MOLECULAR AND CELLULAR BIOLOGY, Sept. 2002, p. 6298–6305
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 17
Male Infertility, Impaired Sperm Motility, and Hydrocephalus in Mice
Deficient in Sperm-Associated Antigen 6
Rossana Sapiro,1Igor Kostetskii,1Patricia Olds-Clarke,2George L. Gerton,1Glenn L. Radice,1and
Jerome F. Strauss III1*
Center for Research on Reproduction and Women’s Health, University of Pennsylvania Medical Center,1and Department of
Anatomy and Cell Biology, Temple University School of Medicine,2Philadelphia, Pennsylvania 19104
Received 25 February 2002/Returned for modification 16 April 2002/Accepted 3 June 2002
Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue
of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for
flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50%
of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile.
Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm
head and disorganization of flagellar structures, including loss of the central pair of microtubules and
disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm
flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The
occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.
Fertilization is the process whereby sperm and eggs interact
reciprocally to begin development. To initiate fertilization,
mammalian sperm cells rely on the propulsive forces generated
by their flagella to reach the site of fertilization in the oviduct
and to penetrate the investments of the egg (8). All flagella
contain an axoneme composed of structural elements and mo-
tor proteins that work in a coordinated and regulated fashion
to produce wave forms that produce progressive movement (3,
4, 6, 8, 15, 21). The axoneme consists of a central pair of
microtubules (central apparatus) surrounded by nine doublets
of microtubules with the associated force-generating dynein
arms. The basic axonemal structure among cilia and flagella is
conserved across species, and much of our understanding of
the structure and function of the axoneme has been derived
from the study of model organisms. Genetic studies on the
green alga, Chlamydomonas, have revealed the importance of
several genes for flagellar assembly, stability of specific axon-
emal structures, and motility (2–6, 15, 21). Inactivation of
PF16, one of these Chlamydomonas genes, results in flagellar
paralysis (2, 20, 21). Moreover, when the flagella from the pf16
mutant are demembranated to produce axonemes, the C1 mi-
crotubule is destabilized and C1-associated polypeptides are
lost. We cloned the human and murine orthologues of PF16,
named sperm-associated antigen 6 (Spag6), and found that the
amino acid sequences of the mammalian and algal proteins
were highly conserved, including the eight armadillo repeats
required for the assembly of PF16 onto the C1 microtubule
and for flagellar function (11, 16, 20, 21). To determine if
Spag6 plays a critical role in the function of the mammalian
axoneme, we inactivated mouse Spag6. Males lacking Spag6
were infertile because their sperm had striking motility defects
and were frequently decapitated and had disorganized flagellar
structures. Approximately 50% of nullizygous males and fe-
males have enlarged heads and smaller bodies and die prema-
turely with hydrocephalus, presumably reflecting abnormalities
in the function of cilia of ependymal cells that facilitate circu-
lation of cerebral spinal fluid. Our findings indicate that Spag6
is essential for sperm flagellar motility and that it may serve as
a scaffold protein that maintains the structural integrity of the
sperm flagella. The occurrence of hydrocephalus strongly sug-
gests a role for Spag6 in ependymal ciliary motility.
