Microtubules play important roles in male fertility and ensure successful cell division during spermatogenesis. Fawcett [13] in his pioneering article on mammalian spermatozoa has discussed the role of microtubular structures in spermatogenesis. Bertalan et al [37] found that the total number of microtubules in the mitotic spindle is a critical controlling factor in cell division. If the number of microtubules significantly changes, this may result in altered cell division with subsequent pathological developments. Microtubule polymers build the mitotic spindle, and numerous associated factors unite to create a dynamic steady state structure [38]. Mitotic spindles in spermatogonia orient perpendicularly to the basement membrane to aid mitotic cell division and are rare in adults, suggesting a faster rate of mitotic division [39].

Spermatogonia, proliferative type A, undergo a series of mitotic divisions to reproduce themselves. While one of these daughter cells restores of type A spermatogonial stock, the other changes to type B spermatogonia in the basal compartment. Then, type B spermatogonia differentiate to form pre-leptotene spermatocytes, which are transported across the seminiferous tubule barrier [40]. After committing to meiosis, two cellular divisions occur where the 4N primary spermatocyte divides to form two 2N (N refers to haploid genome) secondary spermatocytes. Subsequently, each secondary spermatocyte divides to form two N spermatids [41]. The segregation of chromosomes involves three types of microtubules: kinetochore, astral, and polar. The kinetochore microtubules bind to the kinetochore at the chromosomal centromere which is critical for proper chromosomal segregation [38]. Errors in chromosome segregation lead to aneuploidy due to nondisjunction in both mitosis and meiosis. Astral microtubules are cell membrane interacting microtubules and are not connected directly to the chromosome. However, they participate in spindle positioning and cleavage before cytokinesis. Polar microtubules interlace at the midzone of the spindle and aid propelling of the spindle pores apart by the motor proteins [9].

During spermiogenesis, spermatids differentiate into spermatozoa, a process which involves four phases: the Golgi phase, the cap phase, formation of the tail, and the maturation phase. This process was classically described by Holstein [42] using ultrastructural investigations that confirmed the role of microtubules and axonemal structure in spermatid maturation. The Golgi stage of spermiogenesis is characterized by the formation of the head and axoneme. During this stage, sperm head shaping begins with the polarization of round spermatid nucleus to the one side of the cell and then proceed to deviate from a spherical shape as nuclear condensation continue. In addition, the acrosomal cap is formed over the anterior surface of the nucleus [9]. Normal sperm nuclear morphology relies on sperm DNA compaction as well as specialized structures within spermatids that determine the characteristic species-specific sperm head shape. As discussed earlier, the two major structures associated with sperm head shaping, known as the acroplaxome and manchette are closely related to one another as spermatids proceed through the elongation phase [43]. Later in this phase, about 90% of nuclear histones are removed and spermatozoal DNA is packaged first with specific nuclear proteins called transition proteins. Then, these transition proteins are replaced with specialized proteins called protamines [9]. As the acrosome begins to form on one pole of the nucleus, the two pairs of centrioles move toward the opposite pole to initiate axoneme formation. During formation of the sperm tail, the basal body (which is assembled within the spermatid centrosome) attaches with the microtubules. Microtubules present in this region are responsible for trafficking vesicles from the Golgi apparatus to the acrosome [44]. After the initiation of axoneme formation in early spermiogenesis, secondary structures necessary for flagella function (outer dense fibers, the fibrous sheath and the mitochondrial sheath) are assembled during the elongation phase of spermiogenesis [45]. Followed by the formation of the sperm tail, mature spermatozoa get released from Sertoli cells into the lumen of the seminiferous tubule in a process called spermiation [9]. During spermiation, residual bodies are phagocytized by the Sertoli cells [46]. This process involves removal of specialized adhesion structures and remodeling of the spermatid head and cytoplasm [9].

