Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model
Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36...
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MDPI AG
2024-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/4/1953 |
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| author | Estíbaliz Lacalle Estela Fernández-Alegre Belén Gómez-Giménez Manuel Álvarez-Rodríguez Beatriz Martín-Fernández Cristina Soriano-Úbeda Felipe Martínez-Pastor |
| author_facet | Estíbaliz Lacalle Estela Fernández-Alegre Belén Gómez-Giménez Manuel Álvarez-Rodríguez Beatriz Martín-Fernández Cristina Soriano-Úbeda Felipe Martínez-Pastor |
| author_sort | Estíbaliz Lacalle |
| collection | DOAJ |
| description | Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI—median fluorescence intensity—and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified. |
| first_indexed | 2024-03-07T22:29:50Z |
| format | Article |
| id | doaj.art-89f0c16721a241fd81b3a352da733880 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-07T22:29:50Z |
| publishDate | 2024-02-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-89f0c16721a241fd81b3a352da7338802024-02-23T15:19:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-02-01254195310.3390/ijms25041953Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine ModelEstíbaliz Lacalle0Estela Fernández-Alegre1Belén Gómez-Giménez2Manuel Álvarez-Rodríguez3Beatriz Martín-Fernández4Cristina Soriano-Úbeda5Felipe Martínez-Pastor6Institute of Animal Health and Cattle Development (INDEGSAL), University of León, 24071 León, SpainBianor Biotech SL, 24071 León, SpainInstitute of Animal Health and Cattle Development (INDEGSAL), University of León, 24071 León, SpainDepartment of Animal Reproduction, National Institute for Agricultural and Food Research and Technology, Spanish Scientific Research Council (INIA-CSIC), 28040 Madrid, SpainInstitute of Animal Health and Cattle Development (INDEGSAL), University of León, 24071 León, SpainDepartment of Medicine, Surgery and Veterinary Anatomy (Animal Medicine and Surgery), University of León, 24071 León, SpainInstitute of Animal Health and Cattle Development (INDEGSAL), University of León, 24071 León, SpainChromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI—median fluorescence intensity—and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.https://www.mdpi.com/1422-0067/25/4/1953boarspermatozoacooled storageflow cytometrychromatin statusoxidative stress |
| spellingShingle | Estíbaliz Lacalle Estela Fernández-Alegre Belén Gómez-Giménez Manuel Álvarez-Rodríguez Beatriz Martín-Fernández Cristina Soriano-Úbeda Felipe Martínez-Pastor Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model International Journal of Molecular Sciences boar spermatozoa cooled storage flow cytometry chromatin status oxidative stress |
| title | Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model |
| title_full | Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model |
| title_fullStr | Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model |
| title_full_unstemmed | Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model |
| title_short | Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model |
| title_sort | application of flow cytometry using advanced chromatin analyses for assessing changes in sperm structure and dna integrity in a porcine model |
| topic | boar spermatozoa cooled storage flow cytometry chromatin status oxidative stress |
| url | https://www.mdpi.com/1422-0067/25/4/1953 |
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