MATERIALS AND METHODS
Targeted mutation of Spag6. We screened a 129/Sv mouse genomic mouse
library and obtained clones covering approximately 10 kb of the ?80-kb Spag6
gene containing putative exons 3 and 4. We constructed a targeting vector by
substitution of the exon encoding amino acid residues 40 to 96 (GenBank
accession number AF486266) with an internal ribosome entry site (IRES)–lacZ-
Neorfusion gene (9). If the preceding coding sequences were to be expressed, a
39-amino-acid peptide would be produced that lacks the eight contiguous arma-
dillo repeats believed to be essential for Spag6 function (16, 22). For the purpose
of screening, we inserted a BamHI site at the end of the short arm. Embryonic
stem cells derived from 129/Sv mice were transfected with the linearized ?geo
targeting vector, selected in medium supplemented with G-418, and analyzed by
Southern blotting to identify correctly targeted clones. For Southern blotting,
genomic DNA was digested with BamHI and the blots were probed with a 1.5-kb
cDNA containing genomic sequence upstream from the targeted genomic se-
quence. A correctly targeted embryonic stem cell clone was used to generate
chimeric mice, which were crossed with C57BL/6J females to obtain heterozy-
gous mutants. Mice used in these studies were the offspring of crosses between
the F1and/or F2generations (129/SvJ/C57BL/6J genetic background). Mice were
genotyped by PCR. Two sets of primers were used in the PCRs. One set of
primers corresponded to the Neo gene: 5?-CGTGTTCCGGCTGTCAGCGC
A-3? and 5?-CAACGCTATGTCCTGATAGCGGTC-3?. The other set of prim-
ers corresponded to the deleted region of the Spag6 gene: 5?-GACTTAGCAG
AAGCAGTCGTG-3? and 5?-CGGAGA GAAGCTGCTACCAAG-3?.
Assessment of fertility and fecundity. To assess fertility and fecundity, litter-
mate males (?6 weeks old) were placed in cages with two mature wild-type
females for 2 months or more. Littermate females were caged with a wild-type
fertile male for a similar period. The number of mice achieving a pregnancy and
the number of offspring from each mating set or pregnancy were recorded.
Northern blot analysis. Northern blots containing total testicular RNA (30
?g/lane) were probed with a full-length Spag6 cDNA and a cDNA comprising
700 bp of sequence downstream of the targeted exon (16). Similar results were
* Center for Research on Reproduction & Women’s Health, 1354
BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone (215)
898-0147. Fax: (215) 573-5408. E-mail: firstname.lastname@example.org.
obtained with both probes. Blots were stripped and reprobed for mouse Akap82
(1) and 28S rRNA.
Western blot analysis. Equal amounts of testicular protein (40 ?g/lane) were
subjected to Western analysis using antibodies against Spag6 (11, 16) and
Motility assays. Sperm isolation and motility analyses were carried out as
previously described (18). For each observation, four fields from each of two
dilutions of the original sperm suspension were pooled. The IVOS Sperm An-
alyzer (Hamilton-Thorne Research, Beverly, Mass.) was used for all motility
analyses. Only cells with ?16 points in their track and a mean curvilinear velocity
(VCL) of ?50 ?m/s were analyzed. Sperm populations were analyzed as soon as
possible after release from the epididymis.
Histology and immunoelectron microscopy and transmission electron micros-
copy. Cauda epididymal sperm, testes, reproductive tracts, tracheal tissue, and
ependymal tissue were prepared for light and electron microscopy using standard
methods. For immunoelectron microscopy, anti-Spag6 antibody was labeled with
10-nm gold particles as previously described (19).
Targeted disruption of Spag6. We disrupted the Spag6 gene
in murine embryonic stem cells by replacing the third exon with
the fusion gene ?geo (Fig. 1A). This manipulation prevents
expression of protein containing the eight contiguous arma-
dillo repeats that, by analogy to PF16, are predicted to be
essential for Spag6 function (22). To generate chimeras, em-
bryonic stem cells carrying a mutant copy of the Spag6 gene
(Fig. 1B) were injected into blastocysts and implanted into
pseudopregnant mice. Mutant mice were produced from the
chimeric offspring. Disruption of the Spag6 gene was con-
firmed by PCR analysis (Fig. 1C) and Southern blotting (data
not shown). The proportion of wild-type (55 of 189, 29%),
heterozygous (89 of 189, 51%), and nullizygous (36 of 189,
19%) offspring from mating of heterozygous males and fe-
males was not significantly different from the expected Men-
delian pattern of inheritance (chi-square test result ? 4.08; P ?
