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- Original Article -
The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa
Hong-Gang Li, Ai-Hua Liao, Xiao-Fang Ding, Hui Zhou, Cheng-Liang Xiong
Centre of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Abstract
Aim: To investigate the distribution of cation channel of sperm 1 (CATSPER1) protein and the presence of
CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human
sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for
immunocontraception. Methods: Human ejaculated sperm from normozoospermic donors
(n=12) and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse
transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively.
Spermatozoa from normozoospermic donors
(n=12) were individually processed using a swim-up procedure and were then incubated with CATSPER1
antibody at final concentrations of 20, 4 and
0.8mg/mL. After 1, 2 and 6 h incubation, progressive motility and fast
progressive motility were measured by means of computer-assisted semen
analysis. Results: CATSPER1 transcript
was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the
membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1
antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2
and 6 h incubation, and significant dose-dependent changes were observed.
Conclusion: CATSPER1 is meiotically and post-meiotically expressed in human testis tissue.
CATSPER1 mRNA in human ejaculated spermatozoa could be
a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human
CATSPER1 could be a possible target for
immunocontraception. (Asian J Androl 2006 May; 8: 301-306)
Keywords: cation channel of sperm 1; testis; spermatozoa; motility; immunocontraception; human
Correspondence to: Prof. Cheng-Liang Xiong, Centre of Reproductive Medicine, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan 430030, China.
Tel: +86-27-8369-2651, Fax: +86-27-8363-4374
E-mail: clxiong@mails.tjmu.edu.cn
Received 2005-08-20 Accepted 2005-12-23
1 Introduction
Calcium channel activities are involved in the process of sperm maturation, motility, capacitation and sperm-egg
interaction. Several calcium channels have been found to reside in mammalian sperm, including the T-type voltage
operated Ca2+ channels alpha 1G and 1H, the cyclic nucleotide gated channels and the transient receptor potential
(TRP) channel TRP2, the N-type, R-type, and
L-type voltage operated Ca2+ channels, and the pkD2 cation channel.
Recently, four members of a novel channel-like protein family, cation channel of sperm
1(CATSPER1), CATSPER2, CATSPER3 and CATSPER4, unlike other calcium channels, were identified to be exclusively expressed in mice testes
and to be linked to mice sperm mobility and male fertility [1-4].
As the first member of the family, mice CATSPER1 was cloned in 2001 [2]. Recently, its functional
characterization was further described [5]. In mice, CATSPER1 protein expressed exclusively in the testis and not in other
tissues. In mice sperm, CATSPER1 is localized primarily in the tail¡¯s principal piece and is directly associated with
sperm mobility [2]. Moreover, CATSPER1 is required for sperm hyperactivation and penetrating the zona pellucida
[5]. Gene-targeted elimination of the male mouse CATSPER1 results in poor sperm motility and infertility [2].
Based on its restricted expression pattern and important role in sperm mobility and male fertility, CATSPER1 is predicted
to be a potential target for male infertility screening and an ideal target for contraception [1, 2, 5]. However, little is known
about the expression and function of human CATSPER1. Here, we evaluate the distribution of CATSPER1 protein and the
presence of CATSPER1 mRNA in human testis and human ejaculated spermatozoa, and the influence of CATSPER1
antibody upon human sperm progressive motility.
2 Materials and methods
2.1 Materials
Human testis was obtained from Tongji Hospital (Wuhan, China) after obtaining a signed informing consent and
the approval of the Ethical Committee of this University. The tissue was immediately cryostored in liquid nitrogen
after surgery. Human semen samples were obtained from 12 consenting donors who met all the World Health
Organization (WHO) criteria [6] for normozoospermia by masturbation after 3-5 d of abstinence. Samples with
leukocytes and/or immature germ cell concentration greater than
106/mL were excluded.
