Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Predominant localization of ADAMTS1 in follicular granulosa cells of the ovine ovary. ( A ) Hematoxylin and eosin (HE) staining alongside immunohistochemical detection of ADAMTS1 in ovarian tissue. The left panel illustrates HE staining, while the right panel displays ADAMTS1 immunostaining (bar = 50 μm). ( B ) Immunofluorescence analysis of granulosa cells and oocytes. GC-NC: granulosa cells with negative control staining; GC-FSHR: granulosa cells immunostained for FSHR; GC-ADAMTS1: granulosa cells immunostained for ADAMTS1; OC-ADAMTS1: oocytes immunostained for ADAMTS1 (bar = 100 μm).

Article Snippet: Following 24 h culture, cells were fixed in 4% tissue fixative for 30 min, then permeabilized with 0.2% Triton X-100 (Coolaber, Beijing, China) for 20 min. Non-specific binding sites were blocked by treatment with 5% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 3 h. The cells were subsequently incubated overnight at 4 °C with primary antibodies targeting FSHR and ADAMTS1 (Bioss, Beijing, China).

Techniques: Staining, Immunohistochemical staining, Immunostaining, Immunofluorescence, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Design and validation of ADAMTS1 knockdown and overexpression systems in ovarian granulosa cells. ( A ) Four siRNA targeting sequences against ADAMTS1 were designed using an online prediction tool. ( B ) Knockdown efficiency was verified by quantifying ADAMTS1 mRNA and protein levels post-transfection in granulosa cells. Among the four siRNA constructs tested, si-ADAMTS1-2 demonstrated the most robust knockdown efficiency, significantly reducing both ADAMTS1 mRNA and protein expression levels in ovarian granulosa cells. ( C ) Agarose gel electrophoresis confirming successful PCR amplification of the ADAMTS1 coding sequence (CDS). Using the DL10,000 DNA marker, agarose gel electrophoresis confirmed successful amplification of the ADAMTS1 coding sequence (CDS), with the observed band (2903 bp) migrating between 2000 and 4000 bp, consistent with the expected product size. ( D ) Restriction enzyme digestion analysis using EcoR I and BamH I to verify linearized pcDNA3.1-EGFP vector and EGFP-ADAMTS1 recombinant plasmid. Using the DL10000 DNA marker, the linearized pcDNA3.1-EGFP vector migrated as a single band between 7000 and 10,000 bp, consistent with its expected size of 6750 bp. For the EGFP-ADAMTS1 recombinant plasmid, digestion with EcoRI and BamHI yielded fragments of 6750 bp, 994 bp, 900 bp, 475 bp, 387 bp, and 151 bp, all of which matched their predicted sizes by electrophoretic mobility. ( E ) Schematic representation of the ADAMTS1 overexpression vector construct. The size of the overexpression vector EGFP-ADAMTS1 is 9657 bp. ( F ) Overexpression efficacy was assessed by measuring ADAMTS1 transcriptional and translational levels following plasmid transfection. ( G ) Sanger sequencing chromatogram confirming the accurate insertion of EGFP-ADAMTS1 in the expression vector. * for p < 0.05; ** for p < 0.01.

Article Snippet: Following 24 h culture, cells were fixed in 4% tissue fixative for 30 min, then permeabilized with 0.2% Triton X-100 (Coolaber, Beijing, China) for 20 min. Non-specific binding sites were blocked by treatment with 5% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 3 h. The cells were subsequently incubated overnight at 4 °C with primary antibodies targeting FSHR and ADAMTS1 (Bioss, Beijing, China).

Techniques: Biomarker Discovery, Knockdown, Over Expression, Transfection, Construct, Expressing, Agarose Gel Electrophoresis, Amplification, Sequencing, Marker, Plasmid Preparation, Recombinant

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: ADAMTS1 enhances proliferation in follicular granulosa cells. ( A ) Morphological observations of granulosa cells at 0, 24, and 48 h post ADAMTS1 knockdown and overexpression. ADAMTS1 knockdown significantly reduced cell numbers, whereas ADAMTS1 overexpression markedly increased cell numbers and enhanced EGFP fluorescence intensity in transfected ovarian granulosa cells. ( B ) Quantification of granulosa cell numbers at 0, 24, and 48 h following modulation of ADAMTS1 expression. At 48 h post-transfection, ADAMTS1 knockdown significantly reduced granulosa cell numbers by 40.0% ( p < 0.01), while its overexpression enhanced cellular proliferation by 1.13-fold ( p < 0.01). ( C ) Flow cytometric analysis of apoptosis in granulosa cells subjected to ADAMTS1 silencing and overexpression. Relative to control groups, ADAMTS1 knockdown reduced the proliferation rate of EdU-positive granulosa cells (GCs) to 76.3% of baseline levels ( p < 0.01), whereas ADAMTS1 overexpression enhanced proliferative activity to 148.2% ( p < 0.01). ( D ) After knockdown of ADAMTS1 , the mRNA and protein expression levels of Bcl2 ( p < 0.05), Bax ( p < 0.05), and caspase3 ( p < 0.05) in the granulosa cells were significantly increased. ( E ) After overexpression of ADAMTS1 , the mRNA and protein expression levels of Bcl2 ( p < 0.05), Bax ( p < 0.05), and caspase3 ( p < 0.05) in the granulosa cells were significantly decreased. ns for not significant; * for p < 0.05; ** for p < 0.01.

