A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the "9+2" microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice.

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.

Intraflagellar transport (IFT) is a conserved mechanism thought to be essential for the assembly and maintenance of cilia and flagella. However, little is known about its role in mammalian sperm flagella formation. To fill this gap, we disrupted the Ift20 gene in male germ cells. Homozygous mutant mice were infertile with significantly reduced sperm counts and motility. In addition, abnormally shaped elongating spermatid heads and bulbous round spermatids were found in the lumen of the seminiferous tubules. Electron microscopy revealed increased cytoplasmic vesicles, fiber-like structures, abnormal accumulation of mitochondria and a decrease in mature lysosomes. The few developed sperm had disrupted axonemes and some retained cytoplasmic lobe components on the flagella. ODF2 and SPAG16L, two sperm flagella proteins failed to be incorporated into sperm tails of the mutant mice, and in the germ cells, both were assembled into complexes with lighter density in the absence of IFT20. Disrupting IFT20 did not significantly change expression levels of IFT88, a component of IFT-B complex, and IFT140, a component of IFT-A complex. Even though the expression level of an autophagy core protein that associates with IFT20, ATG16, was reduced in the testis of the Ift20 mutant mice, expression levels of other major autophagy markers, including LC3 and ubiquitin were not changed. Our studies suggest that IFT20 is essential for male fertility and spermiogenesis in mice, and its major function is to transport cargo proteins for sperm flagella formation. It also appears to be involved in removing excess cytoplasmic components.

The sperm flagellum is essential for male fertility, and defects in flagellum biogenesis are associated with male infertility. Deficiency of coiled-coil domain-containing (CCDC) 42 (CCDC42) is specifically associated with malformation of mouse sperm flagella. Here, we find that the testis-specific protein CCDC38 interacts with CCDC42, localizing on the manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in a distorted manchette, multiple morphological abnormalities of the flagella of spermatozoa and eventually male sterility. Furthermore, we find that CCDC38 interacts with intraflagellar transport protein 88 (IFT88), as well as outer dense fibrous 2 (ODF2), and the knockout of Ccdc38 reduces transport of ODF2 to the flagellum. Altogether, our results uncover the essential role of CCDC38 in sperm flagellum biogenesis, and suggest that some mutations of these genes might be associated with male infertility in humans.

The sperm flagellum is essential for male fertility. Despite vigorous research progress toward understanding the pathogenesis of flagellum-related diseases, much remains unknown about the mechanisms underlying the flagellum biogenesis itself. Here, we show that the cilia and flagella associated protein 53 (Cfap53) gene is predominantly expressed in testes, and it is essential for sperm flagellum biogenesis. The knockout of this gene resulted in complete infertility in male mice but not in the females. CFAP53 localized to the manchette and sperm tail during spermiogenesis, the knockout of this gene impaired flagellum biogenesis. Furthermore, we identified two manchette and sperm tail-associated proteins that interacted with CFAP53 during spermiogenesis. Together, our results suggest that CFAP53 is an essential protein for sperm flagellum biogenesis, and its mutations might be associated with multiple morphological abnormalities of the flagella (MMAF).

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

The molecular mechanism underlying asymmetric axonemal complexes in sperm flagella is still largely unknown. Here, we showed that the knockout of the coiled-coil domain-containing 176 (CCDC176) in mice led to male infertility due to decreased sperm motility. Ccdc176 knockout specifically destabilized microtubule doublets (MTDs) 1 and 9 during sperm maturation in the corpus epididymis. Single-sperm immunofluorescence showed that most CCDC176 was distributed along the axoneme, and further super-resolution imaging revealed that CCDC176 is asymmetrically localized in the sperm axoneme. CCDC176 could cooperate with microtubule and radial spoke proteins to stabilize MTDs 1 and 9, and its knockout results in the destabilization of some proteins in sperm flagella. Furthermore, as predicted by the sperm multibody dynamics (MBD) model, we found that MTDs 1 and 9 jutted out from the sperm flagellum annulus region in Ccdc176-/- spermatozoa, and these flagellar defects alter sperm flagellar beat patterns and swimming paths, potentially owing to the reduction and disequilibration of bending torque on the central pair. These results demonstrate that CCDC176 specifically stabilizes MTDs 1 and 9 in the sperm flagellum to ensure proper sperm movement for fertilization.

The mammalian sperm-associated antigen 16 gene (Spag16) uses alternative promoters to produce two major transcript isoforms (Spag16L and Spag16S) and encode proteins that are involved in the cilia/flagella formation and motility. In silico analysis of both mouse and human SPAG16L promoters reveals the existence of multiple putative SOX5 binding sites. Given that the SOX5 gene encodes a 48-kDa transcription factor (S-SOX5) and the presence of putative SOX5 binding sites at the SPAG16L promoter, regulation of SPAG16L expression by S-SOX5 was studied in the present work.

Spag17 encodes a protein present in the axoneme central pair complex of motile cilia and flagella. A mutation in this gene has been reported to be associated with infertility caused by defects in sperm motility. Here, we report that Spag17 knockout mice are infertile because of a severe defect in spermatogenesis. The histological evaluation of testis sections from mutant mice revealed seminiferous tubules with spermatogenesis arrested at the spermatid stage and cell debris in the cauda epididymis. The few sperm collected from the cauda epididymis were immotile and displayed abnormal tail and head morphology. Immunofluorescence analysis of Spag17 knockout germ cells showed spermatids with abnormally long manchette structures and morphological defects in the head. Electron microscopy showed altered manchette microtubules, reduced chromatin condensation, irregular nuclear shape, and detached acrosomes. Additionally, the transport of proteins (Pcdp1 and IFT20) along the manchette microtubules was disrupted in the knockout elongating spermatids. Our results show for the first time that Spag17 is essential for normal manchette structure, protein transport, and formation of the sperm head and flagellum, in addition to its role in sperm motility.

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.

Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were observed in the Spag6-deficient MEF cultures. The percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs developed multiple cilia. Furthermore, SPAG6 selectively increased expression of acetylated tubulin, a microtubule stability marker. The Spag6-deficient MEFs were more sensitive to paclitaxel, a microtubule stabilizer. Our studies reveal new roles for SPAG6 in modulation of cell morphology, proliferation, migration, and ciliogenesis.