In a recent study published within the eBioMedicine journal, researchers investigated the genetic underpinning of asthenozoospermia, the leading explanation for male fertility.
Their multidisciplinary examinations were capable of discover adenylate kinase 9 (AK9), an enzyme involved in sperm energy metabolism and cellular nucleotide homeostasis, as essential to fertilization by enabling sperm to swim toward the ovum even in sugar-free media.
Mutations within the AK9 encoding gene were found to cause male infertility, each in murine models and human study participants. While these genetic mutations are life-long, the team discovered that intracytoplasmic sperm injections (ICSI) were capable of rescue afflicted patients from infertility, thereby curing the condition.
Male infertility and asthenozoospermia
Infertility, the shortcoming to conceive even after a yr of frequent unprotected sex, is a typical condition affecting roughly 15% of all couples of childbearing age.
Research suggests that infertility might be brought on by quite a few aspects, including genetics, food regimen, mental well-being, and, especially in women, age. Almost half of all cases of infertility might be attributed to men, with asthenozoospermia being the leading explanation for sterility.
Asthenozoospermia, sometimes called asthenospermia, is a condition wherein sperm motility is severely impaired, making otherwise fertile sperm incapable of successfully reaching the feminine ovum for conception.
Previous studies have identified genetic contributors to asthenozoospermia, including the A-kinase anchoring protein (AKAP), human tRNAGlu (TTC), the dynein axonemal heavy chain (DNAH), and the cilia and flagella associated (CFAP) gene families. Unfortunately, the genetic etiology and molecular pathogenesis underpinning asthenozoospermia remain poorly understood.
Sperm motility is entirely as a consequence of the beating of sperm flagella, an energy-driven process. Adenosine triphosphate (ATP) is the first source of this energy, produced by the flagellum via glycolysis and by the sperm mitochondria via oxidative phosphorylation.
Investigations in male mice models have shown that alterations to any of the aforementioned gene families in sperm end in severe impairment to energy generation and, in turn, sperm motility, leading to infertility and the asthenozoospermia phenotype.
Despite being present in each invertebrates and vertebrates, mammalian sperm is special since it stays motile even within the presence of glycolysis inhibitors, suggesting that along with glycolysis and oxidative phosphorylation, mammalian sperm motility is regulated by other poorly understood energy metabolisms.
Adenylate kinases (Aks) have been suggested to satisfy this role by transferring phosphate groups to adenosine diphosphate (ADP), thereby producing ATP for flagellar use. Nine Aks have been hitherto identified (AK1-AK9), all of which have some role in sperm motility but don’t otherwise affect fertility.
Notably, AK9 is very expressed within the human testis and is involved in maintaining the homeostasis of cellular nucleotides by [catalyzing] the interconversion of nucleoside phosphates. Nevertheless, due to the lack of selective AK inhibitors, the physiological effect of AK9 in sperm and its role within the nucleotide homeostasis and energy metabolism has not been fully uncovered.
Sha et al. (2023)
In regards to the study
In the current study, researchers used a multidisciplinary approach to discover the genetic etiology and molecular pathogenesis of asthenozoospermia, with a concentrate on the results of mutations within the AK9 gene and its encoded AKD2 protein. They used evidence from human asthenozoospermia patients and knockout AK9 (Ak9 KO) mice to discover the role of the gene in affecting sperm motility.
Human recruitment for this study was conducted on the Women and Kid’s Hospital of Xiamen University. 100 and sixty-five Chinese men presenting idiopathic asthenozoospermia (cases) and 200 men with normal fertility (controls) were enrolled. Preliminary tests revealed that in all physical and semen parameters except sperm motility, case and control cohorts were clinically equivalent. Case cohorts presented reduced sperm motility starting from 0 to 32%.
To elucidate genetic mutations involved with the AK9 gene and their impacts on motility, whole-exome sequencing (WES) of participant DNA was carried out. Sanger sequencing of identified mutant genotypes was used for fine-scale data generation.
Wild-type (WT) and mutant AK9 protein structures were predicted and visualized using the AlphaFold database and UCSF Chimera tool, respectively. Semen characteristics analyses of sperm from each cohorts were undertaken as prescribed by the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (5th edition).
