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Understanding molecular mechanisms of male fertility and the link to motile cilia

Reference	BB/V011251/1
Principal Investigator / Supervisor	Professor Hannah Mitchison
Co-Investigators / Co-Supervisors	Professor Andrew Jarman, Dr Dagan Jenkins, Professor Christopher O'Callaghan, Dr Anu Sironen
Institution	University College London
Department	Institute of Child Health
Funding type	Research
Value (£)	919,457
Status	Current
Type	Research Grant
Start date	01/11/2021
End date	31/10/2024
Duration	36 months

Abstract

Our main focus is on cilia related reproductive pathways due to our experience in cilia biology, ciliate organisms and cell culture, in addition to our preliminary results and sample collections. We aim to identify cilio- and sperm flagellogenesis- specific molecular mechanisms underlying male fertility, by examining sperm defects and protein complexes in individuals with cilia related mutations and by investigating the role of male specific motile cilia in mouse mutants. Our preliminary results show that cilia-specific mutations cause extremely low sperm counts, suggesting the correct function of efferent duct (ED) cilia may be crucial for male fertility. We will establish the special characteristics of ED cilia (single cell RNA-Seq, miRNA-Seq) and the possible vesicular crosstalk between cilia and sperm, using Ift80 mutant mice with inhibited intraflagellar protein transport (IFT) that have dose-dependent decreases in male fertility. Effects on ED cilia vesicle content and secretion are studied by transcriptomics and structural microscopy (electron microscopy, immunofluorescence and super resolution). The effects of cilia immotility and malformed sperm tails on cilia/sperm interactions are studied using Dnah11 (cilia-specific dynein) and Tctex1d2 (IFT protein) mutant mice which have defective sperm tail formation, respectively. Dynein complexes will be investigated by protein pull downs and mass spectrometry of mutant human sperm and mouse ED cilia. To elucidate the molecular mechanisms involved in sperm development, we will examine the role of candidate genes identified in whole exome sequencing of azoospermic patients, using Drosophila gene silencing and functional characterization of spermatogenesis, focussing on cilia related genes for further study. The project results will inform us about the specificities (relative contribution, interactions) of male reproductive cilia and flagella and the biological mechanisms required for male germ cell development.

Summary

Reproductive success is crucial for the preservation of species and sustainable agriculture and food, in addition to being essential to human health. In human populations, poor sperm quality is a noteworthy vulnerability that explains around half of infertility cases but remains very little understood. In this study we propose to study the fundamental basis of male factor fertility in order to shed fresh light in this research area. We propose that there is a major but overlooked role of male-specific motile cilia in the production of healthy, fertile sperm. This proposal is focussed on characterizing new genetic factors in male fertility and exploring the balance of sperm and cilia requirements to develop and release healthy male reproductive cells (gametes). Cilia are hair-like organelles extending outside a cell and motile cilia are required in certain specialised areas of the body, for example in our airways beating of cilia lining the lung and upper airways are responsible for mucus flow and pathogen removal. The role of motile cilia in efferent ducts that are unique to male humans, is poorly understood. The efferent ducts are tubules that allow sperm made in the testis to be released into the ejaculatory duct via a structure called the epididymis. Sperm develop in the testis and are transported through the efferent ducts and epididymis, where they undergo maturation as they proceed, with growth of the sperm tail (flagella) giving them motility. Only after this transport do the sperm reach their full fertilizing capacity. In the testis (seminiferous tubules), male germ cells undergo several steps to become highly specialized spermatozoa. This process requires precisely timed gene expression and correct protein function in order to produce fertile sperm and ensure reproductive success. Malfunction of proteins at any time point during this process results in compromised sperm development. Therefore, we aim to characterize the function of genes identified in genetic screens of infertile male patients, using fly as a model organism because spermatogenesis is a conserved process with similar genes coding for proteins in fly and man. Our experiments will help us to define the link between genetics and male fertility in evolutionary conserved processes. The last phase of spermatogenesis involves formation of the sperm tail. The core structure of the motile cilia and sperm tail is almost identical, but with recently identified protein differences. These differences will be elucidated and the effect of specific genetic mutations on male fertility through sperm tail and/or efferent duct cilia functions characterized. To do this, we will examine the sperm quality and structure in patients a disease caused by mutations of the motile cilia in airways, as they have high male fertility that is not well characterised yet. The motility pattern of different cilia types (airway, efferent duct) and sperm are different and therefore we hypothesize that their motility producing complexes also differ. We will investigate motility-producing dynein composition and assembly, in sperm from humans with cilia mutations and in efferent duct cilia from mice with cilia mutations. We speculate that efferent duct cilia may interact with sperm through molecules they release in special packets (vesicles) and we aim to investigate the presence and role of this crosstalk in mouse mutants where the cilia specific protein transport is inhibited. Furthermore, we will develop a cell culture model to study the importance of cilia motility and other characteristics to better explore their role in production of fertile sperm. Overall, these experiments aim to clarify the molecular mechanisms underlying cilia related fertility and identify the influences of cilia versus sperm related mechanisms in successful sperm development in mammals.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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