Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Study Design
2.3. Bovine Testis Collection
2.4. Isolation of Bovine Leydig, Sertoli and Peritubular Myoid Cells
2.5. Three-Dimensional Co-Culture of Testicular Cells
2.6. Testicular Organoid Culture Conditions
2.7. Testicular Organoid Whole-Mounting Staining
2.8. Digital Image Acquisition and Processing
2.9. Q-PCR Analysis
2.10. Fluorescence Activated Cell Sorting (FACS) Analysis
2.11. Testosterone Production Assay
2.12. Statistical Analysis
3. Results
3.1. Isolation and Characterization of Bovine Leydig, Sertoli and Peritubular Myoid Cells
3.2. Formation and Morphological Characterization of Multicellular Bovine TOs
3.3. Testicular Cell Marker Evaluation by QPCR in Bovine TOs
3.4. Confocal Immunofluorescence Analysis of Cell-Specific Testicular Markers in Bovine TOs
3.5. Testosterone Measurements in Testicular Organoids
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Antibody | Cell Marker | Cat.# | P/N Company | Dilution |
---|---|---|---|---|
Anti-COL1 | Peritubular | SC293182 | Santa Cruz Biotechonology | 1:50 |
Anti-STAR | Leydig | AB96637 | Abcam | 1:100 |
Anti-WT1 | Sertoli | AB89901 | Abcam | 1:100 |
IgG FITC | - | AB97050 | Abcam | 1:100 |
IgG Alexa Fluor 488 | - | A32766 | Thermo Fisher | 1:500 |
Gene | Nucleotide Sequence (5′-3′) | Accession Number |
---|---|---|
β-ACTIN | Forward CGCACCACTGGCATTGTCAT Reverse TCCAAGGCGACGTAGCAGAG | NM_173979.3 |
WT1 * | Forward AACCACACCACACCCATCC Reverse ACGCCGCACATCCTGAAT | XM_015466595.1 |
STAR * | Forward GACACGGTCATCACTCACGA Reverse TACGCTCACAAAGTCTCGGG | NM_174189.3 |
αSMA ** | Forward CAGCCGAGAACTTTCAGGGAC Reverse GGTGATGATGCCGTGCTCTA | NM_001034502.1 |
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Cortez, J.; Leiva, B.; Torres, C.G.; Parraguez, V.H.; De los Reyes, M.; Carrasco, A.; Peralta, O.A. Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations. Animals 2022, 12, 2283. https://doi.org/10.3390/ani12172283
Cortez J, Leiva B, Torres CG, Parraguez VH, De los Reyes M, Carrasco A, Peralta OA. Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations. Animals. 2022; 12(17):2283. https://doi.org/10.3390/ani12172283
Chicago/Turabian StyleCortez, Jahaira, Barbara Leiva, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes, Albert Carrasco, and Oscar A. Peralta. 2022. "Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations" Animals 12, no. 17: 2283. https://doi.org/10.3390/ani12172283