ORIGINAL ARTICLE Andrology
Speriolin is a novel human and mouse
sperm centrosome protein
M. Goto1,2, D.A. O’Brien2,*,†, and E.M. Eddy1,†
1Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences,
National Institutes of Health, Research Triangle Park, 111 T.W. Alexander Drive, NC 27709, USA2Department of Cell and Developmental
Biology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA
*Correspondence address. E-mail: firstname.lastname@example.org
Submitted on November 23, 2009; resubmitted on May 4, 2010; accepted on May 10, 2010
background: Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fer-
tilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to
indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice
and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not
seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin
as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the
centrosome in round spermatids.
methods: The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse
were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting.
results: Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal cen-
triole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization
differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in
zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos.
conclusions: Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is trans-
mitted to the mouse zygote and can be detected throughout the first mitotic division.
Key words: centriole / flagellum / fertilization / paternal inheritance / zygote
The perpendicularly oriented pair of centrioles and the surrounding
pericentriolar material (PCM) form the centrosome, an amorphous
cytoplasmic region that serves as the microtubule organizing center
(MTOC) in most cells. In mammals, centrioles are barrel-shaped orga-
nelles with the inner walls lined by a cylinder of nine triplets of micro-
tubules ?0.5 mm in length and 0.2 mm in diameter (Bettencourt-Dias
and Glover, 2007). The centriole pairs duplicate during S-phase with
each cell cycle and along with the PCM have an essential role in orga-
nizing the mitotic spindle in M-phase and completing cytokinesis
(Doxsey et al., 2005; Loncarek and Khodjakov, 2009). The centrioles
also give rise to the basal bodies of cilia and to flagella (reviewed in
Dawe et al., 2006; Nigg and Raff, 2009).
Formation of the mammalian sperm flagellum begins in the round
spermatid when the pair of centrioles localize the side of the cell
facing the seminiferous tubule lumen, with one of the pair lying
against the nuclear envelope (proximal centriole) and the other
(distal centriole) becoming the basal body that nucleates assembly
of the nine-plus-two array of microtubules of the axoneme. A centrio-
lar adjunct develops at the end of the proximal centriole, and nine
longitudinal columns surround the distal centriole and delimit the con-
necting piece region of the sperm. The longitudinal columns extend
from a thickened plate (capitulum) abutting the nuclear envelope
into the flagellum where they are continuous with the outer dense
fibers (reviewed in Eddy, 2006; Kerr et al., 2006). In humans
(Zamboni and Stafanini, 1971; Manandhar et al., 2000), rhesus
monkeys (Manandhar and Schatten, 2000) and most other mammals
(reviewed in Manandhar et al., 2005), the structure of the distal cen-
triole becomes disorganized in late spermatids, while the proximal
centriole retains its structure. However, mice, rats and other
rodents are different in that both the proximal and the distal sperm
†Shared senior authorship.
Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
Human Reproduction, Vol.25, No.8 pp. 1884–1894, 2010
Advanced Access publication on June 11, 2010doi:10.1093/humrep/deq138
centrioles degenerate (Fawcett, 1965; Woolley and Fawcett, 1973;
Manandhar et al., 1998).
An unusual feature of unfertilized mammalian oocytes is their lack of
centrioles which are usually considered essential cell organelles. This
was shown by transmission electron microscopy (TEM) for oocytes
and tubal ova of the rabbit (Zamboni 1970), oocytes undergoing
meiotic divisions in the mouse (Calarco et al., 1972; Szo ¨llo ¨si et al.,
1972), human oocytes (Sathananthan et al., 1988), cow oocytes
(Sathananthan et al., 1997) and sheep oocytes (Crozet et al., 2000).
However, following fertilization, in most species, a sperm aster
develops from the sperm proximal centriole. This replicates during
the pronuclear stage to produce the centrioles at the poles of the
mitotic spindle of the first cleavage and all subsequent cell divisions
(Sathananthan et al., 1996, 1997; Crozet et al., 2000). It remains to
be determined how the single sperm centriole replicates to generate
two centriolar pairs.
Other studies indicated that an intact sperm centriole is associated
with successful ICSI (reviewed in Schatten and Sun, 2009) and that
abnormalities in the human sperm centriolar region are associated
with cases of infertility (Kamal et al., 1999; Rawe et al., 2002),
failure of cleavage after ICSI (Chemes, 2000; Nagy, 2000; Rawe
et al., 2002), failures of fertilization and abnormal early embryonic
cleavages (Nagy, 2000). These observations are consistent with the
hypothesis of the paternal inheritance of centrioles by embryos pro-
posed by Boveri at the turn of the twentieth century. However,
parthenogenetically activated rabbit oocytes can form centrioles
(Szo ¨llo ¨si and Ozil, 1991), and parthenogenetically activated human
oocytes can form multiple asters (Terada et al., 2009), indicating
that sperm components are not necessary for the formation of
asters or centrioles in these species.
