Mehmet O. Atli

Rating of putative housekeeping genes for quantitative gene expression analysis in cyclic and early pregnant equine endometrium

The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 28S rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), at late diestrus (LD, n=4), after luteolyis (AL, n=4) of the cycle and on days 14 (P14; n=3), 18 (P18, n=3) and 22 (P22; n=3) of pregnancy. A model based on REML with support of descriptive statistics was proposed in accordance with experimental design and was further confirmed with principal component analysis (PCA). Results were compared with widely used software including geNorm, BestKeeper, and NormFinder. Results indicated that GAPDH was the most stable HKG and RPL32 was ranked as the second best. 18S and 28S were found to be the least stable. The proposed model, PCA, geNorm, and BestKeeper were in agreement in detecting the most stable and the least stable HKGs in the equine endometrium during the estrous cycle and early pregnancy.

Expression profile of interferon tau–stimulated genes in ovine peripheral blood leukocytes during embryonic death

Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n = 10) and ED group (treated with 75 μg synthetic PGF2α on Day 18, n = 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2α and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2α. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED.

Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy

The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampling days. A mixed model was fitted on the normalized relative mRNA levels, quantified by qPCR in duplicate, and least significant difference test was employed to detect significantly different group(s). In addition, to determine the degree of contribution of each gene to separation of treatment groups, the multivariate projection method partial least square regression discriminant analysis was used. The expression of 5-lipoxygenase mRNA was greater on d0 and LD, declined at AL, and was suppressed by early pregnancy. Leukotriene A4 hydrolase mRNA expression increased at LD and during early pregnancy, but was significantly greater at LD compared with P14. The expression of LT C4 synthase mRNA was only induced at LD. Cysteinyl leukotriene receptors (CysLT1 and CysLT2) mRNA expressions were decreased by both cyclic changes and early pregnancy, whereas 5-lipoxygenase-activating protein and B leukotriene receptor mRNA expressions were not affected by early pregnancy or stages of the estrous cycle. Partial least square discriminant analysis suggests that LT and PTG pathway enzymes and receptors appear to behave similarly in terms of mRNA expression. In conclusion, the expression profiles of LT pathway genes are demonstrated in equine endometrium for the first time by the present study, and the present data suggest that LT pathway mRNA transcriptions are tightly regulated during early pregnancy in mares.

Expression patterns of Toll-like receptors in the ovine corpus luteum during the early pregnancy and prostaglandin F2α-induced luteolysis

The 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 F2α (PGF2α)-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 PGF2α 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 PGF2α 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 (qPCR) 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, TLR10 was downregulated during PGF2α-induced luteolysis and on P16 (P < .05). TLR4 and TLR7 proteins were particularly localized in endothelial cells on C12/PG0h, 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 PGF2α-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.

Effect of multiple low-dose PGF 2 α injections on the mature corpus luteum in non-pregnant bitches

This study investigated molecular regulation in the canine corpus luteums/corpora lutea (CL) following multiple low-dose prostaglandin F2 alpha (PGF2α) injections in non-pregnant bitches around 30-35 days after ovulation. The CL were obtained by ovariohysterectomy 1 h after the last PGF2α injection. The subjects were divided into the following groups: control (no PGF2α injection, n = 4), one PGF2α injection (injection at 0 h, 1PGF, n = 4), two PGF2α injection (injection at 0 and 8 h, 2PGF, n = 4), and three PGF2α injection (injection at 0, 8 and 24 h, 3PGF, n = 4). In the 1PGF group, the steady-state mRNA levels of an immediate early gene (NR4A1) and immune system-related genes (MCP-1 and IL-8) increased. NR4A1 was localized in luteal and endothelial cells. In contrast, MCP-1 was localized in the luteal tissue between the luteal and endothelial cells. LHCGR, CYP11A1, and StAR mRNA expression decreased after the second PGF2α injection. FASLG increased only after the third PGF2α injection. The mRNA levels of PTGFR, PGT, and PTGS2 decreased as the number of PGF2α injections increased. Immunohistochemistry showed a decrease in StAR protein density as the number of PGF2α injections increased. BAX and CASP3 mRNA expression levels were similar among the groups. Serum progesterone (P4) levels decreased dramatically after the PGF2α injections but were still higher than the basal level at the end of the study. In conclusion, repeated low-dose PGF2α injections could induce luteolytic mechanisms in the CL of non-pregnant bitches. Furthermore, it can be concluded that, in non-pregnant bitches, some aspects of the molecular regulation of luteolysis in the CL are similar to some aspects of such regulation in other domestic animals.

Effects of long-term heat stress and dietary restriction on the expression of genes of steroidogenic pathway and small heat-shock proteins in rat testicular tissue

The aim was to investigate the effects of long‐term heat stress and dietary restriction on the expression of certain genes involving in steroidogenic pathway and small heat‐shock proteins (sHSPs) in rat testis. Sprague Dawley rats (n = 24) 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 for 9 weeks. Feed was freely available for Group I and Group III, while Group II and Group IV were fed 60% of the diet consumed by their ad libitum counterparts. At the end of 9 weeks, testicles were collected under euthanasia. Total RNA was isolated from testis tissue samples. Expression profiles of the genes encoding androgen‐binding protein, follicle‐stimulating hormone receptor, androgen receptor, luteinising hormone receptor, steroidogenic acute regulatory protein (StAR), cyclooxygenase‐2 and sHSP genes were assessed at mRNA levels using qPCR. Long‐term heat stress decreased the expression of StAR and HspB10 genes while dietary restriction upregulated StAR gene expression. The results suggested that long‐term heat stress negatively affected the expression of StAR and HspB10 genes and the dietary restriction was able to reverse negative effect of heat stress on the expression of StAR gene in rat testis.