0.131). Approximately 50% of the Spag6?/?mice were smaller
than their heterozygous and wild-type littermates, and these
animals died before 2 months of age with enlarged heads and
hydrocephalus, reflected in dilated lateral and third ventricles
observed in sagittal sections of the brains (Fig. 2). Hydroceph-
alus resulting from impaired circulation of cerebral spinal fluid
is associated with immotile cilia of ependymal cells (7, 10). The
mutant mice displayed no gross abnormalities in organ struc-
ture (e.g., polycystic kidneys) or laterality (situs inversus), an
abnormality associated with defects in ciliogenesis and immo-
tile cilia syndrome (14, 17).
Northern (Fig. 1D) and Western blot analysis (Fig. 1E)
confirmed the absence of Spag6 mRNA and 55,000-Mrprotein
in testes of Spag6?/?mice that survived to sexual maturity. The
FIG. 1. Targeted disruption of the mouse Spag6 gene. (A) Schematic representation of the strategy used to disrupt Spag6. (a) Partial genomic
structure of the Spag6 gene. (b) Structure of the targeting vector. (c) Structure of the mutated allele. Restriction sites: H ? HpaI, X ? XhoI, B
? BamHI, N ? NotI, and S ? SalI. IRES, internal ribosome entry site. (B) Southern blot analysis of transfected embryonic stem cell clones. An
external probe gave rise to a single 8-kb band in wild-type genomic DNA digested with BamHI and a 4-kb band in the mutant allele (box).
(C) Genotyping by PCR. The wild-type allele yielded a 200-bp amplicon that is absent in the homozygous mutant. A 500-bp amplicon representing
the lacZ-Neo cassette was detectable only when a targeted allele was present. (D) Spag6 mRNA is absent in nullizygous mice. Northern blot shows
absence of the two Spag6 transcripts in testicular RNA from Spag6?/?mice (upper panel) but also the presence of Akap82 message (middle panel).
The lower panel shows the probing for 28S rRNA to assess RNA loading. (E) Spag6 protein was not detectable in the testes of Spag6?/?mice.
Western blot demonstrating that the 55,000-MrSpag6 protein is absent from the testis of nullizygous mice and present at approximately half the
level in heterozygous mice (upper panel). Akap82 protein was detected in testes extracts from all genotypes (lower panel). Numbers to the right
of panel E indicate the molecular weights (103) of protein standards.
VOL. 22, 2002Spag6 AND SPERM STRUCTURE AND MOTILITY6299
polyclonal antibody used to perform the Western blot shown in
Fig. 1E, generated against full-length recombinant protein, did
not detect lower-molecular-weight immunoreactive bands that
could have represented truncated Spag6 resulting from trans-
lation of mRNA containing coding sequence upstream from
the targeted exon. The testes of Spag6?/?mice did contain
pro- and mature protein for Akap82, also known as Akap4, the
major component of the sperm flagellum fibrous sheath, and
Akap82 mRNA (Fig. 1D and E).
Spag6 is required for male but not female fertility. Spag6?/?
males mated with wild-type females produced no pregnancies
after more than 2 months of continuous cohabitation even
though vaginal plugs were observed in the females (Table 1).
The Spag6?/?littermates were all fertile, producing as many
offspring per pregnancy as wild-type littermates. Eight of the
10 Spag6?/?females achieved a pregnancy during the obser-
vation period, but the time to establishing a pregnancy was
several weeks longer than for wild-type and heterozygous lit-
Spag6-deficient sperm have motility and structural defects.
The testes of Spag6?/?males surviving to sexual maturity were
of a weight similar to that of wild-type littermates (Spag6?/?:
0.37 ? 0.05 g/100 g of body weight, n ? 5; Spag6?/?: 0.38 ?
0.04, n ? 5, mean ? standard deviation). Likewise, the seminal
vesicles were similar in weight (Spag6?/?: 0.82 ? 0.01 g/100 g,
n ? 5; Spag6?/?: 0.77 ? 0.10, n ? 5, mean ? standard devi-
ation). The reproductive organs were grossly normal, and light
microscopy analysis of histological sections of the testes and
reproductive tract revealed normal architecture of the semi-
niferous tubules and interstitium (Fig. 3A and B). Sperm were
present in the testes and efferent ducts. The three genotypes
were not statistically different in terms of the concentration of
sperm that could be recovered from the caudae epididymides
(Table 2), although the concentration recovered tended to be
lower from Spag6?/?mice, because the recovery process is
aided by sperm motility.
