Mammalian sperm flagella and cilia

In mammals, both sperm flagella and cilia are axoneme-containing organelles. The sperm flagella are major parts of the spermatozoa that are male gametes produced in the testis from progenitor spermatogonia through a process called spermatogenesis. During last stage of spermatogenesis, also called spermiogenesis, an axoneme grows out from one of the two centrioles to form the flagellum. With the growth of the axoneme, accessory components including mitochondria, outer dense fibers, fibrous sheath and annulus are added to the flagellum to form its middle, principal and end pieces. The sperm flagellum is responsible for the sperm motility, and defects in it often cause male infertility. Unlike sperm flagella, cilia contain only axonemes covered with the plasma membrane. Generally two types of cilia exist in mammals: multiple motile cilia and single primary cilia. All cilia extend from basal bodies that are derived from centrioles. Cilia play important physiological roles in the body. Defects in them cause a large number of genetic diseases, such as polycystic kidney disease, retinal degeneration, hydrocephalus, laterality defects, chronic respiratory problems, and even obesity and diabetes. In this thesis, I characterized two proteins related to mammalian sperm flagellum development and ciliogenesis: DNAJB13 and SMC1. Dnajb13 is a type II Hsp40 gene once reported to be highly expressed in testis. By multi-tissue RT-PCR, I found that it was also expressed in several ciliated tissues. In mouse testis, Dnajb13 mRNA was detectable at postnatal week 1 while DNAJB13 was undetectable until postnatal week 4, indicating a translational control for this gene. Although being an Hsp40, DNAJB13 was not heat-inducible. In the cross-sections of seminiferous epithelium, DNAJB13 was first detectable in step2-3 spermatids, peaked at step9-10 and then gradually decreased afterwards. In a mature spermatozoon, DNAJB13 was present in the flagellum throughout the length. Its presence in normal sperm flagellum but not in SDS-resistant sperm tail indicates that it is axonemeassociated, which is further confirmed by its presence in motile cilia of airway and oviduct epithelia. By immunoelectron microscopy DNAJB13 was defined as a radial spoke protein. DNAJB13 is also associated with the annulus spatiotemporally during mouse sperm flagellum development. This association started from the formation of annulus. The annular DNAJB13 increased with the maturation of annulus, and peaked when the annulus reaches the midpiece-principal piece junction of the flagellum, and then gradually disappeared during late spermiogenesis. In annulus-deficient spermatids, DNAJB13 still formed an annulus-like ring in the neck region. In vitro DNAJB13 was colocalized and interacted with an annulus constituent SEPT4. All the data suggest that DNAJB13 may be involved in the assembly and positioning of the annulus during mouse sperm flagellum development. Cohesin protein SMC1 is well known for its roles in sister chromatid cohesion and DNA repair. It was also reported to be present in primary cilia. By immunofluorescence, I found that SMC1 also localized to centrosomes throughout the cell cycle in a microtubule-independent manner. In addition, SMC1 was associated with both centrioles of a centrosome. Biochemically, SMC1 was cofractionated with the centrosomal marker γ-tubulin in centrosomal preparation. In vivo SMC1 localized to the basal bodies of motile cilia. These data suggest that SMC1 is also a centrosome/basal body protein and may play a role in ciliogenesis. LIST OF PUBLICATIONS This thesis is based on the following papers. I. A heat-shock protein 40, DNAJB13, is an axoneme-associated component in mouse spermatozoa. Jikui Guan, Li Yuan* Mol Reprod Dev. 2008 Sep;75(9):1379-86. II. DNAJB13 is a radial spoke protein of mouse ‘9+2’ axoneme. Jikui Guan, Emelie Ekwurtzel, Ulrik Kvist, Kjell Hultenby, Li Yuan* Submitted, 2009 III. Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development. Jikui Guan, Makoto Kinoshita, Li Yuan* BMC Dev Biol. 2009 Mar 19;9(1):23. [Epub ahead of print] IV. Cohesin protein SMC1 is a centrosomal protein. Jikui Guan, Emelie Ekwurtzel, Ulrik Kvist, Li Yuan* Biochem Biophys Res Commun. 2008 Aug 8;372(4):761-4. * Corresponding author