Article Snippet: Following 24 h culture, cells were fixed in 4% tissue fixative for 30 min, then permeabilized with 0.2% Triton X-100 (Coolaber, Beijing, China) for 20 min. Non-specific binding sites were blocked by treatment with 5% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 3 h. The cells were subsequently incubated overnight at 4 °C with primary antibodies targeting FSHR and ADAMTS1 (Bioss, Beijing, China).

Techniques: Knockdown, Over Expression, Fluorescence, Transfection, Expressing, Control, Activity Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Summary of RNA sequencing results and functional enrichment analysis of differentially expressed genes (DEGs). ( A ) Quantification of upregulated and downregulated DEGs in ADAMTS1 knockdown and overexpression groups. ( B ) Volcano plot illustrating DEGs identified between the ADAMTS1 knockdown group and control. Transcriptomic analysis identified 2684 differentially expressed genes (DEGs), comprising 159 upregulated (5.9%) and 2,525 downregulated (94.1%) transcripts, based on the established cutoff criteria ( p < 0.05, |log2 fold change| ≥ 1). ( C ) Volcano plot depicting DEGs between the ADAMTS1 overexpression group and control. Transcriptomic analysis identified 459 differentially expressed genes (DEGs), comprising 271 upregulated (59.0%) and 188 downregulated (41.0%) transcripts, based on the established cutoff criteria ( p < 0.05, |log2 fold change| ≥ 1). ( D ) Gene Ontology (GO) enrichment analysis for DEGs comparing the ADAMTS1 knockdown group with control. ( E ) GO enrichment analysis for DEGs between the ADAMTS1 overexpression group and control. ( F ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs in the ADAMTS1 knockdown versus control comparison. ( G ) KEGG pathway enrichment for DEGs in the ADAMTS1 overexpression versus control comparison.

Article Snippet: Following 24 h culture, cells were fixed in 4% tissue fixative for 30 min, then permeabilized with 0.2% Triton X-100 (Coolaber, Beijing, China) for 20 min. Non-specific binding sites were blocked by treatment with 5% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 3 h. The cells were subsequently incubated overnight at 4 °C with primary antibodies targeting FSHR and ADAMTS1 (Bioss, Beijing, China).

Techniques: RNA Sequencing, Functional Assay, Knockdown, Over Expression, Control, Comparison

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Functional characterization of DEGs. ( A ) Venn diagram illustrating the overlap of DEGs across four groups: downregulated and upregulated genes following ADAMTS1 knockdown, and downregulated and upregulated genes following ADAMTS1 overexpression. Through comparative analysis, it was found that in all comparison groups, there were 17 differentially expressed genes ( p < 0.05, |log2 fold change| ≥ 1). ( B ) Detailed gene information for the 17 co-expressed DEGs (co-DEGs). ( C ) Heatmap depicting the expression patterns of the 17 co-DEGs in the ADAMTS1 knockdown group. ( D ) Heatmap illustrating the expression profiles of the 17 co-DEGs in the ADAMTS1 overexpression group. ( E ) Protein-protein molecular docking analysis between ADAMTS1 (colored blue) and PSAT1 (colored green). ( F ) Predicted interaction interface highlighting critical amino acid residues involved in binding: ADAMTS1 key residues include Ser419, Ser440, Gln441, Asp438, Lys294, Ile541, and Asp530; PSAT1 key residues include Arg336, Asn9, Ala15, His19, Gln26, and Ser38. ( G ) The mRNA expression levels of PSAT1 and SLC6A9 in the ADAMTS1 knockdown group and the overexpression group. ( H ) The protein expression levels of PSAT1 in the ADAMTS1 knockdown group and the overexpression group. * for p < 0.05; ** for p < 0.01.