There were striking differences between the mutant and
wild-type sperm morphology and motility. Light microscopy
examination revealed that 42% of the epididymal sperm from
Spag6?/?was abnormal, as reflected in fragmentation of the
midpiece, truncated flagella, or decapitation, whereas 7% of
heterozygous and wild-type sperm had an abnormal morphol-
ogy (Fig. 4). As expected, Spag6 protein was not detectable
in the tails of permeabilized sperm from Spag6?/?mice
Sperm from wild-type littermates displayed vigorous flagel-
lar activity and progressive forward movement (Table 2 and
movie that can be viewed at http://www.med.upenn.edu/crrwh
/movies/sapiro.mov). In contrast, only a small percentage of the
mutant sperm showed progressive forward motion; flagellar
activity was generally limited to a quaking or twitching motion
(Table 2 and movie on website). Computer-assisted sperm
analysis confirmed that motility parameters were severely im-
paired in the Spag6?/?mutant (Table 2). The percentage of
motile sperm recovered from the epididymis and the VCL of
sperm that were motile, an estimate of instantaneous sperm
swimming speed, were significantly less in Spag6?/?mice. A
mean of 8% of recovered Spag6?/?sperm was motile com-
pared to ?50% motile in heterozygous and wild-type mice.
Interestingly, sperm from Spag6?/?mice had an intermediate
value for VCL, suggesting that, although these animals were
fertile, the inactivation of one Spag6 allele impairs flagellar
activity due to a reduction in Spag6 protein. There were no
differences in linearity of motile sperm, an estimate of the
straightness of the sperm track, between wild-type sperm and
The structural integrity of sperm of Spag6-deficient mice.
The frequent loss of the sperm head in Spag6?/?mice indi-
cated that the absence of Spag6 affected sperm structural in-
tegrity. Immunoelectron microscopy localized Spag6 to the
FIG. 2. Photographs of Spag6?/?mice (A and B) and a wild-type littermate (A) showing small body with a disproportionately large head in the
Spag6-deficient animal. (C) Hydrocephalus in Spag6?/?mice. Sagittal sections of brains from a Spag6-deficient mouse and wild-type mouse
revealing dilatation of the lateral and third ventricles in the mutant brain.
TABLE 1. Fertility and fecundity of Spag6?/?, Spag6?/?, and
6.8 ? 2.4*
7 ? 1.3
5.2 ? 1
7 ? 2
5.8 ? 1.2
aMice of the indicated genotypes were caged with wild-type C57BL/6J mice of
the opposite sex for 2 months or more. The number of fertile animals and litter
sizes were recorded. *, Means ? standard errors.
bShown are the number of fertile mice/total number of mice.
6300SAPIRO ET AL.MOL. CELL. BIOL.
central apparatus of wild-type sperm (Fig. 5). However, we
could not determine whether Spag6 was confined to one of the
central apparatus microtubules, as is the case in Chlamydomo-
nas. Transmission electron microscopy analysis of epididymal
sperm revealed that numerous Spag6?/?sperm samples lacked
the central pair of microtubules of the axoneme and that the
external microtubule doublets and outer dense fibers were
disorganized (Fig. 5). Analysis of 179 transverse sections of
Spag6?/?epididymal sperm revealed an abnormal morphology
in approximately 60% of the flagella (Table 3). The midpiece
was most prominently affected with the central pair of micro-
tubules missing and/or alterations in the fibrous sheaths or
outer dense fibers. Only ?2% of 264 transverse sections of
sperm flagella from wild-type animals had an abnormal ultra-
structure. The flagella of testicular Spag6?/?sperm had fewer
morphological abnormalities and did not display loss of the
central pair of microtubules (Table 3 and Fig. 5), but sacs of
sperm debris were observed in the seminiferous tubules, ap-
parently the result of phagocytosis by Sertoli cells (Fig. 3). This
was not seen in wild-type testes.