Several proteins and their interactions have been reported to play significant roles during sperm development. For example, Hook1 is a protein that binds to microtubules and cargoes in Drosophila [47]. This protein belongs to the family of Hook proteins, consisting of Hook1, Hook2, and Hook3 in mammals. Hook 2 has been reported to play a role in primary cilia morphogenesis while Hook3 has been shown to participate in the localization of Golgi complex [48]. A recent study described the interaction of Hook1 with another protein called coiled-coil domain containing protein 181 (Ccdc181) during localization of the sperm tail and in the formation of motile cilia [49]. Hook1 gene deletion has been reported to cause disruption of the manchette structure and abnormal head morphology in mice. Hook1 gene is suggested as a candidate for decapitation defects and teratozoospermia [50]. The different steps of spermatogenesis and microtubule functions are summarized in Fig. 4.

The tail is a long flagellum, and the central axoneme of this flagellum emerges from a basal body located posterior to the nucleus. For sperm motility, sliding of outer doublet microtubules in the sperm flagellum needs energy generated by ATP hydrolysis [11]. In human sperm, ATP is produced by glycolysis in the principle piece of the flagellum [51] and also, by the mitochondria present in the mid piece [52].

Katanin is a microtubule-severing complex consisting of a p60 severing enzyme and a p80 regulatory subunit [53]. Katanin p80 plays a crucial role in meiotic spindle formation, polarization and depolarization of microtubules and reproduction in male mice [54]. The protein p80 is encoded by Katnb1, and the missense mutation of this gene causes male infertility in mice characterized by oligoasthenoteratozoospermia. Mutation of this gene also causes virtual absence of progressive motility [54]. The expression of katanin p80 (both mRNA and protein levels) was evaluated in testis biopsies from patients with maturation arrest at the level of spermatocyte and spermatogonia and with Sertoli cell only syndrome, and compared with samples showing normal spermatogenesis [55]. These results suggest that KATNB1 plays a role in meiosis, nuclear shaping, and flagellum formation of sperm in humans. It was also reported that expression of p80 occurred only in germ cells and not in stem cells, Sertoli cells or interstitial cells [54].

In mammals, the blood-testis barrier (BTB) creates a unique microenvironment for the development and maturation of germ cells in order to maintain and support spermatogenesis. The BTB is located between Sertoli cells and germ cells, as well as between germ cells [56]. Sertoli cells are dynamic, metabolic epithelial cells that support the seminiferous epithelium. These cells contain microtubules that are oriented in linear arrays parallel to the long axis of the cell, forming a cage-like structure around the nucleus [57]. The shape of Sertoli cell changes according to the requirement of the developing germ cells [9].

The Katanin catalytic subunit A1 like 1 (KATNAL1) gene is a protein coding gene that is expressed in testicular tissue and helps control Sertoli cell microtubule dynamics [58]. Loss of function mutations of KATNAL1 results in disruption of Sertoli cell microtubule dynamics and cause to release of immature sperm within the testis tubules during sperm maturation [59]. Furthermore, it has been reported that induced-expression of a functional KATNAL1 protein raises microtubule severing, thereby impairs Sertoli cell plasticity required for germ cell growth. In the same study, it has also been shown that co-localization of with a Sertoli cell-specific isoform β-tubulin ‘TUBB3’ in Katnal1^1H/1H mutant testes [60]. Localization of TUBB3 showed that microtubule network underwent enormous disruption in the mutant testis [59]. Accordingly, a hypothesis was made that over-expression of functional KATNAL1 disrupts microtubule dynamics, which results in cellular arrest during mitosis that eventually leads to cell death [61]. However, the proper localization, mechanism of nucleation in Sertoli cell microtubules and its control are still not clear.

A-kinase anchor proteins (AKAPs) are regulators of microtubule dynamics and contribute to normal BTB function. Akap9 deletion has been reported to lead to significant changes in the microtubule organization in Sertoli cells and loss of BTB in Akap9 null mice [56].

Male infertility caused by impaired sperm motility and defects in genes that encode for axonemal complexes can be observed in men with primary ciliary dyskinesia (PCD) [62]. It was first described as a syndrome based on the triad of chronic sinusitis, bronchiectasis and situs inversus [63]. Forty years later, in the mid-1970s, Afzelius [64] expanded on this syndrome by observing that these patients had ‘immotile’ cilia and defective ciliary ultrastructure, specifically noting a deficiency of dynein arms, decreased mucociliary clearance, and a lack of ciliary motion. Presently, it is well-known as a heterogeneous and multisystemic disorder that is characterized by structural or functional abnormalities of the motile cilia [65].

