Novel homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia owing to dynein arm complex and mitochondrial sheath defects in flagella

Lanlan Meng; Qiang Liu; Chen Tan; Xilin Xu; Wenbin He; Tongyao Hu; Chaofeng Tu; Yong Li; Juan Du; Qianjun Zhang; Guangxiu Lu; Li-Qing Fan; Ge Lin; Hongchuan Nie; Huan Zhang; Yue-Qiu Tan

doi:10.3389/fcell.2023.1184331

Novel homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia owing to dynein arm complex and mitochondrial sheath defects in flagella

Front Cell Dev Biol. 2023 Jun 1:11:1184331. doi: 10.3389/fcell.2023.1184331. eCollection 2023.

Authors

Lanlan Meng^{1

2}, Qiang Liu^{1

3}, Chen Tan¹, Xilin Xu^{2

4}, Wenbin He^{1

2}, Tongyao Hu¹, Chaofeng Tu^{1

2}, Yong Li¹, Juan Du^{1

2}, Qianjun Zhang^{1

2

4}, Guangxiu Lu^{1

2

4}, Li-Qing Fan^{1

2

4}, Ge Lin^{1

2

4}, Hongchuan Nie², Huan Zhang^{1

2}, Yue-Qiu Tan^{1

2

4}

Affiliations

¹ NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, School of Basic Medical Sciences, Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha, Hunan, China.
² Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China.
³ Hunan Cancer Hospital and the Affiliated Cancer of Xiangya School of Medicine, Central South University, Changsha, Hunan, China.
⁴ College of Life Science, Hunan Normal University, Changsha, Hunan, China.

Abstract

Introduction: Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia. Methods: A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by in silico analysis, and confirmed by in vitro experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy. Results and Discussion: Novel homozygous TTC12 variants (c.1467_1467delG (p.Asp490Thrfs*14), c.1139_1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using in silico prediction tools, and were further confirmed by in vitro functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from TTC12-mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology.

Keywords: TTC12; asthenoteratozoospermia; dynein arm complex defects; male infertility; mitochondrial sheath malformations.

Grants and funding

This work was supported by grants from the National Key Research and Development Program of China (2022YFC2702604), the National Natural Science Foundation of China (82101961, 82171608, 82201773, and 81971447), the China Postdoctoral Science Foundation (2022M711119), and the Graduate Research and Innovation Projects of Central South University and Hunan Province (2022ZZTS0022 and CX20220113).