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Öğe Comparison of five different rna isolation methods from equine endometrium for gene transcription analysis(KAFKAS UNIV, VETERINER FAKULTESI DERGISI, 2010) Kurar, Ercan; Atli, Mehmet Osman; Guzeloglu, Aydin; Ozsensoy, Yusuf; Semacan, AhmetIn this study, five different isolation protocols to extract total RNA from biopsies of equine endometrium were compared in terms of quality and quantity of RNA samples with respect to downstream gene transcription analysis, such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Three phenol-chloroform based protocols (TRIzol, TRItidy, EZ-RNA) and two column based protocols (UltraClean (TM) and E.Z.N.A.(R)) that were commercially available were used. Each protocol yielded good quality total RNA and distinct 28S and 18S rRNA bands were observed in agarose gel electrophoreses. Amount of total RNA isolated was lower for EZ-RNA protocol. Column based protocols had RNA contaminated with great amount of genomic DNA, however, DNAse-I digestion was able to fully clean the DNA contamination from RNA in all the protocols used. Following cDNA synthesis and PCR, GAPDH, a housekeeping gene, bands were amplified from all the samples. In conclusion, all the protocols used extracted good quality but different amounts of total RNA and it is strongly recommended that RNA samples must undergo DNAse-I digestion before RT-PCR to eliminate gDNA contamination..Öğe Early Pregnancy Regulates Expression of Wnt Genes and Their Antagonists in Equine Endometrium(SOC STUDY REPRODUCTION, 2009) Atli, Mehmet Osman; Guzeloglu, Aydin; Dine, Dursun Ali[Abstract not Available]Öğe 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 OsmanIn 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.Öğe 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, NurettinAmaç: 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.Öğe 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, NurettinAim: 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.Öğe Expression of epidermal growth factor (egf) and heparin-binding egf (hb-egf) mrna in mare endometrium(KAFKAS UNIV, VETERINER FAKULTESI DERGISI, 2012) Atli, Mehmet Osman; Guzeloglu, Aydin; Kurar, Ercan; Kayis, Seyit Ali; Aslan, Selim; Semacan, Ahmet; Celik, SefaEGF and HB-EGF play crucial roles in embryonic development and peri-implantation. In this study, aim was to characterize expression profiles of EGF and HB-EGF in mare endometrium during estrous cycle and early pregnancy. Endometrium biopsies were obtained from mares on day of ovulation (d0), late diestrus (LD) and after luteolysis (AL) in the estrus phase. In pregnant groups, biopsies were taken on days 14 (P14), 18 (P18), 22 (P22) and 60 (P60). Relative expression levels of genes were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and Least Significant Difference (LSD) test was employed to determine significantly different group(s). EGF mRNA expression was up-regulated at LD compared to d0 while HB-EGF expression was not changed throughout the cycle. EGF expression was also increased during early pregnancy with the highest expression level observed on P60. Similarly, HB-EGF mRNA level was increased on P60. Pregnancy induced EGF expression on P14 and P18 compared to LD and AL whereas expression of HB-EGF was only significantly higher on P18 than that of AL. These results indicate that EGF expression is up-regulated during the cycle at late diestrus when P4 is high and is increased by pregnancy. HB-EGF expression is induced later in the pregnancy. In conclusion, EGF and HB-EGF appear to involve in the events that happen in the mare endometrium during peri-implantation period.Öğe Expression of Toll-like receptors (TLRs) in the equine peripheral blood mononuclear cells (PBMCs) during the early pregnancy and estrous cycle(Selçuk Ünivesitesi Veterinerlik Fakültesi, 2012) Kurar, Ercan; Atli, Mehmet Osman; Guzeloglu, Aydin; Semacan, AhmetAmaç: 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.Öğe Expression of wingless type (WNT) genes and their antagonists at mRNA levels in equine endometrium during the estrous cycle and early pregnancy(ELSEVIER SCIENCE BV, 2011) Atli, Mehmet Osman; Guzeloglu, Aydin; Dinc, Dursun AliWNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n = 4) and on days of 14 (P14, n = 4), 18 (P18, n = 4), 22 (P22, n = 4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n = 4) and after luteolysis in estrus phase (AL on day of 17.5-18, is = 4) of the cycle. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis. A mixed model was fitted on the normalized data and least significant difference test (alpha = 0.05) was employed. Eleven WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, WNT8A, WNT9B, WNT10B, WNT11 and WNT16) and their antagonists (SFRP1, SFRP2, SFRP5, DKK1, DKK2 and WIF-1) were detected in equine endometrium. Compared to d0, WNT2, WNT5B, WNT7A and SFRP1 expressions were downregulated by the pregnancy while DKK1 was upregulated. WNT5A, WNT11 and WIF-1 were upregulated on P14 and P18. but WNT2B increased only on P14. When LD and P14 were compared, level of WNT8A decreased on P14 while increase in WNT4 level on P14 was slightly significant (P < 0.06). Levels of WNT7A and SFRP1 decreased while DKK1 and WIF-1 increased by the pregnancy on P18 compared to AL Moreover, WNT2B, WNT5A, WNT9B, WNT10B, WNT11, WNT16 DKK1 and WIF-1 were upregulated on LD compared to AL whereas WNT4, WNT7A, SFRP1 were downregulated. In conclusion, the results demonstrate that WNT genes and their antagonists appear to be regulated during early pregnancy in equine endometrium possibly due to embryonic factors and/or maternal progesterone. (C) 2011 Elsevier B.V. All rights reserved.Öğe 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, MuhammetThe 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.Öğe Pomc, inos, pges, il-4, il-5 and il-10 gene expression in peripheral blood mononuclear cells of cyclic and pregnant mares(KAFKAS UNIV, VETERINER FAKULTESI DERGISI, 2011) Kurar, Ercan; Atli, Mehmet Osman; Guzeloglu, Aydin; Semacan, AhmetAlthough a number of different clinical and laboratory methods are used, there is still need for a practical, reliable and economical diagnosis method for early pregnancy in mares. Objective was to provide a preliminary background for understanding immunological modifications at molecular level during early pregnancy in mare and thereby to allow development of a practical pregnancy test. Blood samples were collected from 10 pregnant and 4 cyclic mares on days of ovulation (d0), 4 and 8. Total RNA samples were isolated from peripheral blood mononuclear cells (PBMC) and cDNA synthesis was performed. Pregnancy dependent gene expression profiles of proopiomelanocortin (POMC), nitric oxide synthase (iNOS), prostaglandin E synthase (PGES), interleukin-4 (IL-4), IL-5 and IL-10 were evaluated at mRNA level using semiquantitative Reverse Transcriptase-Polymerase Chain Reaction. Data were analyzed by General Linear Model and possible differences between all mean factors were determined by Tukey's analysis. No gene expressions were observed for PGES, IL-5 and iNOS in PMBCs. Expressions of POMC, IL-4 and IL-10 were not significant on d0, 4 and 8, which suggested that pregnancy or cyclic status had no effect on expression of these genes. Only, IL-10 mRNA expression was lower in pregnant mares than cyclic mares (P < 0.05).Öğe 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.
