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

Abstract

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 (n = 248) 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 Tcte1^+/- 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 (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 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 interests: 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.

Keywords: Fetub; MMAF; N-DRC; TCTE1; haploinsufficiency; male infertility; mt-Co2; oligoasthenoteratozoospermia; sperm mitochondria; sperm motility.

Figure 1.

Characteristics of the Tcte1 gene. (A) Putative homologs of the Tcte1 gene conserved in Bilateria, according to HomoloGene (8434), including collation of proteins and their conserved domains. (B) Sequence alignment between human, mouse, and rat. (C) NCBI Gene schematic representation of the Tcte1 gene in Mus musculus.

Figure 2.

Characteristics of Tcte1 knockout mice. (A) Schematic representation of gRNA localization in CRISPR/Cas9 KO creation. (B) Relative expression of the Tcte1 gene in wild-type (WT), hetero- ^(+/−) and homozygous mice ^(−/−) (Real-time PCR); **** P < 0.0001, ** P < 0.01. (C) Example of genotyping result. Lane 1: 1 kb marker (GeneRuler), lanes 2, 3: wild-type (WT), lanes 4, 5: hetero- ^(+/−), and lanes 6, 7: homozygous mice ^(−/−); lanes: 2, 4, 6 identified the lack (0 bp) or presence of mutation (630 bp), lanes: 3, 5, 7 differentiated hetero- (658 bp) and homozygotes (0 bp).

Figure 3.

Reproductive potential of KO Tcte1 mice. Mating combinations and their results including time of breeding, number of litters and pups, number of male and female pups, and genotypes observed in pups of each combination. (A) Mean time: from mating to first pedigree, and between particular litters. (B) Ratio of males and females per litter in each mating combination. (C) Frequency of males and females and their genotype observed each mating combination; left panel: according to the sex of pups; right panel: according to mating combinations. (D) Ratio of males and females per litter and their genotype observed in each mating combination. ***P < 0.0001, **0.0001 < P < 0.01, *0.01 < P < 0.05.

Figure 4.

Characteristics of Tcte1 wild-type (WT), hetero- ^{(+/ − )} and homozygous mice ^{( − / − )} . (A) Histopathology of testes and epididymis (caput); staining: classic hematoxylin–eosin staining (testis) or Masson–Goldner protocol (epididymis); microscope: Leica DM5500, objective 40$\times$, LASX software with Navigator function. (B) Comparison of body mass, testis weight and size, and epididymis length, followed by gross morphology of gonads (n: no. of mice analyzed). (C) Comparison of the dimensions of testicular tubule sections (n: no. of sections analyzed); ***P < 0.0001, **0.0001 < P < 0.01, *0.01 < P < 0.05.

Figure 5.

Evaluation of germ cell types in testes of Tcte1 wild-type (WT), heterozygous ^{(+/ − )} and homozygous ^{( − / − )} mice. Three germ cell types were evaluated: spermatogonia (stained with intensive green color), preleptotene spermatocytes (intensive red color), and pachytene spermatocytes (medium green and red). Immunofluorescence staining with antibodies: primary: mouse anti-MLH1 (Abcam, Cambridge, UK, cat. no. ab14206), 1:30, rabbit anti-RAD51 (Abcam, Cambridge, UK, cat. no. ab63801), 1:200; secondary: goat anti-mouse-FITC (Sigma Aldrich, St. Louis, MO, USA, cat. no. F2012), 1:400 and goat anti-rabbit-AF594 (Abcam, Cambridge, UK, cat. no. 150080), 1:500.

Figure 6.

Characteristics of spermatozoa of Tcte1 wild-type (WT), heterozygous ^{(+/ − )} and homozygous ^{( − / − )} mice. (A) Sperm parameters: concentration, motility, and morphology comparisons; statistical marks indicate differences according to WT animals; n: number of mice. (B) Frequency of morphological defects according to three parts of the spermatozoa. (C) Examples of the main sperm morphological abnormalities found (all abnormalities observed available in Supplementary Data File S4); Leica DM5500 microscope, objective 63×, CytoVision software. (D) Comparison of the mean length of sperm tail, midpiece and ratio tail:midpiece; n: no. of animals, each with at least 100 spermatozoa analyzed; ***P < 0.0001, **0.0001 < P < 0.01, *0.01 < P < 0.05.

Figure 7.

RNAseq results of gene expression levels found in testes of KO Tcte1^{+/ −} and Tcte1 ^{− / −} mice versus control (WT) mice. Volcano plots: black dots indicate all analyzed genes within the genome, red dots represent genes with significantly changed expression levels. A heatmap and a table describing the genes are also shown. Filtering criteria: P <0.05, log ratio >1 or <−1 (adequate to fold change value of 2 (expression level increased) or 0.5 (expression level decreased)), expression level >8. nd: no data concerning function or disease.

