Study of protein and epigenetic profile of bovine sperm nucleus with impact on in vitro embryo production

Author(s)

Letícia Signori de Castro

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2019-01-31

Examining board members

Mayra Elena Ortiz D'Avila Assumpção; Ricardo Pimenta Bertolla; Marcelo Demarchi Goissis; Marcella Pécora Milazzotto; Felipe Perecin

• Advisor: Mayra Elena Ortiz D'Avila Assumpção

Abstract

In vitro embryo production (IVP) has been highlighted as reproductive biotechnique in bovine herds, but still with some limitations. The fertility difference between bulls is one of them and among the sperm attributes that can impact on IVP, little is known about the composition of the bovine sperm nucleus. Therefore, this work had as main goal to identify which sperm nuclear aspects characterize the difference in the IVP index. Then, the study was divided in 3 experiments in which the database of the company In Vitro Brasil was used for selection of bulls with high (HF) and low (LF) embryo development rate (blastocyst rate/cleavage rate; n=5 bulls/group). In the first experiment, chromatin analysis of susceptibility to acid denaturation (modified SCSA), protamine deficiency (CMA3) and DNA fragmentation (COMET assay) were performed. Only protamine deficiency presented difference between groups, however the percentage of CMA3 positive cells was low and it was not possible to attribute to this alteration the fertility difference between groups. In the second experiment, proteomic analysis of the sperm nucleus of these bulls was performed. For that, samples were incubated with CTAB detergent to remove part of the membrane and tail, submitted to protein extraction, trypsin digestion and mass spectrometry analysis. The amount of the proteins was evaluated by the iBAQ index obtained after the identification of peptides and the average of this index compared to identify the differentially expressed proteins. In this experiment, 196 proteins were identified, 21 differentially expressed, being 17 overexpressed in the HF and 4 overexpressed in LF group. Among them, proteins related to the spermatogenesis, fertilization and motility. In the third experiment, in order to identify the epigenetic marks related to in vitro fertility differences it was performed the evaluation of the nucleosome structures using the MNase enzyme to later utilization of immunoprecipitation of the histone marks technique. However, the presence of nucleosomes in bovine sperm DNA was not identified, unfeasible further analysis. Thus, the evaluation of DNA methylation was chosen as epigenetic mark to be studied. In this case, the RRBS (reduced representation of bisulfite sequencing) technique was used to identify the differentially methylated cytosines (DMC) between the two groups. In this experiment, it was identified 440 DMC, being 327 hypomethylated and 113 hypermethylated in HF group. In addition, 184 genes containing these DMCs were identified, however no biological process enrichment was identified with these genes. In conclusion, this study indicated that in vitro fertility characterization can be multifactorial; small changes in chromatin integrity do not directly affect, but proteins related to spermatogenesis, motility and fertilization may be related to the fertility phenotype studied. Among the epigenetic marks studied, nucleosome structures were absent in bovine spermatozoa, and DNA methylation did not have a significant impact on the fertility profile. (AU)

• FAPESP's process: 15/14146-2 - Study of protein and epigenetic profile of bovine sperm nucleus with effect on in vitro embryo production
• Grantee: Letícia Signori de Castro
• Support Opportunities: Scholarships in Brazil - Doctorate