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From the Department of Science for Laboratory Animal Experimentation,
Research Institute for Microbial Disease, Osaka University, Osaka,
Japan.
*
Present address: Department of Development and Differentiation, Institute for
Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin,
Sakyo-ku, Kyoto, 606-8507, Japan.
| Correspondence to: Dr Hiromitsu Tanaka, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan (e-mail: tanaka{at}biken.osaka-u.ac.jp). |
| Received for publication December 15, 2004; accepted for publication February 17, 2005. |
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Abstract |
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Key words: Sperm, spermatozoa, vesicle, RBCC protein
In testes, the acrosome develops during the late stage of spermatogenesis. During postmeiotic haploid germ cell differentiation, referred to as spermiogenesis, molecules related to chromatin condensation, flagellum development, and acrosome biogenesis are specifically expressed (Tanaka and Baba, 2005). By constructing and screening a subtracted cDNA library enriched for genes expressed in spermatids, we have isolated multiple cDNAs that are specifically expressed in mouse haploid germ cells (Tanaka et al, 1994; Iguchi et al, 1999; Fujii et al, 2002). The mouse haprin gene encoding a novel RING finger, B-box–type zinc finger and coiled-coil domain (RBCC) motif protein has also been cloned from this cDNA library (Kitamura et al, 2003), and it has since been used as a marker for the differentiation of haploid germ cells (Geijsen et al, 2004). The RBCC motif is believed to provide an essential scaffold for homo/hetero-oligomerization by interacting with other proteins to form regulatory complexes required for cellular processes (Reymond et al, 2001). It has been demonstrated that mouse haprin is expressed specifically in the haploid germ cells of the testes, and the Haprin protein is localized to the acrosomal, cytoplasmic region of mature sperm (Kitamura et al, 2003). Inhibition experiments using specific antibodies have indicated that the Haprin protein plays an important role in the formation of the complex for the acrosome reaction, with protein-protein interactions mediated by the RBCC motif (Kitamura et al, 2003).
Infertility affects up to 10% of couples (Dessars and Cochaux, 1999), and an abnormal acrosome reaction could be a cause of male infertility (Liu and Baker, 1994; Liu et al, 2001). To search for the candidate genes and the mechanisms responsible for male infertility, we characterized the expression and protein localization of the human ortholog HAPRIN. The genomic structure and putative transcriptional regulatory elements are also discussed.
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Materials and Methods |
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Reverse Transcription–PCR Analysis
To examine the tissue-specific expression patterns of HAPRIN, we
carried out reverse transcription (RT)–PCR analysis using a
Rapid-ScanTM gene expression panel containing cDNAs from 24 different
human tissues (Origene Technologies, Rockville, Md). The specific forward
primer Fa2 (5'-TGGTTAACAGCTTATGGGCGG-3') was designed to amplify
fragments with deduced sizes of 742 and 507 bp, representing the
HAPRIN-a1 and -a2 splicing isoforms of the transcripts from
exon 1a (Figure 1). The
specific forward primer Fb1 was then designed to amplify fragments with a
deduced size of 807 bp, representing the HAPRIN-b splicing isoform of
the transcript from exon 1b. The reverse primer R2
(5'-GGTCTGAAGTTAGTAGTTGGACC-3') was used to amplify fragments for
splicing isoforms of the transcripts from exons 1a and 1b
(Figure 1). Cycling conditions
were 96°C for 1 minute, followed by 35 cycles of denaturation at 96°C
for 45 seconds, annealing at 62°C for 45 seconds, and extension at
72°C for 60 seconds. As a control, actin was also amplified according to
the manufacturer's protocol.
Human Tissue and Sperm Samples
Human testis fragments were obtained with informed consent from a fertile
middle-aged patient who was castrated for treatment of prostate cancer.
Samples were stored at -80°C until use. Human liver protein samples were
purchased from Clontech (BD Biosciences Clontech, Palo Alto, Calif). Human
semen samples were obtained from fertile male volunteers. After liquefaction,
the semen samples were gently suspended in phosphate-buffered saline (PBS) to
release sperm. After 1 hour, the "swim-up" sperm were centrifuged,
and the pellets were resuspended in either lysis buffer for Western blotting
analysis or in PBS for immunostaining of sperm. For the acrosome reaction,
swim-up sperm were induced with 10 µM calcium ionophore A23187
(Sigma-Aldrich, Tokyo, Japan) for 15 minutes, after a capacitation incubation
with human tubal fluid medium (Quinn et
al, 1985) for 1 hour. Subcellular fractionation of sperm was
performed using a previously described method
(Kitamura et al, 2003).