FIG. 3. Histology and ultrastructure of the testis of Spag6?/?and wild-type mice. (A) Histology of wild-type testis. (B) Histology of Spag6-
deficient mouse testis revealing normal architecture of the seminiferous tubules and interstitial tissue. (C) Ultrastructure of the wild-type testis
seminiferous tubule showing normal sperm flagellar structure. (D) Ultrastructure of a seminiferous tubule of a Spag6-deficient mouse showing
sperm debris (inset) some abnormal sperm tails (arrows) and some normal-appearing sperm tails (arrowheads).
VOL. 22, 2002Spag6 AND SPERM STRUCTURE AND MOTILITY 6301
Spag6, the murine orthologue of Chlamydomonas PF16, is
detectable in the flagella of permeabilized sperm (11, 16).
Immunoelectron microscopy localized the protein to the cen-
tral apparatus, a finding consistent with the known residence of
PF16 (2, 11). A key feature of the domain structure of Spag6
and PF16 is eight contiguous armadillo repeats, motifs that are
involved in protein-protein interaction (15, 20, 21). Smith and
Lefebvre suggested that the unstable C1 microtubule in the
pf16 Chlamydomonas mutant resulted from impairment of crit-
ical protein interactions in the central apparatus (20, 21). Our
observations on sperm of Spag6-deficient mice substantiate a
role for this protein in the maintenance of structural integrity
of the flagella. However, in mice lacking Spag6, the disorgani-
zation of sperm structure extends beyond instability of the
central apparatus microtubules and includes the fragile attach-
ment of the sperm head as well as disarray of the outer micro-
tubule doublets and outer dense fibers. This may imply that
Spag6 has a broader role in maintaining the architecture of the
sperm flagellum or that stability of the mouse sperm central
apparatus is essential for the integrity of other flagellar struc-
tures. However, we cannot formally exclude the possibility that
a putative truncated amino-terminal fragment of Spag6 was
generated in our mutant mice and that this truncated protein
may have led to structural abnormalities in the sperm.
The motility defects in sperm lacking Spag6 probably result
from dysfunction of the central apparatus rather than from the
structural abnormalities in the flagella. The fact that only 8%
of recovered epididymal sperm was motile while ?60% of
transverse sections showed ultrastructural abnormalities in the
flagella suggests that the absence of Spag6 does impair motility
even when the flagella have a normal architecture. Moreover,
the intermediate VCL value for sperm from Spag6?/?mice,
which did not have ultrastructural flagellar abnormalities, also
indicates an important role for Spag6 in sperm motility. How-
ever, because our ultrastructural analyses did not encompass
serial sectioning through the full length of the flagella, we
cannot exclude the possibility that a higher percentage of mu-
tant sperm had regional structural defects that were not de-
The fact that the ultrastructure of ?75% of testicular sperm
of Spag6?/?mice appeared to be generally normal, whereas
60% of the epididymal sperm was abnormal, suggests that
Spag6 is not absolutely essential for flagellar assembly or in-
traflagellar transport. However, the presence of abnormalities
in the organization of the outer dense fibers and fibrous sheath
and debris, reflecting phagocytosis of presumably abnormal
sperm in the testes of Spag6?/?mice, indicates that there is
impairment in spermatogenesis or the maintenance of struc-
tural integrity of testicular sperm flagella. Thus, Spag6 appears
to be important for maintaining the architecture of the central
apparatus of sperm after their release from the testis. This
observation is consonant with the fact that the central appara-
tus is structurally normal in the Chlamydomonas pf16 mutant
and that instability of the C1 microtubule is found when the
axonemes of the mutant flagella are isolated (2, 20, 21).
