MEIG1 is essential for spermiogenesis in mice
Zhibing Zhanga,b,1, Xuening Shena, David R. Gudea,b, Bonney M. Wilkinsonc, Monica J. Justicec, Charles J. Flickingerd,
John C. Herrd, Edward M. Eddye, and Jerome F. Strauss IIIa,b
Departments ofaObstetrics and Gynecology andbBiochemistry, Virginia Commonwealth University, Richmond, VA 23298;cDepartment of Molecular and
Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030;dDepartment of Cell Biology, Center for Research in Contraceptive and
Reproductive Health, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908; andeLaboratories of Reproductive and Developmental
Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709
Edited by Abraham Kierszenbaum, City University of New York, and accepted by the Editorial Board August 6, 2009 (received for review June 9, 2009)
Spermatogenesis can be divided into three stages: spermatogonial
mitosis, meiosis of spermatocytes, and spermiogenesis. During
spermiogenesis, spermatids undergo dramatic morphological
changes including formation of a flagellum and chromosomal
packaging and condensation of the nucleus into the sperm head.
The genes regulating the latter processes are largely unknown. We
previously discovered that a bi-functional gene, Spag16, is essen-
tial for spermatogenesis. SPAG16S, the 35 kDa, testis-specific
isoform derived from the Spag16 gene, was found to bind to
meiosis expressed gene 1 product (MEIG1), a protein originally
thought to play a role in meiosis. We inactivated the Meig1 gene
and, unexpectedly, found that Meig1 mutant male mice had no
obvious defect in meiosis, but were sterile as a result of impaired
spermatogenesis at the stage of elongation and condensation.
Transmission electron microscopy revealed that the manchette, a
microtubular organelle essential for sperm head and flagellar
formation was disrupted in spermatids of MEIG1-deficient mice.
We also found that MEIG1 associates with the Parkin co-regulated
in MEIG1-deficient mice. PACRG is thought to play a key role in
assembly of the axonemes/flagella and the reproductive phenotype
of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. Our
findings reveal a critical role for the MEIG1/PARCG partnership in
manchette structure and function and the control of spermiogenesis.
final step of spermatogenesis. During this stage, the haploid round
spermatids differentiate into species-specific shaped spermato-
zoon, with dramatic morphological changes, including elongation
for this process (3, 4), the underlying mechanisms remains largely
unknown and need to be elucidated.
in a search for mammalian genes potentially involved in meiosis.
Two Meig1 transcripts, 11a2 and 2a2, were identified previously,
both containing three exons. The two transcripts share the same
ORF and 3? UTR, but differ in their 5? UTRs. Each has a unique
non-translated exon 1. The 11a2 message was expressed in somatic
cells in the testis, including Leydig cells, whereas the predominant
2a2 isoform was reported to be germ cell-specific. The 2a2 tran-
script begins to accumulate in the testis at day (d)8–9 of postnatal
(pn) development, coinciding with the entry of germ cells into
stages, when spermatocytes enter the pachytene stage. In situ
hybridization analysis showed that Meig1 expression level was low
in leptotene cells and increased as the cells progressed through
detected in embryonic ovary after d15 of gestation when the cells
entered the pachytene stage of meiosis 1, but not in adult ovary,
suggesting that Meig1 is a meiosis-associated gene (5–9). A recent
transcriptional profile study revealed that the Meig1 message is
also present in Sertoli cells in fetal gonads and a Sertoli cell line
TTE3 (10, 11). Although the molecular weight is 10 kDa
according to its amino acid composition, MEIG1 protein mi-
permatogenesis can be divided into three stages: spermatogo-
nial mitosis, meiosis of spermatocytes, and spermiogenesis, the
MEIG1 protein contains multiple consensus sequences for serine
protein is phosphorylated and forms a dimer in vivo (8). Further-
more, the phosphorylated dimer enters the nucleus during the first
meiotic prophase and binds to meiotic chromatin (9).
The function of MEIG1 remains unknown. Our previous inves-
tigation revealed that MEIG1 associates with SPAG16S, a 35-kDa
nuclear protein essential for spermatogenesis (12). Recent studies
by others also suggested that MEIG1 might be essential for sper-
matogenesis and related to ciliary function. Meig1 message is
dramatically reduced in heat shock transcription factor 2 (Hsf2)
mutant mice, and this may result in impaired spermatogenesis and
reduced fertility of Hsf2 mutant mice (13). A bioinformatic analysis
revealed that Meig1 is most abundantly expressed in tissues rich in
ciliated cells, such as, testis, lung, olfactory sensory neurons, and is,
therefore, predicted to be important for cilia function (14).
