Faculty of Pharmaceutical Sciences, Nagasaki International University, Huis Ten Bosch, Sasebo, Nagasaki, Japan
Department of Urology, Graduate School of Medicine, Osaka University, Yamadaoka, Suita, Osaka, Japan
Department of Urology, Juntendo University Hospital, Hongo, bunnkyouku, Tokyo, Japan
Faculty of Pharmaceutical Sciences
Nagasaki International University
Huis Ten Bosch
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Genetic Polymorphisms within The Intronless ACTL7A and
ACTL7B Genes Encoding Spermatogenesis-Specific Actin-Like
Proteins in Japanese Males.
Int J Fertil Steril.
Actins play essential roles in cellular morphogenesis. In mice, the T-actin1 and 2 genes, which encode actin-like
proteins, are specifically expressed in haploid germ cells. Both T-ACTIN1/ACTLB and T-ACTIN2/ACTL7A have also
been cloned and studied. The orthologous genes in humans are present on chromosome 9q31.3 as intronless genes.
Defects of germ cell-specific genes can introduce infertility without somatic function impairment. We determined T-
ACTIN1 and 2, specifically expressed in the testis using reverse-transcription polymerase chain reaction (RT-PCR).
To examine whether genetic polymorphisms of the T-ACTIN1 and 2 genes are associated with male infertility, we
screened for T-ACTIN1 and 2 polymorphisms by direct sequencing of DNA from 282 sterile and 89 fertile Japanese
men. We identified five and six single nucleotide polymorphisms (SNPs) in the T-ACTIN1 and 2 regions of the sterile
and fertile subjects respectively. Among these genetic polymorphisms was a novel SNP that was not in the National
Center for Biotechnology Information SNP database. Although we could not determine whether these SNPs cause
infertility, the prevalence of these genetic polymorphisms may be useful for analyzing polymorphisms in future large-
scale genetic analyses.
After meiosis, round spermatids undergo a dramatic
change to develop the specific morphology of the mature
sperm. Actin proteins play important functions in this process
(1). We developed a mouse subtracted library including
genes specifically expressed in spermatogenesis and
cloned and characterized these genes (2). Among these
genes were T-actin1 and 2, which encode actin-like proteins
and are specifically expressed in haploid germ cells.
T-actin1 is located in the cytoplasm while T-actin2 is localized
in the nuclei of testicular haploid germ cells and is
present only in the heads and tails of sperm (3). In both the
mouse and human genome, T-ACTIN1 and 2 are positioned
head-to-head and lack introns (4, 5). The resulting amino
acid sequences, genomic construction, and cAMP response
elements (CRE) consensus DNA sequence of the promoters
of T-actin1 and 2 are conserved in mice (4). These
genes have been reported to cause infertility by inducing
autoimmunity to sperm (6). Human T-ACTINs may play
important roles in the specific morphogenesis of spermatozoa
during spermiogenesis, as well as in sperm function.
We investigated genetic polymorphisms in the DNA
sequences of germ cell-specific genes in infertile male
patients and male volunteers with confirmed fertility (7-
19) to identify polymorphisms potentially linked to male
infertility (7, 8, 15, 19). In this study, we report our analysis
of genetic polymorphisms in T-ACTIN1/ACTLB and
T-ACTIN2/ACTL7A in Japanese men.
Defects in germ cell-specific genes may be a cause
of idiopathic infertility. To detect the presence of small
amounts of transcripts, we examined tissue-specific expression
patterns of T-ACTIN1 and 2 by reverse-transcription
polymerase chain reaction (RT-PCR) using cDNA
from various organs and a Rapid-Scan gene expression
panel containing cDNA from different human tissues
(OriGene Technologies, Rockville, MD, USA) (20). The
were designed to amplify fragments from the open reading
frames. Polymerase chain reaction (PCR) was performed
using Gflex Hot Start (Takara, Japan). The cycling
conditions were 96°C for 2 minutes, followed by 35 cycles
of denaturation at 96°C for 45 seconds, annealing at 58°C
for 45 seconds, and extension at 68°C for 90 seconds. As a
control, ß-actin was also amplified using primers:
according to the manufacturer’s protocol. T-ACTINs
were specifically detected only in the testis (Fig .1,).
