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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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    Effect of exogenous progesterone and gonadotropin-releasing hormone application on maintenance of pregnancy in early pregnant ewes after prostaglandin F2 alpha injection
    (Selçuk Üniversitesi Veterinerlik Fakültesi, 2016) Köse, Mehmet; Kaya, Mehmet Salih; Atlı, Mehmet Osman
    Aim: We tested the hypothesis that exogenous progesterone (P4) and gonadotropin-releasing hormone applications (GnRH) would prevent detrimental effect of prostaglandinF2alfa (PGF2?) on early pregnancy in ewes by sustaining high plasma progesterone level. Materials and Methods: For this purpose, nine pregnant ewes (mating=0) were divided into 2 groups on day 18 as follows: (1) PGF2? group (125 mcg of d-cloprostenol injection on day 18, n=5); (2) PGF2? + P4 + GnRH group (125 mcg of d-cloprostenol injection on day 18 + 20 mg flugestone acetate (P4) for 7 days + 10 mg of buserelin acetate injection, on day 22, n=4). P4 was withdrawn on day 25. P4 concentration was measured on days 18 and 19 by ECLIA. Pregnancies were examined by using transrectal ultrasonography on days 18, 22, 25, and 35. Statistical difference between groups was analysed by chi-squared test. Results: P4 concentration declined to below 1 ng/mL on day 19 in both groups. While all pregnancies were terminated in PGF2? group by day 25, P4 prevented pregnancy loss in PGF2? + P4 + GnRH group (P<0.02). When P4 was withdrawn on day 25, half of pregnancies continued after GnRH in PGF2? + P4 + GnRH group until birth. Conclusion: Results suggest that exogenous P4 application is suf-ficient for maintaining early pregnancy in ewes, even if the corpus luteum (CL) is regressed. Furthermore, GnRH application on day 22 might luteinize dominant follicles and could be sufficient to maintain pregnancy after P4 is removed.
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    Effect of long term heat stress and dietary restriction on the expression of small heat shock protein (sHSP) genes in rat liver tissue
    (2016) Bozkaya, Faruk; Atli, Mehmet Osman; Guzeloglu, Aydın; Kayis, Seyit Ali; Kaya, Mehmet Salih; Aydilek, Nurettin
    Amaç: Bu çalışmanın amacı uzun süreli sıcaklık stresi ve yem kısıtla-masının rat karaciğer dokusunda bazı küçük ısı şoku protein (sHSP) genlerinin mRNA düzeyindeki ekspresyonu düzeyleri üzerine etkisi-nin araştırılmasıdır. Gereç ve Yöntem: Bu amaçla on haftalık yaştaki toplam 24 Spra-gue-Dawley rat 4 gruba ayrıldı. Grup I ve Grup II'deki ratlar 22C'lik ortam sıcaklığında, Grup III ve Grup IV'teki ratlar ise 38C'lik ortam sıcaklığında tutuldu. Grup I ve III'teki ratlar ad libitum olarak beslen-di, Grup II ve IV'teki ratlara ise ad libitum grupların tükettiği yemin %60'ı kadar yem verildi. Uygulama 9 hafta sürdürüldükten sonra karaciğer doku örnekleri alınarak sıvı azot içerisinde donduruldu ve RNA izolasyonuna kadar muhafaza edildi. Doku örneklerinden total RNA izole edildikten sonra HspB1, HspB5, HspB6, Hsp10 ve Hsp11 genlerinin ekspresyon düzeyleri gerçek zamanlı nicel polimeraz zin-cir reaksiyonu (RT-qPCR) yöntemi ile incelendi.Bulgular: Sıcaklık stresi HspB2, HspB8 ve Hsp70 genlerinin ekspres-yonunu önemli ölçüde arttığı, HspB1, HspB5, HspB6, Hsp10 ve Hsp11 genlerinin ekspresyonunu ise etkilemediği belirlendi. Yem kısıtla-ması HspB6 geninin expresyonunu arttırırken HspB1, HspB2, HspB5, HspB8 HspB10, HspB11 ve Hsp70 genlerinin ekspresyonunu etkile-mediği gözlendi. Uygulamalar arasında interaksiyon gözlenmedi.Öneri: Çalışmanın sonuçları uzun süreli sıcaklık stresinin rat kara-ciğer dokusundaki sHSP genlerinin ekspresyonlarını değişik düzey-lerde etkilediğini, yem kısıtlamasının sHSP genlerinin sıcaklık stresi tarafından etkilenen ekspresyonlarını değştirmediğini göstermiştir.