Goat polyclonal antibody against human CATSPER1
(clone: H-20, 0.2mg/mL, in 0.01mol/L phosphate-buffered
saline [PBS] containing 0.1% sodium azide and 0.2%
gelatin) was from Santa Cruz Biotechnology incorporation
(California, USA). All reagents for immunohistochemistry were obtained from ZhongShan Golden Bridge
Biotechnology Company (Beijing, China). Percoll was purchased from Sigma Chemical Company (St. Louis, USA). TRI
Reageant was from the Molecular Research Center (Cincinnati, USA). All reagents for reverse transcription
polymerase chain reaction (RT-PCR) were obtained from Toyobo (Osaka, Japan).
2.2 Sperm preparation
1×107 spermatozoa of each sample were used for RNA extraction immediately after liquefaction (30-60min at
room temperature). The remaining spermatozoa were processed using the swim-up procedure [6, 7] and used to
study of influence of CATSPER1 antibody upon sperm motility. For the swim-up procedure, semen samples were
individually fractionated by two-layer (40-80%) Percoll gradient buffered in HEPES-balanced saline (115mmol/L
NaCl, 4mmol/L KCl, 0.5mmol/L MgCl2, 14mmol/L fructose, 25mmol/L HEPES, pH 8.0). After centrifugation at
800×g for 20min, the 80% Percoll fraction was pooled and examined under an optical microscope to verify the
efficacy of the regimen.
2.3 RNA extraction
Total RNA from liquid nitrogen frozen human testis and ejaculated spermatozoa were extracted with TRI Reagent
according to the manufacturer¡¯s instruction. Briefly, approximately 0.1g human testis tissue and pellets containing
approximately 1×107 spermatozoa were repetitively individually pipetted in 1mL TRI Reagent. RNA was separated
by chloroform and precipitated with isopropanol, and then washed with 75% ethanol. After briefly air-drying, RNA
pellets were dissolved in diethyl pyrocarbonate treated water and then stored at -70°C. The purity of RNA samples
was checked spectrophotometrically at 260 nm and 280mm.
2.4 RT-PCR
RT-PCR was performed as described in Sambrook and Russel [8]. Briefly, Total RNA
(2mg) was reverse-transcribed to cDNA as follows: 1 h at 42°C with 100 IU MMLV reverse transcriptase,
500mmol/L of each deoxy-nucleotide-triphosphate,
1mg oligo(dT)18 and 50 IU RNasin in a final volume of
25mL, then 5min at 95°C. The cDNA were further amplified by PCR using selected primers. PCR primers for human
CATSPER1 (GenBank accession number AF407333) were designed using Primer 5 software by professional staff of AuGCT Company (Beijing,
China). To eliminate possible contamination by genomic DNA, primers were chosen in different exons of
CATSPER1 and were as follows: forward 5¡¯-TTTACCTGCCTCTTCCTCTTCT-3¡¯ (1905-1926nt), located in exon 5 and exon 6;
reverse, 5¡¯-ACCAGGTTGA
GGAAGATGAAGT-3¡¯ (2110-2131nt), located in exon 7 and exon 8. PCR primer pairs amplified a 227-bp segment
and the PCR amplification was performed on a Perkin Elmer DNA thermal cycler 480 (Wellesley, USA)
in a final volume of 25mL PCR mixture consisting of 10mmol/L Tris-HCl
(pH 8.3), 50mmol/L KCl, 2mmol/L MgCl2,
200mmol/L of each deoxy-nucleotide-triphos- phate, 1.5IU Taq DNA polymerase and
50 pmol/L of each primer.
0.2mg cDNA reverse-transcribed from human testis or sperm RNA was used as template, and negative control
(human sperm RNA reverse-transcribed without MMLV reverse transcriptase) was included.
β-actin was used in parallel for each run as internal
control. The forward primer (5¡¯-GGGAAATCGTGCGTGACAT-3¡¯) and reverse
primer (5¡¯-TCAGGAGGAGCAATGATCTTG-3¡¯) for β-actin were designed to produce a
385 bp amplicon. PCR products were run on a 1.5% agarose gel stained with ethidium bromide and visualized under ultraviolet transillumination.
