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Öğe Alcoholic extract of Tarantula cubensis induces apoptosis in MCF-7 cell line.(Allied Acad, 2017) Er, Ayse; Corum, Orhan; Corum, Duygu; Hitit, Mustafa; Donmez, Huseyin; Guzeloglu, AydinTarantula cubensis Alcoholic Extract (TCAE) is a homeopathic agent used for treating many disorders. This study aimed to define the effects of TCAE on the breast carcinoma cell line (MCF-7). After various concentrations (10, 20, 40, 80 and 160 mu l/ml) of TCAE were applied to MCF-7 cells and the human embryonic kidney cell line (HEK293), the cells were incubated for 1, 3, 6, 9, 12, 24 and 48 h, followed by analysis by MTT assays. According to the results of the MTT assays, cells treated with 20 or 40 mu l/ml TCAE for 6 h were applied to apoptosis analysis by flow cytometry. Secreted levels of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1 beta, IL-6, IL-10, Interferon-gamma (IFN gamma), Transforming Growth Factor beta (TGF beta), and Nuclear Factor-kappa B (NF-kappa B) were measured using ELISAs. TNF alpha and TGF beta levels increased while IL-6 and IL-10 levels fluctuated in MCF-7 cells. In conclusion, our study suggests that TCAE may change the normal cancer physiology and lead to cell death by activating apoptosis in MCF-7 cells.Öğe Altered luteal expression patterns of genomic and non-genomic progesterone receptors in bitches at different reproductive states(Elsevier Inc., 2024) Uçar, Eyyüp Hakan; Peker, Cevdet; Hitit, Mustafa; Köse, Mehmet; Tatar, Musa; Bozkaya, Faruk; Atlı, Mehmet OsmanThe binding of steroid hormones to their specific receptors is necessary to exert their effects on target cells. Progesterone (P4), a steroid hormone, carries out its effects through both genomic and non-genomic (the cell membrane-associated) receptors. This study aimed to ascertain luteal expression patterns of genomic and non-genomic progesterone receptors in bitches in physiological (early dioestrus and early pregnant) and pathological (pyometra) reproductive states. Luteal tissue was collected from the bitches at early dioestrus (ED, n = 5), early pregnant (EP, n = 5), and pyometra (PY, n = 5). The expression profiles of Steroidogenic Acute Regulator Protein (STAR), Progesterone Receptor (PGR), Membrane Progestin Receptors (PAQR5, PAQR7 and PAQR8), and Progesterone Membrane Components (PGMRC1 and PGMRC2) were examined at the mRNA levels using Real-Time Polymerase Chain Reaction (RT-PCR). Protein levels of PGR, PGMRC1 and PGMRC2 were detected by western blotting (WB). The STAR expression was found in all groups, with a statistical difference observed between EP and PY groups (P < 0.05). The protein level of PGR was determined to be highest in the EP group and lowest in the PY group. The expression of PAQR8 increased in the EP group (P < 0.05). The PAQR5 exhibited high expression in the EP group and low expression in the PY group (P < 0.05). PGRMC1 was more elevated in the EP group and lower in the PY group (P < 0.05). Protein levels of PGMRC1 and PGMRC2 were also observed at the highest expression in EP group. According to the altered expression profiles for examined receptors, we suggest that those progesterone receptors have roles in early pregnancy or pyometra in bitches.Öğe Cell-specific expression pattern of toll-like receptors and their roles in animal reproduction(Springer Science and Business Media Deutschland GmbH, 2022) Atlı, Mehmet Osman; Hitit, Mustafa; Özbek, Mehmet; Köse, Mehmet; Bozkaya, Faruk; 0000-0001-9853-5334; 0000-0001-5636-061X; 0000-0003-0070-8458Toll-like receptors (TLRs), a part of the innate immune system, have critical roles in protection against infections and involve in basic pathology and physiology. Secreted molecules from the body or pathogens could be a ligand for induction of the TLR system. There are many immune and non-immune types of cells that express at a least single TLR on their surface or cytoplasm. Those cells may be a player in a defense system or in the physiological regulation mechanisms. Reproductive tract and organs contain different types of cells that have essential functions such as hormone production, providing an environment for embryo/fetus, germ cell production, etc. Although lower parts of reproductive organs are in a relationship with outsider contaminants (bacteria, viruses, etc.), upper parts should be sterile to provide a healthy pregnancy and germ cell production. In those areas, TLRs bear controller or regulator roles. In this chapter, we will provide current information about physiological functions of TLR in the cells of the reproductive organs and tract, and especially about their roles in follicle selection, maturation, follicular atresia, ovulation, corpus luteum (CL) formation and regression, establishment and maintenance of pregnancy, sperm production, maturation, capacitation as well as the relationship between TLR polymorphism and reproduction in domestic animals. We will also discuss pathogen-associated molecular patterns (PAMPs)-induced TLRs that involve in reproductive inflammation/pathology.