Briefly, sperm were suspended in ice-cold TN buffer (20 mM Tris-HCl, pH 7.0,
and 130 mM NaCl) and sonicated. The cell lysate was centrifuged at 10 000
x g for 10 minutes. The supernatant, the membrane fraction, was
further ultracentrifuged at 100 000 x g for 2 hours at 4°C
using an SW41 rotor (Beckman Coulter Inc, Fullerton, Calif). The precipitant
was used as the membrane fraction. The membrane fraction was extracted with 4%
Triton X-100, 1.5 M NaCl, 2 M urea, or 100 mM Na2CO3 (pH
11.5). The samples were solubilized in sodium dodecyl sulfate (SDS) sample
buffer and subjected to Western blot analysis.
Western Blot Analysis
Each sample containing 50 µg of protein was subjected to
SDS-polyacrylamide gel electrophoresis using 5%–20% polyacrylamide
gradient gels (ATTO, Tokyo, Japan) and was then electroblotted onto
polyvinylidene difluoride membrane filters (Millipore, Bedford, Mass). The
filters were blocked with 5% nonfat dried milk for 30 minutes and washed for
15 minutes with Tris-buffered saline-Tween (TBS-T: 50 mmol/L Tris-HCl, pH 7.5;
150 mmol/L NaCl; and 0.05% Tween-20). The filters were then reacted with
polyclonal rabbit anti-mouse Haprin (C-terminal region) antiserum, diluted
1:500 (Kitamura et al, 2003)
in TBS, for 1 hour at room temperature and were then washed 3 times in TBS-T
for 5 minutes each time. Finally, the filters were incubated with polyclonal
peroxidase-conjugated anti-rabbit immunoglobulin antibody (Amersham Pharmacia
Biotech, Tokyo, Japan; diluted 1:500) for 1 hour at 25°C. After further
washing, reactive bands were visualized by development with a peroxidase
staining kit (Wako, Osaka, Japan).
Immunofluorescence Microscopy of Human Sperm
Human sperm samples were spotted onto silane-coated Superfrost glass
microslides (Matsunami Glass Ind Ltd, Osaka, Japan) and treated with 70%
methanol on ice for 10 minutes. For indirect immunofluorescence staining, the
slides were blocked with 5% normal donkey serum for 1 hour and incubated with
rabbit anti-mouse Haprin antiserum (diluted 1:300 in PBS) for 16 hours at
4°C. After washing, the slides were treated with fluorescein-conjugated
donkey anti-rabbit antibody (Amersham; diluted 1: 500), incubated for 2 hours
at room temperature, washed with PBS, and observed under a fluorescence
microscope (Olympus BX50; Olympus, Tokyo, Japan). Acrosome status was
evaluated by staining with fluorescein isothiocyanate-conjugated peanut
agglutinin (Sigma-Aldrich).
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Results |
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Expression of HAPRIN Transcripts
We examined the expression of the HAPRIN mRNA in different organs
using RT-PCR analysis. As 3 mRNA variants (HAPRIN-a1/TRIM36,
HAPRIN-a2, and HAPRIN-b) were transcribed from distinct first
exons (1a and 1b), we designed 2 forward primers (Fa2 and Fb1 in
Figure 1) and a reverse primer
at exon 3 (R2 in Figure 1) to
amplify transcripts from exons 1a and 1b (Figures
1 and
3). All of the mRNA variants of
HAPRIN were expressed at the expected sizes in testes, but not in any
other organs examined (Figure
4). Although exons 1a and 1b both had a start ATG codon, the amino
acid sequences of the two isoforms differed (21 and 9 amino acids for exons 1a
and 1b, respectively) at the N-terminal end of the RBCC motif
(Figure 1). Sequence data for
an additional 507-bp amplified band from testes using the exon 1a primer
indicated that another splicing variant removing exon 2 of HAPRIN was
generated in the transcription of the HAPRIN gene (HAPRIN-a2
in Figure 2).
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Comparison of Mouse Haprin and Human HAPRIN Proteins
A stop codon was present 15 bp (HAPRIN-a1) or 42 bp
(HAPRIN-b) upstream from the first ATG of human HAPRIN cDNA
(Figure 1), and the deduced
amino acid sequences of human HAPRIN-a1 and -b were,
respectively, 92.5% and 92.1% identical to the orthologous mouse protein
(GenBank accession no. AB103063). The human HAPRIN protein also contained
several conserved regions consisting of a RING finger domain, two
B-box–type zinc-binding domains, and an 
-helical coiled-coil
domain known as an RBCC tripartite motif. The fibronectin type III and the
Spla kinase and ryanodine receptor (SPRY) domains were also conserved in the
C-terminal regions of the mouse and human proteins
(Figure 1). In the HAPRIN-a2
N-terminal region, the RING finger domain was truncated by the loss of exon 2,
although it is transcribed (Figures
1,
2, and
4).
Expression and Localization of the HAPRIN Protein
Western blot analysis using anti-mouse Haprin antiserum produced a major
positive band with a molecular weight of 82 000 and a faint band of 40 000
from human testes (Figure 5a).