- mRNA expression of T-ACTIN1 and 2 in various human organs. Multiple
human tissue cDNAs were subjected to polymerase chain reaction
analysis. Fragments of T-ACTIN1 and 2 were specifically detected in the
testes. Numbers in the right-hand margin indicate the lengths of the amplified
fragments and DNA ladder makers. The expression of actin mRNA
was also examined as a control.
The entire coding sequences of T-ACTIN1 and 2 (National
Center for Biotechnology Information [NCBI]
accession number: chromosome 9, NC_000009.12
(108862228..108863755), Fig.2) are intronless, similar to
mouse T-actin1 and 2. As T-ACTINs are expressed at high
levels in the human testis (Fig .1,), we investigated whether
genetic polymorphisms in T-ACTINs are associated with
- Schematic view of the T-ACTIN1 and 2 genes. The T-ACTIN1 and 2
intronless genes are located on chromosome 9 (NCBI accession number:
NC_000009.12). The box indicates the transcribed region of the T-ACTIN1
and 2 genes. The open reading frame is shaded. T-ACTIN1 is transcribed
to the right and T-ACTIN2 to the left. The small horizontal arrows in the
box indicate the locations of the polymerase chain reaction (PCR) and
DNA-sequencing primers. The arrowheads indicate the positions of genetic
polymorphisms. The NCBI accession numbers of T-ACTIN1 and 2 are
CCDS4295.1 and CCDS6772.1, respectively.
Infertile Japanese subjects (n=282) were divided into
subgroups according to the degree of defective spermatogenesis:
192 patients (68%) had non-obstructive azoospermia,
and 90 (32%) had severe oligospermia (<5×106
cells/mL), according to the criteria of the World Health
Organization (Table 1,). All patients had idiopathic infertility
based on cytogenetic analysis and no history of other
medical conditions, including cryptorchidism, recurrent
infections, trauma, orchitis, varicocele, and others. The
control group consisted of fertile males who had fathered
children born at a maternity clinic (n=89). All donors were
informed of the purpose of the study and gave permission
for use of their blood for genomic DNA data. This study
was approved by the institutional review board and independent
ethics committee of Osaka University.
Genomic DNA was isolated from blood samples by protease
treatment and phenol extraction. T-ACTIN1 and 2
sequences were amplified through PCR using the following
for T-ACTIN 2 (Fig .2,).
PCR was performed using Ex Taq Hot Start (Takara,
Japan) and consisted of 40 cycles of 96ºC for 45 seconds,
65ºC for 45 seconds, and 72ºC for 90 seconds. PCR-amplified
fragments were purified using SUPREC PCR spin
columns (Takara). The resulting DNA fragments were sequenced
independently from both ends by the same PCR
protocol using thermal cycle sequencing kits (Applied
Biosystems, Foster City, CA, USA). Internal primers:
were used to confirm T-ACTIN genes with ambiguous
identifications. The reaction products were analyzed using
an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems).
Differences in variables between the experimental
and control conditions were compared using Fisher’s exact
Nucleic acid base exchanges introducing one nonsense
mutation and four silent mutations were found in the
T-ACTIN1 open reading frame (Table 2,). Single nucleotide
polymorphisms (SNPs) were found in three silent
mutations (48C>T, 561C>T, 870C>T) as minor genotypes
in the entire Japanese cohort. The minor 1137
C>T homozygous alleles on T-ACTIN1 was not detected
in the infertile group. One nonsense mutation was
found in the volunteer group. The translated region of
T-ACTIN1 is 1248 bp long, and the nonsense mutation
appears at base pair 1,171, near the C-terminus. This
mutation thus has little influence on the function of the
translated protein, making it unlikely to be a cause of
Prevalence of single nucleotide polymorphisms (SNPs) in T-ACTIN1 in infertile or proven fertile populations
Number (%) of SNP
Proven fertile (%)
(NCBI dbSNP rs#)
In present study
R; Arginine, C; Cysteine, P; Proline, V; Proline, M; Methionine, and *; The nucleotide positions relative to the first methionine.
