Analysis of the sperm flagellar axoneme using gene-modified mice.

Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.

Introduction

Cilia and flagella are evolutionarily conserved organelles that extend from the cell surface and are involved in sensing and locomotion. Defects in these organelles in humans lead to a group of disorders called ciliopathies that affect many tissues, such as lung, kidney, and brain tissues [4, 20, 43]. Primary ciliary dyskinesia (PCD) is one ciliopathy where the formation or motility of motile cilia is impaired, leading to abnormal mucociliary clearance and recurrent respiratory infections. Testes can be affected in PCD as well, and this can result in impairment of the formation and function of sperm flagella, leading to male infertility [22, 28, 55].

Spermatozoa comprise a head region, which contains the paternal genetic information, and flagellum, which is important for migration of spermatozoa in the female reproductive tract and passage through the zona pellucida, an extracellular matrix that surrounds the oocyte [8, 14, 40]. In contrast to cilia, sperm flagella possess unique accessory components that divide the flagellum into three parts: the midpiece, principal piece, and endpiece (Fig. 1) [13]. The midpiece contains the mitochondrial sheath and outer dense fibers, while the principal piece contains the fibrous sheath and outer dense fibers. No accessory structures are found in the endpiece. The accessory structures of the midpiece and principal piece regulate sperm motility through elastic forces and/or scaffolding molecules that are involved in energy production and/or signaling [13]. The axoneme is a conserved component of cilia and flagella that runs through the entire flagellum and is composed of a “9+2” structure consisting of a central pair (CP) of two singlet microtubules surrounded by nine doublet microtubules (DMTs) (Fig. 1) [19, 64]. In addition to these structures, the axoneme contains several macromolecular complexes, such as outer and inner dynein arms (ODAs and IDAs), radial spokes (RSs), and nexin-dynein regulatory complex (N-DRC).

Fig. 1.

Mouse sperm flagellum. Flagella can be divided into three parts: the midpiece, principal piece, and endpiece. Cross sections of each part are shown. The axoneme runs through the flagellum, which consists of several macromolecular complexes. CP, central pair; DMT, doublet microtubules; IDA, inner dynein arm; N-DRC, nexin-dynein regulatory complex; ODA, outer dynein arm; RS, radial spoke.

The components of the axoneme and their functions have been studied extensively in unicellular flagellates such as Chlamydomonas [19, 64]. These axonemal proteins are evolutionarily conserved in species that possess cilia and flagella, including mice; however, there are several molecules whose function in mouse spermatozoa remain unknown. One reason for this is the difficulty and cost in generating gene-modified mice using homologous recombination in embryonic stem (ES) cells; however, recent developments in genome-editing technology made it possible to manipulate mouse genomes with ease at low cost [1, 31, 39, 65]. Another reason for this is that knockout (KO) mouse lines often die before sexual maturation because of impaired ciliary motility, such as hydrocephalus, which hampers the analysis of mature spermatozoa. Here, we introduced phenotypes of mouse lines with KO of CP components and methods of overcoming premature lethality caused by impaired ciliary motility.

Structure and Components of the CP

The CP is composed of two singlet microtubules, C1 and C2, that are structurally and biochemically distinguishable [38, 57, 59]. There are several projections on each singlet microtubule and their precise localization has been analyzed with cryo-electron tomography (Fig. 2) [6]. The most extended projections on C1 are termed C1a and C1b, and 4 short projections, C1c, C1d, C1e, and C1f, are located between C1a and C1b projections. For C2, there are two long projections, C2a and C2b, and three short projections, C2c, C2d, and C2e. In addition, there is a bridge that connects C1 and C2 microtubules. The molecular components of these projections have been identified and shown to be critical for regulating flagellar motility in Chlamydomonas [38, 57, 59]. These components are conserved in mice, and several KO mouse lines have been generated (Table 1); these mouse lines are introduced next.

Fig. 2.

Structure and components of the CP. There are two singlet microtubules, C1 and C2, that contain several projections (C1a-C1f and C2a-C2e). There is also a bridge that connects C1 and C2 microtubules. Chlamydomonas CP components (top) with mouse orthologs (bottom) are indicated.

Table 1.

Conservation of CP components and phenotypes of KO mice.

