The KLF14 Variant is Associated with Type 2 Diabetes and HbA1C Level.

The purpose of this study was to scan variants in coding region of Krȕppel like factor14 (KLF14) locus and assess association related to type 2 diabetes (T2D) in Iranian population. We sequenced the coding region of KLF14 to scan variants in case-sibling study (92 individuals with T2D and 92 healthy older siblings). To confirm, we analyzed rs76603546 association with T2D in a larger unrelated case-control study by PCR-RFLP (475 cases and 512 controls). We analyzed the association of rs76603546 with HbA1C, BMI, fat mass, waist circumference, fasting glucose, cholesterol and HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) using one-way ANOVA analysis. Also, association of genotypes with T2D adjusted for confounding variables was analyzed using logistic regression. HaploReg v 4.1 was used to predict rs76603546 possible function. Sequencing results analysis revealed the association of C allele of rs76603546, synonymous variant C>T, [OR 2.10 (1.38-3.20), P value < 0.001] and CC genotype of rs76603546 [OR 4.3 (1.79-10.23), P value = 0.001] with susceptibility to T2D. PCR-Restriction Fragment Length Polymorphism (RFLP) results analysis confirmed the association of rs76603546 with T2D [C allele, OR 1.91 (1.59-2.29), P value = 0.002, CC genotype, OR 3.27 (2.26-4.73), P value = 0.002 and TC genotype, OR 1.74 (1.31-2.31), P value = 0.001]. The CC genotype of rs76603546 is associated with HbA1C level (P value < 0.001) and BMI (P value = 0.02). After adjustment with confounding variables, we observed association of CC genotype with T2D [OR 2.542 (1.25-3.77), P value = 0.03]. Among over 220 SNPs, rs76603546 was associated with T2D, HbA1C and BMI in our study.

Expression profile of FGF receptors in preimplantation ovine embryos and the effect of FGF2 and PD173074.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increasingly recognized as important regulators of embryo development in mammals. This study investigated the importance of FGF signaling during in vitro development of ovine embryo. The mRNAs of four FGFR subtypes were detected throughout preimplantation development of in vitro fertilized (IVF) embryos, peaked in abundance at the morula stage, and decreased significantly at the blastocyst stage. To gain insight into the role of these mRNAs in embryo development, IVF embryos were cultured in the presence of FGF2 (100 or 500 ng/ml: beginning from days 1 or 4 to 7) or PD173074 (1 µM: beginning from days 1 to 7) as usual treatments for activation or inhibition of FGFRs, respectively. FGF2-supplementation did not affect the percentage of embryos that developed to the blastocyst, blastocyst cell count and the proportion of cells allocated in inner cell mass (ICM) and trophectoderm (TE) compared to control (p > 0.05). Also, increasing the dosage or duration of FGF2 treatment did not significantly alter blastocyst yield or differential cell count (p > 0.05). PD173074-mediated inhibition of FGFRs did not significantly affect blastocyst yield (p > 0.05). Assessment of expression profiles of lineage-associated markers revealed that FGF2 (500 ng/ml) supplementation: (i) significantly increased expression of putative hypoblast marker (GATA4), (ii) significantly decreased expression of putative epiblast (EPI) marker (NANOG) and (iii) did not change TE markers (CDX2 and IFNT) and pluripotency makers (OCT4, SOX2 and REX1). In summary, FGF2-mediated activation of FGFRs may promote a switch in transcriptional profile of ovine ICM from EPI- to hypoblast-associated gene expression.

Dynamics of The Expression of Pluripotency and Lineage Specific Genes in The Pre and Peri-Implantation Goat Embryo.