Öğe Circulating miRNAs in maternal plasma as potential biomarkers of early pregnancy in sheep(Frontiers Media Sa, 2022) Hitit, Mustafa; Kose, Mehmet; Kaya, Mehmet Salih; Kirbas, Mesut; Dursun, Sukru; Alak, Ilyas; Atli, Mehmet OsmanMicroRNA (miRNA) plays an important role in the control of gene expression and is implied in many biological functions, including embryo implantation and development. The aim was to assess plasma miRNA profiles during the peri-implantation and ascertain potential candidate miRNA markers for early pregnancy diagnosis in ovine plasma. The plasma samples were obtained from a total of 24 ewes on days 12 (pre-implantation; P12, n = 4), 16 (implantation; P16, n = 4) and 22 (post-implantation; P22, n = 4) after mating, and on their corresponding days of 12 (Pre-C; C12, n = 4), 16 (Imp-C; C16, n = 4) and 22 (Post-C; C22, n = 4) of the estrous cycle. The miRNA profiles in plasma were assessed by microarray technology. We detected the presence of 60 ovine-specific miRNAs in plasma samples. Of these miRNAs, 22 demonstrated a differential expression pattern, especially between the estrous cycle and early pregnancy, and targeted 521 genes. Two miRNAs (oar-miR-218a and oar-miR-1185-3p) were confirmed using RT-qPCR in the ovine plasma samples. Protein-protein interaction (PPI) network of target genes established six functional modules, of which modules 1 and 3 were enriched in the common GO terms, such as inflammatory response, defense response, and regulation of immune response. In contrast, module 2 was enriched in the developmental process involved in reproduction, embryo development, embryonic morphogenesis, and regulation of the developmental process. The results indicate that miRNAs profiles of plasma seemed to be modulated during the peri-implantation stage of pregnancy in ewes. Circulating miRNAs could be promising candidates for diagnosis in early ovine pregnancy.Öğe Effects of essential oil mixtures on expression of genes involved in lipogenesis and fatty acid oxidation in geese (Anser anser)(Springer Science and Business Media B.V., 2024) Aydın, Özlem Durna; Hitit, Mustafa; Usta, Zafer; Yıldız, Gültekin; Saçaklı, Pınar; Kaplan, Oktay; Merhan, OğuzThe use of essential oils has recently increased in the poultry sector. The aim of this study was to investigate the effects of essential oil mixture (juniper, mint, oregano and rosemary oil) on fatty acid oxidation and lipogenic gene expression in geese. Research groups were formed as C (control; no additives), EK1 (0.4 ml/l essential oil mixture supplemented) and EK2 (0.8 ml/l essential oil mixture supplemented). Relative expression levels of genes included in lipogenesis (ACCα, ChREBP, FASN, LXRα and SREBP-1) expression levels of genes included in fatty acid oxidation (ACOX1, CPT1, CPT1A, PPARα and PPARγ) were measured using RT-qPCR. Group EK1 upregulates the mRNA expression levels of genes involved in lipogenesis such as ACCα, ChREBP and SREBP-1, while it downregulates the mRNA expression in levels of all genes involved in fatty acid oxidation. Group EK2 increases the mRNA expression levels of genes involved in lipogenesis such as ACCα, FASN and SREBP-1, while it decreased mRNA expression at the levels of all genes involved in fatty acid oxidation, as in the other group. In the study, adding an essential oil mixture to drinking water is predicted to increase fatty liver because it upregulates genes related to fat synthesis (lipogenesis) and downregulates genes related to fat degradation (fatty acid oxidation).