A role for Spag6 as a scaffold protein mediating protein-
protein interactions and structural stability is consistent with
our observations that Spag6 and fusion Spag6-green fluores-
cent protein associate with high affinity to microtubules in
transfected COS-1 and Chinese hamster ovary cells and that
microtubules decorated with Spag6-green fluorescent protein
are bundled and stabilized (i.e., resistant to nocodazole or
cooling to 4°C) [16; R. Sapiro, J. M. Murray, M. Zhang, E. J.
Blanchette-Mackie, and J. F. Strauss III, 34thAnnu. Meet. Soc.
Study Reprod., 28 July to 1 August 2001, Ottawa, Canada;
Biol. Reprod. 64(Suppl. 1):106, 2001]. The identities of the
proteins other than polymerized tubulin that could be directly
or indirectly associated with Spag6, remain to be determined.
Interestingly, three polypeptides are missing from pf16 flagella
(2), and these molecules, if orthologues exist in mice, are
candidates for interacting proteins.
We deduce that Spag6 is important for the motility of cilia,
as reflected in the occurrence of hydrocephalus in Spag6-defi-
cient mice, an indication of a motility defect in ependymal cell
cilia (7, 10). Low levels of Spag6 mRNA are present in the lung
(11), and the mRNA is expressed in the medulla as docu-
mented in expressed-sequence-tag data for UniGene Cluster
Hs.158213 (Spag6). Hydrocephalus, retarded postnatal growth,
and early death have been previously described in mice with
mutations affecting axonemal structure and function (7). While
the ultrastructural appearance of tracheal and ependymal cell
cilia of Spag6?/?mice was normal, we did not directly examine
the motility of ependymal cell cilia and can only infer from the
occurrence of hydrocephalus that ciliary motility is affected.
Mice with defects in Tg737, the homologue of the Chlamydo-
monas IFT88 intraflagellar transport protein, die shortly after
birth from polycystic kidney disease, resulting from shorter
than normal primary cilia in the kidneys of these mice (14).
This renal lesion was not observed in Spag6-deficient mice.
Some epithelial cells of the female reproductive tract are
ciliated, and normal beating of these organelles could be im-
portant for gamete and embryo transport. The fact that fe-
males lacking Spag6 were fertile suggests that either ciliary
TABLE 2. Motility characteristics of sperm from Spag?/?, Spag?/?, and Spag6?/?micea
Sperm concn (106/ml)
(mean ? SD)
% Motile sperm (mean ? SD)
(range of mean)
VCL (mean ? SD)
(range of mean)
LIN (mean ? SD)
(range of mean)
82 ? 31b
65 ? 35b
26 ? 9b
52 ? 6b(46–58)
56 ? 5b(52–62)
8 ? 9c(0.3–18)
341 ? 33b(313–378)
252 ? 37c(218–292)
136 ? 8d(127–142)
35 ? 5b,c(30–40)
38 ? 4c(34–42)
27 ? 3b(24–30)
aComputer-assisted sperm analysis was performed as described in the text. The number of motile sperm samples analyzed for each genotype ranged from 289 to
1,107 for wild-type mice, 425 to 635 for heterozygous mice, and 11 to 366 for nullizygous mice. n ? number of males tested; VCL, mean curvilinear velocity; LIN ?
linearity, the best estimate of the straightness of a sperm cell’s track (100 ? a straight line). Means with different superscripts (b, c, and d) in the same column are
statistically significantly different: P ? 0.05 by analysis of variance and Newman-Keuls multiple-comparison test.
6302SAPIRO ET AL.MOL. CELL. BIOL.
FIG. 4. Morphology of epididymal sperm from Spag6?/?mice. (A) Wild-type sperm. (B) Sperm cell from a Spag6?/?mouse showing a
disruption of the midpiece. (C) Headless sperm from a Spag6?/?mouse. (D) Sperm cell with a truncated flagellum and abnormal midpiece from
a Spag6?/?mouse. (E) Phase-contrast figure of wild-type sperm. (F) Immunostaining of Spag6 in Triton X-100-permeabilized wild-type sperm
showing staining along the tail. (G) Phase-contrast image of Spag6-deficient sperm. (H) Absence of Spag6 staining in Spag6-deficient sperm.
