Bi-allelic variants in human TCTE1/DRC5 cause asthenospermia and male infertility

Abstract

Asthenozoospermia (AZS) is a common male infertility phenotype, accounting for 18% of infertile patients. The N-DRC (Nexin-dynein Regulatory Complex) complex is the motor regulating device in the flagellum, which is found in most eukaryotic organisms with flagellum. The deletion of TCTE1 (T-Complex-Associated Testis-Expressed 1), a component of the N-DRC complex also known as DRC5 (Dynein regulatory complex subunit 5), has been shown to cause asthenospermia in mice. This study mainly introduces a clinical case of male infertility with normal sperm count, normal morphological structure, but low motility and weak forward movement. By whole-exome sequencing, we found that TCTE1 became a frameshift mutant, ENST00000371505.5: c.396_397insTC (p.Arg133Serfs*33), resulting in the rapid degradation of TCTE1 protein and male infertility. This phenotype is similar to the Tcte1^-/- (Tcte1 knockout) mice, which showed structural integrity but reduced motility. Further, different from mice, in vitro Fertilization (IVF) could successfully solve the patient's problem of infertility. Our data provides a better understanding of the biological functions of TCTE1 in human flagellum assembly and male fertility.

Fig. 1. Schematic representation of the mutated TCTE1 variant and its expression profile.

A The sterile patient pedigree. B Sanger sequencing confirming the TCTE1 variant, c.396_397ins TC. The proband harbored the homozygous variant. The patient’s parents possessed the heterozygous allele, whereas his elder sister carried wild-type alleles. The variant located is represented by a black arrow and the inserted bases are highlighted with a red box. C the TCTE1 antibody could specifically identify the overexpressed TCTE1-FLAG fusion protein; β-TUB, β-TUBULIN. D TCTE1 is missing in spermatozoa from the patient. E Cycloheximide exposure and subsequent Western Blot analysis illustrating the rapid degradation of the truncated TCTE1 protein, expressed by the TCTE1^TCins-FLAG vector, in relation to the control TCTE1 protein, expressed by the TCTE1-FLAG vector at time points 0 min, 15 min, and 30 min after dosing.

Fig. 2. Morphological observations of the patient sperm using TEM and immunofluorescence.

A A cross-sectional model of the axonemal components of the sperm flagellum; N-DRC, nexin-dynein regulatory complex; CPC, central-pair complex; ODA, out dynein arm; IDA, inner dynein arm; At, A-tubule; Bt, B-tubule; Rs2, radial spoke 2. B Immunofluorescent evaluation of acetylated-TUBULIN (green), Peanut arachis hypogaea agglutinin (PNA, red), and Hoechst (blue) in control and patient sperms; scale bar = 10 μm. C The flagellar cross sectional structure of control human and patient using TEM. The N-DRC complex is represented by a red arrow; scale bar = 200 nm.

Fig. 3. Expression and localization of N-DRC components in the human and mice sperm flagellum.

A–F Immunofluorescence depicting different DRCs’ locations on the human sperm flagella. TCTE1/DRC5 was not detected in the patient sample (A). However, DRC1 (B), CCDC65/DRC2 (C), DRC3(D), GAS8/DRC4(E), and DRC7 (F) were detected. DRCs (red), acetylated-TUBULIN (green), and Hoechst (blue); scale bar = 10 μm. G Western Blot evaluation of mice sperm proteins. DRC1, CCDC65/DRC2, DRC3, GAS8/DRC4, and DRC7 were expressed, but not TCTE1/DRC5.

Fig. 4. Mice IVF experiment and movement analysis.

A, B Mice IVF assessment. Images of egg fertilization with spermatozoa from wild-type and Tcte1^−/− mice; scale bar = 500 μm. A Column chart depicting the drastically low fertilization rates of Tcte1^−/− mice, as compared to control mice (n = 5) (B). C The Bohboh analysis software revealing the movement mode of spermatozoa from both wild-type and Tcte1^−/− mice.

Fig. 5. Morphological observation of brain and tracheal cilia in mice.

A The respiratory epithelial cells were dual-stained with an antibody marker specific for the ciliary axoneme (acetylated-tubulin, green) and TCTE1. The TCTE1 could be detected in the Tcte1^+/+ group, while no signal was evident in Tcte1^−/− samples. B TCTE1 expression could be detected in the Tcte1^+/+ respiratory epithelial cells using Western blot. C Coronal mice brain sections stained with HE. Lateral ventricles (LV), third ventricles (3 V), and fourth ventricles (4 V) are marked in corresponding positions on the images; scale bar = 1 mm. D Immunofluorescent evaluation of acetylated-tubulin (green) and Hoechst (blue) in the tracheal ciliated columnar epithelial cells; scale bar = 10 μm. (E) Harvested respiratory cilia length evaluation from wild-type and Tcte1^−/− mice. Each dot represents the average cilia length of one analyzed specimen (n = 71 vs. 68 cells). AC-TUB, acetylated-TUBULIN; ns, no significance.