Öğe Expression of circulating oar-miR-485-5p and oar-miR-493-5p during the estrous cycle and early pregnancy in ovine plasma(Brazilian Coll Animal Reproduction, 2024) Ucar, Eyyup Hakan; Hitit, Mustafa; Kose, Mehmet; Atli, Mehmet OsmanIn the current study, we aimed to assess the expression levels of two circulating microRNAs (miRNA) (oar-miR-485-5p and oar-miR-493-5p) in the ovine plasma during the peri-implantation. After mating, we collected the plasma samples from a total of 8 ewes on day 22 of pregnancy (P22; n = 4) and day 22 of the estrous cycle (C22; n=4). We used mature miRNA sequences for oar-miR-485-5p and oar-miR-493-5p out of one hundred fifty, which were retrieved from our microarray results of previous study. We showed that the miRNA expression of oar-miR-485-5p and oar-miR-493-5p were upregulated in P22 (P<0.05) when compared to C22. Those two miRNAs targeted 311 target genes in the peri-implantation period of pregnancy. Furthermore, we revealed 151 GO/pathway terms in biological process (BP) and 25 GO/pathway terms in molecular function (MF), while we demonstrated 13 GO/pathway terms in cellular component (CC). We revealed three hub genes as interleukin 2 (IL2), interleukin 18 (IL18), and C -X-C Motif Chemokine Ligand 10 (CXCL10). In conclusion, both miR-485-5p and oar-miR-493-5p have the potential to be a biomarker to understand peri-implantation of the ovine pregnancy in the aspect of pregnancy -reflected changes in maternal plasma.Öğe Expression pattern and cellular localization of two critical non-nuclear progesterone receptors in the ovine corpus luteum during the estrous cycle and early pregnancy(Elsevier, 2022) Atli, Mehmet O.; Akbalik, Mehmet Erdem; Kose, Mehmet; Alak, Ilyas; Atli, Zehra; Hitit, MustafaThe study aimed to investigate the expression and cellular localization of two critical non-nuclear progesterone receptors, including membrane-associated-progesterone-receptor-component-1 (PGRMC1) and progestin and adipoQ receptor family member 7 (PAQR7) throughout the estrous cycle and early pregnancy in ovine corpus luteum (CL). Ewes were randomly grouped into cyclic (C, n = 4 per group) or pregnant (P, n = 4 per group) groups. Following slaughtering, the CL was obtained from both cyclic and pregnant ewes on days 12 (C12 and P12), 16 (C16 and P16), and 22 (C22 and P22). Western blotting and RT-qPCR were utilized to assess the expression levels of PGRMC1 and PAQR7, whereas immunohistochemistry was performed to determine the localization of PGRMC1 and PAQR7 in CL. Data were evaluated by one-way ANOVA, and the P < 0.05 was considered a significant difference. PGRMC1 was shown to be expressed in both small and large luteal cells and endothelial cells in CL, while PAQR7 expression was only found in small and large luteal cells. Compared to cycle days, pregnancy increased the expression of PGRMC1. PAQR7 did not differ during early pregnancy but reduced during the functional luteolysis stage (C16). mRNA and protein expression patterns for PGRMC1 and PAQR7 were similar on the studied days. This is the first study that demonstrates the expression and cellular localization of PGRMC1 and PAQR7 in ovine CL. We suggest that these receptors could execute a significant role in the ovine CL life span in both cyclic changes and the establishment of pregnancy.Öğe Expression pattern of microRNAs in ovine endometrium during the peri-implantation(Elsevier Science Inc, 2022) Kose, Mehmet; Hitit, Mustafa; Kaya, Mehmet Salih; Kirbas, Mesut; Dursun, Sukru; Alak, Ilyas; Atli, Mehmet OsmanMicroRNA (miRNA), acting as the transcriptional regulator of gene expression, has been widely demonstrated to be involved in many biological functions, including embryo implantation and devel-opment. The objective of the current study was to illuminate the expression pattern of microRNAs (miRNAs) in the endometrium during the peri-implantation in ewes. Intercaruncular endometrial samples was obtained from a total of 24 ewes on days of 12 (pre-implantation, n = 4), 16 (implantation, n = 4) and 22 (post-implantation, n = 4) of pregnancy following mating, and on their corresponding days of 12 (n = 4), 16 (n = 4) and 22 (n = 4) of the estrous cycle. The miRNA profiles were examined in the endometrium by microarray technology. We detected 116 ovine specifics miRNAs in the endometrium. Of these, nineteen were differentially expressed in early pregnancy. Four miRNAs (oar-miR-370-3p, oar-miR-411b-5p, oar-miR-379-3p and oar-miR-411a-3p) that had the most differential fold change were confirmed by RT-qPCR in ovine endometrium. The differentially expressed miRNAs targeted a total of 315 genes, resulting in 39 GO terms in molecular function, 353 in biological process, and 17 in the cellular component. The construction of the PPI network of target genes established two functional modules mostly enriched in the innate immune system, toll receptor cascades in module 1, whereas genes in module 2 were associated with GMCSF-mediated signaling events, insulin pathway, and mTOR signaling pathway. Based on the results, we may imply that miRNAs modulate ovine endometrium during the peri-implantation.