OBJECTIVE: Two critical points of early development are the first and second lineage segregations, which are regulated by a wide spectrum of molecular and cellular factors. Gene regulatory networks, are one of the important components which handle inner cell mass (ICM) and trophectoderm (TE) fates and the pluripotency status across different mammalian species. Considering the importance of goats in agriculture and biotechnology, this study set out to investigate the dynamics of expression of the core pluripotency markers at the mRNA and protein levels. MATERIALS AND METHODS: In this experimental study, the expression pattern of three pluripotency markers (Oct4, Nanog and Sox2) and the linage specific markers (Rex1, Gata4 and Cdx2) were quantitatively assessed in in vitro matured (MII) oocytes and embryos at three distinctive stages: 8-16 cell stage, day-7 (D7) blastocysts and D14 blastocysts. Moreover, expression of Nanog, Oct4, Sox2 proteins, and their localization in the goat blastocyst was observed through immunocytochemistry. RESULTS: Relative levels of mRNA transcripts for Nanog and Sox2 in D3 (8-16 cell) embryos were significantly higher than D7 blastocysts and mature oocytes, while Oct4 was only significantly higher than D7 blastocysts. However, the expression pattern of Rex1, as an epiblast linage marker, decreased from the oocyte to the D14 stage. The expression pattern of Gata4 and Cdx2, as extra embryonic linage markers, also showed a similar trend from oocyte to D3 while their expressions were up-regulated in D14 blastocysts. CONCLUSION: Reduction in Nanog, Oct4, Sox2 mRNA transcription and a late increase in extra embryonic linage markers suggests that the developmental program of linage differentiation is retarded in goat embryos compared to previously reported data on mice and humans. This is likely related to late the implantation in goats.

Stage-Specific Profiling of Transforming Growth Factor-ß, Fibroblast Growth Factor and Wingless-int Signaling Pathways during Early Embryo Development in The Goat.

OBJECTIVE: This research intends to unravel the temporal expression profiles of genes in- volved in three developmentally important signaling pathways [transforming growth factor-ß (TGF-ß), fibroblast growth factor (FGF) and wingless/int (WNT)] during preand peri-implan- tation goat embryo development. MATERIALS AND METHODS: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the down- stream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 (D7) blastocysts and day-14 (D14) blas- tocysts] were separately prepared in triplicate for comparative real-time reverse tran- scriptase polymerase chain reaction (RT-PCR) using the selected gene sets. RESULTS: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-ß, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-ß, FGF and WNT components with renewal thereafter. CONCLUSION: The results suggested that TGF-ß, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of preand peri- implantation goat embryo development.

Expression Profile of Developmentally Important Genes in preand peri-Implantation Goat Embryos Produced In Vitro.

BACKGROUND: Little is understood about the regulation of gene expression during early goat embryo development. This study investigated the expression profile of 19 genes, known to be critical for early embryo development in mouse and human, at five different stages of goat in vitro embryo development (oocyte, 8-16 cell, morula, day-7 blastocyst, and day 14 blastocyst). MATERIALS AND METHODS: In this experimental study, stage-specific profiling using real time-quantitative polymerase chain reaction (RT-qPCR) revealed robust and dynamic patterns of stage-specific gene activity that fall into four major clusters depending on their respective mRNA profiles. RESULTS: The gradual pattern of reduction in the maternally stored transcripts without renewal thereafter (cluster-1: Lifr1, Bmpr1, Alk4, Id3, Ctnnb, Akt, Oct4, Rex1, Erk1, Smad1 and 5) implies that their protein products are essential during early cleavages when the goat embryo is silent and reliant to the maternal legacy of mRNA. The potential importance of transcription augment at day-3 (cluster-2: Fzd, c-Myc, Cdc25a, Sox2) or day- 14 (cluster-3: Fgfr4, Nanog) suggests that they are nascent embryonic mRNAs which intimately involved in the overriding of MET or regulation of blastocyst formation, respectively. The observation of two expression peaks at both day-3 and day-14 (cluster-4: Gata4, Cdx2) would imply their potential importance during these two critical stages of preand periimplantation development. CONCLUSION: Evolutionary comparison revealed that the selected subset of genes has been rewired in goat and human/goat similarity is greater than the mouse/goat or bovine/goat similarities. The developed profiles provide a resource for comprehensive understanding of goat preimplantation development and pluripotent stem cell engineering as well.