Expressionregulation And Function Of TIMP3 In Dairy Goat Ovarian Granulosa Cells

Matrix metalloproteinases(MMPs) play a key role in various processes such as follicular growth, ovulation and corpus luteum formation. As the inhibitor of the MMPs, TIMP3 might be involved in the cooperation of the bio-events in the follicular growth cycle. However, less is known about the molecular mechanism in the dairy goat follicle regarding TIMP3 expression level, expression pattern, pathway and the effects of TIMP3 on follicular hormone synthesis and granulosa cell apoptosis. In the present study, the follicle and granular cell of Guanzhong dairy goat were treated as the studying material. On the basis of cloning and synthesizing the cDNA of TIMP3, we analyzed the expression pattern of TIMP3 in the follicular growth cycle; meanwhile, we explored the molecular mechanism of LH/hCG and miRNA regulating the expression of TIMP3 and TIMP3 controlling follicular hormone synthesis and granulosa cell apoptosis, elucidating the function of TIMP3. The main results were as follows:1. Molecular cloning and bioinformaticsanalysis of TIMP3 geneRACE technology was used to obtain the cDNA sequence of TIMP3 from dairygoat ovary. The sequencing results showed that the length of TIMP3 cDNA was 2208 bp, and TIMP3 cDNAsequence contained one 348 bp 5’UTR region, one 636 bpCDs region(coding protein of 211 animo acids), and one 1224 bp3’UTR region. Sequence alignment analysis showed that the homology was very high between animo acid sequence of TIMP3 in goat and those in sheep, cows, pigs, human, mice and rats, with the similarity of 100%, 99%, 100%,99%, 97% and 96%, respectively. Amino acid sequence analysis found that the predicted goat amino acid sequence contained one N-terminal domain and one C-terminal domain, and each domain contained6 conservative cysteine residues.2. The expression analysis of TIMP3 gene in goat ovary and follicle cellsThe results of Real-time PCR showed that TIMP3 was differentially expressed in the organisms examined in this study. And, TIMP3 was relatively highly expressed in the oviduct.In the follicle, the mRNA abundance level of TIMP3 was gradually increasing with the development of the follicle with highest expression level in the corpus luteum. In addition, the expression level was significantly higher in the polytocous goats(3-4 kids per pregnancy)compared with the monotocous goats(one kid per pregnancy), demonstrating close relationship between TIMP3 gene and the dairygoat litter size.3. Molecular mechanism of LH/hCG regulating the expression of TIMP3The granulosa cell cultured was treated by hCG. The results of real-time PCR showed that the abundance level of TIMP3 mRNAshowed a peak after the treatment with hCG for 4h and 24 h, respectively. Western Blot analysis showed that the protein level of TIMP3 was increasing with the extension of the treating time, with the highest level at 24 h. The blocking analysis suggested that the increased expression of TIPM3 induced by hCG was influenced by the pathway PKA, PKC, MAPK and PI3 K. We furtherly analyzed the transcriptional region of TIMP3 gene and found that the upstream(1-122bp) had 3 binding sites for Sp1 gene.Site-specific mutagenesis analysis results demonstrated that the mutation occurring at region67-58 bp and 74-65 bp could greatly reduce the transcriptional avtivity of TIMP3. As the activator, FSK was used to treat the granulosa cell, the expression of Sp1 was increased and the transcription of TIMP3 was enhanced, which were in agreement with EMSA, ChIP,over-expression and silencing Sp1 experiments, showing that cAMP could regulate TIMP3 expression through controlling the transcriptional factor Sp1.4. The molecular mechanism of miRNAsregulating the expression of TIMP3 geneBased on the bioinformatics analysis results, we established the luciferase report vectors of miR-21, miR-221, miR-222 and mi R-181, which were then respectively used to transfect the granulosa cell. Real-time PCR results showed that, compared with NC group, miR-21 and miR-181 b could significantly reduce the expression of TIMP3 mRNA, while miR-221 and miR-222 did not affect the expression of TIMP3 m RNA. Western blot analysis results suggested that, compared with NC group, miR-21, miR-181 b, miR-221 and miR-222 all significantly reduced the expression of TIMP3 protein, clarifying that miR-21, miR-221,miR-222 and mi R-181 couldspecifically combine with the binding sites of 3’UTR region of TIMP3 gene, influencing the expression of TIMP3. Of the 4 miRNAs, miR-21 and miR-181 b regulated TIMP3 by degrading mRNA, while miR-221 and miR-222 regulated TIMP3 by translation inhibition after transcription.5. The molecular mechanism of TIMP3 expression regulating granulosa cell apoptosis and hormone synthesisOver-expression of TIMP3 can inhibit the proliferation of granulosa cells and promote cell apoptosis; down-regulation of TIMP3 could inhibit the expression of steroidogenic enzymes(StAR, p450 scc and HSD3B), thus reducing the progesterone production. In addition,TIMP3 could inhibit ADAM17 expression and increase the expression of gene FN and DCN,which were associated with ECMprotein.In conclusion, cAMP pathway could be activated through LH/hCG binding to the receptor LHR of the granulosa cell, increasing the expression of transcriptional factor Sp1.Binding to the responsive element of TIMP3, gene Sp1 could improve the activity of TIMP3.In addition, TIMP3 protein could regulate the synthesis of progesterone through promoting the expression of StAR,p450 scc and HSD3 B, which could increase the expression of FN and DCN affecting the reconstruction of ECM, repress the expression of ADAM17, inhibit the activity of granulosa cell, promote the apoptosis of granulosa cell and regulate the follicular growth cycle. Accordingly, our results provided theoretical and experimental evidence to explain the molecular mechanism of TIMP3 regulating follicular growth cycle and reveal the mechanism of follicle development and maturation, and ovulation, furtherly giving directions for controlling the reproduction process of goat.