(c) 2022 Elsevier Inc. All rights reserved.Öğe Expression patterns of genes in steroidogenic, cholesterol uptake, and liver x receptor-mediated cholesterol efflux pathway regulating cholesterol homeostasis in natural and PGF2? induced luteolysis as well as early pregnancy in ovine corpus luteum(Elsevier, 2022) Hitit, Mustafa; Kose, Mehmet; Kocak, Nadir; Atli, Mehmet OsmanThe aim was to evaluate the expression of genes of steroidogenic, cholesterol uptake, and liver X receptor (LXR) mediated cholesterol efflux pathway in ovine corpus luteum (CL) during natural and prostaglandin F2 alpha (PGF2 alpha) induced luteolysis and early pregnancy. For this study, two experiments were carried out 1); ewes were grouped into two sub-groups as cyclic 12 (C12, n = 4) and 16 (C16, n = 4) and pregnant 12 (P12, n = 4), 16 (P16, n = 4), and 22 (P22, n = 4). Additionally, 2) ewes were grouped into four groups following treatment of PGF2 alpha, the duration of PGF2 alpha challenge at 1 (PG1, n = 4), 4 (PG4, n = 4), and 16 (PG16, n = 4) hours on day 12 of the cycle was compared with 0 h. The corpus luteum tissue samples were collected on the corresponding estrus cycle and pregnancy days, and RNA was extracted using Trizol. mRNA expression levels of the steroidogenic (StAR, CYP11A1, and HSD3B1) and cholesterol uptake receptors (SCARB1 and LDLR) and LXR pathway (NR1H3, NR1H2, ABCA1, and ABCG1) were assessed using quantitative PCR (qPCR), and protein of LXR pathway was investigated using western blot. In-situ hybridization was used to detect mRNA localization. Steroidogenic and cholesterol uptake mRNAs were decreased in C16, while NR1H2 and ABCG1 were increased in C16, compared to C12. Steroidogenic and cholesterol uptake mRNA was greater in P16 than in C16. NR1H2 and ABCA1 protein expression were higher in P16 than in C16. LDLR mRNA was higher in P22 than in P12, while SCARB1 was higher in P16 than in P12. NR1H2 mRNA was greater in P22 than in P12. Steroidogenic and cholesterol uptake mRNA were decreased in PGF2 alpha-induced luteolysis groups against C12. ABCG1 mRNA was higher in PG16 than in PG4 and PG1. The reduction of lipoprotein receptors rather than LXR-mediated reverse transport may contribute to the decline in progesterone (P4) in natural and functional luteolysis.Öğe Expression patterns of Toll-like receptors in the ovine corpus luteum during the early pregnancy and prostaglandin F2?-induced luteolysis(Elsevier Science Inc, 2018) Atli, Mehmet O.; Kose, Mehmet; Hitit, Mustafa; Kaya, M. Salih; Bozkaya, FarukThe aim of this study was to elucidate the expression profiles of Toll-like receptors (TLRs) in the ovine corpus luteum (CL) during early pregnancy and prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. For this purpose, multiparous Anatolian Merino ewes were selected and randomly allotted into cyclic (including those in the induced luteolysis group, n = 20) and pregnant (n = 12) groups. All of the ewes were scheduled to be slaughtered for predetermined days/hour during the estrous cycle, early pregnancy, and PGF2 alpha induced luteolysis. The CLs were collected from both cyclic and pregnant ewes on days 12 (C12 and P12; n = 8) and 16 (C16 and P16; n = 8) and pregnant ewes on day 22 (P22; n = 4). For the induced luteolysis model, ewes were injected with PGF2 alpha on day 12 of the estrous cycle and CLs were collected at 1 h (PG1h; n = 4), 4 h (PG4h; n = 4), and 16 h (PG16h, n = 4) after injection. Quantitative polymerase chain reaction (VCR) was used to evaluate the expression profiles of TLR2, TLR4, TLR6, TLR8, and TLR10, while free-floating in situ hybridization and immunohistochemistry were used to define the spatial localization of TLR2, TLR4, and TLR7 in the CL. Data were then analyzed by one-way ANOVA and were considered statistically significant when P values were lower than 0.05. Expression of TLR2 was upregulated in both early and late stages of luteolysis (P < .05). An upregulation of TLR4 was detected at PG16h, while TLR6 was decreased at PG4h (P < .05). Expression of TLR7 and TLR8 was significantly