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 ?
matogenesis, primer sets were designed so that the three isoforms
1, the forward primers are in individual non-translated exons (Fig.
performed using cDNAs reversely transcribed from total RNA
isolated from different tissues. Meig1?v1 message, which was re-
including lung, liver, testis, and oocytes; Meig1?v2, which was
reported to be germ cell-specific isoform, was present in almost
all of the tissues analyzed (Fig. 1B). The other isoform we
identified, Meig1?v3 was only expressed in the testis (Fig. 1B).
Expression patterns of the three isoforms were also investigated
during the first wave of spermatogenesis (Fig. 1C). Meig1?v1
message was present from d20 after birth, Meig1?v2 was present
throughout the whole process of spermatogenesis, Meig1?v3 was
detectable from d12 after birth, and the message was abundant
in a late stage of spermiogenesis.
The Ensemble Genome Browser shows five distinct Meig1 tran-
scripts in the mouse: Meig1-001, 002, 003, 004, and 005. Sequences
of these five transcripts were compared with the three Meig1
transcripts we identified. Meig1-002 is identical to Meig1-v1; Meig1-
001 is identical to Meig1-v2, except that it lacks sequence I; and
Meig1-003 and Meig1-v3 share the same coding exons and the
nontranslated exon 1c, but Meig1-003 contains an extra nontrans-
lated exon, sequence III in 1b. Meig1-004 contains the same coding
sequence as the other Meig1 transcripts, but a shorter 3?UTR, in
addition, its nontranslated exon is located between 1b and 1c (Fig.
S2). Specific primers were designed for PCR amplification of
Meig1-004 and Meig1-005. Meig1-004 is only present in the testis.
Besides being highly expressed in the testis, Meig1-005 was present
in most of the tissues tested, including brain, lung, and oviduct (Fig.
S2Ba). During the first wave of spermatogenesis, Meig1-004 is
To investigate the role of the Meig1 gene in vivo, we generated
upstream and downstream of the first coding exon, exon 1. The
targeting construct was linearized by ClaI digestion at the plasmid/
gene insert junction and transfected into the XY ES cells by
electroporation. The ES clones were screened by PCR with a
forward primer located in the Neo cassette of the target construct,
and a reverse primer located downstream of the right arm; or a
forward primer located upstream of the left arm, and a reverse
primer located in the Neo cassette (as shown in Fig. S3A). Five
positive ES cell clones were identified by PCR and were further
confirmed by Southern blotting using a probe as shown in Fig. S3A
corresponding to an upstream region flanking the mutation site.
and a 7-kb DNA in the targeted allele when DNA was digested by
BamH I (Fig. S3B). ES cells from 2 different clones verified to
contain the properly Meig1-mutated gene were injected into blas-
tocysts to produce chimeric mice. The chimeric male mice were
bred to wild-type female mice to create heterozygous mice without
deletion of any genomic sequence (Fig. S3C). These heterozygous
mice were crossed to FLP recombinase transgenic mice to delete
the Neo cassette, and the resulting mice (Meig1floxmice) were
crossed with CMV-Cre transgenic mice expressing Cre recom-
binase and thus deleting exon 1 of the Meig1 gene during
When Meig1floxmice were crossed to CMV-Cre transgenic mice,
it was found that the mutant allele could be transmitted from both
Meig1 heterozygous males and females, and that homozygous mice
female mice (Fig. 2A). Both male and female null mice were viable
in mice, including failure to thrive, hydrocephalus, situs inversus,
sinus, and pulmonary infection. Northern blot and RT-PCR anal-
yses revealed that the Meig1 messages were undetectable in the
testis, and all other somatic tissues evaluated (Fig. 2 B and C and
Fig. S2Bc). Western blot analysis using testis extracts demonstrated
that MEIG1 protein was absent in the Meig1-null mice (Fig. 2D).
To test fertility of the Meig1 mutant mice, mature nullizygous
mice (?6 weeks of age) were mated with wild-type mice. All of the
wild-type (Meig1flox), heterozygous and homozygous female mice
tested delivered pups with normal litter sizes. Wild-type and
heterozygous males were also fertile. However, all nine homozy-
gous mutant male mice tested were sterile through 4 months of
breeding (Table 1).
The testis weight of the nullizygous mice euthanized at 6 months
of age was comparable to age-matched wild-type mice (Table 1).
mice, all stages of spermatogenesis were present (Fig. 2E and Fig.
