Identification of molecular role of Pelota protein in proliferation and differentiation of male germ stem cells by analysis of conditional knock-out mice
Molecular role of Pelota on male germ stem cells in mouse
by Priyadharsini Raju
Date of Examination:2015-05-11
Date of issue:2015-05-27
Advisor:Prof. Dr. Dr. Bertram Brenig
Referee:Prof. Dr. Wolfgang Engel
Referee:Prof. Dr. Ibrahim Adham
Persistent Address:
http://dx.doi.org/10.53846/goediss-5095
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Abstract
English
Pelota (Pelo) is an evolutionally conserved gene, which has been characterized in various species. In Drosophila, Pelo deficiency affects both male and female fertility. The molecular function of PELO has been extensively characterized in yeast, where PELO ortholog Dom34 and its interacting partner Hbs1 participate in RNA quality control mechanism named No-Go decay (NGD). In mice, genetic ablation of Pelo leads to embryonic lethality at the early implantation stage as a result of the impaired development of extra-embryonic endoderm (ExEn). The in vitro culture of Pelo-null blastocysts revealed that PELO may have a role in the regulation of the cell cycle or the self-renewal of a pluripotent inner cell mass (ICM) or embryonic stem cells (ESCs). The overall aim of this study was to investigate the role of Pelo on male germ cell development. To define the consequences of Pelo deletion on male germ cells, we temporally deleted the gene at both embryonic and postnatal stages. Deletion of Pelo in adult mice resulted in a complete loss of whole germ cell lineages after 45 days of deletion. The absence of newly emerging spermatogenic cycles in mutants confirmed that spermatogonial stem cells (SSCs) were unable to maintain spermatogenesis in the absence of PELO. However, germ cells beyond the undifferentiated SSC stage were capable of completing spermatogenesis and producing spermatozoa, even in the absence of PELO. The spermatozoa lacking Pelo were also capable of fertilizing oocytes. Following the deletion of Pelo during embryonic development, we found that PELO is dispensable for maintaining gonocytes. However, embryonic depletion of Pelo disrupts the transition of gonocytes to SSCs. Immunohistological and protein analyses revealed the attenuation of FOXO1 transcriptional activity, which induces the expression of many SSC self-renewal genes. These results indicate that the role of Pelo in SSC maintenance is mediated through the activation of the FOXO1. The decreased transcriptional activity of FOXO1 in mutant testes was due to enhanced activity of the PI3K/Akt signaling pathway, which led to phosphorylation and cytoplasmic sequestration of FOXO1. The inactivation of FOXO1 in the Pelo mutants resulted in the failure of establishment of SSCs in the postnatal life. These results suggest that PELO negatively regulates the PI3K/Akt pathway and that the enhanced activity of PI3K/Akt and subsequent FOXO1 inhibition are responsible for the impaired development of SSCs in mutant testes. Taken together, our study has revealed the indispensable role of Pelo in male fertility and also identified the molecular pathway that is responsible for the male infertility from Pelo deletion. Further to identify the putative role of Pelo in mammals we identified the interacting proteins of PELO by yeast two-hybrid (Y2H) screening of ESC cDNA library. We have identified ribosomal protein, RACK1 and elongation factor 2 as interactional partners of PELO through direct Y2H. In the second part of the study, we found that Pelo is dispensable for the self-renewal of ESCs but is essential for the differentiation into extra embryonic endoderm (ExEn). At molecular level, our observations showed alterations in expression of components of the bone morphogenetic protein (BMP) signaling pathway. To further verify the attenuated activity of BMP signaling in Pelo-null cells, we have established a BMP responsive reporter cell line (PeloF/- BRE-FFLuc). The relative FFLuc activity in control cells was significantly higher than that of mutant cells after the treatment with BMP. Collectively, the results of this study further confirmed that the mutant Pelo∆/- EBs produced extracellular modulators of BMP signaling activity.
Keywords: Pelo; SSCs; PI3K/Akt