Spermiogenesis is the longest phase of spermatogenesis, with dramatic morphological changes and a final step of spermiation, which involves protein degradation and the removal of excess cytoplasm; ...

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism that is indispensable for the formation and maintenance of cilia and flagella; however, the implications and functions of IFT81 remain unknown. In this study, we disrupted IFT81 expression in male germ cells starting from the spermatocyte stage. As a result, homozygous mutant males were completely infertile and displayed abnormal sperm parameters. In addition to oligozoospermia, spermatozoa presented dysmorphic and nonfunctional flagella. Histological examination of testes from homozygous mutant mice revealed abnormal spermiogenesis associated with sloughing of germ cells and the presence of numerous multinucleated giant germ cells (symblasts) in the lumen of seminiferous tubules and epididymis. Moreover, only few elongated spermatids and spermatozoa were seen in analyzed cross sections. Transmission electron microscopy showed a complete disorganization of the axoneme and para-axonemal structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers. In addition, numerous vesicles that contain unassembled microtubules were observed within developing spermatids. Acrosome structure analysis showed normal appearance, thus excluding a crucial role of IFT81 in acrosome biogenesis. These observations showed that IFT81 is an important member of the IFT process during spermatogenesis and that its absence is associated with abnormal flagellum formation leading to male infertility. The expression levels of several IFT components in testes, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but not IFT140, were significantly reduced in homozygous mutant mice. Overall, our study demonstrates that IFT81 plays an essential role during spermatogenesis by modulating the assembly and elongation of the sperm flagella.

Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the ‘9 + 2’ axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we reexamined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2 and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6-binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners. Abstract Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the ‘9 + 2’ axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we reexamined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2 and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6-binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. IFT172 is a component of the IFT complex. Global disruption of mouse Ift172 gene caused typical phenotypes of ciliopathy. Mouse Ift172 gene appears to translate two major proteins; the full-length protein is highly expressed in the tissues enriched in cilia and the smaller 130 kDa one is only abundant in the testis. In male germ cells, IFT172 is highly expressed in the manchette of elongating spermatids. A germ cell-specific Ift172 mutant mice were generated, and the mutant mice did not show gross abnormalities. There was no difference in testis/body weight between the control and mutant mice, but more than half of the adult homozygous mutant males were infertile and associated with abnormally developed germ cells in the spermiogenesis phase. The cauda epididymides in mutant mice contained less developed sperm that showed significantly reduced motility, and these sperm had multiple defects in ultrastructure and bent tails. In the mutant mice, testicular expression levels of some IFT components, including IFT20, IFT27, IFT74, IFT81 and IFT140, and a central apparatus protein SPAG16L were not changed. However, expression levels of ODF2, a component of the outer dense fiber, and AKAP4, a component of fibrous sheath, and two IFT components IFT25 and IFT57 were dramatically reduced. Our findings demonstrate that IFT172 is essential for normal male fertility and spermiogenesis in mice, probably by modulating specific IFT proteins and transporting/assembling unique accessory structural proteins into spermatozoa. Abstract Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. IFT172 is a component of the IFT complex. Global disruption of mouse Ift172 gene caused typical phenotypes of ciliopathy. Mouse Ift172 gene appears to translate two major proteins; the full-length protein is highly expressed in the tissues enriched in cilia and the smaller 130 kDa one is only abundant in the testis. In male germ cells, IFT172 is highly expressed in the manchette of elongating spermatids. A germ cell-specific Ift172 mutant mice were generated, and the mutant mice did not show gross abnormalities. There was no difference in testis/body weight between the control and mutant mice, but more than half of the adult homozygous mutant males were infertile and associated with abnormally developed germ cells in the spermiogenesis phase. The cauda epididymides in mutant mice contained less developed sperm that showed significantly reduced motility, and these sperm had multiple defects in ultrastructure and bent tails. In the mutant mice, testicular expression levels of some IFT components, including IFT20, IFT27, IFT74, IFT81 and IFT140, and a central apparatus protein SPAG16L were not changed. However, expression levels of ODF2, a component of the outer dense fiber, and AKAP4, a component of fibrous sheath, and two IFT components IFT25 and IFT57 were dramatically reduced. Our findings demonstrate that IFT172 is essential for normal male fertility and spermiogenesis in mice, probably by modulating specific IFT proteins and transporting/assembling unique accessory structural proteins into spermatozoa.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

According to the World Health Organization (WHO), infertility is the failure of a couple to achieve pregnancy after one year of consistent unprotected sex. Male factors contribute to about 50 percent of all infertility cases. Male infertility is a multifactorial disease that can stem from multiple etiologies which can be congenital or acquired. Due to the complex nature of male infertility, a multifaceted approach is crucial for the diagnosis and treatment of the condition. But, in most cases, the cause of infertility is idiopathic. The diagnosis and management of male infertility is a comprehensive and stepwise process that involves history, physical examinations, and semen analyses which is the gold standard for evaluating male fertility in the clinic. The outcomes of semen analyses will determine the next step of investigation which may include hormone profiling, imaging, and genetic testing to identify the mechanism of infertility. Through advances in fertility research, Assisted Reproductive Technology has revolutionized the treatment approach for male infertility. ARTs like IVF and Intracytoplasmic sperm Injection have been useful in helping couples achieve pregnancy when all other treatment options have failed. Despite advances in fertility research, there are still challenges to be overcome such as improved access to fertility care, optimization of ART to achieve 100 percent clinical pregnancy, deeper understanding of etiologies of male infertility with emphasis on idiopathic male infertility. This review summarizes current knowledge on the etiologies, diagnosis, and therapeutic interventions as well as recent advances in basic and clinical research on male infertility.