2.5 Immunolocalization of CATSPER1 protein in human testis and sperm
Smears of human ejaculated spermatozoa and 10mm thin frozen sections of human testis
were used to study the immunolocalization of CATSPER1. Immunohistochemistry was performed as previously described [9]. Briefly,
Goat anti-human CATSPER1 polyclonal antibody at 1: 50 dilution was used as the primary antibody.
Fluorescein-isothiocyanate-labeled rabbit anti-goat antibody
was used as the secondary antibody at a 1: 100 dilution.
0.01mol/L phorsphoric acid buffer solution (PBS,
pH7.4) instead of primary antibodiy was used as the negative control. The
human testis was stained by Hoechest (Beyotime Biotechnology, China) to discriminate nucleus of the cells in the
seminiferous tubules.
2.6 Determination of CATSPER1 antibody influence upon sperm motility
Spermatozoa pooled in 80% Percoll layer described above were diluted to
50×106 cells/mL in Biggers-Whitten-Whittingham medium devoid of bicarbonate and BSA and containing 1mmol/L
CaCl2 and 25mmol/L HEPES (pH8.0) [10]. Aliquots of
90mL were incubated with 10mL CATSPER1 antibodies
(200mg/mL, 40mg/mL, 8mg/mL, diluted in 0.01mol/L PBS, pH7.4, containing 0.1% sodium azide and 0.2% gelatin) at final concentrations of 20,
4 and 0.8mg/mL. 10mL 0.01mol/L PBS (pH7.4, containing 0.1% sodium azide and 0.2% gelatin) instead of antibody was used as the
negative control. After 1, 2 and 6 h at 37°C with 5%
CO2, progressive motility (WHO motility class a+b) and fast
progressive motility (WHO motility class a) was measured using a computer-assisted motion analyzer (Beijing Weili,
China) at 37°C. At least 200 spermatozoa in randomly chosen fields of vision were analyzed.
2.7 Statistical analysis
The data are presented as mean±SD. Statistical analyses were performed using the SPSS software (version
11.5) by paired sample t-tests after checking for normal distribution by means of the Kolmogorov-Smirnov test. All
statistical tests were two-tailed and P <0.05 was considered as statistically significant.
3 Results
3.1 CATSPER1 expression
3.1.1 mRNA expression
In RNA isolated from human testis and 12 cases of human spermatozoa, robust PCR products of the expected
227 bp for CATSPER1 and 385 bp for
β-actin were identified following analysis on agarose gels (Figure1).
There was no detectable signal in samples without reverse transcriptase.
The primers were designed to amplify nucleotides
1905-2131nt. Comparison with the genomic sequence of the human
CATSPER1 indicated that this region encompasses 496 bp of DNA, including exons 5-8 and the intronic sequences. Therefore, it is highly unlike that the
CATSPER1 RT-PCR products in the human testis and in ejaculated spermatozoa were derived from genomic DNA.
3.1.2 Protein expression
In human testis, abundant CATSPER1 protein was detected in the membrane of spermatid (Figure2). In human
ejaculated spermatozoa, CATSPER1 protein was localized in the principal piece of the sperm tail (Figure3).
3.2 Influence of CATSPER1 antibody upon motility of human sperm
As shown in Table1, significant and dose-dependent changes in the percentage of progressively motile sperm
were observed after 1, 2 and 6 h incubation with CATSPER1 antibody at all concentrations used in the present study.
Moreover, under the influence of CATSPER1 antibody, similar changes of the
percentage of fast progressively motile sperm were observed (Table2). The percentage of slow progressively motile sperm (WHO motility class b) was not
affected by this treatment (data not show). Therefore, the decline of progressive motility caused by CATSPER1
antibody was mainly ascribed to the changes of fast progressive motility.
4 Discussion
The recent cloning and characterization of CATSPER1 is a matter of significance to the study
of male reproduction and infertility [1, 5]: 1) CATSPER1 is the first calcium ion channel required for sperm mobility and hyperactivation.