Chlamydomonas	Localization	Mouse	Phenotype of KO mice		Human diseases
			Lethalityb)	Spermatozoa
CPC1	C1b	Spef2	Premature lethality	Impaired tail formation [54]	PCD and MMAF [29, 30, 52, 63]
FAP42	C1b	Guk1	NA	NA	NA
FAP46	C1d	Cfap46	NA	NA	NA
FAP54	C1d	Cfap54	Premature lethality	Impaired tail formation [33]	NA
FAP69	C1b	Cfap69	Viable	Impaired tail formation [12, 17]	MMAF [12, 17]
FAP74	C1d	Cfap74	NA	NA	NA
FAP101	C1a	Tcte1a)	Viable	Impaired motility [7]	NA
FAP114	C1a	Ccdc189	NA	NA	NA
FAP119	C1a	Ccdc189	NA	NA	NA
FAP174	C1b	Mycbp	NA	NA	NA
FAP221	C1d	Pcdp1	Premature lethality	Impaired tail formation [27]	NA
FAP297	C1d	Wdr93	NA	NA	NA
HYDIN	C2b	Hydin	Premature lethality	Impaired tail formation [47]	PCD [46]
KLP1	C2c/d	Kif9	Viable	Impaired motility [41]	NA
PF6	C1a	Spag17	Neonatal demise	Impaired tail formation [23]	NA
PF16	C1	Spag6	Premature lethality	Impaired tail formation [51]	NA
PF20	Bridge	Spag16l	Viable	Impaired motility [71]	NA

a)Tcte1 is also the Chlamydomonas ortholog of DRC5, which is a component of the N-DRC. b)The most severe phenotypes are shown for cases in which analyses were performed in different genetic backgrounds.

Analysis of Mature Spermatozoa Using 129 Backgrounds

Chlamydomonas FAP221 (flagellar associated protein 221) is a component of the C1d projection and can bind to calmodulin in a calcium-dependent manner [10, 11]. The mouse ortholog of FAP221 is Pcdp1 (primary ciliary dyskinesia protein 1), which was identified by analyzing nm1054 mutant mice that possess an approximately 400-kb deletion on chromosome 1 containing 6 genes [27]. Nm1054 mutant mice exhibit anemia and cutaneous phenotypes that are caused by Steap3 (STEAP family member 3) and Dbi (diazepam binding inhibitor), respectively, as well as hydrocephalus that is caused by Pcdp1. Nm1054 mutant mice on the C57BL/6 background that possess a Steap3 transgene to rescue anemia die at around 3 to 5 weeks of age because of hydrocephalus, which hampers the analysis of mature spermatozoa [27]. Intriguingly, mutants on the 129S6/Sv background develop either mild or no hydrocephalus, suggesting that there are genetic modifiers that characterize these two genetic backgrounds [27]. When the testes of mature nm1054 mutant mice on the 129S6/Sv background were analyzed, no flagella were observed, indicating that Pcdp1 is essential for flagellar formation in mice. In contrast to the abnormal flagellar formation, cilia in sinus and tracheal epithelial cells can form with a normal ultrastructure, although the cilia beat slowly, which leads to sinusitis. These results indicate that there are differences in the formation of flagella and cilia.

Milder hydrocephalus phenotypes in 129 backgrounds were observed in other mouse lines with KO of CP components as well. ChlamydomonasCPC1 (central pair complex 1) is a component of the C1b projection and contains an adenylate kinase domain [37, 69]. The mouse ortholog of CPC1 is Spef2 (sperm flagellar protein 2). Spef2 mutant mice were generated by N-ethyl-N-nitrosourea (ENU) mutagenesis on the C57BL/6 background and named big giant head (bgh) because of severe hydrocephalus. Most bgh homozygous mice on the C57BL/6 background die at around weaning age because of hydrocephalus, while bgh homozygous mice on an F1 mixed background (C57BL/6-129S6/Sv) show no evidence of gross hydrocephalus or no premature mortality [54]. The bgh homozygous male mice on the F1 mixed background were infertile because of immotile short flagella. Analyses with transmission electron microscopy (TEM) indicate disorganization or absence of the CP in round spermatids and a complete disorganization of the axoneme, mitochondrial sheath, and outer dense fibers in early elongating spermatids, indicating that SPEF2 is essential for the formation of the CP and axoneme in flagella. In contrast, the ultrastructure of tracheal cilia is normal, although their cilia beat slowly [54], supporting the idea that there are differences in flagella and cilia formation.

Mouse CFAP54 (cilia and flagella associated protein 54), an ortholog of Chlamydomonas FAP54 that is a component of the C1d projection [5], was also analyzed using a mixed background. Cfap54 mutant mice on the C57BL/6 background exhibit severe hydrocephalus, while mutant mice on the F1 mixed background (C57BL/6-129S6/Sv) do not develop severe hydrocephalus [33]. Cfap54 mutant male mice were infertile due to short tails. TEM analysis indicates that mutant flagella are highly disorganized, with axonemal structures mostly absent. Consistent with nm1054 and bgh mutant mice, Cfap54 mutant mice develop sinusitis, although airway epithelial cilia are present and organized. TEM analysis indicates that the ciliary axoneme is normally organized, but there is an absence of electron-dense material, indicating a loss of the C1d projection in the CP of Cfap54 KO mice [33].