Arrowheads indicate abnormalities.
VOL. 22, 2002 Spag6 AND SPERM STRUCTURE AND MOTILITY6303
function is not impaired in Spag6-deficient females or that
normal ciliary function is not essential for female reproduc-
tion. However, the finding that only 80% of the nullizygous
females conceived in our study and that the time to pregnancy
was delayed suggest that there may be subtle deficits in
reproductive function in Spag6?/?females. Moreover, the nul-
lizygous mice that died early with severe hydrocephalus may
have had more profound defects in ciliary function and could
have demonstrated impaired fertility had they survived to ma-
Human infertility associated with absence of the central pair
of microtubules, “9 ? 0” immotile sperm, has been reported by
several authors (12, 13, 23). Other ultrastructural defects have
been described in the flagella of these cases. The underlying
causes of these abnormalities remain largely unknown. Our
findings suggest that mutations in the SPAG6 gene could be
In summary, we have shown that a conserved orthologue of
a Chlamydomonas central apparatus protein plays a key role in
regulating the function of ependymal cilia and in maintaining
the motility and organization of the mouse sperm flagellum
and, consequently, male fertility.
FIG. 5. Ultrastructure of epididymal and testicular sperm from Spag6?/?mice. (A) Immunoelectron microscopy localization of Spag6 in
wild-type epididymal sperm using colloidal gold-labeled antibody. Spag6 is localized in the central apparatus. Control sections processed in the
absence of primary antibody showed no specific localization (not shown). Inset, transverse section through a wild-type epididymal sperm viewed
by transmission electron microscopy. (B) Ultrastructure of Spag6?/?testicular sperm revealing normal flagellar architecture (C) Transverse section
from a Spag6?/?epididymal sperm lacking a central apparatus (arrowhead). (D) Transverse sections through Spag6?/?epididymal sperm showing
intact central apparatus microtubules but supernumerary (a) and disorganized outer dense fibers (b).
6304 SAPIRO ET AL.MOL. CELL. BIOL.
This research was supported by NIH grants HD37416 (J.F.S.),
HD06274 (J.F.S. and G.L.G.), and HD15045 (P.O.-C.). R.S. was a
visiting scholar from the Department of Histology and Embryology,
Faculty of Medicine, University of the Republic, Montevideo, Uru-
guay, and supported by the Fogarty International Center (D43-TW/
We thank Stuart Moss and Vargheese Chennathukuzhi and Melanie
Lieberman for their technical advice and comments on this work. The
Biomedical Imaging Core Laboratory of the University of Pennsylva-
nia Diabetes Center supported by DK19525 is also recognized for
assistance with the ultrastructural analysis. We acknowledge the advice
and helpful comments of Pete Lefebvre (University of Minnesota)
during the course of this work.
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TABLE 3. Structural abnormalities in the flagella of wild-type and Spag6?/?sperma
% Normal flagella
(mean ? SE) for:
% Flagella lacking central
pair (mean ? SE) for:
% Flagella with FS and/or
(mean ? SE) for:
98 ? 2
99 ? 1
40 ? 15
68 ? 12
77 ? 11
86 ? 3
2.3 ? 2
32 ? 11*
8 ? 6*
0 28 ? 4
24.3 ? 3
23 ? 11
14 ? 3
1 ? 2
aTransverse sections of flagella from wild-type and Spag6?/?sperm were examined by transmission electron microscopy. Each cross-section was scored for flagellar
abnormalities (absence of central pair of microtubules or alterations in the fibrous sheath and/or outer dense fibers). Sperm from three wild-type and three Spag6?/?
mice was examined. A total of 264 Spag6?/?and 179 Spag6?/?epididymal sperm and 247 Spag6?/?and 166 Spag6?/?testicular sperm were examined. *, P ? 0.001,
chi-square test. MP, midpiece; PP, principal piece; FS, fibrous sheath; and ODF, outer dense fibers.
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