CATSPER1-deficient mice are infertile as a result of an impairment of sperm mobility and an inability to fertilize intact
oocytes; 2) The vital role of mice CATSPER1 in male fertility and the restricted loca-lization of CATSPER1 to the
mice testis and mature sperm suggest that
CATSPER1 gene could be a potential target for male infertility screening
and treatment, and it might be an excellent target for contraception. The study of the evolution of CATSPER1 in
primates and rodents also suggests its possibly important physiological role in sperm competition [11, 12]. Frequent
incidences of indel substitutions were discovered in the evolution of the first exon of
CATSPER1 gene. Because exon 1 is supposed to be an important functional part of this gene, it is reasonable to conclude that these indel substitutions,
particularly larger indels which are significantly more prevalent in exon 1 than in the neutral genomic regions, are
positively selected for the evolution of
CATSPER1, and positive selection on this gene suggests its important
physiological role.
To further explore the role and significance of CATSPER1, the expression and function of human CATSPER1
need to be addressed. In the present study, the expression profiles of CATSPER1 in human testis and ejaculated
spermatozoa were investigated for the first time. Consistent with the meiotic and postmeiotic expression in mice
testis [2, 13], CATSPER1 protein expressed in the membrane of spermatid. In ejaculated spermatozoa, CATSPER1
protein was highly localized to the principal piece of the sperm tail. The same expre-ssion and localization of CATSPER1
in humans as in mice indicates the important role of CATSPER1 in human male fertility.
To further elucidate the function of human CATSPER1 and to estimate its possibility as a target for potential
contraception, we studied the influence of CATSPER1 antibody upon human sperm motility
in vitro. Significant and dose-dependent changes in progressive motility and fast progressive motility after 1, 2 and 6 h incubation with
CATSPER1 antibody suggested that, consistent with the function of its mice counterpart, human CATSPER1, which
was highly localized to the principal piece of the sperm tail, play an important role in the progressive motility of human
spermatozoa. The inhibition effect of CATSPER1 antibody upon human sperm also implies that CATSPER1 could be
a possible target for immunocontraception.
CATSPER1 was implicated as a potential target for male infertility screening and treatment. Recently, the profiles
of CATSPER1 mRNA expression in testis biopsy of subfertile patients were investigated [13]. Compared with
patients whose infertility cannot be ascribed to a deficiency in motility, a significant reduction in the level of
CATSPER1 gene expression among patients who lack sperm motility was observed. However, testicular puncture is invasive and
inconvenient and might not be accepted by patients because of ethical considerations. The presence of
CATSPER1 mRNA in human testis and ejaculated spermatozoa were involved in the present study, and we found that
CATSPER1 mRNA was not only detected in human testis, but also, as a post-meiotically active gene,
CATSPER1 mRNA existed in human ejaculated spermatozoa. In fact, mRNA of some other CatSper family members were detected in human
ejaculated spermatozoa by our group (unpublished data). During the last decade, findings from several studies
support the conclusion that spermatozoa contain a complex repertoire of mRNA, including mRNA of some ion
channels [7, 14-17]. Their putative roles are unknown, but the most common idea is to consider that these
transcripts represent remnants of stored mRNA from post-meiotically active genes [15-17]. The study of mRNA in
sperm could reflect past events of spermatogenesis and/or spermiogenesis, and could be used as a clinical
assay to provide a panoramic view of testis gene expression that can be difficult to achieve from a testicular biopsy [15, 17].
Therefore, if CATSPER1 were to be evaluated as a target for male infertility screening and treatment, it is apparently
more feasible to study its mRNA in ejaculated spermatozoa than in testicular biopsy.
Taken together, the work here provides insights into expression and function of CATSPER1 in human testis and
ejaculated spermatozoa. Abundant CATSPER1 mRNA in human ejaculated spermatozoa might be a more feasible
target than testis biopsy for male infertility scree-ning and study on human CATSPER1.
Acknowledgment
We are grateful to Prof. Ming-Da Zuo, Ms Xue-Bing Pang and Ms Shuang Li for their excellent technical assistances
and advice. The investigation was partly supported by grants from the National Key Technologies R&D Program for
the Tenth Five-year plan, China (No. 2004BA720A33-01) and the Hubei province health department project (No.
JX2B03).
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