These studies suggest that the 129 background can be used to overcome premature lethality caused by hydrocephalus. However, genetic modifiers that segregate these strains remain unknown [34], and it is still unclear if the 129 background can rescue all cases. For example, Spag6 (sperm associated antigen 6), a mouse ortholog of Chlamydomonas PF16 that contains armadillo repeats and is associated with the C1 microtubule [56], was mutated on the mixed background (C57BL/6-129/Sv); however, about 50% of the Spag6 KO mice died from hydrocephalus before 2 months of age [51]. Spag6 KO males surviving to sexual maturity were infertile due to impaired flagellar morphology and sperm motility. It is important to understand genetic modifiers that segregate these strains, which may uncover pathways that underlie susceptibility to severe hydrocephalus.

Analysis of Mature Spermatozoa Using the Conditional KO Approach

Mouse lines with KO of axonemal components can be lethal due to not only hydrocephalus but also other factors. In such cases, it is difficult to overcome lethality using the 129 background. Mouse SPAG17 is an ortholog of Chlamydomonas PF6 that is a component of the C1a projection [15, 49]. Spag17 KO mice on the C57BL/6-129S4/Sv mixed background exhibit profound respiratory distress associated with lung fluid accumulation and neonatal demise within 12 h of birth, in addition to hydrocephalus [60]. More severe phenotypes of Spag17 KO mice than other CP component KO mice are probably caused by the proximity of SPAG17 to other macromolecular complexes, such as RSs. To prevent neonatal lethality, the conditional KO approach was utilized with transgenic mouse lines that express CRE recombinase under the Sox2 promoter [23]. Obtained mutant mice were infertile due to immotile short flagella. TEM analysis indicates that some flagellar axonemes lack one CP microtubule. These results indicate that SPAG17 is essential for sperm flagellar formation. There are several transgenic mouse lines that express CRE specifically in the testis [32, 44, 62], and the conditional KO approach can be an excellent way to overcome lethality and analyze mature spermatozoa. However, it should be noted that CRE-dependent recombination is not 100% efficient [50, 62], which may make the interpretation of results difficult.

Analysis of Mature Spermatozoa Using Chimeric Mice

Conditional KO approaches can be used to overcome lethality; however, it takes time to generate mouse models and breed them with transgenic mouse lines that express recombinases such as CRE or FLP. To analyze phenotypes of KO spermatozoa faster, chimeric analysis can be utilized, which can analyze mature spermatozoa in one generation (Fig. 3) [45]. In this method, homozygous mutations of target genes are introduced into ES cells using genome-editing technology, and chimeric mice are generated using the KO-ES cells. Because chimeric mice possess wild-type (WT) cells that are derived from host embryos, premature lethality can be rescued. If ES cells express fluorescent proteins, KO spermatozoa derived from the ES cells can be recognized using the fluorescence as a marker, and their phenotypes can be analyzed. This approach was used to analyze HYDIN, which is a component of the C2b projection [26] and whose mutation leads to premature lethality due to hydrocephalus (FVB background) [9, 25, 35, 48]. Hydin was mutated in ES cells that were established by mating 129/Sv mice with RBGS (Red Body Green Sperm) transgenic mice [B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb] whose spermatozoa exhibit green fluorescence in the acrosome, a secretory vesicle localized in the head, and red fluorescence in the mitochondria [16]. Chimeric mice that were generated with Hydin KO ES cells could reach sexual maturity, and KO spermatozoa with fluorescence signals exhibited short, immotile flagella, indicating that HYDIN is essential for sperm flagellar formation [47]. The next generation (F1) was obtained by injecting KO spermatozoa into B6D2F1 mouse oocytes (intracytoplasmic sperm injection, ICSI). KO mice that were obtained from F1 × F1 mating could not reach sexual maturity, indicating that the KO allele is lethal even in the mixed background.

Fig. 3.

Chimeric analysis. A gene of interest is knocked out in ES cells that express fluorescent proteins. Chimeric mice are generated with the ES cells, and phenotypes of KO cells that are derived from ES cells are analyzed, such as spermatozoa or blood cells, using the fluorescence as a marker. Because chimeric mice possess wild-type cells derived from host embryos, the mice could overcome premature lethality.

