Mutation analysis of genes involved in sperm motility: A study on patients with total sperm immotility

The spermatozoon is divided into two fundamental parts, the head and the tail (flagellum). The axoneme (Ax) is the flagellar motor of the sperm cell and extends throughout the flagellum. The Ax is composed by 9 peripheral doublets of microtubules (an internal (A) and an external (B)), linked to each other by nexin bridges, and connected by radial spokes (RS) to a single pair of central microtubules. The microtubule A has two dynein arms (DA), outer (ODA) and inner (IDA). The central microtubules are linked by a fibrilar bridge and are surrounded by a fibrilar central sheath, constituting the central pair complex (CPC). The CPC functions like a distributor of motility signal. The CPC gets the signal and distributes to the RS that, allied with the dynein regulatory complex (DRC), transmit regulatory signals that selectively activate the DA. DA converts the chemical energy contained in ATP, through ATPase, into the mechanical energy which will allow the flagellar movement. The outer dense fibre and the fibre sheath (FS) surround the Ax. The FS influences/modulates the bending and the flagellar beat and provides energy for motility. In addition, it contains glycolytic enzymes that provides energy for motility. Infertility is a current significant problem that affects about 10-17% of couples worldwide. A male contribution to infertility is found in 30–45% of the cases. There are several causes for male infertility being the sperm immotility one of the major. The genetic basis and molecular mechanisms that control sperm motility are not fully understood. Primary ciliary dyskinesia (PCD) and dysplasia of the fibrous sheath (DFS) are considered the two main disorders associated with sperm immotility. PCD is an autosomal recessive disease causing male infertility due to sperm immotility associated to abnormalities in the Ax. Kartagener syndrome (KS), characterized by a combination of situs inversus, chronic sinusitis, and bronchiectasis, occurs in about 50% of PCD patients. DFS is a disease mostly characterized by hyperplasia and disorganization of the FS. Several genes have been described as associated to these syndromes and to the assembly and function of the normal sperm flagellum. The total immotile spermatozoa of five Portuguese patients with asthenozoospermia were previously analysed by transmission electron microscopy. This ultrastructural analysis revealed 4 cases with DFS associated with disrupted CPC, RS, DA and doublets; and one patient with situs inversus who presented absence of DA and nexin bridges (compatible with KS). Therefore, the objective of the present work was to identify genetic alterations that could explain patients’ phenotypes. In that way, we searched for genes associated to DFS and PCD, as well as, with the ultrastructural defects in sperm cells of these patients. Seven genes were initially selected for analysis: CCDC39 and CCDC40 (DRC and IDA), DNAH5 and DNAI1 (ODA), RSPH1 (RS), AKAP3 and AKAP4 (FS). We designed primers for the exonic regions (plus intronic boundaries) of each gene. Then, these regions were amplified by polymerase chain reaction (PCR) and sequenced by Sanger’s method. Additionally, we sequenced all the exonic regions of the genome (exome) in the patient with situs inversus using next-generation sequencing (NGS) technology. As result, we identified 9 new DNA sequence variants (CCDC39:c.2540A>G, CCDC40:c.2620-92C>T, CCDC103:c.104G>C, DNAH5:c. 5882+133A>G e c.11570+124G>A, DNAH6:c.3167A>T, GAS8:c.828C>G, INSL6:c.262_263delCC e SPAG17:c.4445G>T); and 10 rare variants (CCDC39:c.233G>A; CCDC40:c.2682G>A; DNAH5:c.1537-102T>A, c.1537-100_1537-99delTT, c.3835-3delT, c.7408-84_7408-83delAT, c.10282-81delT, c.10872+84T>C; DNAH10:c.7895C>T e DNAI1:c.81+61A>G). The variants in CCDC103 and INSL6 are the most promising to have a role in sperm immotility. Our work demonstrates the genetic heterogeneity and complexity of DFS, PCD and sperm immotility. We highlight the difficulties for the identification of the genetic causes using conventional (Sanger) sequencing, especially due to the labour and costs involved. We also demonstrate the importance of exome sequencing, as broad mutation screening method, very useful to understand the genetics of highly complex processes as formation/assembly of sperm flagellum and sperm motility. With these work we intended to initiate the genetic diagnosis of this patients, and help to identify potential genetic markers for individuals with this kind of pathology. Understanding which genes are involved in sperm motility, how they are correlated as well as the exactly protein’s function and how these variants may affect its function, is essential to plan and develop, in the future, a gene therapy.