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    Effect of long term heat stress and dietary restriction on the expression of small heat shock protein (sHSP) genes in rat liver tissue
    (Selçuk Üniversitesi, 2016) Bozkaya, Faruk; Atli, Mehmet Osman; Kayis, Seyit Ali; Kaya, Mehmet Salih; Aydilek, Nurettin
    Aim: Investigation of the effects of dietary restriction on expression of certain small heat shock protein (sHSP) genes at mRNA level in liver tissue of rats reared under long-term heat stress. Material and Method: Sprague-Dawley rats (n=24) 10 weeks of age, were equally divided into four groups. Group I and II were kept at an ambient temperature of 22°C, while Groups III and IV were reared at 38°C. Groups I and III were fed ad libitum, while Groups II and IV were fed 60% of the diet consumed by their ad libitum counterparts. The treatment continued for 9 weeks. At the end of the treatment, liver tissue samples were taken. Total RNA was isolated and mRNA expression level of the HspB1, HspB2, HspB5, HspB6, HspB8, Hsp10, Hsp11 and HspA1A genes were assessed by Real-Time PCR analysis. Results: Heat stress significantly up regulated mRNA expressions of HspB2, HspB8 and Hsp70 genes, while it did not change mRNA expressions of HspB1, HspB5, HspB6, Hsp10 and Hsp11 genes. Dietary restriction (DR) did not significantly affect the expression of HspB1, HspB2, HspB8 HspB10, HspB11 and Hsp70, while it increased mRNA expression of HspB6 gene. No interaction between treatments was observed. Conclusion: The results suggested that long term heat stress differentially affected the sHSP genes studied and DR had no affect on the heat stress mediated changes in the expression of sHSP.
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    Expression profiles of interferon-tau stimulated genes (isgs) in peripheral blood leucocytes (pbls) and milk cells in pregnant dairy cows
    (KAFKAS UNIV, VETERINER FAKULTESI DERGISI, 2014) Kose, Mehmet; Gorgulu, Murat; Kaya, Mehmet Salih; Aydilek, Nurettin; Bozkaya, Faruk; Bayril, Tahir; Kurar, Ercan
    In previous reports, it was indicated that measurement of activity of Interferon-tau Stimulated Genes (ISGs) in Peripheral Blood Leucocytes (PBLs) may be used as an alternative early pregnancy detection method in dairy cows. However, there are no data showing the expression profiles of ISGs in other body fluids containing leucocytes such as milk. In the present study, it was hypothesized that leucocytes in milk samples may reflect the increases in expression profiles of ISGs as shown in PBLs. For this purpose, nine pregnant lactating Holstein cows were used. Insemination day was accepted as day zero (day 0). Blood and milk samples were collected on day 0 and 18 after insemination for cell isolation. Total RNA was extracted from isolated cells and converted to cDNA. Steady state levels of Interferon-tau Stimulated Gene 15 (ISG15), Myxovirus (influenza virus) resistance 1 (MX1) and 2 (MX2) mRNA transcripts were assayed by using real-time reverse transcriptase PCR. Relative Expression Software Tool (REST2009) was used for statistical analyses. There was no statistical significant difference for expression levels of ISG15, MX1 and MX2 mRNAs between days 0 and 18 in milk samples. However, when compared to day 0, levels of ISG15 and MX2 transcripts were increased 6.97 +/- 0.68 fold and 5.84 +/- 1.27 fold on day 18 in PBLs in pregnant cows, respectively (P<0.05). According to this result, it may be suggested that milk cells are not suitable measurement of expression profiles of ISGs to detect early pregnancy in lactating dairy cows.
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    Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle
    (BRAZILIAN COLL ANIMAL REPRODUCTION, 2017) Atli, Mehmet Osman; Kose, Mehmet; Kaya, Mehmet Salih; Aydilek, Nurettin; Guzeloglu, Aydin; Wiltbank, Milo C.
    The aim of this study was to elucidate the presence of components of the innate immune system in the bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3-6), mature CL (mCL; n = 5, approx. days 8-12), and regressing CL (rCL; n = 5, approx. days 17-19). An upregulation of TLR2 mRNA was detected only in rCL (P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCL and rCL compared to dCL (P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.