Seyit Ali KAYIS, Mehmet Osman ATLI, Ahmet SEMACAN, Ercan KURAR, Aydin GUZELOGLU

Expression of Toll-like receptors (TLRs) in the equine peripheral blood mononuclear cells (PBMCs) during the early pregnancy and estrous cycle

Amaç: Günümüzde kısraklarda birçok gebelik teşhis metodu olmasına rağmen, mümkün olabilecek en erken zamanda gebeliğinin teşhis edilmesi için pratik, güvenilir ve ekonomik bir yönteme halen ihtiyaç bulunmaktadır. Allojenik fötusa karşı immun tolerans erken gebelik esnasında kritik bir öneme sahiptir. Toll-like reseptörlerinin (TLRs) doğal immun sistemin önemli bir parçası olduğu bilinmektedir. Sunulan çalışmada, kısraklarda erken gebelikte periferal kan mononükler hücrelerinde (PKMH) TLRs ekspresyonunun incelenmesi amaçlandı. Gereç ve Yöntem: Kan numuneleri aynı üç kısraktan östrus siklusunun ve gebeliğin 0 ve 8. günlerinde toplandı. Total RNA örnekleri PKMH’lerinden izole edildi ve cDNA sentezlendi. mRNA düzeyindeki TLRs ekspresyonları iki defa realtime PZR kullanılarak kuantifiye edildi. İstatistiksel analiz için Relative Expression Software Tool (REST2009) kullanıldı. Bulgular: RT-PZR ile PKMH’lerde TLRs (1-10) ekspresyonları tespit edildi. Bununla beraber, real-time PZR ölçümleri siklik veya gebelik durumunun TLRs ekspresyonu üzerine etkisini göstermekte başarısız kaldı. Öneri: Bu sonuçlara bakıldığında, atlarda gebeliğin erken döneminde PKMH’lerinde TLR genlerinin ekspresyonlarının gebeliğin erken anlaşılması için iyi bir markör olmadığı söylenebilir.

Atların periferal kan mononükler hücrelerinde (PKMH) erken gebelik ve östrus siklusu sürecinde Toll-like reseptörlerinin (TLRs) ekspresyonu

Aim: Practical, reliable and economical pregnancy diagnosis method for as early as possible time of pregnancy in mares is still requirement. Immune tolerance against allogenic fetus is critically important during the early pregnancy. Tolllike Receptors (TLRs) are known to be an important part of innate immune system. Aim was to evaluate expression of TLRs in peripheral blood mononuclear cells (PBMCs) during the early pregnancy in mare. Material and Methods: Blood samples were collected on days of ovulation (d0) and 8 during estrous and pregnancy from three same mares. Total RNA samples were isolated from PBMCs and cDNA synthesis was performed. Expressions of TLRs at mRNA levels were quantified by using realtime RT-PCR in duplicates. Relative Expression Software Tool (REST2009) was used for statistical analysis. Results: TLRs (1 to 10) were found to be expressed in PMBCs by RT-PCR. However, real-time PCR measurements failed to show effects of pregnant or cyclic status on expression of TLRs. Conclusion: According to these results, it may suggest that determination of TLRs gene expression in equine PBMCs during early pregnancy is not a good marker in understanding early equine pregnancy.

