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Mol. Hum. Reprod. Advance Access originally published online on March 8, 2007
Molecular Human Reproduction 2007 13(5):299-306; doi:10.1093/molehr/gam009
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility

Hong-Gang Li1, Xiao-Fang Ding2, Ai-Hua Liao1, Xiang-Bing Kong1 and Cheng-Liang Xiong1,3

1 Centre of Reproductive Medicine and Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Centre of Reproductive Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

3 To whom correspondence should be addressed at: Centre of Reproductive Medicine and Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. E-mail: clxiong{at}mails.tjmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
CatSper is a unique sperm cation channel-like protein family exclusively expressed in the testis and plays important roles in sperm functions. The temporal expression profiles of CatSper1–4 mRNAs in the mouse testis during post-natal development through adulthood were investigated using real-time RT–PCR. The CatSper2 transcript was present in the testis of the 8-day-old mice, and was repressed in the adult testis after two sharp up-regulations at day 18 and 35. CatSper1 and CatSper3, 4 mRNAs were detectable in the testis of 18-day and 15-day-old mice, respectively. After sharp up-regulation at day 25 and 35, respectively, they were maximal at the adult testis stage. The differences between the temporal expression profiles of the CatSper transcripts in post-natal mouse testis development suggest different regulation to their transcription, and potentially contribute to the possibility of forming heteromeric channels among these four CatSper family members. CatSper1–3 transcripts were identified to be present in the human ejaculated spermatozoa by RT–PCR. Significantly higher levels of CatSper2 and CatSper3 mRNAs revealed by real-time RT–PCR were observed in the high-motile spermatozoa than in the low-motile fraction and suggests that CatSper2 and CatSper3 transcripts in the human ejaculated spermatozoa could be the potential targets for further study and male infertility screening.

Key words: calcium/developmental biology/messenger RNAs/spermatozoa/testis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Calcium channel activities play important roles in different aspects of mammalian sperm functions such as sperm motility, capacitation and the acrosome reaction (for reviews see: Darszon et al., 2006; Jimenez-Gonzalez C et al., 2006). The research of ion channels in sperm is now focusing on the identification and characterization of genes and proteins. Multiple Ca2+-permeable channels have been found to reside in mammalian sperm, including the T-type voltage-operated Ca2+ channels alpha 1G and 1H (Espinosa et al., 1999; Son et al., 2000; Jagannathan et al., 2002; Trevino et al., 2004), the N-type, R-type (Wennemuth et al., 2000) and the L-type (Goodwin et al., 1997) voltage-operated Ca2+ channels, the voltage-operated Ca2+ channels alpha 1A, 1C and 1E plus beta subunits (Lievano et al., 1996; Serrano et al., 1999), the cyclic nucleotide gated (CNG) channels (Wiesner et al., 1998), the transient receptor potential (TRP) channel TRP2 (Jungnickel et al., 2001; Castellano et al., 2003), the pkD2 cation channel (Gao et al., 2003) and the high voltage-gated calcium channels (Trevino et al., 2004).

Recently, four members of a unique sperm cation channel-like protein family, named CatSper (Cation channel of Sperm) were cloned and characterized (Ren et al., 2001; Quill et al., 2001; Lobley et al., 2003). These four members are solely expressed in the testis and differentially localized in the sperm. CatSper1 and CatSper2 were identified to be essential for mouse sperm motility and male fertility (Carlson et al., 2003, 2005; Quill et al., 2003, 2006). Recently, it was revealed that CatSper1 is required for the Ca2+ current activation by alkalinization during sperm cell capacitation (Kirichok et al., 2006). The study of evolution of CatSper1 in primate and rodent also suggests its important physiological roles, possibly in sperm competition (Podlaha and Zhang, 2003; Podlaha et al., 2005). As for CatSper3 and CatSper4, a recent study has suggested their roles in acrosome reaction and male fertility (Jin et al., 2005).

Although these studies have demonstrated that CatSper family members play roles in controlling sperm functions, their actions and mechanisms as well as their performing units remain poorly understood. The failure to achieve functional expression of CatSper1 and CatSper2 singly or in conjunction with heterogonous expression systems suggests that CatSper1 and CatSper2 proteins require additional subunits and/or interaction partners to function (Quill et al., 2001; Ren et al., 2001). The search for the accessory proteins led to the in silico identification of two further members of the CatSper family, CatSper3 and CatSper4 (Lobley et al., 2003). Based upon their similar domain structure, these four CatSper proteins were predicted to form a functional hetero-tetrameric channel in sperm (Lobley et al., 2003). However, this hypothesis is disproved by the distinct localization patterns between CatSper1/2 and CatSper3/4 revealed recently by Jin et al. (2005). Moreover, the transcription of CatSper1 and CatSper2 is discovered to be initiated at a different time during spermatogenesis (Schultz et al., 2003; Shima et al., 2004). More recently, the interactions of the voltage-gated T-type calcium channel Cav3.3 with CatSper1 and CatSper2 has been reported (Zhang et al., 2006). These findings blurred the relationship between these four members of the CatSper family.

