Deficiency in AK9 causes asthenozoospermia and male infertility by destabilising sperm nucleotide homeostasis

Abstract

Background: Asthenozoospermia is the primary cause of male infertility; however, its genetic aetiology remains poorly understood. Adenylate kinase 9 (AK9) is highly expressed in the testes of humans and mice and encodes a type of adenosine kinase that is functionally involved in cellular nucleotide homeostasis and energy metabolism. We aimed to assess whether AK9 is involved in asthenozoospermia.

Methods: One-hundred-and-sixty-five Chinese men with idiopathic asthenozoospermia were recruited. Whole-exome sequencing (WES) and Sanger sequencing were performed for genetic analyses. Papanicolaou staining, Haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were used to observe the sperm morphology and structure. Ak9-knockout mice were generated using CRISPR-Cas9. Sperm adenosine was detected by liquid chromatography-mass spectrometry. Targeted sperm metabolomics was performed. Intracytoplasmic sperm injection (ICSI) was used to treat patients.

Findings: We identified five patients harbouring bi-allelic AK9 mutations. Spermatozoa from men harbouring bi-allelic AK9 mutations have a decreased ability to sustain nucleotide homeostasis. Moreover, bi-allelic AK9 mutations inhibit glycolysis in sperm. Ak9-knockout male mice also presented similar phenotypes of asthenozoospermia. Interestingly, ICSI was effective in bi-allelic AK9 mutant patients in achieving good pregnancy outcomes.

Interpretation: Defects in AK9 induce asthenozoospermia with defects in nucleotide homeostasis and energy metabolism. This sterile phenotype could be rescued by ICSI.

Funding: The National Natural Science Foundation of China (82071697), Medical Innovation Project of Fujian Province (2020-CXB-051), open project of the NHC Key Laboratory of Male Reproduction and Genetics in Guangzhou (KF202004), Medical Research Foundation of Guangdong Province (A2021269), Guangdong Provincial Reproductive Science Institute Innovation Team grants (C-03), and Outstanding Young Talents Program of Capital Medical University (B2205).

Keywords: AK9; Asthenozoospermia; Energetic metabolism; Intracytoplasmic sperm injection; Nucleotide homeostasis.

Fig. 1

Pedigree-based identification of the bi-allelic AK9 mutations from five infertile patients with asthenozoospermia. (a–e) Pedigree chart of the five patients with asthenozoospermia affected by bi-allelic AK9 mutations. The black squares represent the affected individuals. Sanger sequencing results are shown below the pedigrees. The mutation positions are indicated by red arrows or red rectangles. (f) The location of the mutated base sites on the genome of AK9.

Fig. 2

Effect of mutant sites on the structure of the AK9 protein. (a) The position of the amino acid substitutions on the secondary structure of AK9. The dark green rectangles represent the “adenylate kinase” domain, the light green rectangles represent the “NMPbind” domain, and the orange rectangles represent the “LID” domain. (b) The three-dimensional structure of wild-type (WT) AK9 (AlphaFoldDB: AF-Q5TCS8-F1) protein. (c–d) Three-dimensional structure of AK9 residues after homozygous frameshift insertion mutations. (e–g) Local magnification of changes in amino acid residues between the WT and mutated AK9 protein in compound heterozygous patients.

Fig. 3

Morphology and ultrastructure of the spermatozoa from patients affected by bi-allelic AK9 mutations. (a) Morphological analysis of the spermatozoa from a control subject and the patients with bi-allelic AK9 mutations. Scale bar: 20 μm. (b) Field emission-scanning electron microscopy (FE-SEM) analysis of the morphological characteristics of the patients' spermatozoa. Scale bar: 5 μm. (c) Ultrastructural analysis of the spermatozoa flagella from the patients at the mid-piece. Scale bar: 100 μm. (d) Immunofluorescence analysis of AK9 expression in the patients’ spermatozoa flagella. Scale bar: 20 μm.

Fig. 4

Metabolite analysis of the spermatozoa from the patient F013/II-1 affected by a bi-allelic AK9 mutation. (a) Changes in the nucleotide metabolites in the spermatozoa from the AK9 mutated patient F013/II-1 compared with the control subjects (∗p < 0.05, ∗∗p < 0.01). (b) Changes in the glycolytic metabolites in the spermatozoa from the AK9 mutated patient F013/II-1 compared with the control subjects. (c) The percentages of β-ATP phosphoryl labelled with ¹⁸O in the spermatozoa from the AK9 mutated patient F013/II-1 compared with the control subjects. (d) Heat map of differentially expressed proteins in the spermatozoa from the AK9 mutated patient F013/II-1 compared with the control subjects. Orange represents high expression, and green represents low expression. (e) Signalling pathways involved in the downregulated expression of proteins in the spermatozoa from the AK9 mutated patient F013/II-1 compared with the control subjects. (f) Signalling pathways involved in the upregulated expression of proteins in the spermatozoa from AK9 mutated patient F013/II-1 compared with the control subjects. (g) Western blot confirmed the protein levels of proteomics analyses.

Fig. 5

The phenotype of the Ak9 knockout mice. (a) Morphological analysis of the sperm from wild-type (WT) and the Ak9 knockout (KO) mice. Scale bar: 10 μm. (b) Ultrastructural analysis at the mid-piece of the flagella from the Ak9 KO mice. Scale bar: 200 μm. (c) Ultrastructural analysis at the principal piece of the flagella from the Ak9 KO mice. Scale bar: 100 μm. (d) Percentages of motile spermatozoa in the cauda epididymidis from the wild-type and Ak9 KO mice. (e) Changes in the nucleotide metabolites in the spermatozoa from Ak9 KO mice compared with WT controls. (f) Changes in the glycolytic metabolites in the spermatozoa from Ak9 KO mice compared with WT controls. (g) The percentages of β-ATP phosphoryl labelled with ¹⁸O in the spermatozoa from the WT and Ak9 KO mice. (h) The percentages of the 2 PN rate (% of 2PN/total) and birth rate (% of live offspring/transferred embryos) of intracytoplasmic sperm injection (ICSI) of the WT and Ak9 KO mice. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig. 6

Morphology of embryogenesis for the implanted embryos. The embryogenesis of the AK9 mutated patients on day 1, day 2, and day 3.

Fig. 7

Schematic model of asthenozoospermia caused by AK9 deficiency.

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