Analysis Of Extracellular Matrix Related Genes In Testis Of Guizhou Black Goat Based On Transcriptome And Proteome

Testicular development regulated by gene and protein expression and characterized by dynamic and stage-specific characteristics.Testicular extracellular matrix（ECM）is involved in various biological activities such as spermatogenesis,testicular development,blood testosterone barrier and testicular cord formation.Therefore,it is of great significance to identify the expression regulation of testicular extracellular matrix at different growth stages for the study of reproductive biology of goats.However,there is still little understanding on the molecular biology of goat testis extracellular matrix.In this study,the testicular tissues of Guizhou Black Goats at three physiological stages of lamb（3 months old）,young goat（6 months old）and post-sexual maturity（12 months old）were used as research objects to explore the gene and protein expressions of mRNA,mRNA,LncRNA,miRNA,CircRNA and protein in the testicular tissues of goats at different growth and development stages by integrating transcriptome and proteome techniques.These results provide theoretical basis for the regulation mechanism of extracellular matrix formation in goat testis.The main results are as follows:1.The HE histomorphological study of testis and epididymis of Guizhou Black Sheep showed that the germ cells in testicular tissue at the age of 3 months had not differentiated,and the lumen was not yet presented,and the spermatogenic tubules were still in the spermatic cord state.At 6 months of age,the lumen of spermatogenic tubules of testis has formed initially.There are many differentiated germ cells in spermatogenic epithelium,including secondary spermatocytes and elongated sperm cells,but the histomorphological observation of epididymis shows that there is no sperm storage in epididymis.At the age of12 months,the lumen and epithelium of spermatogenic tubules have formed,and there are a lot of germ cells in spermatogenic tubules,including sperm cells.A large number of spermatozoa were found in the epididymal head tissue of goats aged 12 months.It is suggested that the formation of testicular extracellular matrix is critical before 6 months of age in goats,and the formation of testicular extracellular matrix in 12 months of age in Guizhou black goats has reached the stage of sexual maturity.2.There were 161（105 up-regulated,56 down-regulated）,12（9 up-regulated,3down-regulated）and 665（512 up-regulated,153 down-regulated）differentially expressed mrnas at 3 VS 6,6 VS 12 and 3 VS 12 months of age,respectively.Differentially expressed lncrnas were 23（5 up-regulated,18 down-regulated）,8（5 up-regulated,3 down-regulated）and 135（33 up-regulated,102 down-regulated）,respectively.Analysis of protein PPI interaction network of differentially expressed genes in significantly enriched GO terms indicated that MMP2 was the core gene of extracellular matrix related biological events.LncRNA tcons-0002139 and Tcons-00093342 were co-expressed with MMP2 and COL4A4.LncRNA TCONS₀0093342 was expressed with IGFBP6.LncRNA Tcons-0002139 was also co-expressed with COL4A1.The expression levels of IGF2,IGFBP6 and MMP2 were the highest at 3 months of age,and then decreased gradually with the increase of months.3.Differentially expressed mirnas were 112（49 up-regulated,63 down-regulated）,37（18 up-regulated,19 down-regulated）and 154（83 up-regulated,71 down-regulated）at 3 VS6,6 VS 12 and 3 VS 12 months of age,respectively.GO term analysis related to extracellular matrix showed that biological processes（BP）had the highest proportion（70.97%）and molecular functions（MF）had the lowest proportion（9.68% and 0.00%）in testicular tissues at 3 VS 6 and 6 VS 12 months of age.Combined analysis of lncr Na-mir NA-mrna showed that ACTG2 had a targeted regulatory relationship with Mir-15b-5p and Mir-16a-5p,while GRK1 had a targeted regulatory relationship with Mir-241-5p.It is speculated that Mir-241-5p,Mir-15B-5p and Mir-16a-5p play an important role in the formation of extracellular matrix in goat testis by regulating ACTG2 and GRK1,respectively.4.199 circrnas（93 up-regulated,106 down-regulated）,145（50 up-regulated,95down-regulated）and 269（134 up-regulated,135 down-regulated）were differentially expressed between 3 VS 6,6 VS 12 and 3 VS 12 months of age,respectively.Protein interaction network analysis of differentially expressed circr NA-derived genes at 3 VS 6months of age showed that CHD7 and HIPK3 were core genes of extracellular matrix related biological events.GO analysis showed that two genes corresponding to GO term associated with extracellular matrix were laminin subunit 3（LAMA3）gene.The circr Na-mir NA-mrna integration analysis showed that ACTG2 was co-expressed with Both Chi-Mir-16a-5p and Chi-Mir-15b-5p,while Chx_CIRC-0004920 was co-expressed with Chi-Mir-15b-5p.It is speculated that there is a targeted regulatory relationship between CHX_CIRC-0004920 and Chi-Mir-15B-5p and ACTG2,and that ch X_CIRC-0004920 plays a regulatory role in testicular extracellular matrix.5.The number of differentially expressed proteins between 3 VS 12 months of age was809,of which 386 proteins were up-regulated（47.71%）and 423 proteins were down-regulated（52.29%）.GO analysis showed that two GO terms were directly related to extracellular matrix（GO:0031012）and protein extracellular matrix（GO:0005578）.The corresponding differentially expressed proteins were A9LLG1（SPARC）,A0A452G224（GPC1）,A0A452E9H3（SPOCK2）.The pathways enriched to extracellular matrix by KEGG analysis are adherens junction and focal adhesion,respectively.In this study,differentially enriched proteins associated with adhesive plaques included LAMA3,DOCK1,PTK2 and ITGA9.PPI interaction network analysis of differentially expressed up-regulated and down-regulated proteins showed that the differentially expressed up-regulated proteins were mostly associated with spermatogenesis,while the differentially expressed down-regulated proteins were mostly associated with extracellular matrix.6.Transcriptome and proteome integration analysis showed that 56 genes（4up-regulated and 52 down-regulated）showed consistent expression trends at mRNA and protein levels.PPI interaction network analysis showed that ITGA6,HSPG2,SPARC,VWA1 and LEPREL1 played important roles in the regulation of testicular extracellular matrix formation.ENS expression trends at mRNA and protein levels are opposite,and this gene may be regulated by LncRNA TCONS₀0111208,TCONS₀0107391 and TCONS₀0107411.PPI network analysis showed that NES was co-expressed with extracellular matrix associated TIGA6 and SPARC.Chx_circ₀011820 and CHX_cir C₀015823 may regulate extracellular matrix formation by targeting P3H2 and DNAH3,respectively.Only mRNA differentially expressed genes were associated with extracellular matrix,while only protein differentially expressed genes were associated with spermatogenesis.In conclusion,this study obtained the expression profiles of mRNA,LncRNA,miRNA,CircRNAs and proteins in goat testis tissues at three different physiological stages,and preliminarily explored the expression regulation of differentially expressed genes and proteins in goat testis extracellular matrix formation.Furthermore,some mRNA,LncRNA,miRNA and CircRNAs with targeted regulation related to extracellular matrix formation in goat testis were identified.The expression of most of these genes may be regulated by miRNAs and/or CircRNAs in response to extracellular matrix formation in testicular tissue.These findings further enrich our understanding of goat testicular tissue,particularly the regulation of expression associated with extracellular matrix.This study will provide theoretical basis and new insights for further research on testicular tissue of goats and other mammals.