To investigate the function of the Meig1 gene, we generated a
Meig1-conditional knockout mouse model, and crossed it to CMV-
Cre transgenic mice so that the Meig1 gene was deleted globally in
vivo. Our studies with the globally targeted Meig1-knockout mice
indicate that homozygous mice are viable, but the males are sterile,
producing only a few sperm that are morphologically abnormal.
and condensation. These studies indicate that MEIG1 is a key
protein in the regulation of spermiogenesis.
To predict potential function of the MEIG1 protein, the mouse
NCBI conserved domain program (http://www.ncbi.nlm.nih.gov/
tified. However, the MEIG1 protein sequences were found to be
highly conserved among different species (Fig. S1).
with a Meig1 specific reverse primer localized in exon 1. The PCR
products were cloned into pCR2.1 TA vector, and 20 inserts were
fully sequenced. Three isoforms were identified, Meig1?v1,
Meig1?v2, and Meig1?v3. Meig1?v1 and Meig1?v2 correspond to 11a2
and 2a2, respectively, Meig1?v3 is a previously unidentified isoform.
by their non-translated exons. To further investigate the tissue
distribution and expression patterns during the first wave of sper-
Author contributions: Z.Z. designed research; Z.Z., X.S., D.R.G., and B.M.W. performed
research; B.M.W. and M.J.J. contributed new reagents/analytic tools; Z.Z., C.J.F., J.C.H.,
E.M.E., and J.F.S. analyzed data; and Z.Z. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
October 6, 2009 ?
vol. 106 ?
no. 40 ?
transfected into an XY ES line (TL) by electroporation. Positive ES cells were
Two positive ES cell clones were injected to generate chimeric mice.
Southern Blot Analysis. Fifteen micrograms of DNA from ES cells was digested
with BamHI and separated by 0.8% agarose gel electrophoresis. The DNA was
transferred to nylon membranes and probed with a Meig1 gene-specific probe
upstream of the homologous recombination region.
lane) were probed with a full-length coding region of mouse Meig1 cDNA or
other cDNAs for genes involved in sperm function.
subjected to Western blot analysis by using indicated antibodies.
Histology and Transmission Electron Microscopy. Testes were prepared for light
and electron microscopy by using standard methods (42).
Flow Cytometry. Fresh isolated mixed germ cells were washed twice in PBS and
stained cells were analyzed by using a FACScan flow cytometer.
Real-Time PCR. Total RNA was isolated from whole testes using TRIzol reagent
was screened with the full-length Meig1 coding region as bait following the
protocol provided in the kit.
cloned into the BamH1/XhoI site of the pTarget vector, Pacrg cDNA was cloned
into cloned into the EcoRI/BamHI sites of the pEGFP-C1vector, creating Meig1/
pTarget and Pacrg/pEGFP-C1plasmids.
Co-immunoprecipitation. COS-1 cells were transfected with Meig1/pTarget and
Pacrg/pEGFP-C1 plasmids. Forty-eight hours later, the cells were harvested into im-
munoprecipitation buffer (150 mM NaCl/50 mM Tris?HCl, pH 8.0/5 mM EDTA/1%
through a 20-gauge needle. After centrifugation at 11,600 ? g for 5 min, the
tants were then incubated with preimmune serum (negative control) or an anti-
MEIG1 polyclonal antibody at 4°C for 2 h, and protein A beads were added with a
blotting with monoclonal anti-GFP antibody and anti-MEIG1 antibody. Co-
incubated with anti-PACRG polyclonal antibody or preimmune rabbit serum, and
ACKNOWLEDGMENTS. We thank Glenn L. Radice and Igor Kostetskii for their
suggestions of making Meig1 conditional knockout construct; Dr. Rita Shiang
scopic histology preparations. This work was supported by National Institutes of
in part by the Intramural Research Program of the National Institutes of Health,
National Institute of Environmental Health Sciences Grant ZO1-ES070076 (to
E.M.E.) and National Institute of Child Health and Human Development F31
ing were performed in Histology Core Facility of Virginia Commonwealth Uni-
versity. Transmission electron microscopy was performed in the Imaging Core of
Virginia Commonwealth University (5P30NS047463).
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