776
34. Nishimura M, Naito S: Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharmacol Bull 2005;28:886-892. doi: 10.1248/bpb.28.886
35. Girling JE, Hedger MP: Toll-like receptors in the gonads and reproductive tract: emerging roles in reproductive physiology and pathology. Immunol Cell Biol 2007;85:481-489. doi: 10.1038/sj.icb.7100086
36. Astakhova NM, Perelygin AA, Zharkikh AA, et al: Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes. Immunogenetics 2009;61:529-539. doi: 10.1007/s00251-009-0381-z
37. Feng X, Ma BF, Liu B, et al: The involvement of the chemokine RANTES in regulating luminal acidification in rat epididymis. Front Immunol 2020;11:583274. doi: 10.3389/fimmu.2020.583274
38. Naz RK, Leslie MH: Immunobiologic implication of RANTES in seminal plasma of fertile, infertile and immunoinfertile men. Am J Reprod Immunol 2020;44:197-204. doi: 10.1111/j.8755-8920.2000.440402.x
39. Caflisch CR, DuBose Jr, TD: Cadmium-induced changes in luminal pH in testis and epididymis of the rat in vivo. J Toxicol Environ Health 1991;32:49-57. doi: 10.1080/15287399109531464
40. Chandel HS, Pandey SP, Roy S, et al: TLR-CD40 cross-talk in anti-leishmanial immune response. Front Immunol 2014;5:220. doi: 10.3389/fimmu.2014.00220
41. Karimi MH, Pourfathollah AA: CD40 and tolerance induction. Iran J Allergy Asthma Immunol 2012;11:1-13.
42. Tuettenberg A, Fondel S, Steinbrink K, et al: CD40 signaling induces IL-10-producing, tolerogenic dendritic cells. Exp Dermatol 2010;19:44-53. doi: 10.1111/j.1600-0625.2009.00975.x
43. Yi Z, Stunz LL, Bishop GA: CD40-mediated maintenance of immune homeostasis in the adipose tissue microenvironment. Diabetes 2014;63:2751-2760. doi: 10.2337/db13-1657
44. Palladino MA, Savarese MA, Chapman JL, et al: Localization of toll-like receptors on epididymal epithelial cells and spermatozoa. Am J Reprod Immunol 2008;60:541-555. doi: 10.1111/j.1600-0897.2008.00654.x
45. Abdel-maksoud FM, Zayed AE, Abdelhafez EA, et al: Seasonal variations of the epididymis in donkeys (Equus asinus) with special reference to blood epididymal barrier. Microsc Res Tech 2024;87:326-338. doi: 10.1002/jemt.24436
46. Raetz M, Kibardin A, Sturge CR, et al: Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin. J Immunol 2013;191:4818-4827. doi: 10.4049/jimmunol.1301301
2. Stejskalova K, Janova E, Splichalova P, et al: Twelve toll-like receptor (TLR) genes in the family Equidae – comparative genomics, selection and evolution. Vet Res Comm 2024;48:725-741. doi: 10.1007/s11259-023-10245-4
3. Irvine KL, Hopkins LJ, Gangloff M, et al: The molecular basis of recognition of bacterial ligands at equine TLR2, TLR1 and TLR6. Vet Res 2023;44:50. doi: 10.1186/1297-9716-44-50
4. Chen JQ, Szodoray P, Zeher M: Toll-like receptor pathways in autoimmune diseases. Clin Rev Allergy Immunol 2016;501-17. doi: 10.1007/s12016-015-8473-z
5. Crisan TO, Netea MG, Joosten LAB: Innate immune memory: implications for host responses to damage-associated molecular patterns. Eur J Immunol 2016;46:817-828. doi: 10.1002/eji.201545497
6. Tarlinton RE, Alder L, Moreton J, et al: RNA expression of TLR10 in normal equine tissues. BMC Res Notes 2016;9:353. doi: 10.1186/s13104-016-2161-9
7. de Laat MA, Clement CK, McGowan CM, et al: Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses: implications for laminitis. Vet Immunol Immunopathol 2014;157:78-86. doi: 10.1016/j.vetimm.2013.10.010
8. Kennedy R, Lappin DF, Dixon PM, et al: Gingival toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health. Equine Vet 2017;49:294-299. doi: 10.1111/evj.12597
9. Gornik K, Moore P, Figueiredo M, et al: Expression of toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva. Vet Ophthalmol 2011;14:80-85. doi: 10.1111/j.1463-5224.2010.00844.x
