De novo transcriptome assembly and annotation of the isopod Idotea balthica

The Baltic Sea is a unique environment that contains unique genetic populations. In order to study these populations on a genetic level basic molecular research is needed. The aim of this thesis was to provide a basic genetic resource for population genomic studies by de novo assembling a transcriptome for the Baltic Sea isopod Idotea balthica. RNA was extracted from a whole single adult male isopod and sequenced using Illumina (125bp PE) RNA-Seq. The reads were preprocessed using FASTQC for quality control, TRIMMOMATIC for trimming, and RCORRECTOR for error correction. The preprocessed reads were then assembled with TRINITY, a de Bruijn graph-based assembler, using different k-mer sizes. The different assemblies were combined and clustered using CD-HIT. The assemblies were evaluated using TRANSRATE for quality and filtering, BUSCO for completeness, and TRANSDECODER for annotation potential. The 25-mer assembly was annotated using PANNZER (protein annotation with z-score) and BLASTX. The 25-mer assembly represents the best first draft assembly since it contains the most information. However, this assembly shows high levels of polymorphism, which currently cannot be differentiated as paralogs or allelic variants. Furthermore, this assembly is incomplete, which could be improved by sampling additional developmental stages.

Family rivalry in the testis: Retinoblastoma protein and E2F transcription factors in testicular development and adult spermatogenesis

Disorders of male reproductive health are becoming increasingly prevalent globally. These defects, ranging from decreasing sperm counts to an increasing rate of infertility and testicular cancer, have a common origin in the early phases of testicular development, but the exact mechanisms that cause them remain unknown. Testicular development and adult spermatogenesis are complex processes in which different cell types undergo mitosis, meiosis, differentiation and apoptosis. The retinoblastoma protein family and its associated E2F transcription factors are key regulators of these cellular events. In the present study, the functions of these factors in postnatal testicular development and adult spermatogenesis were explored using different animal models. In addition, a new application of flow cytometry to study testicular cell dynamics was developed. An ablation of retinoblastoma protein in mouse Sertoli cells resulted in their cell cycle re-entry in adult testes, dedifferentiation and a severe spermatogenic defect. We showed that deregulated E2F3 contributed to these changes. Our results indicated that the E2F1 transcription factor is critical for the control of apoptosis in the developing postnatal testis. In the adult testis, E2F1 controls the maintenance of the spermatogonial stem cell pool, in addition to inhibiting apoptosis of spermatocytes. In summary, this study elucidated the complex interdependencies of the RB and E2F transcription factor families in the control of postnatal testicular development and adult spermatogenesis. Furthermore, this study provided a new methodology for the analysis of testicular cells.