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    Early pregnancy-related changes in toll-like receptors expression in ovine trophoblasts and peripheral blood leukocytes
    (ELSEVIER SCIENCE INC, 2017) Kaya, Mehmet Salih; Kose, Mehmet; Guzeloglu, Aydin; Kiyma, Zekeriya; Atli, Mehmet Osman
    In the present study, we aimed to 1) demonstrate the presence of all 10 toll-like receptors (TLRs) in ovine trophoblasts, and 2) investigate the expression profiles of TLR1 - 10 mRNAs in peripheral blood leukocytes (PBLs) in ewes during early pregnancy. For those purposes, ovine trophoblasts (n = 6) were collected from pregnant ewes on day 13. PBLs were collected from non-pregnant (n = 6) and pregnant ewes (n = 17) on days of mating (d) 0 and 18. TLR mRNAs in ovine trophoblasts were visualized by free-floating in situ hybridization (ISH). To assess the expression profiles of TLR1-10 in PBLs, total RNA was isolated and transcribed to cDNA. TLR1-10 mRNA levels were determined by real-time PCR in triplicate. The Relative Expression Software Tool (REST 2009) was used for statistical analysis. We detected mRNAs for TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR10 but not for TLR1, TLR3, and TLR9 in trophoblasts. TLR2, TLR5, TLR6, TLR7, TLR8, and TLR10 mRNAs were expressed by all trophoblasts, whereas TLR4 mRNA and protein in trophoblasts were more limited. In PBLs, TLR expression did not differ between day 0 and day 18 in non-pregnant ewes; however, ewes in early pregnancy exhibited significantly upregulated expression of TLR2 (23-fold), TLR4 (3.1-fold), TLR6 (1.7-fold), and TLR8 (2.2-fold) on day 18 compared with day 0. In contrast, TLR10 was downregulated (2-fold) on day 18 by pregnancy. Similar results were also obtained for TLR2, TLR4, TLR6, TLR8 and TLR10 from the comparison between day 18 non pregnant and day 18 pregnant groups. According to these results, the presence of TLRs in early ovine trophoblasts suggests that these cells play an immunological role at the maternal fetal interface. The results also suggest that tight regulation of some components of TLRs in PBLs due to embryo- and/or pregnancy related factors is necessary for successful establishment of early pregnancy in ewes. (C) 2017 Elsevier Inc. All rights reserved.
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    Investigation of interferon-tau stimulated genes (ISGs) simultaneously in the endometrium, corpus luteum (CL) and peripheral blood leukocytes (PBLs) in the preluteolytic stage of early pregnancy in ewes
    (ELSEVIER SCIENCE BV, 2016) Kiyma, Zekeriya; Kose, Mehmet; Atli, Mehmet Osman; Ozel, Caglayan; Hitit, Mustafa; Sen, Gonca; Kaya, Muhammet
    The aim of the present study was to investigate the expression profiles of Interferon-tau (IFN-T) stimulated genes (ISGs) at the mid-luteal stage of the cycle (on day 13) in the uterine endometrium and extra-uterine tissues, such as the corpus luteum (CL), and in the peripheral blood leukocytes (PBLs) of pregnant and non -pregnant ewes, The objective was to evaluate the possibility of using the regulation of ISG expression in PBLs as a possible early pregnancy indicator in ruminants. For this purpose, multiparous ewes were synchronized and either allowed to mate (pregnancy group) or detected in estrus (cyclic group; day 0). The ewes were slaughtered on day 13, and the PBLs, endometrium and luteal tissues were collected. Total RNA was isolated from eight cyclic and eight pregnant ewes, and qPCR was employed to detect the steady state levels of Interferon -stimulated gene 15 (ISG15), Myxovirus (influenza virus) resistance 1 (Mxl) and Receptor transporter protein 4 (RTP4) mRNAs. The expressions of ISG15, Mxl and RTP4 were detected in the endometrium, CL and PBLs on day 13 of the estrous cycle and pregnancy. The expressions of these ISGs were upregulated only in the endometrium of pregnant ewes compared to non-pregnant ewes, but this stimulation was not observed in the CL and PBLs. The results suggest that the embryo stimulates ISGs only in the endometrium, and the effects are not evident in the extra-uterine tissues on day 13 of pregnancy. This study suggests that the measurement of the ISG expression in the PBLs is not a reliable detection method of early pregnancy in ewes, which are in the preluteolytic stage of early pregnancy. (C) 2016 Elsevier B.V. All rights reserved.