S4). However, in Meig1 homozygous mutant mice, microscopy
revealed that most spermatids developed only up to steps 12–13, a
few to steps 14–15, and beyond that most had been shed (Fig. 2E
and Fig. S4). Even though spermiogenesis is arrested before
in the DNA content of cell populations between wild-type and
indicating no defects in meiosis (Fig. 2F). In epididymides from
Exons 1a 1 2
I II III
H B Sp L u Li Sm K T Oc Ov
6 8 12 16 20 30 42
Days after birth
Meig1 gene. Solid boxes indicate translated exons (1 and 2), open, notched,
and crossed boxes indicate nontranslated exons (1a, 1b, and 1c). The arrows
indicate location of RT-PCR primers. The red solid boxes indicate 3?UTR of the
Meig1 transcripts. I, II, III under 1b represent three consecutive sequences in
the exon. (B) Tissue distribution of the three Meig1 transcripts. H: heart; B:
brain; SP: spleen; Lu: lung; Li: liver; Sm: skeletal muscle; K: kidney; T: testis; Oc:
oocyte; Ov: oviduct. (C) mRNA expression of the three Meig1 transcripts
during the first wave of spermatogenesis (RT-PCR analysis).
Expression patterns of the three Meig1 isoforms. (A) Structure of the
www.pnas.org?cgi?doi?10.1073?pnas.0906414106Zhang et al.
wild-type adult mice, typical adult sperm concentrations were
found (Fig. 2G), but epididymides from Meig1 homozygous mutant
mice only contained debris and degenerating sperm, none of which
were motile as examined by visual inspection with a light micro-
scope (Fig. 2 G–I). Histological evaluation of the lung, brain, heart,
kidney and liver revealed normal tissue architecture. Transmission
electron microscopy demonstrated that spermatids in wild-type
formation and chromatin condensation and have a normal
manchette structure (Fig. 3 A–D and Fig. S5). However, in Meig1-
deficient mice, there was failure of flagellar formation, disorgani-
zation of sperm axonemes, and deformed sperm heads, no obvious
or disrupted manchette structures were seen in spermatids (Fig. 3
E–L and Fig. S6). We also observed condensed Sertoli cell cyto-
plasm. A normal ‘‘9 ? 2’’ axoneme arrangement was not present in
the flagellum of MEIG1-deficient spermatids, and some flagella
contained multiple axoneme structures. Interestingly, flagellar
components such as microtubules and outer dense fibers could be
detected but were not assembled correctly. This is consistent with
the normal expression of genes encoding axoneme and fibrous
+/- +/+ +/+ -/- +/- +/-
+/+ -/- +/- -/-
+/+ -/- +/- -/- +/+ -/-
B Li Lu Sp K H T T
Meig1 mutant wild-type
+/+ +/- -/-
Sperm count (x106)
50 µ µm 50 µ µm
50 µ µm 50 µ µm
25 µ µm 25 µ µm
25 µ µm
25 µ µm
25 µ µm
25 µ µm
heterozygous (?/?), wild-type (?/?), and homozygous (?/?) genotypes. (B) Northern blot analysis of testicular Meig1 mRNA expression in wild-type, heterozygous
and homozygous mice with Meig1 full-length cDNA as the probe. (C) All of the three Meig1 transcripts are disrupted in all of the tissues in Meig1/CMV-Cre mice as
(F) Analysis of haploid (D), diploid (2D), and tetraploid cells (4D) cell populations in wild-type and knockout mice by flow cytometry. (G) Representative images of
(I) Sperm count of wild-type (?/?), heterozygous (?/?), and homozygous (?/?) mice. n ? 8 for each group. *: P ? 0.001.
Phenotype of Meig1/CMV-Cre mutant mice. (A) Representative PCR results using a primer set (P3 and P4 in Fig. S3A, primer sequences in Table S1) showing
Table 1. Fertility, fecundity, and testis weight of wild-type and Meig1 mutant mice
(n ? 8)
(n ? 8, mg/g)
(n ? 8)
8.6 ? 0.6
8.4 ? 0.4
8.15 ? 0.41
8.05 ? 0.35
7.95 ? 0.41
9.4 ? 0.5
9.6 ? 0.4
10.1 ? 0.6
To test fertility, sexually mature mice were bred to wild-type animals for four months. Litter size was recorded
for each mating. Testis weight and body weight were measured from adult mice.
Zhang et al.PNAS ?
October 6, 2009 ?
vol. 106 ?
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sheath proteins in the MEIG1-deficient mice (Fig. S7) and the
observation that TNP2 failed to translocate into nuclei of sperma-
tids in Meig1-deficient mice (Fig. S8).