increased during early pregnancy, at both PG16h and regressed groups (C16, P < .05). In contrast, 711210 was downregulated during PGF2 alpha-induced luteolysis and on P16 (P < .05). TLR4 and TLR7 proteins were particularly localized in endothelial cells on C12/PGOh, but prominent signals corresponding to TLR4 and TLR7 were detected in luteal cells at PG16h. The results suggest an involvement of TLRs in the luteolytic mechanism in ovine CL, as indicated by differential expression levels of TLRs during PGF2 alpha-induced luteolysis. Moreover, the present study indicates that early pregnancy-mediated changes in TLR expression in the CL may contribute to the establishment and maintenance of ovine pregnancy. (C) 2018 Elsevier Inc. 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, 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 Kısrak endometriyumunda Phosphatase and Tensin Homolog (PTEN) geni mRNA ekspresyonunun araştırılması(2015) Kayış, Seyit Ali; Güzeloğlu, Aydın; Şen, Gonca; Hitit, Mustafa; Atlı, Mehmet Osman; Kurar, Ercan; Özel, ÇağlayanAmaç: Bu çalışmanın amacı, siklüs ve erken gebelik dönemlerinde dinamik bir yapıya sahip olan kısrak endometriyumunda phosphatase and tensin homolog (PTEN) gen ekspresyonunun mRNA düzeyinde belirlenmesidir.Gereç ve Yöntem: Araştırmada her güne 4 farklı kısrak olacak şekilde (n=4/gün) siklik kısraklardan ovulasyon gününde (d0), geç diöstrusta (LD) ve luteolizis sonrası östrusta (AL), gebe kısraklardan ise gebeliğin 14. (P14), 18. (P18) ve 22. (P22) günlerinde endometriyum biyopsi örnekleri toplandı. Doku örneklerinden total RNA elde edildi ve cDNA'ya dönüştürüldü. PTEN ekspresyonlarında siklus ve gebeliğe bağlı muhtemel değişiklikler mRNA seviyesinde kantitatif polimeraz zincir reaksiyonu (qPZR) kullanılarak araştırıldı. Referans gen olarak GAPDH ekspresyonu ile normalize edilen veriler karışık model kullanılarak analiz edildi. Farklı olan grup(lar) Asgari Önemli Fark (LSD) testi ile tespit edildi.Bulgular: PTEN ekspresyonu çalışmaya konu olan tüm siklüs ve erken gebelik dönemlerinde at endometriyumunda mRNA düzeyinde tespit edildi. LD'e göre AL'de ekspresyon seviyesinde anlamlı olmayan bir düşüş gözlendi. Benzer şekilde d0'a göre araştırılan P14, P18 ve P22 erken gebelik günlerinde PTEN ekspresyonunun baskılanmış olduğu tespit edildi. Ancak fark istatistiksel olarak anlamlı bulunmadı (P>0.10).Öneri: PTEN aktivitesinin ve protein düzeyinde ekspresyonun araştırılmasının anlamlı olacağı kanaatine varılmıştır.Öğe Relative abundance and localization of interferon-stimulated gene 15 mRNA transcript in intra- and extra-uterine tissues during the early stages of pregnancy in sheep(Elsevier, 2020) Alak, Ilyas; Hitit, Mustafa; Kose, Mehmet; Kaya, M. Salih; Ucar, Eyyup Hakan; Atli, Zehra; Atli, Mehmet O.The aim of this study was to investigate relative abundance and localization of ISG15 mRNA transcript in intra-uterine (trophoblast, endometrium) and extra-uterine (hypothalamus, anterior pituitary, corpus luteum) tissues before and during the period of conceptus implantation. Multiparous ewes (n = 16) were randomly allotted into four groups: pregnant or estrous cyclic on days of 12 and 16 (n = 4 per group) following estrus. Relative abundances of ISG15 mRNA transcript were determined in the endometrium, corpus luteum, hypothalamus, and anteriorpituitary using real time quantitative PCR. Localization of ISG15 mRNA transcript was evaluated using in situ hybridization. The presence of ISG15 mRNA transcript was only visualized in intrauterine tissues including the endometrium and trophoblast on day 12 of pregnancy. The ISG15 mRNA transcript was detected in all tissues evaluated on day 16 of pregnancy. The abundance of ISG15 mRNA transcript was greater in the endometrium on day 12 of pregnancy than at other days when evaluations occurred while in all other tissues except the hypothalamus there were large abundances of ISG15 mRNA on day 16 of pregnancy. It is concluded that the ISG15 mRNA transcript is only present in intra-uterine tissues before conceptus implantation. The ISG15 mRNA transcript, however, is present in extra-uterine tissues of ewes during implantation probably due to an increased amount of interferon-tau in blood circulation that is produced by the developing embryo. Results also indicate, for the first time, that pregnancy is associated with an intra-hypothalamus and anterior pituitary increased abundance of ISG15 mRNA transcript in ewes.