Two nucleic acid base exchanges introducing amino
acid substitutions and four silent mutations were found
in the T-ACTIN2 open reading frame (Table 3,). The frequency
of minor genotypes associated with T-ACTIN2 nucleotide
polymorphisms was low in Japanese males. One
silent mutation, 153A>G, in T-ACTIN2 was not registered
in the NCBI SNP database (dbSNP), marking a novel discovery
in our Japanese cohort.
Logistic regression modeling of the prevalence of haplotypes,
including SNPs, revealed no significant differences
between major and minor alleles lacking the 1,018
G>A on T-ACTIN2 SNP in males proven to be fertile. The
minor 1,018 G>A homozygous alleles on T-ACTIN2 in
males proven to be fertile is considered to be due to an
error made by the sequencer.
The appearance of 48 C>T and 561 C>T in T-ACTIN1
was linked; as was the appearance of 657 G>A and 1,018
G>A in T-ACTIN2. Thus, the SNPs in these two genes
may have the same origin.
Although many SNPs have been registered in the NCBI
dbSNP, we detected only 11 genetic polymorphisms in
the open reading frames of the T-ACTIN genes among
371 Japanese men. Finally, z χ2-test was used to compare
genotype distributions between infertile males and proven
fertile controls. There were no significant differences for
the minor genotypes (P>0.05).
Our research group has focused on cloning and analyzing
germ cell-specific genes. Chromosome mapping
of these genes revealed that they are distributed across
various chromosomes, and that many are intronless (21).
T-ACTINs are among these intronless genes and are specifically
expressed in the testis (Fig .2,). The dysfunction
of germ cell-specific genes does not affect ontogeny and
may be a cause of unexplained male infertility. The dysfunction
of these genes in mice has been shown to lead
to infertility (22). Dominant-negative gene mutations are
not passed on to the next generation, however other gene
mutations can be inherited from a heterozygous male parent
or from the female parent. More than 20% of married
couples in Japan are affected by infertility and the male
partner is responsible in two-thirds of these cases (23).
We undertook an extensive analysis of genetic polymorphisms
in germ cell-specific genes and of the relationship
between gene polymorphisms and infertility (7-19).
We found potential relationships between infertility in
Japanese men and genetic polymorphisms or mutations
in PRM2, TP1, PGAM4, and SCOT-T (7, 8, 15, 19). We
analyzed SNPs in germ cell-specific genes and found that
some included genetic polymorphisms with single amino
acid substitutions, whereas other specific genes had few
genetic polymorphisms. Most genes having several genetic
polymorphisms encoded in proteins were involved
in signal transduction or regulation, whereas those with
few genetic polymorphisms were more likely to encode
structural proteins (12). In this study, we discovered
several different SNPs in T-ACTIN1 and 2 in a cohort
of Japanese men. The similar frequencies of these polymorphisms
between the fertile and infertile groups in this
study imply that these mutations are not associated with
male infertility. However, the prevalence data for these
genetic polymorphisms might be useful when analyzing
the association of traits and genetic polymorphisms in further
large-scale genetic analyses.
We thank Ms. H. Nishimuna, Mr. H. Kawaguchi, and Ms. M. Kanzaki for their technical assistance with the sequence analysis of human DNA samples. The funding sources played no role in the study design; the collection, analyses, or interpretation of data; or in the writing or publication of the manuscript. The authors declare no conflict of interest.
H.T.; Contributed to conception and design, all experimental work, data and statistical analysis, and interpretation of data, and wrote the manuscript. Y.M., A.T., M.W.; Contributed to materials and analyzed the data. All authors read and approved the final manuscript.
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