The ontogeny of centrioles in the embryos of mice and other
rodents is different from that in most mammals. In addition to the
lack of centrioles in oocytes and the absence of distinct centrioles in
the sperm at the time of fertilization, centrioles are not present in
mouse embryos until they form de novo in the late morula (Szo ¨llo ¨si
et al., 1972) or early blastocyst stage of development (Gueth-Hallonet
et al., 1993). It was seen by TEM that microtubules forming the
meiotic spindles in oocytes emanate from several dense fibrillar aggre-
gates comparable in appearance to MTOCs (Calarco et al., 1972;
Szo ¨llo ¨si et al., 1972). In addition, multiple rounds of cell division can
occur in mouse embryos developing parthenogenetically without the
contribution of sperm components (Schatten et al., 1991; Schatten,
1994; Hiiragi and Solter, 2004). These and other observations have
led to the belief that centrioles are maternally inherited in the
mouse (reviewed in Sun and Schatten, 2007).
The PCM of centrosomes contains a variety of proteins critical for
their functions. The list of proteins associated with centrosomes con-
tinues to grow from studies using indirect immunofluorescence
microscopy (IIF) and immunoelectron microscopy (IEM), identifying
their interactions with known centrosomal proteins, the application
of proteomics and the tools of genetic analysis (reviewed in Andersen
et al., 2003; Doxsey et al., 2005; Bettencourt-Dias and Glover, 2007).
Although most studies focus on the basic roles of centrosomes in cell
cycle regulation, some centrosomal proteins have been found to be
associated with a variety of human diseases (reviewed in Badano
et al., 2005; Nigg and Raff, 2009; Bettencourt-Dias and Glover,
2009). For example, mutations in pericentrin are associated with
Seckel syndrome (Griffith et al., 2008) and Majewski osteodysplastic
primordial dwarfism type II (Rauch et al., 2008). Although usually a
component of the centrosome, pericentrin is not detected in sperma-
tids or in sperm in mice (Manandhar et al., 1998; Goto and Eddy,
2004) and apparently does not have an essential role in the centro-
somes of these cells.
Despite the considerable amount of knowledge about centrosomal
proteins in other cell types, immunolocalization studies have identified
only a few of these proteins in mammalian gametes and early embryos.
The MTOCs of meiotic spindles stain for g-tubulin in mouse oocytes
(Gueth-Hallonet et al., 1993; Palacios et al., 1993; Lee et al., 2000),
but not in human, bovine (Simerly et al., 1999) or pig oocytes
(Manandhar et al., 2006). Pericentrin and centrin staining are seen at
the spindle poles of mouse oocytes (Hiraoka et al., 1989; Carabatsos
et al., 2000) but not of pig oocytes (Manandhar et al., 2006), further
indicating the differences between mice and other species. In addition,
NEDD1 (Ma et al., 2010) and proteins recognized by the human scler-
oderma autoantiserum 5051 (Calarco-Gillam et al., 1983; Hiraoka
et al., 1989) and rabbit autoantiserum NRS-01 (Hiraoka et al., 1989)
are localized to MTOCs of mouse oocytes, and the human autoanti-
serum SPJ stains the spindle poles in hamster oocytes (Hewitson et al.,
The centrosomes in round and elongating spermatids have been
found to contain g-tubulin, often detectable by IIF as two spots and
by IEM in association with the proximal and distal centrioles. This
has been seen in mice (Fouquet et al., 1998; Manandhar et al.,
1998), rats, hamsters, guinea pigs (Fouquet et al., 1998), rabbits
(Fouquet et al., 1998; Tachibana et al., 2009), bulls (Simerly et al.,
1999), rhesus monkeys (Fouquet et al., 1998; Manandhar and
Schatten, 2000) and humans (Tachibana et al., 2005). The g-tubulin
is shed from late spermatids in the residual bodies in mice (Manandhar
et al., 1998) and monkeys (Manandhar and Schatten, 2000) and is no
longer detectable by IIF or IEM in sperm of most species. Although it is
seen by IIF in ,5% of human sperm, g-tubulin can be detected in
human and bull sperm by immunoblotting (Simerly et al., 1999).