PDF

___

Abrahams VM, Mor G, 2007. Toll-like Receptors and their role in the trophoblast. Placenta, 26, 540-547.
Aflatoonian R, Tuckerman E, Eliot SL, Bruce C, Aflatoonian A, Li TC, Fazeli A, 2007. Mestrual cycle-dependent changes of Toll-like receptors in endometrium. Hum Reprod, 22, 586-593.
Akira S, 2003. Toll-like receptor signaling. J Biol Chem, 278, 38105-38118.
Allen WR, Antczak DF, 2000. Reproduction and Modern Breeding Technologies in the Mare, in; The Genetics of the Horse, Eds; Bowling AT, Ruvinsky A, CAB International, NY, USA, pp: 314-316.
Antczak DF, Allen WR, 1989. Maternal immunological recognition of pregnancy in equids. Reprod Fertil Suppl, 37, 69-78.
Aslan S, Fındık M, Izgür H, Çelebi M, Çelebi M, 1997. Pregnancy diagnosis by using PMSG-Latex Test in mares. Tr J Vet Anim Sci, 21, 195-199.
Atli MO, Kurar E, Kayis SA, Aslan A, Semacan A, Celik S, Guzeloglu A, 2010a. Expression of Toll-like receptors (TLRs) in the equine endometrium during the estrous cycle. Reprod Dom Anim, 45, 58.
Atli MO, Kurar E, Kayis SA, Aslan S, Semacan A, Celik S, Guzeloglu A, 2010b. Expression of Toll-like receptors (TLRs) is regulated by early pregnancy in the mare endometrium. Biol Reprod, 83, 349.
Atli MO, Kurar E, Kayis SA, Aslan S, Semacan A, Celik S, Guzeloglu A, 2010c. Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy. Anim Reprod Sci, 122, 124-132.
Dixit VD, Parvizi N, 2000. Lymphocytic secretion of adrenocorticotropic hormone throughout pregnancy in cows and its possible physiological role. Reprod Dom Anim, 3, 171.
Dixit VD, Parvizi N, 2001. Pregnancy stimulates secretion of adrenocorticotropin and nitric oxide from peripheral bovine lymphocytes. Biol Reprod, 64, 242-248.
Foristall KM, Roser JF, Liu IKM, Lasley B, Munro CJ, Carneiro GF, 1998. Development of a Tandem Hormone Assay for the Detection of Pregnancy in the Miniature Mare, 44th Annual Convention of the American Association of Equine Practitioners, Baltilmore, Maryland, USA.
Gifford CA, Racicot K, Clark DS, Austin KJ, Hansen TR, Lucy MC, Davies CJ, Ott TL, 2007 Regulation of interferonstimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows. J Dairy Sci, 90, 274-280.
Gonzales J, Xu H, Ofori E, Elovitz MA, 2007. Toll-like receptors in the uterus, cervix and placenta: is pregnancy an immunosuppressed stare? Am J Obstetric Gynecol, 197, 1-6.
Green JC, Okamura CS, Poock SE, Lucy MC, 2010. Measurement of interferon-tau (IFN-tau) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18–20 d after insemination in dairy cattle. Anim Reprod Sci, 121, 24-33.
Hicks BA, Etter SJ, Carnahan KG, Joyce MM, Assiri AA, Carling SJ, Kodali K, Johnson GA, Hansen TR, Mirando MA, Woods GL, Vanderwall DK, Ott TL, 2003. Expression of the uterine Mx protein in cyclic and pregnant cows, gilts, and mares. J Anim Sci, 81, 1552-1561.
Holmlund U, Cebers G, Dahlfors AR, Sandstedt B, Bremme K, Ekström ES, Scheynius A, 2002. Expression and regulation of the pattern recognition receptors Toll-like receptor- 2 and Toll-like receptor-4 in the human placenta. Immunol, 107, 145-151.
Hyland JH, 1990. Reproductive endocrinology: it’s role in fertility and infertility in the horse. Br Vet J, 14, 1-16.
Jiang SP, Vacchio MS, 1998. Multiple mechanisms of peripheral T cell tolerance to the fetal “allograft”. J Immunol, 160, 3086-3090.
Kannaki TR, Shanmugan M, Verma PC, 2011. Toll-like receptors and their roles in animal reproduction. Anim Reprod Sci, (Article in press).
Kılıçarslan MR, Soylu MK, Şenünver A, Kırşan İ, Carioğlu B, 1999. Kısraklarda erken gebelik tanısında ultrasonografik tekniklerin kullanımı. Kafkas Univ Vet Fak Derg, 2, 147-150.
Koga K, Mor G, 2008. Expression and function of toll-like receptors at the maternal- fetal interface. Reproductive Sci, 15, 231-242.
Kurar E, Atli MO, Guzeloglu A, Ozsensoy Y, Semacan A, 2010. Comparison of five different RNA isolation methods from equine endometrium for gene transcription analysis. Kafkas Univ Vet Fak Derg, 16, 851-855.
Kurar E, Atli MO, Guzeloglu A, Semacan A, 2011. POMC, iNOS, PGES, IL-4, IL-5 and IL-10 gene expression in Peripheral Blood Mononuclear Cells of cyclic and pregnant mares. Kafkas Univ Vet Fak Derg, 17, 319-323.
Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the method. Methods, 25, 402-408.
Mann GE, Lamming GE, 2001. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction, 121, 175-180.
Metcalf ES, McCue P, Jasko DJ, Roor JB, 2004. Evaluation of a test for equine early conception factor. 50th Annual Convention of the American Association of Equine Practitioners, Denver, Colorado, USA.
Meyer F, Ramanujam KS, Gobert AP, James SP, Wilson WT, 2003 Cutting edge: cyclooxygenase-2 activation suppresses Th1 polarization in response to Helicobacter pylori. J Immunol, 171, 3913-3197.
Mor G, 2008. Inflamation and pregnancy; the role of toll-like receptors in trophoblast-immune interaction. Ann NY Acad Sci, 1127, 121-128.
Ott TL, Yin J, Wiley AA, Kim HT, Gerami-Naini B, Spencer TE, Bartol FF, Burghardt RC, Bazer FW, 1998. Effects of the estrous cycle and early pregnancy on uterine expression of mx protein in sheep (Ovis aries). Biol Reprod, 59, 784-794.
Pfaffl MW, Horgan GW, Dempfle L, 2002. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in realtime PCR. Nucl Acid Res, 30, 1-10.
Roberts RM, Liu L, Alexenko A, 1997. New and atypical families of type I interferons in mammals: comparative functions, structures, and evolutionary relationships. Prog Nucleic Acid Res Mol Biol, 56, 287-325.
Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H, 2006. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula. J Mol Med, 84, 901-910.
Spencer TE, Bazer FW, 2002. Biology of progesterone action during pregnancy recognition and maintenance of pregnancy. Front Biosci, 7, 1879-1898.
Tirumurugaan KG, Dhanasekaran S, Raj GD, Raja A, Kumanan K, Ramaswamy V, 2010. Differential expression of toll-like receptor mRNA in selected tissues of goat (Capra hircus). Vet Immunol Immunopathol, 133, 296- 301.
Weetman P, 1999. The immunology of pregnancy. Thyroid, 9, 643-646.
Wilder RL, 1998. Hormones, pregnancy and autoimmune diseases. Ann NY Acad Sci, 84, 45-50.
Young SL, Lyddon TD, Jorgenson RL, Misfeldt ML 2004. Expression of toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immun, 52, 67-73.
Zarember KA, Godowski PJ, 2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol, 16, 8554-8561.