The core axoneme structure of both the motile cilium and sperm tail has the same ultrastructural 9 + 2 microtubular arrangement. Thus, it can be expected that genetic defects in motile cilia also have an effect on sperm tail formation. However, recent studies in human patients, animal models and model organisms have indicated that there are differences in components of specific structures within the cilia and sperm tail axonemes. Primary ciliary dyskinesia (PCD) is a genetic disease with symptoms caused by malfunction of motile cilia such as chronic nasal discharge, ear, nose and chest infections and pulmonary disease (bronchiectasis). Half of the patients also have situs inversus and in many cases male infertility has been reported. PCD genes have a role in motile cilia biogenesis, structure and function. To date mutations in over 40 genes have been identified cause PCD, but the exact effect of these mutations on spermatogenesis is poorly understood. Furthermore, mutations in several additional axonemal genes have recently been identified to cause a sperm-specific phenotype, termed multiple morphological abnormalities of the sperm flagella (MMAF). In this review, we discuss the association of PCD genes and other axonemal genes with male infertility, drawing particular attention to possible differences between their functions in motile cilia and sperm tails.

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Sperm defects in primary ciliary dyskinesia and related causes of male infertility.

Motile cilia and sperm flagella share an evolutionarily conserved axonemal structure. Their structural and/or functional defects are associated with primary ciliary dyskinesia (PCD), a genetic disease characterized by chronic respiratory-tract infections and in which most males are infertile due to asthenozoospermia. Among the well-characterized axonemal protein complexes, the outer dynein arms (ODAs), through ATPase activity of their heavy chains (HCs), play a major role for cilia and flagella beating. However, the contribution of the different HCs (γ-type: DNAH5 and DNAH8 and β-type: DNAH9, DNAH11, and DNAH17) in ODAs from both organelles is unknown. By analyzing five male individuals who consulted for isolated infertility and displayed a loss of ODAs in their sperm cells but not in their respiratory cells, we identified bi-allelic mutations in DNAH17. The isolated infertility phenotype prompted us to compare the protein composition of ODAs in the sperm and ciliary axonemes from control individuals. We show that DNAH17 and DNAH8, but not DNAH5, DNAH9, or DNAH11, colocalize with α-tubulin along the sperm axoneme, whereas the reverse picture is observed in respiratory cilia, thus explaining the phenotype restricted to sperm cells. We also demonstrate the loss of function associated with DNAH17 mutations in two unrelated individuals by performing immunoblot and immunofluorescence analyses on sperm cells; these analyses indicated the absence of DNAH17 and DNAH8, whereas DNAH2 and DNALI, two inner dynein arm components, were present. Overall, this study demonstrates that mutations in DNAH17 are responsible for isolated male infertility and provides information regarding ODA composition in human spermatozoa.

https://hal.archives-ouvertes.fr/hal-02315263/document

Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.

Spermatozoa contain highly specialized structural features reflecting unique functions required for fertilization. Among them, the flagellum is a sperm-specific organelle required to generate the motility, which is essential to reach the egg. The flagellum integrity is, therefore, critical for normal sperm function and flagellum defects consistently lead to male infertility due to reduced or absent sperm motility defined as asthenozoospermia. Multiple morphological abnormalities of the flagella (MMAF), also called short tails, is among the most severe forms of sperm flagellum defects responsible for male infertility and is characterized by the presence in the ejaculate of spermatozoa being short, coiled, absent and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous which is consistent with the large number of proteins (over one thousand) localized in the human sperm flagella. In the past 5 years, genomic investigation of the MMAF phenotype allowed the identification of 18 genes whose mutations induce MMAF and infertility. Here we will review information about those genes including their expression pattern, the features of the encoded proteins together with their localization within the different flagellar protein complexes (axonemal or peri-axonemal) and their potential functions. We will categorize the identified MMAF genes following the protein complexes, functions or biological processes they may be associated with, based on the current knowledge in the field.

The genetic architecture of morphological abnormalities of the sperm tail.

