BIOLOGY OF REPRODUCTION 77, 864–871 (2007)
Published online before print 15 August 2007.
A Heterozygous Mutation Disrupting the SPAG16 Gene Results in Biochemical
Instability of Central Apparatus Components of the Human Sperm Axoneme1
Zhibing Zhang,2,3Maimoona A. Zariwala,4Maha M. Mahadevan,5Pedro Caballero-Campo,7Xuening Shen,3
Estelle Escudier,8Be ´ne ´dicte Duriez,9Anne-Marie Bridoux,9Margaret Leigh,4George L. Gerton,10
Marcus Kennedy,6Serge Amselem,8Michael R. Knowles,4and Jerome F. Strauss III3
Department of Obstetrics & Gynecology,3Virginia Commonwealth University, Richmond, Virginia 23298
Pulmonary and Critical Care Medicine,4University of North Carolina, Chapel Hill, North Carolina 27599
Department of Obstetrics & Gynecology,5and Pulmonary and Critical Care,6
University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Laboratorio de Andrologı´a,7Unidad de Reproduccio ´n Humana, Clı´nica Tambre, 28002 Madrid, Spain
INSERM U. 428, Faculte de Medecine,8Universite Paris-Descartes, 75006 Paris, France
INSERM U. 654, Ho ˆpital Henri-Mondor,994010 Cre ´teil cedex, France
Center for Research on Reproduction and Women’s Health,10University of Pennsylvania,
Philadelphia, Pennsylvania 19104
The SPAG16 gene encodes two major transcripts, one for the
71-kDa SPAG16L, which is the orthologue of the Chlamydomo-
nas rheinhardtii central apparatus protein PF20, and a smaller
transcript, which codes for the 35-kDa SPAG16S nuclear protein
that represents the C-terminus (exons 11–16) of SPAG16L. We
have previously reported that a targeted mutation in exon 11 of
the Spag16 gene impairs spermatogenesis and prevents trans-
mission of the mutant allele in chimeric mice. In the present
report, we describe a heterozygous mutation in exon 13 of the
SPAG16 gene, which causes a frame shift and premature stop
codon, affording the opportunity to compare mutations with
similar impacts on SPAG16L and SPAG16S for male reproduc-
tive function in mice and men. We studied two male
heterozygotes for the SPAG16 mutation, both of which were
fertile. Freezing-boiling of isolated sperm from both affected
males resulted in the loss of the SPAG16L protein, SPAG6,
another central apparatus protein that interacts with SPAG16L,
and the 28-kDa fragment of SPAG17, which associates with
SPAG6. These proteins were also lost after freezing-boiling
cycles of sperm extracts from mice that were heterozygous for
an inactivating mutation (exons 2 and 3) in Spag16. Our findings
suggest that a heterozygous mutation that affects both SPAG16L
and SPAG16S does not cause male infertility in man, but is
associated with reduced stability of the interacting proteins of
the central apparatus in response to a thermal challenge, a
phenotype shared by the sperm of mice heterozygous for a
mutation that affects SPAG16L.
axoneme, central apparatus, mutation, sperm motility, SPAG16,
Cilia and flagella are evolutionarily conserved eukaryotic
organelles that extend from the cell body. They comprise a
microtubular backbone, the axoneme, which is surrounded by a
plasma membrane and organized by the basal body, a
cylindrical structure that consists of nine microtubule triplets
and that is located underneath the cell membrane [1–3].
Depending on their function, cilia can be divided into motile
and primary (nonmotile) cilia. Motile cilia are characterized by
a typical ‘‘9þ2’’ architecture, with nine outer microtubule
doublets and a central pair of microtubules. These 9þ2
structures are present in sperm and ciliated cells of the lung,
brain, oviduct, and other tissues . Attached to the outer nine
microtubules are the outer and inner dynein arms. The outer
arms generate power and adjust beat frequency [5, 6], while the
inner arms generate the axonemal waveform [7, 8]. The radial
spokes and central pair structures are key regulators of dynein
activity . More than 200 different polypeptides have been
identified in the axoneme [10–12], and cDNAs encoding some
of them have been cloned from Chlamydomonas rheinhardtii,
trypanosomes, and mammals [13–18]. Mutations in some of
these genes result in paralyzed flagella and cilia.
Polypeptide mapping of the Chlamydomonas central pair
suggests the presence of at least 23 different proteins in
addition to tubulin , including PF6 (SPAG17), PF16
(SPAG6), and PF20 (SPAG16) [17, 18, 20–25]. SPAG6,
SPAG16, and SPAG17 form part of an interactome, with
SPAG6 binding to SPAG16 and to SPAG17. SPAG16L has
contiguous tryptophan and aspartic acid (WD) repeats in the C-
terminus, which mediate interactions with SPAG6. These
proteins are essential for flagellar motility [26–28]. Targeted
mutations of the Spag16 and Spag6 genes in mice cause male
infertility due to severe defects in sperm motility.
The mouse Spag16 gene encodes a 2.5-kb transcript from
exons 1 to 17 and a 1.4-kb transcript from mouse exons 11–17,
and these transcripts are expressed in different patterns during
spermatogenesis, yielding proteins of 71 kDa (SPAG16L) and
35 kDa (SPAG16S), respectively. The SPAG16L protein is
expressed meiotically (in diploid cells) and is incorporated into
the central apparatus of the sperm tail. The SPAG16S protein,
which is derived from a testis-specific transcript in the mouse,
accumulates in both meiotic and postmeiotic (haploid) male
1Supported by National Institutes of Health Grants HD37416,
HD06724, and U54 RR019480.
2Correspondence: Zhibing Zhang, Department of Obstetrics & Gyne-
cology, Virginia Commonwealth University, 1101 East Marshall Street,
Room 11-028A, Richmond, VA 23298. FAX: 804 828 0573;
Received: 1 June 2007.
First decision: 26 June 2007.
Accepted: 30 July 2007.
? 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
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SPAG16 MUTATION AND CENTRAL APPARATUS ABNORMALITIES