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Human Genetics

, Volume 139, Issue 2, pp 257–271 | Cite as

Novel mutations in SPEF2 causing different defects between flagella and cilia bridge: the phenotypic link between MMAF and PCD

  • Chaofeng Tu
  • Hongchuan Nie
  • Lanlan Meng
  • Weili Wang
  • Haiyu Li
  • Shimin Yuan
  • Dehua Cheng
  • Wenbin He
  • Gang Liu
  • Juan Du
  • Fei Gong
  • Guangxiu Lu
  • Ge Lin
  • Qianjun ZhangEmail author
  • Yue-Qiu TanEmail author
Original Investigation

Abstract

Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that SPEF2 mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in SPEF2 (8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in SPEF2 were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a Spef2 knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female Spef2−/− mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of SPEF2 mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.

Notes

Acknowledgements

The authors would like to thank all families and individuals participated in this study. We are grateful to the excellent technical support provided by Junpu Wang, as well as support from the clinical and nursing staff at the Reproductive and Genetic Hospital of CITIC-Xiangya. We also want to thank Director Hong Luo and Dr. Ting Guo in the Department of Respiratory, Second Xiangya Hospital of Central South University for their generous help with PCD diagnosis, and Zhuoyao Guo and Weicheng Chen at the Department of Respiratory, Children's Hospital of Fudan University for their generous help with the high-speed video microscopy for tracheal epithelial cilia and oviduct cilia. We also acknowledge the support of the National Natural Science Foundation of China (81971447 and 81771645 to YQ.T), the National Key Research & Developmental Program of China (2018YFC1004900 to YQ.T), the science and technology major project of the ministry of science and technology of Hunan Province (2017SK1030 to YQ.T), and the China Postdoctoral Science Foundation Funded Project (2019M662786 to CF.T).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

439_2020_2110_MOESM1_ESM.pdf (266 kb)
Figure S1. Analysis for the mutation identified in family 1 and longitudinal ultrastructure of spermatozoa in the affected individuals.a Homozygosity mapping of individuals II-2 and II-3 from family 1. Homozygous regions with a remarkable signal are colored in red. The asterisk indicates the area where SPEF2 is located. b Upper panel: schematic representation of aberrant splicing by skipping SPEF2 exon 17 caused by the variant c.2507+5delG. Lower panel: splice mechanisms of SPEF2 exons associated with the variant c.2507+5delG. The variant leads to a premature termination codon (PTC) in exon 18 (p.A800E fs*3). c TEM photographs of longitudinal sections of SPEF2-mutant individuals. Compared to normal control, the principal piece of mutant sperm flagella (F1: II-3) appears without CPC or RS (red asterisk) and hyperplastic FS (red bracket). Short sperm tails embraced by cytoplasmic residuals (red arrow) with poorly assembled components were observed in the spermatozoa of SPEF2-mutant individuals (F4: II-1). CPC, central pair complex (red arrows); FS, fibrous sheath (blue arrows). Scale bars =1 µm
439_2020_2110_MOESM2_ESM.pdf (61 kb)
Figure S2. TUNEL staining of respiratory epithelia in SPEF2-mutant individual (F4: II-1). TUNEL staining of apoptotic cells in respiratory epithelia of normal control (NC), and SPEF2-mutant individual (F4: II-1). Red arrows indicate apoptotic germ cells (left). Scale bar = 25 μm
439_2020_2110_MOESM3_ESM.pdf (197 kb)
Figure S3. Generation of Spef2-knockout mice. a Gene-targeting construct for Spef2. b Sanger sequencing using PCR products of Spef2−/− mice revealed a large fragment deletion between introns 2-6. c RT-PCR confirmed the lack of exon 4 with primers amplifying exons 1-4, and a shorter product was found with primers amplifying exons 1-9. Subsequently, Sanger sequencing identified a shorter product that lacks exon 3-6. d-e SPEF2 staining is present in the respiratory cilia and whole-length sperm flagella of Spef2+/− mice but absent in that of Spef2−/− mice. Scale bars: (d) 25 μm; (e) 5 μm
439_2020_2110_MOESM4_ESM.pdf (137 kb)
Figure S4. Ultrastructure of spermatozoa in Spef2−/− mice.a-c: TEM photographs of longitudinal-sections of sperms in Spef2+/− (a) and Spef2−/− mice (b-c). Disorganized axoneme or short tails with cytoplasmic residuals containing multiple flagellar components, such as tubulin or fibrous-like elements (asterisk), were observed in the sperm flagella of Spef2−/− mice. Abbreviations: Ax, axoneme; Mi, mitochondria (blue arrow) and FS, fibrous sheath (green arrow). Scale bars for the longitudinal sections =250 nm
439_2020_2110_MOESM5_ESM.pdf (242 kb)
Figure S5. Haploid sperm differentiation was destroyed at stage XI and XII in Spef2−/− mice during spermiogenesis. a H & E staining of cross-sections of testes from Spef2+/− and Spef2−/− mice at 2 months old. A major difference was detected at stages XI and XII during spermiogenesis with a constricted head shape, where sperm tails were absent (asterisk) in the tubular lumen of Spef2−/− mice. Numerous highly condensed nuclei (red arrow) in the seminiferous epithelium at stage IX-XII were observed in Spef2−/− mice, seemingly to be phagocytosed by Sertoli cells. St, Sertoli; Sg, spermatogonia; P, pachytene spermatocyte; RS, round spermatid; ES, elongated spermatid. Scale bar represent 20 μm. b TUNEL staining of apoptotic cells in Spef2+/− and Spef2−/− mice testis sections. Red arrowheads indicate apoptotic germ cells (left). Quantification of apoptotic cells in Spef2+/− and Spef2−/− mice testes (right). Apoptotic cells were counted in 30 random seminiferous tubules. Scale bar = 50 μm. ***p < 0.001
439_2020_2110_MOESM6_ESM.doc (114 kb)
Supplementary file6 (DOC 114 kb)
439_2020_2110_MOESM7_ESM.mp4 (240 kb)
Movie S1. Analysis of high-speed video microscopy imaging of Spef2+/− mice tracheal epithelial cilia. Cilium beat frequency (CBF) is calculated in beats per second (Hz)
439_2020_2110_MOESM8_ESM.mp4 (820 kb)
Movie S2. Analysis of high-speed video microscopy imaging of Spef2−/− mice tracheal epithelial cilia. Cilium beat frequency (CBF) is calculated in beats per second (Hz)
439_2020_2110_MOESM9_ESM.mp4 (1.4 mb)
Movie S3. Analysis of high-speed video microscopy imaging of Spef2+/− mice oviduct cilia. Cilium beat frequency (CBF) is calculated in beats per second (Hz)
439_2020_2110_MOESM10_ESM.mp4 (1.8 mb)
Movie S4. Analysis of high-speed video microscopy imaging of Spef2−/− mice oviduct cilia. Cilium beat frequency (CBF) is calculated in beats per second (Hz)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Institute of Reproductive and Stem Cell Engineering, School of Basic Medical ScienceCentral South UniversityChangshaChina
  2. 2.Reproductive and Genetic Hospital of CITIC-XiangyaChangshaChina
  3. 3.National Engineering and Research Center of Human Stem CellChangshaChina
  4. 4.Key Lab of MOE for Development Biology and Protein Chemistry, The Center for Heart Development, College of Life SciencesHunan Normal UniversityChangshaChina

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