Gene Expressions Related To Smad Signaling Molecule And Its Relationship With Fecundity In Hu Sheep

Hu sheep is famous for its hyper-prolificacy in the world, and the members of Smad pathway are shown to have close relationships with reproduction and follicular development with roles of regulating ovulation and litter size in sheep. In order to investigate the mechanism of high fecundity in Hu sheep and its relationships with Smad pathway, tissue mRNA distributions of genes encoding Smad pathway molecules were detected by RT-PCR, and the differential expression levels in ovaries, pituitaries and antral follicles of high-fecundity (HF) and low-fecundity (LF) Hu sheep were further investigated by real-time PCR. In addition, SPSS software was used to analyze the relationship between the gene expression level and ovulation number in Hu sheep. Our data show the first evidence that genes of Smad pathway in the mRNA levels may be correlated with regulation of ovulation number in Hu sheep and provide the reference for finding candidate genes of high fecundity and understanding the molecular mechanism of hyper-prolificacy in Hu sheep.1. Tissue Expression Profiles of BMP/Smad Pathway Molecule Gene mRNAs in Hu SheepTissue expression profiles of BMP/Smad pathway molecule gene mRNAs in Hu sheep were detected using RT-PCR. The results showed that cDNA fragments were successfully cloned at expected sizes for genes encoding BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP 15), BMP receptors (BMPRIA, BMPRIB and BMPRII) and intracellular transducers (Smadl, Smad 4, Smad 5 and Smad 7), and tissue mRNA distributions indicated that BMP2, BMP4, BMP7, BMPRIA, BMPRII, Smadl, Smad4 and Smad7 mRNA exited in the ovary and other tissues, including hypothalamus, pituitary, uterus, heart, liver, spleen, lung, kidney, muscle and oviduct, but BMP6 mRNA was only detected in the ovary, kidney, muscle and oviduct, and Smad5 mRNA in uterus, lung, muscle and oviduct were not detected, but BMP15 mRNA was only found in the ovary.lt is suggested that BMP/Smad pathway might have roles in pituitary-ovary axis for prolificacy of Hu sheep.2. BMP/Smad Pathway Gene Expression Levels in Pituitaries and Associations with Ovulation Number in Hu Sheep The present study was performed to determine the expressional differences of mRNAs encoding BMP/Smad pathway molecules, including BMP2, BMP4, BMP7, BMPRIA,BMPRIB, BMPRII, Smadl, Smad5 and Smad4 in pituitaries of Hu sheep during 24 and 36 hours after estrus diagnosis between high-fecundity (HF) and low-fecundity (LF) and to further investigate the relationships between their expressions and ovulation number. The results showed that there were no significant differences in BMP2,BMP7, BMPRIA,BMPRII,Smadl and Smad4 mRNA abundance in the pituitary between HF and LF group (P>0.05) respectively. But BMP4, BMPRIB and Smad5 mRNA abundances in the pituitary were respectively higher in HF group than those in LF group (P<0.01) and negatively correlated with ovulation number. It is suggested that the expressional differences of BMP4, BMPRIB and Smad5 in the pituitary between high-fecundity and low-fecundity group might be one of factors for high fecundity in Hu sheep.3. BMP/Smad Pathway Gene Expression Levels in Ovaries and Associations with Ovulation Number in Hu SheepThe mRNA transcript expression differences for members of the BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP 15), BMP receptors (BMPRIA, BMPRIB and BMPRII) and intracellular transducers (Smadl, Smad5 and Smad4) in the ovary of High- fecundity(HF group,) and Low-fecundity (LF) Hu Sheep between 24 and 36 hours after estrus diagnosis were measured by real-time PCR. The results showed that