Article Snippet: Following 24 h culture, cells were fixed in 4% tissue fixative for 30 min, then permeabilized with 0.2% Triton X-100 (Coolaber, Beijing, China) for 20 min. Non-specific binding sites were blocked by treatment with 5% bovine serum albumin (BSA) (Servicebio, Wuhan, China) for 3 h. The cells were subsequently incubated overnight at 4 °C with primary antibodies targeting FSHR and ADAMTS1 (Bioss, Beijing, China).

Techniques: Functional Assay, Knockdown, Over Expression, Comparison, Expressing, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Predominant localization of ADAMTS1 in follicular granulosa cells of the ovine ovary. ( A ) Hematoxylin and eosin (HE) staining alongside immunohistochemical detection of ADAMTS1 in ovarian tissue. The left panel illustrates HE staining, while the right panel displays ADAMTS1 immunostaining (bar = 50 μm). ( B ) Immunofluorescence analysis of granulosa cells and oocytes. GC-NC: granulosa cells with negative control staining; GC-FSHR: granulosa cells immunostained for FSHR; GC-ADAMTS1: granulosa cells immunostained for ADAMTS1; OC-ADAMTS1: oocytes immunostained for ADAMTS1 (bar = 100 μm).

Article Snippet: Sections were incubated overnight at 4 °C with a primary antibody targeting ADAMTS1 (Biorbyt, Beijing, China), followed by incubation with an appropriate secondary antibody (Kangwei, Taizhou, China).

Techniques: Staining, Immunohistochemical staining, Immunostaining, Immunofluorescence, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Design and validation of ADAMTS1 knockdown and overexpression systems in ovarian granulosa cells. ( A ) Four siRNA targeting sequences against ADAMTS1 were designed using an online prediction tool. ( B ) Knockdown efficiency was verified by quantifying ADAMTS1 mRNA and protein levels post-transfection in granulosa cells. Among the four siRNA constructs tested, si-ADAMTS1-2 demonstrated the most robust knockdown efficiency, significantly reducing both ADAMTS1 mRNA and protein expression levels in ovarian granulosa cells. ( C ) Agarose gel electrophoresis confirming successful PCR amplification of the ADAMTS1 coding sequence (CDS). Using the DL10,000 DNA marker, agarose gel electrophoresis confirmed successful amplification of the ADAMTS1 coding sequence (CDS), with the observed band (2903 bp) migrating between 2000 and 4000 bp, consistent with the expected product size. ( D ) Restriction enzyme digestion analysis using EcoR I and BamH I to verify linearized pcDNA3.1-EGFP vector and EGFP-ADAMTS1 recombinant plasmid. Using the DL10000 DNA marker, the linearized pcDNA3.1-EGFP vector migrated as a single band between 7000 and 10,000 bp, consistent with its expected size of 6750 bp. For the EGFP-ADAMTS1 recombinant plasmid, digestion with EcoRI and BamHI yielded fragments of 6750 bp, 994 bp, 900 bp, 475 bp, 387 bp, and 151 bp, all of which matched their predicted sizes by electrophoretic mobility. ( E ) Schematic representation of the ADAMTS1 overexpression vector construct. The size of the overexpression vector EGFP-ADAMTS1 is 9657 bp. ( F ) Overexpression efficacy was assessed by measuring ADAMTS1 transcriptional and translational levels following plasmid transfection. ( G ) Sanger sequencing chromatogram confirming the accurate insertion of EGFP-ADAMTS1 in the expression vector. * for p < 0.05; ** for p < 0.01.

Article Snippet: Sections were incubated overnight at 4 °C with a primary antibody targeting ADAMTS1 (Biorbyt, Beijing, China), followed by incubation with an appropriate secondary antibody (Kangwei, Taizhou, China).

Techniques: Biomarker Discovery, Knockdown, Over Expression, Transfection, Construct, Expressing, Agarose Gel Electrophoresis, Amplification, Sequencing, Marker, Plasmid Preparation, Recombinant

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: ADAMTS1 enhances proliferation in follicular granulosa cells. ( A ) Morphological observations of granulosa cells at 0, 24, and 48 h post ADAMTS1 knockdown and overexpression. ADAMTS1 knockdown significantly reduced cell numbers, whereas ADAMTS1 overexpression markedly increased cell numbers and enhanced EGFP fluorescence intensity in transfected ovarian granulosa cells. ( B ) Quantification of granulosa cell numbers at 0, 24, and 48 h following modulation of ADAMTS1 expression. At 48 h post-transfection, ADAMTS1 knockdown significantly reduced granulosa cell numbers by 40.0% ( p < 0.01), while its overexpression enhanced cellular proliferation by 1.13-fold ( p < 0.01). ( C ) Flow cytometric analysis of apoptosis in granulosa cells subjected to ADAMTS1 silencing and overexpression. Relative to control groups, ADAMTS1 knockdown reduced the proliferation rate of EdU-positive granulosa cells (GCs) to 76.3% of baseline levels ( p < 0.01), whereas ADAMTS1 overexpression enhanced proliferative activity to 148.2% ( p < 0.01). ( D ) After knockdown of ADAMTS1 , the mRNA and protein expression levels of Bcl2 ( p < 0.05), Bax ( p < 0.05), and caspase3 ( p < 0.05) in the granulosa cells were significantly increased. ( E ) After overexpression of ADAMTS1 , the mRNA and protein expression levels of Bcl2 ( p < 0.05), Bax ( p < 0.05), and caspase3 ( p < 0.05) in the granulosa cells were significantly decreased. ns for not significant; * for p < 0.05; ** for p < 0.01.

