Biology subjects

Ray, P. F.

Publications and source records attributed to Ray, P. F..

3 recordsLinked to original sources

bioRxiv2023

Sperm motility restoration in mice suffering from oligo-astheno-teratozoospermia by in vivo injection and electroporation of naked mRNA

Oligo-astheno-teratozoospermia (OAT), a recurent cause of male infertility, is the most frequent disorder of spermatogenesis with a probable genetic cause. Patients and mice bearing mutations in the ARMC2 gene have a decreased sperm concentration, and individual sperm show multiple morphological defects and a lack of motility - a canonical OAT phenotype. Intracytoplasmic sperm injection (ICSI) is required to treat such a condition but it is associated with a small increase in birth defects in comparison to pregnancies not involving assisted conception. Consequently, new targeted treatments are needed to restore fertility. Here, a combination of in vivo injection and electroporation of capped and poly-A-tailed naked mRNA is tested as a strategy to treat ARMC2-related infertility in mouse. mRNAs coding for several reporter genes are tested and the efficiency and the kinetic of expression are assessed using in vivo and in vitro 2D and 3D imaging experiments. We show that mRNA-coded reporter proteins are detected for up to 3 weeks in germ cells, making the use of mRNA possible to treat infertility. We compare these results with those obtained with a non-integrative plasmid Enhanced Episomal Vector (EEV), which induces low and transient expression in spermatogenic cells. Consequently, injection and electroporation of naked mRNA-Armc2 into the testes of Armc2-deficient males were performed and we show the presence of normal and motile sperm in the epididymis. These motile sperm were able to produce embryos by IVF and ICSI. This study demonstrates, for the first time, that mRNA electroporation can restore sperm motility and partially fertilizing ability, providing a proof-of-concept for mRNA-based strategies to correct monogenic causes of male infertility and opening new avenues for male infertility treatment.

molecular biology↗

bioRxiv2023

Novel axonemal protein ZMYND12 is required for flagellum function and male fertility

Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66 and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous ZMYND12 variants. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

genetics↗

bioRxiv2023

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Genetic mutations are a recurrent cause of male infertility. Multiple morphological abnormalities of the flagellum (MMAF) syndrome is a heterogeneous genetic disease, with which more than 50 genes have been linked. Nevertheless, for 50% of patients with this condition, no genetic cause is identified. From a study of a cohort of 167 MMAF patients, pathogenic bi-allelic mutations were identified in the CCDC146 gene in two patients. This gene encodes a poorly characterized centrosomal protein which we studied in detail here. First, protein localization was studied in two cell lines. We confirmed the centrosomal localization in somatic cells and showed that the protein also presents multiple microtubule-related localizations during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To better understand the function of the protein at the sperm level, and the molecular pathogenesis of infertility associated with CCDC146 mutations, two genetically modified mouse models were created: a Ccdc146 knock-out (KO) and a knock-in (KI) expressing a HA-tagged CCDC146 protein. KO male mice were completely infertile, and sperm exhibited a phenotype identical to our two MMAF patients phenotype with CCDC146 mutations. No other pathology was observed, and the animals were viable. CCDC146 expression starts during late spermiogenesis, at the time of flagellum biogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets, suggest that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 affected the formation, localization and morphology of all microtubule-based organelles such as the manchette, the head-tail coupling apparatus (HTCA), and the axoneme. Through this study, we have characterized a new genetic cause of infertility, identified a new factor in the formation and/or structure of the sperm axoneme, and demonstrated that the CCDC146 protein plays several cellular roles, depending on the cell type and the stages in the cell cycle.

genetics↗