10. Park J-W, Kim K-H, Choi J-K, et al: Regulation of toll-like receptors expression in muscle cells by exercise-induced stress. Anim Biosci 2021;34:1590. doi: 10.5713/ab.20.0484
11. Waller AP, Huettner L, Kohler K, et al: Novel link between inflammation and impaired glucose transport during equine insulin resistance. Vet Immunol Immunopathol 2012;149:208-215. doi: 10.1016/j.vetimm.2012.07.003
12. Schöniger S, Gräfe H, Wipplinger M, et al: Expression of toll-like receptors 2, 4 and 6 in the equine chorioallantois. Vet Immunol Immunopathol 2018;206:49-53. doi: 10.1016/j.vetimm.2018.11.010
13. Schöniger S, Gräfe H, Schoon HA: Expression of toll-like receptors 2, 4 and 6 in different cell populations of the equine endometrium. Vet Immunol Immunopathol 2017;185:7-13. doi: 10.1016/j.vetimm.2017.01.002
14. Siemieniuch MJ, Szóstek AZ, Gajos K, et al: Type of inflammation differentially affects expression of Interleukin 1β and 6, Tumor Necrosis Factor-α and Toll-like receptors in subclinical endometritis in mares. PLoS One 2016;11:e0154934. doi: 10.1371/journal.pone.0154934
15. El-Sheikh Ali H, Dini P, Scoggin K, et al: Transcriptomic analysis of equine placenta reveals key regulators and pathways involved in ascending placentitis. Biol Reprod 2021;104:638-656. doi: 10.1093/biolre/ioaa209
16. Marth CD, Firestone SM, Glenton LY, et al: Oestrous cycle-dependent equine uterine immune response to induced infectious endometritis. Vet Res 2016;47:110. doi: 10.1186/s13567-016-0398-x
17. Hedger MP: Toll-like receptors and signaling in spermatogenesis and testicular responses to inflammation - a perspective. J Reprod Immunol 2011;88:130-41. doi: 10.1016/j.jri.2011.01.010
18. Bhushan S, Schuppe HC, Fijak M, et al: Testicular infection: microorganisms, clinical implications and host–pathogen interaction. J Reprod Immunol 2009;83:164-167. doi: 10.1016/j.jri.2009.07.007
19. Michailidis G, Anastasiadou M, Guibert E, et al: Activation of innate immune system in response to lipopolysaccharide in chicken Sertoli cells. Reproduction 2014;148259-270. doi: 10.1530/REP-14-0064
20. Zhang X, Wang T, Deng T, et al: Damaged spermatogenic cells induce inflammatory gene expression in mouse Sertoli cells through the activation of Toll-like receptors 2 and 4. Mol Cell Endocrinol 2013;365:162-173. doi: 10.1016/j.mce.2012.10.016
21. Fujita Y, Mihara T, Okazaki T, et al: Toll-like receptors (TLR) 2 and 4 on human sperm recognize bacterial endotoxins and mediate apoptosis. Human Reprod 2011;26:2799-2806. doi: 10.1093/humrep/der234
22. Liu Z, Zhao S, Chen Q, et al: Roles of toll-like receptors 2 and 4 in mediating experimental autoimmune orchitis induction in mice. Biol Reprod 2015;92:1-11. doi: 10.1095/biolreprod.114.123901
23. Card C: Cellular associations and the differential spermiogram: making sense of stallion spermatozoal morphology. Theriogenology 2005;64:558-567. doi: 10.1016/j.theriogenology.2005.05.014
24. Heckenbichler S, Deichsel K, Peters P, et al: Quality, and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology 2011;75:849-856. doi: 10.1016/j.theriogenology.2010.10.027
25. Morrell JM, Johannisson A, Dalin A-M, et al: Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates. Acta Vet Scand 2008;50:2. doi: 10.1186/1751-0147-50-2
26. Pickett BW, Voss JL, Bowen RA, et al: Seminal characteristics and total scrotal width (T.S.W.) of normal and abnormal stallions. Proc Am Assoc Equine Pract 1987;33:487-518.
27. Kenney RM, Kingston RS, Rajamannon AH, et al: Stallion semen characteristics for predicting fertility. Proc Am Assoc Equine Pract 1971;17:53-67.