Background Infertility is a major issue in human reproductive health, affecting an estimated 15% of couples worldwide. Infertility can result from disorders of sex development (DSD) or from reproductive endocrine disorders (REDs) with onset in infancy, early childhood or adolescence. Male infertility, accounting for roughly half of all infertility cases, generally manifests as decreased sperm count (azoospermia or oligozoospermia), attenuated sperm motility (asthenozoospermia) or a higher proportion of morphologically abnormal sperm (teratozoospermia). Female infertility can be divided into several classical types, including, but not limited to, oocyte maturation arrest, premature ovarian insufficiency (POI), fertilization failure and early embryonic arrest. An estimated one half of infertility cases have a genetic component; however, most genetic causes of human infertility are currently uncharacterized. The advent of high-throughput sequencing technologies has greatly facilitated the identification of infertility-associated gene mutations in patients over the past 20 years. Objective and rationale This review aims to conduct a narrative review of the genetic causes of human infertility. Loss-of-function mutation discoveries related to human infertility are summarized and further illustrated in tables. Corresponding knockout/mutated animal models of causative genes for infertility are also introduced. Search methods A search of the PubMed database was performed to identify relevant studies published in English. The term 'mutation' was combined with a range of search terms related to the core focus of the review: infertility, DSD, REDs, azoospermia or oligozoospermia, asthenozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF), primary ciliary dyskinesia (PCD), acephalic spermatozoa syndrome (ASS), globozoospermia, teratozoospermia, acrosome, oocyte maturation arrest, POI, zona pellucida, fertilization defects and early embryonic arrest. Outcomes Our search generated ∼2000 records. Overall, 350 articles were included in the final review. For genetic investigation of human infertility, the traditional candidate gene approach is proceeding slowly, whereas high-throughput sequencing technologies in larger cohorts of individuals is identifying an increasing number of causative genes linked to human infertility. This review provides a wide panel of gene mutations in several typical forms of human infertility, including DSD, REDs, male infertility (oligozoospermia, MMAF, PCD, ASS and globozoospermia) and female infertility (oocyte maturation arrest, POI, fertilization failure and early embryonic arrest). The causative genes, their identified mutations, mutation rate, studied population and their corresponding knockout/mutated mice of non-obstructive azoospermia, MMAF, ASS, globozoospermia, oocyte maturation arrest, POI, fertilization failure and early embryonic arrest are further illustrated by tables. In this review, we suggest that (i) our current knowledge of infertility is largely obtained from knockout mouse models; (ii) larger cohorts of clinical cases with distinct clinical characteristics need to be recruited in future studies; (iii) the whole picture of genetic causes of human infertility relies on both the identification of more mutations for distinct types of infertility and the integration of known mutation information; (iv) knockout/mutated animal models are needed to show whether the phenotypes of genetically altered animals are consistent with findings in human infertile patients carrying a deleterious mutation of the homologous gene; and (v) the molecular mechanisms underlying human infertility caused by pathogenic mutations are largely unclear in most current studies. Wilder implications It is important to use our current understanding to identify avenues and priorities for future research in the field of genetic causes of infertility as well as to apply mutation knowledge to risk prediction, genetic diagnosis and potential treatment for human infertility.

Molecular genetics of infertility: loss-of-function mutations in humans and corresponding knockout/mutated mice

Male infertility is a major concern affecting human reproductive health. Asthenoteratospermia can cause male infertility through reduced motility and abnormal morphology of spermatozoa. Several genes, including DNAH1 and some CFAP family members, are involved in multiple morphological abnormalities of the sperm flagella (MMAF). However, these known genes only account for approximately 60% of human MMAF cases. Here, we conducted further genetic analyses by using whole-exome sequencing in a cohort of 65 Han Chinese men with MMAF. Intriguingly, bi-allelic mutations of TTC21A (tetratricopeptide repeat domain 21A) were identified in three (5%) unrelated, MMAF-affected men, including two with homozygous stop-gain mutations and one with compound heterozygous mutations of TTC21A. Notably, these men consistently presented with MMAF and additional abnormalities of sperm head-tail conjunction. Furthermore, a homozygous TTC21A splicing mutation was identified in two Tunisian cases from an independent MMAF cohort. TTC21A is preferentially expressed in the testis and encodes an intraflagellar transport (IFT)-associated protein that possesses several tetratricopeptide repeat domains that perform functions crucial for ciliary function. To further investigate the potential roles of TTC21A in spermatogenesis, we generated Ttc21a mutant mice by using CRISPR-Cas9 technology and revealed sperm structural defects of the flagella and the connecting piece. Our consistent observations across human populations and in the mouse model strongly support the notion that bi-allelic mutations in TTC21A can induce asthenoteratospermia with defects of the sperm flagella and head-tail conjunction.

Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice.

Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.

https://hal.archives-ouvertes.fr/hal-02003335/document

Nathalie Rives

Papers

Bi-allelic Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia Due to Sperm Flagellum Malformations in Humans and Mice