Schistosomiasis remains a devastating disease in many tropical and subtropical countries worldwide. Its pathology is caused by the eggs laid by constantly paired adult schistosome females. Without pairing, female worms remain virgin-like, or they will lose their fecundity, which includes the de-differentiation of their gonads if they have been paired but get separated from their male partners.To come closer to answers to the unsolved questions about the molecular basis of male-female interaction of schistosomes, my study aimed at the application of RNA- Seq to elucidate the transcriptomes of male and female worms of S. mansoni and, in addition, of testes and ovaries that were obtained by an organ-isolation method established in our laboratory. In this context, the effects of gender, pairing-status, and tissue-origin on gene transcription were analysed. Furthermore, based on the organ-isolation approach I isolated and characterised vitellarium tissue and vitelline cells of different differentiation stages.RNA-Seq analyses were performed on testes (T) isolated from paired (b) and unpaired (s) males (= bT, sT) and ovaries (O) from paired and unpaired females (= bO, sO), as well as their whole worm counterparts (bM, sM and bF, sF). The effects of gender, pairing-status, and tissue origin on gene expression among these samples were compared. The data sets revealed that tissue origin (organ versus worm) has a large effect on gene expression in general, which resulted in 4,534-5,478 (35% - 51%) differentially expressed genes (DEGs; 1.5-fold difference). In addition, 1,012 genes were shown to be up-regulated in all gonads compared to whole worms (> 1.5-fold change). Gene ontology analyses showed that these genes were mainly involved in processes like RNA biosynthesis, cell cycle and ribosome biogenesis, which are essential for gonad development.The number of DEGs induced by gender varied, with the lowest number of 1,445 (out of 8,590; 7.8%) for sM/sF comparison to 4,629 (out of 7,970; 41.3%) for the bT/bO comparison (1.5-fold difference). Furthermore, detailed bioinformatics analyses revealed that sF, which had never been paired before, were much closer related to males (bM or sM) than to paired females at the transcriptome level, based on the fact that 841 genes were transcribed preferentially in bM/sM/sF. This important finding sheds also new light on the question of hermaphroditism versus dioeciousness and provides a molecular fundament for the actual hypothesis of schistosome evolution and its roots originating from the Spirorchidae. In these hermaphroditic worms the male gonad develops ahead of the female gonad (protandry). This principle appears to be conserved in schistosomes as well, although apportioned to two genders.When comparing the male and female gonads, 182 and 33 genes were identified as testis- and ovary-specifically transcribed markers, respectively, which can be used in future studies as reference genes for different kinds of molecular analyses such as comparative qPCRs.Furthermore, investigating the pairing effect in detail demonstrated its fundamental impact on molecular processes in males and females, and more importantly, in their gonads. Of note, transcription of only 243 (1.4%) and 426 (1.7%) genes was influenced by pairing in testes and males, respectively. However, for ovaries and females the numbers of DEGs were 3,748 for females and 3,600 for ovaries (about 30% each). Consistent with former studies, a number of egg biosynthesis-related genes were found to be up-regulated in paired females, including egg-shell protein genes (e.g. p14 and p48) and genes encoding tyrosinases that are involved in egg-shell formation. Pairing-affected transcripts in the gonads might have functions in spermatogenesis (e.g. synaptotagmin 1 and cdc25), oogenesis (e.g. synaptotagmin 2 and cpeb), or both processes (e.g. melk).Moreover, a number of 15 genes were found to be equally transcribed among all samples, and, therefore, represented potential house-keeping genes useful in future studies.Furthermore, the RNA-Seq data also aided in the prediction in the biological context of the activities of hypothetical genes. Thus the occurrence of 238 gonad-preferentially transcribed genes indicated their hypothetical roles in testis-and/or ovary-associated functions.Finally, with the help of a modified version of the organ-isolation protocol established in our group, I succeeded in the isolation and purification of S1-S4 vitelline cells. These cells were characterized at the molecular level by RT-PCR experiments and morphologically analysed by various microscopic techniques including bright field, fluorescent, and electron microscopy. Their vitality and physiological activity was confirmed by cytological staining procedures and Ca2+-imaging analyses. It was even possible to enrich subpopulations of S1 to S4 vitelline cells by FACS analysis, which is a prerequisite for further studies. Thus S1-cell enrichment provides a basis for novel approaches towards cell-culture establishment.In summary the results obtained in my study provide novel and diverse insights into the developmental processes of the gonads as well as a broad basis for future studies aiming to unravel the role of specific factors controlling the unusual reproductive biology of schistosomes.
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