PCD is a rare autosomal recessive disorder with an estimated prevalence of 1:10,000 to 1:20,000 in the general population [66]. In 1975, Afzelius et al [67] first reported PCD as a genetic disorder involving production, assembly, or attachment of dynein arm in spermatozoa in two brothers who were not twins. Pedersen and Rebbe [68] reported total immobility of spermatozoa in a patient with absent dynein arms in the axoneme of spermatozoa. Approximately, one half of patients presenting with PCD have situs inversus, a condition where the major organs appear mirrored to their normal position, which is called Kartagener syndrome [69]. Males with PCD are infertile due to immotile sperm and are at an increased risk of having respiratory problems such as lung infections caused by paralysis of the cilia [70]. Nearly 30 genes are involved in PCD pathogenesis in humans [71].

Various defects in the cilia structure are present in patients with PCD, including radial spoke abnormalities, outer and/or IDAs defects and the absence of a central microtubule [30]. Morphological changes always accompany one of these defects [72]. Abnormalities of the cilia can be classified as (1) dynein arm abnormalities with the most common cilia abnormality [73] involving either partial or complete absence of ODA/IDA [74]; (2) radial spoke abnormalities, including the absence of spokes or deviated central microtubules [75]; (3) inappropriate directionality of cilia, caused by a partial or complete absence of the central microtubules or defects in planar cell polarity [74]; and (4) an abnormal number of peripheral microtubules such as, 8+1, 8+2, 8+3, or 7+2 arrangements of the cross-section (Fig. 5) [76].

PCD occurs as a result of genetic mutations in genes encoding for ciliary proteins [77]. Many structural and regulatory proteins are present in the cilia, and hundreds of genes control these proteins. As a result, mutation in any of the genes encoding for these proteins may lead to abnormalities in ciliary functions [78].

Several studies have focused on identifying genes responsible for PCD [2979] using genetic linkage analysis in familial cases and candidate gene approaches, and proteomic analyses [80]. The most commonly studied genes are the dynein axonemal intermediate chain 1 (DNAI1) and dynein axonemal heavy chain 5 (DNAH5) genes [2980]. Mutations in these two genes result in abnormalities in ciliary structure and motor functions [2980]. DNAI2 is expressed in the trachea and testes and its mutations can lead to PCD [81]. Mutations in dynein axonemal beta heavy chain 9 (DNAH9) and dynein axonemal heavy chain type 11 (DNAH11) were found to be involved in PCD [79]. Mazor et al [82] reported a homozygous point mutation of dynein axonemal light chain 1 (DNAL1) that reduced the axonemal dynein light chain 1 stability and damaged its interactions with tubulin and with dynein heavy chain, subsequently resulting in PCD. Three additional mutations have been identified in DNAAF1, DNAAF2, and DNAAF3 all of which play important roles in the preassembly of dynein into complexes before their transport into cilia [83].

Castleman et al [84] reported two novel mutations in radial spoke head 9 homolog (RSHP9) and radial spoke head 4 homolog A (RSPH4A) encoding radial spoke head proteins with defects of the central microtubular pair related with PCD. These authors performed homozygosity mapping and identified the location of these genes on the long arm of chromosome 6. This study also revealed that RSPH9 is important in maintaining normal movement of motile ‘9++2’ structure of flagella. Ultrastructure analysis showed transposition defects with the absence of CP and 9+0 or 8+1 microtubule configuration.

Most genes implicated in PCD were thought to cause structural defects in the axoneme through mutations inducing CP defects. Recently, an association between a homozygous loss-of-function mutation in DNAJB13, a heat-shock protein, and PCD has been shown. DNAJB13 plays an essential role in preserving the integrity of the CP in humans [85]. Gene mutations and their consequences for axonemal structure in human and functional defects in PCD are summarized in (Table 1).