28. Bielanski W: The evaluation of stallion semen in aspects of fertility control and its use for artificial insemination. J Reprod Fertil Suppl 1975;23:19-24.
29. Dott HM: Morphology of stallion spermatozoa. J Reprod Fertil Suppl 1975;23:19-24.
30. Stout TAE, Colenbrander B: Reproductive parameters of Draft Horse, Friesian, and Warmblood stallions. In: McKinnon AO, Squires EL, Vaala WE, et al: editors. Equine Reproduction. 2nd edition, Ames, IA; Wiley Blackwell: 2011. p. 1362-1366.
31. Trapnell C, Williams BA, Pertea G, et al: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotech 2010;28:511-515. doi: 10.1038/nbt.1621
32. Anders S, Huber W: Differential expression analysis for sequence count data. Genome Biol 2010. doi: 10.1186/gb-2010-11-10-r106
33. Wu H, Wang H, Xiong W, et al: Expression patterns and functions of toll-like receptors in mouse Sertoli cells. Endocrinology 2008;149:4402-4412. doi: 10.1210/en.2007-1776
34. Nishimura M, Naito S: Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharmacol Bull 2005;28:886-892. doi: 10.1248/bpb.28.886
35. Girling JE, Hedger MP: Toll-like receptors in the gonads and reproductive tract: emerging roles in reproductive physiology and pathology. Immunol Cell Biol 2007;85:481-489. doi: 10.1038/sj.icb.7100086
36. Astakhova NM, Perelygin AA, Zharkikh AA, et al: Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes. Immunogenetics 2009;61:529-539. doi: 10.1007/s00251-009-0381-z
37. Feng X, Ma BF, Liu B, et al: The involvement of the chemokine RANTES in regulating luminal acidification in rat epididymis. Front Immunol 2020;11:583274. doi: 10.3389/fimmu.2020.583274
38. Naz RK, Leslie MH: Immunobiologic implication of RANTES in seminal plasma of fertile, infertile and immunoinfertile men. Am J Reprod Immunol 2020;44:197-204. doi: 10.1111/j.8755-8920.2000.440402.x
39. Caflisch CR, DuBose Jr, TD: Cadmium-induced changes in luminal pH in testis and epididymis of the rat in vivo. J Toxicol Environ Health 1991;32:49-57. doi: 10.1080/15287399109531464
40. Chandel HS, Pandey SP, Roy S, et al: TLR-CD40 cross-talk in anti-leishmanial immune response. Front Immunol 2014;5:220. doi: 10.3389/fimmu.2014.00220
41. Karimi MH, Pourfathollah AA: CD40 and tolerance induction. Iran J Allergy Asthma Immunol 2012;11:1-13.
42. Tuettenberg A, Fondel S, Steinbrink K, et al: CD40 signaling induces IL-10-producing, tolerogenic dendritic cells. Exp Dermatol 2010;19:44-53. doi: 10.1111/j.1600-0625.2009.00975.x
43. Yi Z, Stunz LL, Bishop GA: CD40-mediated maintenance of immune homeostasis in the adipose tissue microenvironment. Diabetes 2014;63:2751-2760. doi: 10.2337/db13-1657
44. Palladino MA, Savarese MA, Chapman JL, et al: Localization of toll-like receptors on epididymal epithelial cells and spermatozoa. Am J Reprod Immunol 2008;60:541-555. doi: 10.1111/j.1600-0897.2008.00654.x
45. Abdel-maksoud FM, Zayed AE, Abdelhafez EA, et al: Seasonal variations of the epididymis in donkeys (Equus asinus) with special reference to blood epididymal barrier. Microsc Res Tech 2024;87:326-338. doi: 10.1002/jemt.24436
46. Raetz M, Kibardin A, Sturge CR, et al: Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin. J Immunol 2013;191:4818-4827. doi: 10.4049/jimmunol.1301301
Published
2025-10-28
How to Cite
Ferrer , M., Moran , K., Bilbao , M., Barletta , M., Williams , J., Gordon , J., & Bartolomé , J. (2025). Constitutive expression of toll-like receptor genes and signaling pathways in stallion testis and epididymis. Clinical Theriogenology, 17. https://doi.org/10.58292/CT.v17.12705
Section
Research Reports
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