Figure 8.

STRING analyses of potential interactions between proteins with observed differences in gene expression level in RNAseq from mouse testis of the KO model for Tcte1 gene. Genes revealed by the RNAseq are bracketed. (STRING database, 24 February 2021).

Figure 9.

Immunolocalization of the nexin–dynein regulatory complex (N-DRC) proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) within mouse sperm cells. (A) Localization of Tcte1 in spermatozoa from wild-type (WT), hetero- (^+/−) and homozygous (^−/−) animals. (B) Localization of other proteins (Drc7, Fbxl13, Eps8l1) building the nexin–dynein regulatory complex (N-DRC), responsible for the coordination of the dynein arm activity and stabilization of the doublet microtubules attachment, in wild-type (WT) spermatozoa. Antibodies: primary (all anti-rabbit 1:100; Biorbyt, Cambridge, UK): Tcte1 (orb357083); Drc7 (orb58695); Fbxl13 (orb678278); Eps8l1 (orb382538); secondary: goat anti-rabbit-AF594 conjugated, 1:500, ab150160, Abcam, Cambridge, UK. Fluorescence microscope: Leica DM5500, filters: DAPI, TxR, FITC, BGR, magnification 630$\times$ (with immersion); software: LASX or CytoVision. Bar represents 10 µm.

Figure 10.

ATP levels in wild-type (WT), heterozygous (^{+/ −} ) and homozygous ( ^{− / −} ) Tcte1 mice. Measurements made at two time points (0 and +60 min) showed a decrease of ∼35% in luminescence in all three groups over time. Mean measured values in the homozygous group were ∼2.4-fold lower. Dots: particular animals; long dash: mean value for the group; short dashes, standard deviation; RLU, relative luminescence unit. ***P < 0.0001, **0.0001 < P < 0.01.

Figure 11.

Immunolocalization of caspase 3 and TUNEL assay results within mouse testicular tissue of the KO Tcte1 model. Caspase 3 (A) is one of the apoptotic indicators because of its engagement in the induction, transduction, and amplification of intracellular apoptotic signals. Caspase 3 signals were found only in spermatogonia of all three genotypes (wild-type (WT), hetero- (^+/−), and homozygous (^−/−)). No differences between the level of fluorescent signals were found between evaluated mouse genotypes, indicating similar levels of apoptosis. Antibodies: primary (1:100) rabbit anti-Casp3 (Biorbyt, Cambridge, UK); secondary (1:500): goat anti-rabbit-AF594 conjugated (ab150160, Abcam, Cambridge, UK). (B) TUNEL assay revealed no differences between zygosities (positive BrdU signal). Kit used: ab66110 TUNEL Assay Kit: BrdU-Red; Abcam, Cambridge, UK. Fluorescent microscope: Leica DM5500, filters: DAPI, TxR, magnification 630$\times$ (with immersion); software: LASX. Bar represents 5 µm. Analyzed: three independent males per zygosity, on 15–20 tubules each.

Figure 12.

Homology modeling of TCTE1. (A) Secondary structure diagram and associated confidence scheme generated by I-TASSER (Roy et al., 2010; Yang et al., 2015; Yang and Zhang, 2015], Phyre2 (Kelley et al., 2015], and AlphaFold intensive mode. Positions of amino acids of interest are indicated. Green, predicted unstructured regions; yellow rectangles, predicted helices; orange arrows and arrowheads, predicted strands. The confidence scale ranges from white to cyan, with the probability of the secondary structure increasing with increasing intensity of cyan. (B) Cartoon diagram of the homology model of human TCTE1 generated by AlphaFold with residues of interest shown as spheres and labeled. N, amino terminus. C, carboxy terminus. (C–F) Detailed views of indicated residues and mutations: (C) Ile-125 resides deep within a hydrophobic pocket, which would be disturbed upon substitution with arginine. (D) Mutation R133C would impact the hydrogen bond interactions that organize the architecture of the globular domain N-terminal to the tandem leucine-rich repeats. (E) Mutation of Glu-157 to alanine would destabilize the charge of the surface of this globular region, likely leading to unfolding. (F) Gly-350 allows for the compact interactions of tandem helices of the LRR. Depending on the rotamer it adopted, a serine side chain in this position would sterically clash with residues D321, A324, or V346 in the neighboring helix.

Figure 13.

Summary of findings revealed for abnormal sperm tail beating in the mouse knockout model of the Tcte1 gene, a component of the N-DRC complex in the sperm tail. Three major groups of factors required for proper sperm tail beating are marked with bold color arrows (green, yellow, and blue). Documented connections (solid grey arrows) were revealed on the basis of RNAseq data for testis tissues, protein in silico predictions for mutated Tcte1 protein, and measurements of ATP level and sperm cell components in mouse spermatozoa. Unresolved possible linkages (cause/effect) are marked with a question (?) mark.