Dysplasia of the fibrous sheath (DFS) is an autosomal recessive genetic condition and appears in severe asthenozoospermic patients, which are characterized by dysplastic development of the axonemal and peri-axonemal cytoskeleton [86]. DFS is diagnosed by marked hypertrophy and hyperplasia of the fibrous sheath in association with deficiency of the annulus, mitochondria, the CP complex (50% of the cases) and/or the dynein arms [86]. The genetic origin of this disorder has been reported by numerous researchers [8788].

The AKAPs, which are the most abundant structural proteins of the fibrous sheath and engaged in organizing and assembling the basic structure of protein (AKAP3, 4) [89] have been reported to cause DFS on its mutation [88]. In humans, two other forms of these sperm fibrous sheath proteins, hAKAP82 and its precursor pro-hAKAP82, were cloned and characterized [90]. DFS is prevalent in consanguineous marriages [87] and is associated with PCD (in 20% of the cases), transmission of unbalanced gametes [8891], and increased sperm ubiquitination [92].

Several studies have reported a correlation between sperm flagellar abnormalities and increased frequency of gonosomal disomies and diploidies in spermatozoa [93]. In addition, numerous studies have found an elevated aneuploidy rate in multiple morphological abnormalities of the sperm flagella (MMAF), which might be associated with the sperm centrosome, the mitotic spindle and the flagella [94]. An elevated aneuploidy rate suggests that abnormalities of various centrosome-associated proteins may affect flagellum formation and mitotic/meiotic spindle assembly during spermatogenesis and lead to numerical chromosomal aberrations in spermatozoa [93]. However, these chromosomal aneuploidies have not been observed in all MMAF patients, suggesting that these flagellar and spindle defects may not directly cause chromosomal aneuploidies in all cases [95], thus requiring further investigation to specifically analyse the effects of flagellar and spindle defects on chromosomal aberrations in spermatozoa.

Recently, a study has demonstrated a high pregnancy rate and low numerical chromosomal aberration rate as well as good nuclear sperm quality in MMAF patients with DNAH1 mutations [74]. Assisted reproductive technologies have also been used to achieve a successful pregnancy in a few PCD cases. Intracytoplasmic sperm injection was performed in MMAF patients with and without DNAH1 mutations and compared with age-matched non-MMAF patients during the same period. Despite the significant increased rate of disomy XY and chromosome 18, no significant difference in the frequencies of either chromosomes 13, 21, XX or YY disomies or diploidy in sperm from patients with DNAH1 mutation were found compared with non-MMAF patients [74]. Thus, findings from this study suggests that deep genetic investigation using high-throughput sequencing to identify the mutations of genes coding for microtubular proteins and their impact on male infertility are to be focused upon in immediate future.

Mutations of several genes required for sperm development or absence of the respective proteins have been reported to be associated with male infertility by disrupting microtubular functions during formation of sperm head and/or the manchette, and by affecting normal sperm motility [549697]. Deficiencies in the proteins associated with formation of the sperm head (Sept12, SUN, and KASH domain proteins etc.) result in disorganized sperm head formation [98]. Centrobin, an essential centriole protein for centrosome duplication, along with other proteins, such as, ODF1/HSP10, Oazt/Oaz-3, SPATA6 etc. are involved in proper assembly and rigid head-tail conjunction of the sperm [99]. Mutation of these genes causes the acephalic spermatozoa syndrome, fragile necks, and dramatically increased susceptibility to decapitation [97]. A number of proteins, as discussed earlier, is involved in the formation of the manchette (Katnb1, Lrguk1, SUN domain proteins, Hook1, Kif3A etc.). Some mutations of these proteins lead to an elongated and knobbed-like manchette with disorganized microtubules [154754100]. Sperm associated antigen-6 (Spag6), Katnb1, Sept12, Lrguk1, testitin, Ift88, and Kif3A are involved in sperm flagellar motility and maintenance of the structural integrity of mature human sperm. Deficiencies of these proteins cause marked motility defects and morphologically abnormal and disorganized of flagellar structures, including loss of the CP of microtubules and disorganization of the outer dense fibers and fibrous sheath (Table 2) [155496100].