To explore possible mechanisms of MEIG1 action, we
carried out a yeast two-hybrid screen to identify interacting
partners. Among the 70 positive clones sequenced, 18 repre-
sented PACRG. This interaction was confirmed by co-
immunoprecipitation from lysates of MEIG1 and PACRG
co-transfected COS-1 cells (Fig. 4 A and B), and testicular
extracts (Fig. S9). Interestingly, testicular PACRG protein,
but not mRNA expression, was reduced in Meig1 mutant
mice. However, in quaking mutant mice in which the Pacrg
gene is deleted, testicular MEIG1 protein was unchanged
(Fig. 4 C–E).
and Meig1 mutant mice. Representative transmission
electronic microscopy images from an adult wild-type
mouse (A–D) and a Meig1 homozygous mutant mouse
(E–L). A and B show normal spermatogenesis and ax-
oneme structure in wild-type testes, C and D shows
normal condensing spermatids heads and manchette
structure (arrow heads) in wild-type testes. E shows
failure of spermatogenesis as evaluated by absence of
sperm in the lumen of seminiferous tubule. F repre-
sents highly condensed Sertoli cell cytoplasm (arrow).
G and H represent some condensing spermatids that
(C and D). Inserts in F and G represent two deformed
sperm heads. I–L and inserts show disorganized fla-
gella. Note that the flagella components, such as mi-
crotubules and outer dense fibers seem to be made
Some flagella contain multiple axonemal structure (J).
Arrows point to disorganized flagella.
Testicular ultrastructure in adult wild-type
COS-1 pEGFP-C1 PACRG
COS-1 pEGFP-C1 PACRG
Cell lysate Pre MEIG1 Ab
v/v TG testis
Meig1 mutant testis
Pacrg relative expression
v/v TG testis
Meig1 mutant testis
ac g e at ve exp ess o
(B) Co-immuniprecipitation of MEIG1 and PACRG. COS-1 cells were co-transfected with PACRG/pEGFP-C1 and MEIG1/pTarget plasmids. Forty-eight hours after
an anti-MEIG1 antibody (ii). (C) Representative Western blot analysis of testicular PACRG protein expression in Meig1 mutant mice. v/v: quakingviablemutant mice; v/v
TG: Pacrg-rescued qkvmice. (D) Real-time PCR analysis of Pacrg mRNA expression in Meig1 mutant mice. (E) Representative Western blot analysis of testicular MEIG1
protein expression in Pacrg mutant mice.
MEIG1 associates with PACRG. (A) Expression of PACRG in COS-1 cells. COS-1 cells were transfected with the PACRG/pEGFP-C1 or empty pEGFP-C1 vector.
www.pnas.org?cgi?doi?10.1073?pnas.0906414106Zhang et al.
The tissue distribution of the three Meig1 tanscripts we identified is
different. Meig1?v1 is expressed in several tissues, including testis,
lung, liver, and oocytes; it is the most abundant in the testis.
Meig1?v2 is expressed in almost all of the tissues tested, and the
abundance is comparable in these tissues, suggesting that Meig1?v2
is not a germ cell-specific transcript. Meig1?v3 is only present in the
testis, indicating that this is a testis-specific transcript.
The expression pattern of the three transcripts during the first
wave of spermatogenesis is also different. Meig1?v2 is stably ex-
pressed during the whole process of spermatogenesis, indicating it
is a house-keeping isoform. This result contrasts with the results of
previous studies, which suggested that 2a2 (Meig1?v2) was a germ
cell-specific isoform (5–9). Meig1?v1 and Meig1?v3 are expressed
from d20 and d12 after birth, respectively, indicating that that they
are late-meiosis and postmeiosis-expressed isoforms, suggesting a
in vivo suggests that the protein might have multiple functions
depending upon cell type and timing of expression.
The phenotype of the Meig1-deficient mice generated using
CMV-Cre excision was surprising, since previous studies suggested
that MEIG1 might play a role in meiosis (5–9). However, we found
that spermatocytes completed meiosis and spermatogenesis was
arrested in the elongation and condensation stage, revealing that
the MEIG1 is not necessary for meiosis, but is required for the
completion of spermiogenesis.
With the exception of male infertility, no other phenotype
major feature of infertility is failure of spermiogenesis rather
than sperm motility. Thus, MEIG1 is not a major candidate for
immotile cilia syndrome.
