Human sperm transcriptome: characterization, biological relevance, and biomarker functionality
- Autores: Celia Corral Vázquez
- Directores de la Tesis: Joan Blanco Rodríguez (dir. tes.), Ester Antón (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2019
- Idioma: español
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
The biological relevance of sperm contribution to the embryo has been shown to go beyond a mere transmission of the paternal genome. Several findings revealed that human spermatozoa carry a complex population of coding and non-coding RNAs with potential implications in multiple fertility-related pathways. Accordingly, the consideration of these molecules as simple residual pools of earlier processes has been left behind. This new paradigm also opens the possibility for potential applications in the field of male fertility biomarkers. However, sperm transcriptomic analysis has several limitations due to the heterogeneity and delicate nature of these molecules, besides the small amount of RNA contained in spermatozoa.
In this context, the objective of this Doctoral Thesis is to characterize the human sperm transcriptome to set up the basis for developing new biomarkers of male fertility. Within this goal, the following aims were undertaken: 1) To optimize specific methodologies of sperm RNA analysis using qRT-PCR and RNA-seq strategies; 2) To provide an integrative profiling and functional characterization of sperm mRNAs and lncRNAs by RNA-seq technologies; and 3) To establish new fertility biomarkers among the transcriptomic cargo of the human spermatozoa.
For this purpose, the experimental protocols and data analysis were adapted to the inherent limitations of sperm RNA and to the used transcriptomic technology. Therefore, methods for the elimination of non-sperm cells from semen samples were implemented, together with strict quality controls for ensuring the absence of DNA and non-sperm RNA. Besides, an organic solvent-based method was used for qRT-PCR studies, and non-organic solvent kits were employed for RNA-seq.
The obtained data were normalized by specific methods depending on the used technique. In particular, the normalization of sperm miRNA qRT-PCR singleplex studies required the determination of a suitable set of normalizing miRNAs molecules. This was achieved by comparing the results derived from a sperm miRNA expression dataset normalized by: i) the reference Mean Centering Restricted (MCR) method; and ii) the expression level of different miRNAs. The miRNAs hsa-miR-100-5p and hsa-miR-30a-5p showed ubiquitous and stable expressions, and data normalized by their mean expression led to results with an appropriate quality when compared to MCR. Therefore, this miRNA combination was suggested as the most suitable choice for data normalization in further sperm singleplex studies.
RNA-seq analysis was used to characterize the sperm transcriptome cargo of fertile individuals. Results revealed a complex network of mRNAs and lncRNAs with a high fragmentation status, but containing a host of ubiquitous transcripts. Gene ontology analyses of the whole set of expressed mRNAs showed an enrichment of spermatogenesis and reproduction processes, which was more significant in the sets of highly expressed, ubiquitous, and highly stable mRNAs. Additionally, the functional profiling of potential cis-target genes of the observed lncRNAs showed a significant involvement in embryo development and cell adhesion. This implication became more evident in those cis-target genes that were not present among the sperm mRNA cargo.
Finally, the detection of ubiquitous transcripts and pairs of RNAs with correlated expressions suggested a potential use of these molecules as fertility biomarkers. Accordingly, the presence of sperm miRNA pairs with a correlated expression in fertile individuals that was disrupted in infertile patients of different ethiologies (asthenozoospermia, teratozoospermia, oligozoospermia, and Unexplained Male Infertility or UMI) was evaluated and validated by qRT-PCR. The hsa-miR-942-5p/hsa-miR-1208 pair allowed correctly classifying the 85.71% of infertile individuals, thus achieving the highest potential for discerning infertility cases with seminal alterations. Additionally, the pair hsa-miR-34b-3p/hsa-miR-93-3p was highlighted due to its high potential for discerning UMI patients. Besides, several pairs of ubiquitous lncRNAs and mRNAs were also observed to display a correlated expression in fertile individuals, becoming potential candidates for further biomarker studies.
