Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
Abstract
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 axoneme-associated, 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 midpieceprincipal 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 gamma-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