The Clustered Commonly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) endonuclease (Cas9) (CRISPR-Cas9) technology was used to construct male Ak9 KO mice, murine representatives of the asthenozoospermia phenotype. Electron microscopy (each scanning [SEM] and transmission [TEM]) of human and murine sperm was undertaken for spermatozoa ultrastructure visualization.
Immunofluorescence and western blotting assays were employed to discover AK9/AKD2 protein concentrations in sperm samples. The sperm chromatin structure assay (SCSA) and flow cytometry were used for sperm DNA stainability and fragmentation detection. Excised Ak9 KO mice testicles were then stained using hematoxylin and eosin dyes to visualise the testes’ structure.
Liquid chromatography-mass spectrometry (LC/MS) was used to detect and characterize sperm adenosines and measure phosphotransfer rates. In tandem with flow cytometry, the mitochondrial membrane potential (MMP) assay kit detected the mitochondrial membrane potential of sperm samples.
Finally, for men identified as having AK9 mutations as their causes of infertility and asthenozoospermia, intracytoplasmic sperm injection (ICSI), a process via which sperm from a donor is extracted and directly inserted into an ovum, was carried out.
Study findings
Of the 165 case males presenting the idiopathic asthenozoospermia phenotype, genetic evaluation revealed five with bi-allelic mutations of their AK9 gene. Of those, two presented homozygous mutations and were found to be from unrelated consanguineous families.
One had a homozygous frameshift insertion mutation, while the opposite had a unique homozygous frameshift insertion mutation, a heterozygous non-frameshift deletion mutation, and a stop-loss mutation.
In silico evaluation of the human AK9 transcript revealed that each the above-referenced mutations significantly altered the three-dimensional structure of the conventional AK9/AKD2 protein. Cross-referencing these obtained structures against the ExAC, 1000 Genomes Project, gnomAD _exome (All), and GnomAD _exome (East Asian) databases revealed that these mutations were absent or rare across a sizeable global populace, suggesting that the AK9 gene is very conserved.
Taken together, these findings imply that AK9 gene mutations are inherited from parental heterozygous carriers following Mendelian patterns, and the mutations are autosomally recessive.
Comparisons of physical, secondary sexual, and, surprisingly, sperm morphology characteristics from case and control cohorts for each humans and mice revealed that phenotypes were equivalent between cohorts. SEM and TEM images revealed that AK9 WT and mutants were indistinguishable from one another even on the ultrastructure level.
Targeted metabolomic evaluation revealed, nonetheless, that functionally, AK9-deficient individuals showed significantly reduced AMP and ADP levels in comparison with their WT counterparts.
On account of the unique role of AK in-phosphotransfer, we evaluated the phosphoryl moiety in ATP. Surprisingly, O-labelled-ATP was significantly reduced, indicating insufficient AK-catalysed phosphotransfer within the sperm of patients with AK9 deficiency. These results suggest that bi-allelic mutations in AK9 disrupt glycolytic metabolic homeostasis and inhibit AK-catalysed phosphotransfer in human sperm.
Sha et al. (2023)
Mass spectrometry analyses of AK9 mutated sperm elucidated that 211 proteins were upregulated, and 195 proteins were downregulated in mutant sperm in comparison with WT. Gene ontology analyses of those differentially expressed proteins revealed that they’re involved in energy production and conversion, carbohydrate transport and metabolism, secondary metabolite biosynthesis, signal transduction, and catabolism.
Finally, ICSI performed on each mice asthenozoospermia models and human patients showed successful results. Three of the five human patients with asthenozoospermia participated within the study, with all cases leading to a successful pregnancy and the delivery of healthy babies.
This highlights that AK9 affects only sperm motility and energy modalities but doesn’t alter the fertilization ability of the spermatozoa. ICSI can thus be utilized in future clinical trials as a rescue from male infertility for couples whose male partner has mutations in his AK9 gene.
Conclusions
In the current study, researchers investigated the genetic etiology and molecular pathogenesis of asthenozoospermia, the first explanation for male infertility.
Their results highlight that mutations within the AK9 gene severely reduce sperm motility and alter protein expression while leaving sperm structure and fertility unchanged. Their findings highlight the genetic underpinnings of the condition and present ICSI as a possible rescue for couples wherein the male partner suffers from asthenozoospermia.