Centrin has been identified by IIF in the centrosomes and by IEM
associated with the proximal and distal centrioles in spermatids.
This was reported for mice (Hart et al., 1999; Manandhar et al.,
1999), rabbits (Tachibana et al., 2009), pigs (Manandhar et al.,
2006), rhesus monkeys (Manandhar and Schatten, 2000) and
humans (Simerly et al., 1999; Tachibana et al., 2005; Tachibana
et al., 2009). While centrin is not detectable by IIF in mouse sperm
(Manandhar et al., 1999), it is seen as two spots at the junction
between the head and flagellum and in association with the tip of
the flagellum separated from the sperm head in pigs (Manandhar
et al., 2006), humans (Simerly et al., 1999), rhesus monkeys (Mana-
ndhar and Schatten, 2000) and rabbits (Tachibana et al., 2009). In
addition, centrin has been detected by immunoblotting in human
sperm (Simerly et al., 1999). The centrin in rhesus monkey spermatids
was localized in two spots corresponding to the location of the cen-
trioles. The spot associated with the proximal centriole has a substan-
tially higher intensity than the spot associated with the distal centriolar
vault, suggesting that centrin loss correlates with the loss of centriolar
organization (Manandhar and Schatten, 2000).
We have identified speriolin as a novel spermatogenic cell-specific
protein that co-localizes with pericentrin in the centrosome in
mouse spermatocytes and continues to be present in spermatid
Speriolin in the sperm centrosome
reference to membranes and the extracellular space. J Ultrastruct Res
Kerr JB, Loveland KL, O’Bryan MK, de Kretser DM. Cytology of the testis
and intrinsic control mechanisms. In: Neill JD (ed). Physiology of
Reproduction. San Diego, CA: Elsevier Academic Press, 2006, 827–947.
Lee J, Miyano T, Moor RM. Spindle formation and dynamics of g-tubulin
and nuclear mitotic apparatus protein distribution during meiosis in
pig and mouse oocytes. Biol Reprod 2000;62:1184–1192.
Loncarek J, Khodjakov A. Ab ovo or de novo? Mechanisms of centriole
duplication. Mol Cells 2009;27:135–142.
Ma W, Baumann C, Viveiros MM. NEDD1 is crucial for meotic spindle
stability and accurate chromosome segregation in mammalian oocytes.
Dev Biol 2010;339:439–450.
Manandhar G, Schatten H. Centrosome reduction during rhesus
spermiogenesis: g-tubulin, centrin, and centriole degeneration. Mol
Reprod Dev 2000;56:502–511.
Manandhar G, Sutovsky P, Joshi HC, Stearns T, Schatten G. Centrosome
reduction during mouse spermiogenesis. Dev Biol 1998;203:424–434.
Manandhar G, Simerly C, Salisbury JL, Schatten G. Centriole and centrin
degeneration during mouse spermiogenesis. Cell Motil Cytoskeleton
Manandhar G, Simerly C, Schatten G. Highly degenerated distal centrioles
in rhesus and human spermatozoa. Hum Reprod 2000;15:256–263.
Manandhar G, Schatten H, Sutovsky P. Centrosome reduction during
gametogenesis and its significance. Biol Reprod 2005;72:2–13.
Manandhar G, Feng D, Yi Y-J, Letko J, Laurincik J, Sutovsky M, Slisbury JL,
Prather RS, Schatten H, Sutovsky P. Centrosomal protein centrin is not
detectable during early pre-implantation development but reappears
during late blastocyst stage in porcine embryos. Reproduction 2006;
Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways.
Nat Cell Biol Suppl 2002;4:s41–s49.
Nagy ZP. Sperm centriole dysfunction and sperm immotility. Mol Cell
Nakagawa Y, Yamane Y, Okanoue T, Tsukita S, Tsukita S. Outer dense
fiber 2 is a widespread centrosome scaffold component preferentially
associated with mother centrioles: its identification from isolated
centrosomes. Mol Biol Cell 2001;12:1687–1697.
Nigg EA, Raff JW. Centrioles, centrosomes, and cilia in health and disease.
Palacios MJ, Joshi HC, Simerly C, Schatten G. g-Tubulin reorganization
during mouse fertilization and early development. J Cell Sci 1993;
Palermo GD, Colombero LT, Rosenwaks Z. The human sperm
centrosome is responsible for normal syngamy and early embryonic
development. Rev Reprod 1997;2:19–27.