Although chimeric analysis can be used to analyze the function of axonemal components within a short period, it should be noted that analyses using bulk cell populations are difficult because of the contamination of WT cells derived from host embryos. In this case, chimeric mice that are generated by injecting male ES cells into female embryos can be used because all spermatogenic cells of the chimeric mice are derived from ES cells [21]. Another problem of chimeric analysis is that the next generation cannot be obtained to maintain the line if the gene of interest is essential for male fertility. In that case, assisted reproductive technologies can be utilized to rescue the fertility as ICSI was used for Hydin KO spermatozoa [47]. ICSI can often rescue male fertility in mouse lines with KO of axonemal components that exhibit short sperm tails [2, 42, 61, 68].

KO Mouse Lines that Do Not Exhibit Premature Lethality

Although KO mouse lines that show premature lethality have been introduced so far, there are some KO mouse lines that have been reported to be viable. Chlamydomonas PF20 possesses five WD repeats that are important for protein interactions and is a component of a bridge that connects C1 and C2 microtubules [58]. The mouse ortholog of PF20 is Spag16, which encodes two major transcripts, Spag16l (2.5 kb) and Spag16s (1.4 kb) [72]. SPAG16L is localized in the central pair of sperm flagella, while SPAG16S is localized abundantly in the nuclei of round and condensing spermatids [70, 72]. Deletion of Spag16 exon 11, which disrupts both Spag16l and Spag16s, causes impaired spermatogenesis in chimeric mice due to haploinsufficiency [70]. In contrast, deletion of exons 2 and 3, which disrupts only Spag16l, leads to male infertility due to impaired sperm motility with a normal axoneme ultrastructure (mixed background C57BL/6-129/Sv) [71]. Spag16l KO mice do not show hydrocephalus or sinusitis [71]. This mild phenotype might be due to a mixed background, or it is possible that SPAG16L does not play critical roles in regulating ciliary motility.

Milder phenotypes were also observed in Kif9 (kinesin family member 9) KO mice (B6D2 background). KIF9 contains a motor domain like other kinesin superfamily proteins [18] and is an ortholog of Chlamydomonas KLP1 (kinesin like protein 1), which is a component of the C2c/d projections [3, 67]. Kif9 KO male mice were subfertile due to impaired sperm motility, while no overt hydrocephalus was observed [41]. This mild phenotype may be because KIF9 does not play critical roles in axonemal formation and/or motility. It is also possible that KIF6, a paralog of KIF9, plays more important roles in regulating ciliary motility [24]. Two paralogs playing separate roles in cilia and flagella have been reported in other axonemal components. For example, Rsph4a (radial spoke head 4 homolog A) is essential for regulating ciliary motility, disruption of which leads to hydrocephalus [53, 66], while its paralog, Rsph6a, is essential for sperm flagellar formation [2].

Mice with KO of Cfap69, an ortholog of Chlamydomonas FAP69, which is a component of the C1b projection [36], could survive to sexual maturity as well and were infertile due to abnormal flagellar formation (mixed background C57BL/6-FVB) [12]. Variations in CFAP69 were also found in infertile men who exhibited severe flagellar abnormalities, including flagella being short, coiled, absent, and of irregular caliber, which is referred to as multiple morphological abnormalities of the flagella (MMAF) [12, 17]. Variations in another CP component, SPEF2, were found in MMAF patients as well [29, 30, 52, 63], suggesting that CP components are important for sperm flagellar formation not only in mice but also humans.

Conclusion

Generation and analysis of KO mice with various methods indicate that most CP components were essential for sperm flagellar formation. These phenotypes in flagella are different from those in cilia that can form but exhibit impaired motility. The difference could be because sperm tail elongation is associated with the formation of accessory structures such as mitochondrial sheaths, fibrous sheaths, and outer dense fibers that are not found in cilia. Supporting this idea, mice with KO of CP components exhibit not only a disrupted axoneme but also the absence or disorganized accessory structures. Further, Spef2 KO mice exhibit more disorganized axonemes in round spermatids when accessory structures are being built [54]. Recently, more candidate components of the CP were identified in Chlamydomonas with proteomics analysis [73]. Understanding the function of these CP components may lead us to understand how CP components are involved in flagellar formation and to uncover the causes of male infertility, such as MMAF.

In this review, we introduced three methods that could overcome premature lethality: 129 backgrounds, the conditional KO approach, and chimeric analysis. These methods can be applied to understand the functions of not only CP but also other axonemal components. For example, chimeric analysis has been utilized to analyze DNAJB13, a component of RS [45]. Chimeric analysis can even be applied to examine other cell types, such as blood cells or immune cells, if analysis in mature KO mice is difficult to perform due to lethality (Fig. 3). Using the methods introduced here and genome-editing technology, the regulatory mechanism of flagellar formation and motility in mammals will be unveiled further.