The faint signal indicates a truncated HAPRIN protein translated from another
initiation signal of HAPRIN mRNA (eg, the methionine in the
coiled-coil domain; Figure 1)
or some processed products. Western blot analysis of the membrane fraction of
sperm showed 1 positive band with a molecular weight of 82 000 that was partly
extracted with 2 M urea, 1.5 M NaCl, or at pH 11.5, but that was not extracted
with 4% Triton X-100 (Figure
5b). These results indicate that the major component of HAPRIN
protein is localized in the sperm membrane and as membrane-associated
cytoskeletal elements. Immunofluorescence staining of ejaculated sperm with
the antiserum showed that the expression of the HAPRIN protein was restricted
to the acrosomal region of the sperm head
(Figure 6a through c). After
inducing the acrosome reaction with calcium ionophore, HAPRIN protein
disappeared from sperm heads in association with the loss of the acrosome
marker PNA labeling (Figure 6d through
i).
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Discussion |
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We have previously demonstrated that the mouse Haprin protein is exclusively expressed in haploid germ cells in testes and that its RBCC motif plays an important role in the acrosome reaction (Kitamura et al, 2003). In this study, we found that the HAPRIN gene was also expressed in human testes and that the product was localized to the acrosome of mature sperm. The fractionation studies of sperm proteins recovered the HAPRIN protein in the membrane-associated cytoskeletal fraction (Figure 5b), as was the case in the mouse. These results indicate that the Haprin protein is located in the plasma and outer acrosomal membrane and that it has a similar acrosome reaction function to its mouse ortholog. The amino acid sequence of HAPRIN is highly conserved between human and mouse. This is notable because a large number of genes involved in sex and male reproduction have been demonstrated to be undergoing rapid evolution (Wyckoff et al, 2000; Swanson and Vacquier, 2002); these include proteins involved in sperm–egg binding, such as ADAM2 and SAM-1, nuclear proteins such as protamine-1, and metabolic enzymes such as GAPDS (Torgerson et al, 2002). On the other hand, genes involved in the mechanisms of intracellular membrane fusion, such as the SNARE complex components, are highly conserved in eukaryotic cells (Ferro-Novick and Jahn, 1994). The high degree of sequence similarity between human and mouse HAPRIN indicates the importance of the protein in fertilization and reflects its indispensable function in intracellular membrane fusion during the sperm acrosome reaction.
RBCC family genes, such as the HAPRIN gene, are reported to be scattered throughout the human genome, except at 2 clusters at 6p21–23 and 11p15 (Reymond et al, 2001). The mouse haprin gene was mapped to 22.0 cM of chromosome 18 (Kitamura et al, 2003), and the human HAPRIN gene was mapped to chromosome 5q23.1, syntenic to the region of mouse haprin (Gregory et al, 2002). This also supports the hypothesis that HAPRIN is the human ortholog of the mouse haprin gene. RT-PCR analyses showed that the isoforms transcribed from 2 distinct putative first exons were expressed in the testes. HAPRIN-a1 and HAPRIN-b are transcribed from different first exons and encode different N-terminal amino acid sequences (21 and 9 amino acids, respectively), but this difference does not reach the consensus cysteine residue of the RING finger motif (Figure 1). These 2 isoforms were indistinguishable in the Western blot analysis (Figure 5). The HAPRIN-a2 transcript did not encode the complete HAPRIN protein because of the lack of exon 2, which caused a premature termination codon via a frame-shift (Figure 1). However, it is possible that another methionine codon may be used as a translation initiation signal, thereby producing a truncated HAPRIN protein. We found a faint band with a molecular weight of 40 000 from the testes in a Western blot analysis using anti-Haprin C-terminal region antiserum (Figure 5). The functional differences between HAPRIN-a1 and HAPRIN-b and the biological roles of the HAPRIN-a2 transcripts remain to be elucidated. Upstream from the putative transcription initiation site of both exons 1a and 1b are sequences with high GC content and high CpG ratios (Figure 3). However, these regions lack a typical TATA box or CRE sequence, which is recognized by the transcription factor CRE modulator tau (CREMt) and is essential for the transcription of several testis-specific genes. Recently, Han et al (2004) grouped germ cell–specific promoters with high GC content and CG dinucleotides. Some of these promoters lacked a TATA-like sequence: calmegin (Watanabe et al, 1995), PIASx (Santti et al, 2003), CaMII (Ikeshima et al, 1994), c-kit (Albanesi et al, 1996), and HSP70-2 (Dix et al, 1996). Male germ cell–specific expression of some of these genes is controlled by the methylation of CpG islands (Trasler et al, 1990; Ariel et al, 1991; De Smet et al, 1999).
The high level of evolutionary conservation of amino acid sequences and similar localization of HAPRIN protein indicate that the protein plays a crucial role in the regulatory mechanism of the acrosome reaction in human as well as in mouse sperm. Anomalies in HAPRIN function could result in male infertility. Further studies are currently in progress both to elucidate the precise molecular function of Haprin in mice and to screen for mutations or single nucleotide polymorphisms in the HAPRIN gene in infertile men.
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