Peters JM. The anaphase promoting complex: proteolysis in mitosis and
beyond. Mol Cell 2002;9:931–943.
Rauch A, Thiel CT, Schindler D, Wick U, Crow YJ, Ekici AB, van Essen AJ,
Goecke TO, Al-Gazali L, Chrzanowska KH et al. Mutations in the
pericentrin (PCNT) gene cause primordial dwarfism. Science 2008;
Rawe VY, Terada Y, Nakamura S, Chillik CF, Olmedo SB, Chemes HE. A
pathology of the sperm centriole responsible for defective sperm
aster formation, syngamy and cleavage. Hum Reprod 2002;17:
Sathananthan AH, Trounson A, Freemann L, Brady T. The effects of
cooling human oocytes. Hum Reprod 1988;3:968–977.
Sathananthan AH, Ratnam SS, Ng SC, Tarin JJ, Gianarili L, Trounson A.
The sperm centriole: its inheritance, replication and perpetuation in
early human embryos. Hum Reprod 1996;11:345–356.
Sathananthan AH, Tatham B, Dharmawardena V, Grills B, Lewis I,
Trounson A. Inheritance of sperm centrioles and centrosomes in
bovine embryos. Arch Androl 1997;38:37–48.
Schalles U, Shao X, van der Hoorn FA, Oko R. Developmental expression
of the 84-kDa ODF sperm protein: localization to both the cortex and
medulla of outer dense fibers and to the connecting piece. Dev Biol
Schatten G. The centrosome and its mode of inheritance: the reduction of
the centrosome during gametogenesis and its restoration during
fertilization. Dev Biol 1994;165:299–335.
Schatten H, Sun QY. The role of centrosomes in mammalian fertilization
and its significance for ICSI. Mol Hum Reprod 2009;15:531–538.
Schatten G, Simerly C, Schatten H. Maternal inheritance of centrosomes in
mammals? Studies on parthenogenesis and polyspermy in mice. Proc
Natl Acad Sci USA 1991;88:6785–6789.
Schmel ML, Graham EF. Ultrastructure of the domestic tom cat (Felis
Simerly C, Zoran SS, Payne C, Dominko T, Sotovsky P, Navara CS,
Salisbury JL, Schatten G. Biparental inheritance of g-tubulin during
human fertilization: molecular reconstitution of functional zygotic
centrosomes in inseminated human oocytes and in cell-free extracts
nucleated by human sperm. Mol Biol Cell 1999;10:2955–2969.
Sun Q-Y, Schatten H. Centrosome inheritance after fertilization and
nuclear transfer in mammals. Adv Exp Med Biol 2007;591:58–71.
Szo ¨llo ¨si D, Ozil J-P. De novo formation of centrioles in parthenogenetically
activated, diploidized rabbit embryos. Biol Cell 1991;72:61–66.
Szo ¨llo ¨si D, Calarco P, Donahue RP. Absence of centrioles in the first and
second meiotic spindles of mouse oocytes. J Cell Sci 1972;11:521–541.
Tachibana M, Terada Y, Murkawa H, Murakami T, Yaegashi N,
Okamura K. Dynamic changes in the cytoskeleton during human
spermiogenesis. Fertil Steril 2005;84:1241–1248.
Tachibana M, Terada Y, Ogonuki N, Ugajin T, Ogura A, Marakami T,
Yaegashi N, Okamura K. Functional assessment of centrioles of
spermatozoa and spermatids microinjected into rabbit oocytes. Mol
Reprod Dev 2009;76:270–277.
Terada Y, Hasegawa H, Ugajin T, Murakami T, Yaegashi N, Okamura K.
Microtubule organization during human parthenogenesis. Fertil Steril
Woolley DM, Fawcett DW. The degeneration of disappearance of the
centrioles during the development of the rat spermatozoon. Anat Rec
Xu B, Hao Z, Jha KN, Zhang Z, Urekar C, Digalio L, Pulido S, Strauss JF III,
Flickinger CJ, Herr JC. TSKS concentrates in spermatid centrioles during
flagellogenesis. Dev Biol 2008;319:201–210.
Yan W. Male infertility caused by spermiogenic defects: lessons from gene
knockouts. Mol Cell Endocrinol 2009;306:24–32.
Zamboni L. Ultrtrastructure of mammalian oocytes and ova. Biol Reprod
Zamboni L, Stafanini M. The fine structure of the neck of mammalian
spermatozoa. Anat Rec 1971;169:155–172.
Goto et al.