Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility?
SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular
molecular network, leading to male reproductive failure.
WHAT IS KNOWN ALREADY: Recent data have 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 the DRC1–DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a
candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective
observations.
STUDY DESIGN, SIZE, DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created
on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm
ATP, and testis apoptosis level measurements were then assessed, 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 (n¼248) was applied for screening
of TCTE1 variants.
PARTICIPANTS/MATERIALS, SETTING, METHODS: To check the reproductive potential of KO mice, adult animals were crossed for
delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were
performed for wild-type (WT, control group), heterozygous Tcte1þ/− and homozygous Tcte1−/− male mice. Gross anatomy was
performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice
testis tissues. STRING interactions were checked for protein–protein interactions, based on changed expression levels 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 amount of
ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence 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 (n¼137
with non-obstructive azoospermia or cryptozoospermia, n¼111 samples with a spectrum of oligoasthenoteratozoospermia,
including n¼47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction
modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed.
MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have
oligoasthenoteratozoospermia, while heterozygous animals were fertile but 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 or linked with apoptosis or spermatogenesis. In Tcte1−/− males, the protein was
revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC
components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:
midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia
only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five
ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the 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 disrupted interactions of TCTE1 with its binding partners located within the axoneme.
LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request.
LIMITATIONS, REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked.
WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the ‘male infertility list’ because of its crucial role in spermatogenesis and proper sperm functioning.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/ NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.