To further investigate the mechanisms of MEIG1 in the regula-
tion of spermatogenesis, a yeast two-hybrid screen was conducted,
and PACRG was identified to be a major partner of MEIG1 in the
testis. The Pacrg gene is co-regulated with the Parkin gene, a gene
involved in Parkinson’s disease, and it is a reverse strand gene
located upstream of the Parkin gene (17). In the quaking mouse,
promoter region, the first five coding exons of Parkin, and the
a neurological phenotype, including a severe lack of myelin and the
tremor of voluntary movements from which quaking mice get their
name, but also in male infertility (18, 19), a phenotype that mirrors
that of the Meig1 mutant mice described here. A subsequent study
demonstrated that the infertility phenotype of the chromosome 17
deletion is due to deletion of the Pacrg gene (20).
26), further investigation conclusively established its role in cilio-
genesis. Proteomics and biochemical studies revealed that it is a
component of Chlamydomonas reinhardtii centriole/basal bodies
(27, 28). In Trypanosoma brucei, two PACRG proteins localize
along the full length of the axoneme. Ablation of both proteins by
RNA interference knockdown experiments produced slow growth
and paralysis of the flagellum due to disorganized axoneme struc-
ture (29). Of particular interest, variation in the human PACRG
promoter was demonstrated to be a risk factor associated with
azoospermia (30). A recent study demonstrated that PACRG
associates with tubulin and microtubules (31).
The similar phenotypes of Meig1 and Pacrg mutant mice, the
that PACRG protein is markedly reduced in MEIG1-deficient
mice, suggests that the two proteins regulate spermiogenesis
and/or serve as a co-factor for PACRG action. Notably, the impact
of the absence of these two proteins is on formation of the sperm
flagella and not more broadly on cilia, as might have been antici-
pated from the studies on Chlamydomonas and trypanosomes. The
a failure of intramanchette transport, which plays a key role in
shaping the spermatid head, centrosome, and tail (32), and it has
been shown that centrosome and associated microtubules play an
essential during spermiogenesis (33–35). The manchette is a tran-
sient microtubular structure assembled concurrently with the elon-
the manchette can sort structural proteins to the centrosome and
the developing sperm tail through a mechanism of intramanchette
transport (IMT) (32). In addition, IMT can also transport cargo
proteins necessary for spermatid nuclear condensation (38). Dis-
ruption of manchette structure in Meig1 deficient mice may impair
the intramanchette transport system, resulting in the failure of
spermatid head shaping and the formation of flagella. Indeed, our
ultrastructural analysis of testis of MEIG1-deficient mice revealed
disrupted, whereas they were observed in wild-type testis. The
by a failure to produce components of the axoneme and fibrous
sheath, since the expression of flagellar genes, including Spag6,
Spag16L, Spag17, and Akap4 was not affected in MEIG1-deficient
mice (Fig. S7). Moreover, genes involved in the remodeling that
forms the sperm head or that encode proteins that localize to the
sperm head such as transition protein 2 (Tnp2), testis-specific
histone H1 (Histone H1t) and Spag16S were not altered in Meig1
mutant mice. However, we cannot rule out the possibility that the
expression of other genes playing important roles in flagellar
formation and remodeling of the sperm head are altered in the
absence of MEIG1. This possibility must be entertained since
MEIG1 has been reported to bind chromatin, and because MEIG1
is also present in somatic cells of the testis, where it might affect
cells and/or Leydig cells. In the same yeast two hybrid screen, a
protein that is present in the Chromatoid body was identified seven
that MEIG1 might, in addition, play a role in RNA processing,
which is required for spermiogenesis (39, 40).
In summary, we discovered that MEIG1, possibly through an inter-
action with PACRG, is essential for spermiogenesis, rather than its
originally proposed role in meiosis. The profound sperm head and tail
implicates MEIG1 as a critical gene for manchette structure and
function and the control of spermiogenesis.
Rapid Amplification of cDNA Ends (RACE). 5?- and 3?- RACE were carried out to
define the 5?- and 3?-untranslated region sequence of the mouse Meig1 using
according to the manufacturer’s instructions.
Generation of an Anti-MEIG1 Antibody. AcDNAencodingfull-lengthMEIG1was
protein was used to generate polyclonal antisera in rabbits by a commercial
Targeted Disruption of Meig1. The 1.1-kb deletion region (exon 1 and flanking
region) was amplified by PCR, and BamHI and LoxP sites were added to the left
end and a NotI site to the right end. The PCR product was digested with BamHI/
NotI, and cloned into the pBluescript plasmid (this construct was named M2/
pBlue). A 5-kb left arm (red line in Fig. S3A) was amplified and cloned into
and cloned into M3/pND1 to finish the final construct. The resulting targeting
Zhang et al.PNAS ?
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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