| Germ cell nuclear factor (GCNF) and Steroidogenic factor1(SF-1) were orphan nuclear receptor members of the nuclear receptor superfamily that regulate key aspects of embryo development, gonad differentiation and endocrine regulation. In the current study, we focused on swamp buffalos and swines as models to clone GCNF and SF-1coding sequences by RT-PCR, then analyzing expression pattern of GCNF and SF-1in different buffalo tissues by qRT-PCR and characterizing the localization of them in ovaries and testis by immunohistochemistry. Moreover, we further analysed expression pattern of GCNF and SF-1during folliculogenesis and early embryo development. Meanwhile, we explored regulation of GCNF and SF-1gene expression from methylation and miRNA levels. The main achievements of our studies were as following:1. Cloning GCNF and SF-1coding sequences. Sequence analysis indicated that GCNF genes of buffalo and swine were1391bp and1572bp in length, which contain opening reading frames (ORF) and encode435and450amino acids respectively. The buffalo GCNF amino acid sequence was aligned with pig, goat, mouse, human and pan troglodytes similarities were97%,97%,97%,96%and97%, respectively. SF-1genes of buffalo and swine were1490bp and2002bp in length, which contain opening reading frame (ORF) and encode461amino acids. The buffalo SF-1amino acid sequence was aligned with pig, goat, mouse, human and pantroglodytes similarities were96%,98%,94%,94%and95%respectively. The results indicated that nucleotides and amino acid sequences of GCNF and SF-1were highly conservative. The secondary structure of GCNF and SF-1protein were dominate by random coil, and also contain beta-turn, helix and sheet. GCNF and SF-1proteins structure also contained zinc finger and ligand binding domains that suggest these two proteins had basic characteristics and functions of nuclear receptor.2. Furthermore, the GCNF and SF-1gene expressions in buffalo various tissues were analyzed by qRT-PCR. For GCNF, the expression in testis (relative expression was24.9±0.02) and ovary (24.1±0.16) were higher than skin(1.0±0.29),kinde(0.77±0.1),brain(0.48±0.26),liver(0.46±0.02),genitalridge (0.45±0.02),muscle(0.43±0.01),skeleton(0.31±0.26) pituitary tissues(0.25±0.01). For SF-1, the expression in testis (17.8±0.16),ovary (12.1±0.03) and genital ridge (7.7±0.02) were higher than other tissues. It also observed in brain (3.1±0.01), liver(1.8±0.12), skin(1.8±0.11)and pituitary(1.0±0.3). However, the expression in kindey (0.79±0.68),skeleton (0.65±0.03) and muscle(0.58±0.18) were less.The immunohistochemical result showed that GCNF had positive staining in oocytes and granule cells of primordial follicle and primary follicle.In secondary follicle and antral follicle, the strong positive staining of GCNF were found in theca cells and granule cells,but the weakly positive staining were found in cumulus cells and oocytes.SF-1were strong positive staining in oocytes of primordia follicle.In primary and secondary follicle,it was positive sign in oocytes and granule cells. In antral follice,SF-1protein existed in cumulus cells.In addition, GCNF and SF-1protein were all location in spermatogenic cell. These result suggested that GCNF and SF-1might be important in folliculogenesis and spermatogenesis.3. The GCNF and SF-1expression pattern in cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) was investigated by Cells to cDNA and qRT-PCR. The results showed expression of GCNF and SF-1in COCs decreased after IVM.In early pig parthenogenetic embryonic development, the highest expression of GCNF gene is in the two cell period (1.0±0.03), then the amount of expression presents the trend of slow increase after reduction at first, while the amount of expression suddenly dropped to almost no expression in the blastocyst stage(0.01±0.04).The amount of SF-1gene expression between the2cell stage to the8cell stage has no obvious change, the amount of expression between the8cell stage to morula stage significantly increased to the peak (6.89±0.35),and the amount of expression suddenly dropped to almost no expression in the blastula stage(0.001±0.3). Furthermore, we analyzed methylation level of GCNF and SF-1gene during COCs maturation by bisulfite genomic sequencing.The results showed GCNF methylation level increase after COCs maturation and SF-1methylation level had no changes.4. The3’UTR sequence of GCNF and SF-1genes were successfully cloned using3’RACE technology, and the length of them is1816bp and893bp respectively. Two miRNAs which maybe act on the3’UTR of GCNF and SF-1were forecasted and screened using Microinspector software,they were ssc-miR-181a and ssc-miR-615respectively.The expression level of the two miRNAs in early parthenogenetic embryos of pig was detected using the miRNA qRT-PCR.Compared to the expression pattern of GCNF and SF-1,the results showed that there was a reciprocal relationship between the expression of miRNA and its target genes,thus we predicted that ssc-miR-181a and ssc-miR-615maybe be involved in the expression regulation of GCNF and SF-1in the the process of early embryonic development. |
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