Article Snippet: Sections were incubated overnight at 4 °C with a primary antibody targeting ADAMTS1 (Biorbyt, Beijing, China), followed by incubation with an appropriate secondary antibody (Kangwei, Taizhou, China).

Techniques: Knockdown, Over Expression, Fluorescence, Transfection, Expressing, Control, Activity Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Summary of RNA sequencing results and functional enrichment analysis of differentially expressed genes (DEGs). ( A ) Quantification of upregulated and downregulated DEGs in ADAMTS1 knockdown and overexpression groups. ( B ) Volcano plot illustrating DEGs identified between the ADAMTS1 knockdown group and control. Transcriptomic analysis identified 2684 differentially expressed genes (DEGs), comprising 159 upregulated (5.9%) and 2,525 downregulated (94.1%) transcripts, based on the established cutoff criteria ( p < 0.05, |log2 fold change| ≥ 1). ( C ) Volcano plot depicting DEGs between the ADAMTS1 overexpression group and control. Transcriptomic analysis identified 459 differentially expressed genes (DEGs), comprising 271 upregulated (59.0%) and 188 downregulated (41.0%) transcripts, based on the established cutoff criteria ( p < 0.05, |log2 fold change| ≥ 1). ( D ) Gene Ontology (GO) enrichment analysis for DEGs comparing the ADAMTS1 knockdown group with control. ( E ) GO enrichment analysis for DEGs between the ADAMTS1 overexpression group and control. ( F ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs in the ADAMTS1 knockdown versus control comparison. ( G ) KEGG pathway enrichment for DEGs in the ADAMTS1 overexpression versus control comparison.

Article Snippet: Sections were incubated overnight at 4 °C with a primary antibody targeting ADAMTS1 (Biorbyt, Beijing, China), followed by incubation with an appropriate secondary antibody (Kangwei, Taizhou, China).

Techniques: RNA Sequencing, Functional Assay, Knockdown, Over Expression, Control, Comparison

Journal: Animals : an Open Access Journal from MDPI

Article Title: Transcriptome Analysis Reveals the Molecular Mechanisms by Which ADAMTS1 Influences the Proliferation of Ovarian Granulosa Cells in Sheep

doi: 10.3390/ani15162354

Figure Lengend Snippet: Functional characterization of DEGs. ( A ) Venn diagram illustrating the overlap of DEGs across four groups: downregulated and upregulated genes following ADAMTS1 knockdown, and downregulated and upregulated genes following ADAMTS1 overexpression. Through comparative analysis, it was found that in all comparison groups, there were 17 differentially expressed genes ( p < 0.05, |log2 fold change| ≥ 1). ( B ) Detailed gene information for the 17 co-expressed DEGs (co-DEGs). ( C ) Heatmap depicting the expression patterns of the 17 co-DEGs in the ADAMTS1 knockdown group. ( D ) Heatmap illustrating the expression profiles of the 17 co-DEGs in the ADAMTS1 overexpression group. ( E ) Protein-protein molecular docking analysis between ADAMTS1 (colored blue) and PSAT1 (colored green). ( F ) Predicted interaction interface highlighting critical amino acid residues involved in binding: ADAMTS1 key residues include Ser419, Ser440, Gln441, Asp438, Lys294, Ile541, and Asp530; PSAT1 key residues include Arg336, Asn9, Ala15, His19, Gln26, and Ser38. ( G ) The mRNA expression levels of PSAT1 and SLC6A9 in the ADAMTS1 knockdown group and the overexpression group. ( H ) The protein expression levels of PSAT1 in the ADAMTS1 knockdown group and the overexpression group. * for p < 0.05; ** for p < 0.01.

Article Snippet: Sections were incubated overnight at 4 °C with a primary antibody targeting ADAMTS1 (Biorbyt, Beijing, China), followed by incubation with an appropriate secondary antibody (Kangwei, Taizhou, China).

Techniques: Functional Assay, Knockdown, Over Expression, Comparison, Expressing, Binding Assay