BMP4, BMP15, BMPRIB, BMPRII and Smad4 mRNA abundances in the ovary of HF group were respectively higher than those in LF group (P<0.05 or P <0.01) and also correlated with ovulation number(P<0.05 or P<0.01). But there were no significant differences in BMP2, BMP6, BMP7, BMPRIA, Smadl and Smad5 mRNA abundances in the ovary between LF and HF group (P>0.05). It is suggested that BMP4,BMP15,BMPRIB,BMPRII and Smad4 might play roles in ovulation number and might be candidate genes for high fecundity in Hu sheep. These findings may help to improve the molecular breeding of Hu sheep.4.Differential Expression of mRNAs Encoding BMP/Smad Pathway Molecules in Antral Follicles of High-and Low-fecundity Hu SheepIn order to further investigate the mechanism of high fecundity in Hu sheep and its relationship with the BMP/Smad pathway, antral follicles were punctured for high-fecundity (HF) and low-fecundity (LF) animals. The gene expression levels of mRNAs encoding BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP15), BMP receptors (BMPRIA, BMPRIB and BMPRⅡ) and intracellular transducers (Smad1, Smad5 and Smad4) in antral follicles were detected by real-time PCR. The results showed that BMP4, BMPRIB, BMPRⅡand Smad4 mRNAs were more abundant in the antral follicles of HF animals than those of LF animals (P<0.05 or P<0.01), but BMP15 mRNA was less abundant (P<0.05).The expression levels of BMP2, BMP6, BMP7, BMPRIA, Smad1 and Smad5 did not differ between the HF and LF groups (P>0.05).This suggests that the expressional differences of BMP4, BMP 15, BMPRIB, BMPRⅡand Smad4 in the antral follicles between high-fecundity and low-fecundity group might be one of factors for high fecundity in Hu sheep.5. Tissue Expression Profiles of TGF-β/Smad Pathway Molecule Gene mRNAs in Hu SheepTissue expression profiles of TGFβ/Smad pathway molecule gene mRNAs encoding TGF-β1,TGF-β2,TGF-β3,TGF-βRⅠ,TGF-βRⅡ,Smad2 and Smad7 in Hu sheep were detected using RT-PCR. The results showed that cDNA fragments were successfully cloned at expected sizes for the genes and tissue mRNA distributions indicated that TGF-β1, TGF-β2,TGF-β3,TGF-βRⅠ,TGF-βRⅡ,Smad2 and Smad7 mRNA exited in the ovary and other tissues, including hypothalamus, pituitary, uterus, heart, liver, spleen, kidney, muscle and oviduct, and TGF-β1, TGF-β2,TGF-β3 and TGF-βRⅠmRNA were not only detected in the lung, but TGF-βRⅡ,Smad2 and Smad7 mRNA exited in lung. It is suggested that genes related toTGF-β/Smad pathway expressed extensively in Hu sheep and the obvious existences of their mRNAs in the ovary might play roles in the ovary for prolificacy in Hu sheep.6. TGF-β/Smad Pathway Gene Expression Levels in Ovaries and Associations with Ovulation Number in Hu SheepThe mRNA transcript expression differences for members of the TGFβ/Smad pathway molecules including TGF-β1,TGF-β2,TGF-β3 and their receptors (TGF-βRⅠand TGF-βRⅡ) and intracellular transducers (Smad2 and Smad7) in the ovary of High-fecundity(HF) and Low-fecundity (LF) Hu Sheep between 24 and 36 hours after estrus diagnosis were measured by real-time PCR. The results showed that TGF-β1,TGF-β2, TGF-β3,TGF-βRⅠand TGF-βRⅡmRNA abundances in the ovary of HF group were respectively higher but Smad7 was lower than LF group (P<0.05 or P<0.01), and there were no significant differences in Smad2 mRNA abundances in the ovary between HF and LF group (P>0.05), and the correlation analysis also indicated that TGF-βRⅡpositively and Smad7 negatively correlated with ovulation number(P<0.05 and P<0.01). It is suggested that TGF-βRⅡand Smad7 might play roles in ovulation number and might be candidate genes for high fecundity in Hu sheep.These findings may help to improve the molecular breeding of Hu sheep.