The zona-free eggs were rapidly washed five times and?kept at 37C under 5% CO2 in air flow for 2 to 3 3?h to recover their fertilization ability. Capacitated sperm preparation: mouse spermatozoa were obtained from the cauda epididymides of C57BL6/J male mice (8- to 10-week-old) and capacitated at 37C under 5% CO2 for 90?min in a 500?L drop Ferticult medium supplemented with 3% BSA, under mineral oil. fertilization: cumulus-intact and zona-free eggs were inseminated with capacitated spermatozoa for 3?h in a 100?L drop of Ferticult medium 3% BSA at a final concentration of 106 or 105 per mL, respectively. in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we recognized bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and recognized five individuals with homozygous truncating variants in loss-of-function models in the flagellated protist and in the TPR structural motifs, highly conserved between the analyzed orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that mutations are a cause of male sterility due to MMAF. (MIM: 603332),14, 15, 16 (MIM: 603333),17 (MIM: 617558),18,19 (MIM: 617559),18, 19, 20 (MIM: 617949),21 (MIM: 618146),22,23 (MIM: 615796),24 (MIM: 618424),25 (MIM: 618304),26 (MIM: 611430),27 and (MIM: 610172)28 in unrelated MMAF-affected subjects. AMG 487 S-enantiomer In addition, mutations in (MIM: 614270),19 (MIM: 611423),29 and (MIM: 615364)30 were reported in single familial MMAF-affected case subjects. With the aim to identify additional genetic causes of human asthenozoospermia related to MMAF, we further analyzed whole exome sequencing data from a cohort of 167 MMAF individuals previously established by our team25 and statement the identification and characterization of bi-allelic truncating mutations in five unrelated individuals. In addition, by performing studies, using and mutant models, we demonstrate that TTC29 is a conserved axonemal protein required for correct flagellar beating and motility in three evolutionary distant species. Material and Methods Study Participants and Whole-Exome Sequencing (WES) We analyzed data obtained by WES performed for a total of 167 men affected by main infertility associated with a MMAF phenotype.25 WES and bioinformatics analyses were performed according to our previously explained protocol using the human genome assembly GRCh38 as a reference sequence.18 All the recruited individuals displayed isolated infertility with no other clinical features; in particular, CD244 main ciliary dyskinesia (PCD) syndrome was excluded. In this cohort, 83 individuals originated from North Africa (mainly from Algeria, Libya, and Tunisia) and sought consultation for main infertility at the Clinique des Jasmins in Tunis, 52 individuals originated from the Middle East (Iran) and were treated in Tehran at the Royan Institute (Reproductive Biomedicine Research Center) for main infertility, and 32 individuals were recruited in France, mainly at the Reproductive Department at Cochin Hospital in Paris. All individuals presented with a typical MMAF phenotype, which is characterized by severe asthenozoospermia (total sperm motility below?10%; normal value over 40% according to the World Health Organization research values,6 in association with increased level of the following sperm flagellar abnormalitiesshort, absent, coiled, bent, or irregular flagellain comparison with the normal ranges AMG 487 S-enantiomer observed in control fertile individuals13). Informed consent was obtained from all the individuals participating in the study according to local protocols and the principles of the Declaration of Helsinki. The study was approved by local ethics committees, and samples were then stored in the CRB Germethque (certification under ISO-9001 and NF-S 96-900) according to a standardized process or were part of the Fertithque collection declared to the French Ministry of Health (DC-2015-2580) and the French Data Protection Expert (DR-2016-392). Sanger Sequencing The selected mutations in were validated by Sanger sequencing performed on ABI 3130XL (Applied Biosystems); analyses were performed using SeqScape software (Applied Biosystems). Sequences of primers used and expected product sizes are summarized in Table S2. Semen Analysis Semen samples were obtained by masturbation after a period of 2 to 7?days of sexual abstinence. Semen samples AMG 487 S-enantiomer were incubated at 37C for 30?min for liquefaction; ejaculate volume and pH, sperm concentration, vitality, morphology, and motility were evaluated according to World Health Business (WHO) guidelines.6 Sperm vitality was assessed by eosin staining, and sperm morphology was analyzed on Schorr stained semen smears according to Davids classification.31 Transmission Electron Microscopy Analysis of Sperm Cells Human AMG 487 S-enantiomer or mouse sperm cells (10 millions) were fixed by incubation in 0.1?M phosphate buffer (pH 7) supplemented with 3% glutaraldehyde (Grade I; Sigma-Aldrich) for 2?h at room temperature. The samples were washed twice in PBS and resuspended in 0.2?M sodium cacodylate buffer. The samples were.