男性不妊の遺伝的知見：TCTE1の役割

タイトル: Tcte1 knockout influence on energy chain transportation, apoptosis and spermatogenesis - implications for male infertility

概要: STUDY QUESTIONIs Tcte1 mutation causative for male infertility? SUMMARY ANSWERCollected data underline the complex and devastating effect of the single-gene mutation on testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADYLatest data revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity, and is built from DRC1-DRC7 proteins. DRC5 (TCTE1) - one of N-DRC element, has been already reported as a candidate for abnormal sperm flagella beating, however, only in restricted manner with no clear explanation of respective observations. STUDY DESIGN, SIZE, DURATIONUsing CRISPR/Cas9 genome editing technique, mouse knockout line of Tcte1 gene was created on the basis of C57Bl/6J strain. Then, the mouse reproductive potential, semen characteristics, testicular gene expression level, sperm ATP and testis apoptosis level measurements have been performed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n=248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS, SETTING, METHODSTo check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild type (WT - control group), heterozygous Tcte1+/-, and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) has been done for mice testis tissues. STRING interactions have been checked for protein-protein interactions, based on changed expression level of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the ATP amount in spermatozoa, the luminescence level was measured. Also, immunofluorescent in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n=137 non-obstructive azoospermia or cryptozoospermia, n=111 samples with spectrum of oligoasthenoteratozoospermia, including n=47 with asthenozoospermia) has been extracted to perform genomic sequencing (WGS, WES or Sanger). Protein prediction modeling of human identified variants and the exon 3 structure deleted in mouse knockout has been also performed. MAIN RESULTS AND THE ROLE OF CHANCENo progeny at all was found for homozygous males with revealed oligoasthenoteratozoospermia, while heterozygous animals (fertile) manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing, linked with apoptosis, or spermatogenesis. In Tcte1-/- males the protein revealed only residual amounts in sperm head nucleus, and was not transported to sperm flagella, as other N-DRC components. Decreased ATP level (2.4-fold lower) was found in spermatozoa of homozygous mice, together with disturbed tail:midpiece ratio, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at similar level in all three mouse genotypes. Mutation screening of human infertile males revealed 1 novel and 5 ultrarare heterogeneous variants (predicted as disease causing) in 6.05% of patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus, suggesting the disrupted TCTE1 interaction with its binding partners located within the axoneme. What does this mean for patients?Abnormal semen parameters (sperm count, motility and morphology) are known as one of the first symptoms that may be related to male fertility problems. This study aimed to determine the role of Tcte1 in male infertility using mouse knockout model. Tcte1 protein is one of the structural elements building N-DRC, a complex within an axoneme that is responsible for coordination of the sperm flagella elements activity, strictly related to sperm motility. We have found that mutations in mouse Tcte1 knockout model revealed two phenotypes, dependent on zygosity: infertile oligoasthenoteratozoospermic homozygotes, and fertile oligozoospermic heterozygotic males, suggesting haploinsufficiency mechanism. Pilot study on human samples with TCTE1 variants revealed a wide spectra of semen quality (from non-obstructive azoospermia, via cryptozoospermia, to severe oligoasthenozoospermia). Thus, TCTE1 gene is the next one that should be added to the male infertility list because of its crucial role in spermatogenesis influencing the variety of testicular molecular networks (incl. energy machinery processing) and proper sperm function.