Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigating the function of RBMXL2 in spermatogenesis in humans and using mouse models(Saudi Digital Library, 2023-11-20) Aldalaqan, Saad; Elliott, DavidRBMX RBMXL2 and RBMY belong to a family of RNA-binding proteins. How important these proteins are in whole animal biology, what their global RNA targets and mechanisms of action are, and to what extent these proteins overlap in function has been poorly understood. Using histology I found that Rbmxl2 knockout phenotypes differed between mouse strains (C57BL/6 and Sv/129). Both strains, however, were infertile, with no sperm found, which indicates the importance of RBMXL2 in spermatogenesis. I performed an iCLIP analysis to investigate RBMXL2-RNA binding, which revealed that RBMXL2 directly binds to exons and introns of protein-coding genes. The strongest RBMXL2 binding was observed in genes associated with spermatogenesis and reproduction, specifically meiosis, such as Meioc and Esco1. Binding was also enriched within ultra-long exons to prevent the selection of cryptic splice sites. I attempted minigene experiments to try and model RBMXL2 function in transfected cells, but these experiments did not replicate RBMXL2 function in vivo. As an alternative I developed a stable cell line “rescue” approach to over-express RBMXL2 and test if this could replace endogenous RBMX. Using this approach, I demonstrated that RBMXL2 is likely to replace RBMX's function in meiosis when the X and Y are inactive. RBMXL2's ability to restore splicing control is dependent on its disordered domain not the RRM. RBMY, which is distantly related to RBMX, also replaces RBMX function in somatic cells.18 0Item Restricted Whole Exome Investigation Of Infertility In Men With Azoospermia(Saudi Digital Library, 2023-10-01) Sindi, Emad Rashad B; Jayasena, ChannaMale infertility is a disabling condition that affects 7% of men worldwide. The most severe form of male infertility is non-obstructive azoospermia (NOA) and approximately 20% of men with NOA have no identified cause. These patients are classified as idiopathic NOA. There is some data suggesting that the majority of men with idiopathic NOA have a genetic cause. The development of newer genetic sequencing technologies have allowed researchers to gain a better understanding of the genes contributing to spermatogenesis and those potentially causing NOA. In the present work, I have investigated clinical, biochemical, genetic and histological findings in men with NOA. I recruited 101 participants with azoospermia, and 48 of this cohort were diagnosed with idiopathic NOA. The cohort included 19 men with known causes of NOA and 24 men with obstructive azoospermia. Whole exome sequencing was performed on blood samples of men with idiopathic NOA for identification of candidate variants. I also recorded the available clinical factors, hormone profiles and testicular histopathology for all participants. The analysis of the sequenced data from the 48 men with idiopathic NOA identified 11 novel candidate variants. There were seven novel candidate Loss of Function (LoF) variants in men with NOA (MEIOB c.1140_1143del; FKBP6 c.469−2A>T; ANKRD36B c.2715T>G; FAM47C c.1536del; PKD2L2 c.1690C>T; FAM122C, c.427C>T; MCMDC2 c.108del) and four missense variants (YBX2 c.200G>A; MEIOB c.988A>C, PKD2L2 c.1391C>T; MCMDC2 c.119A>G). Two of the LoF (MEIOB and FKBP6) and two of the missense variants (MEIOB and YBX2) have functional evidence to support their association with azoospermia. The identified genes in NOA subjects showed a testicular phenotype matching the genes expression site during spermatogenesis. There was a significant decrease in testicular volume (p= 0.0392) and a significant increase in serum FSH (p=0.0014) in men with NOA compared to men with obstructive azoospermia (OA). Additionally, data is showing that identified variants have testicular histology phenotypes matching their site of expression. FKBP6, MEIOB, PKD2L2, and MCMDC2 genes are normally expressed in spermatocytes. Patients identified with variants in these genes were found to have histology phenotypes of maturation arrest at spermatocyte level. This could potentially be used to predict the histology phenotypes without surgical intervention. I have identified novel genetic variants in men with idiopathic NOA but further studies are needed to confirm causality. Genetic analysis can potentially improve our understanding of fertility by identifying genes involved in spermatogenesis and novel variants in men with infertility.17 0