Sperm ion channels are difficult to study using conventional electrophysiological methods, because of their smaller size, complex geometry and motile nature. During the last decade, investigations have revealed the presence of a complex population of mRNA (Dadoune et al., 2005; Miller et al., 2005; Miller and Ostermeier, 2006), including the transcripts of some ion channels (Goodwin et al., 2000) in mature spermatozoa of mammals. The mRNA of ion channels might be a feasible target to study ion channels in spermatozoa. First, although the origin and the role of the mRNAs found in the human mature spermatozoa are yet to be discussed, the most common idea is to consider that these transcripts represent the remnants of stored mRNA from genes activated during spermatogenesis (Dadoune et al., 2005; Miller et al., 2005; Miller and Ostermeier, 2006). The study of mRNAs in spermatozoa could reflect past events of spermatogenesis and/or spermiogenesis, and could be used as a tool for study and a clinical assay to provide a panoramic view of testis gene expression that can be difficult to achieve from a testicular biopsy (Ostermeier et al., 2002; Lambard et al., 2004; Martins and Krawetz, 2005; Zhao et al., 2006). Secondly, although transcription and translation are generally considered to have been terminated (Kierszenbaum and Tres, 1975) in mammal mature spermatozoa, there are many indications to the contrary (Premkumar and Bhargava, 1972; Hecht and Williams, 1978; Alcivar et al., 1989; Naz et al., 1998; Gur and Breitbart, 2006). The decrease of c-myc transcripts in capacitated sperm compared with uncapacitated sperm has been reported (Lambard et al., 2004). The optional use of mRNAs for protein synthesis may occur in mature spermatozoa during their residence in the female reproductive tract, and the protein translation is essential for sperm functions, such as motility, capacitation and acrosome reaction (Gur and Breitbart, 2006). In addition, it has been reported that sperm mRNAs can be transferred to the oocyte after fertilization (Ostermeier et al., 2004). It is also validated that the sperm quality is related to its mRNA distribution. Differences in the levels of some transcripts coding for molecules involved in sperm function have been observed between high- and low-motile spermatozoa (Lambard et al., 2004). These results could explain the difference in motility between the two populations. Moreover, considering motility as an important physiological marker, the analysis of mRNA levels in high- and low-motile sperm could be an additional diagnostic tool and have prognostic value to fertilization and pregnancy (Lambard et al., 2004).

Herein, the expression patterns of these four CatSper mRNAs in post-natal mouse testis development were investigated by real-time RT–PCR to shed light on the relationship of the CatSper family members and the further study of their precise roles. Aimed to provide a tool to study CatSpers in sperm in which relatively fewer methods could be applied, the presence of four CatSper mRNAs in the human ejaculated spermatozoa was examined and the levels of their mRNAs in the high- and low-motile spermatozoa were compared.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
A C57BL/6 mouse colony was maintained in a temperature- and humidity-controlled animal facility with free access to water and food. Testes were individually collected from male neonates at day 8, 11, 15, 18, 21, 25, 28, 35, 42, 56 and 120 post-partum. At each age group, three mice from different breeding pairs were used to obtain the testes. They were rapidly killed by cervical dislocation and both testes were removed. One testis was fixed in Bouin's fixative and processed for histological analysis. The other one was immediately cryostored in liquid nitrogen for subsequent RNA extraction and RT–PCR analysis.

All protocols and experimental procedures for the use of animals in this study were performed in accordance with the NIH Guiding Principles in the Care and Use of Animals.

Human semen samples
Human semen samples (n = 17; mean age: 29.5 ± 2.9 years) were obtained from healthy donors, who met all the criteria of World Health Organization (WHO) (1999) for normozoospermia, by masturbation after 3–5 days of abstinence. Informed patient consent was obtained for the use of sperm samples in this study. Samples with leukocytes and/or immature germ cell concentration greater than 106 ml–1 were excluded (Lambard et al., 2004). Samples were allowed to liquefy for 30–60 min at room temperature before processing. Five semen samples (mean age: 31.4 ± 1.5 years) were used to study the presence of CatSper mRNAs, and other 12 semen samples (mean age: 28.7 ± 3.0 years) were used to isolate high- and low-motile sperm.

Sperm purification
To rule out the possibility of contamination by residual cells, the five samples used for the study of the presence of CatSper mRNAs in spermatozoa were individually purified by two sequential centrifugations (20 min at 300g, 25°C) through 40:80 discontinuous gradients of Percoll (Sigma, MO, USA) as described elsewhere (Ostermeier et al., 2002). C-Kit, a positive marker for testicular germ cells (Lambard et al., 2004), was included to control the quality of sperm purification as well as high- and low-motile spermatozoa isolation described subsequently. The 80% fraction of the second centrifugation through the Percoll gradient was pooled for RNA extraction after a microscopic examination and a hypotonic treatment. For the microscopic examination, 50 µl sperm fraction was stained for 30 min by an equal volume of 0.4% eosin solution (Sigma) and was observed under a light microscope to confirm no remaining round cells and cytoplasmic droplets (Lambard et al., 2003). For hypotonic treatment, sperm fraction was washed with a solution of 0.5% Triton X-100 (Sigma) (Ostermeier et al., 2002).

High- and low-motile spermatozoa isolation
High- and low-motile sperm fractions were isolated as described by Lambard et al. (2004). The motility of these two fractions was examined by computer-assisted semen analysis. Sperm of the high-motile fractions showing motility <90% and sperm of the low-motile fraction showing motility >30% were excluded. Five microlitre sperm fraction was stained by an equal volume of 0.4% eosin solution (Sigma in 0.9% NaCl) and was observed for 15 s under a light microscope to determine the viability of the high- and low-motile spermatozoa (Lambard et al., 2003, 2004). A microscopic examination and a hypotonic treatment were performed as described earlier (Ostermeier et al., 2002) before sperm fractions were used for RNA extraction.

RNA extraction
Total RNAs from liquid nitrogen frozen mouse testes and purified spermatozoa were extracted with TRI Reagent (Molecular Research Center, Cincinnati, USA) and RNeasy Mini kit (Qiagen, Hilden, German), respectively, according to the manufacturer's instruction.

The purity of total RNA from both mouse testis and purified spermatozoa was checked spectrophotometrically at 260 and 280 nm. A minimum optical density (OD260/280) ratio 1.80 was required for the following RT–PCR.

Reverse transcription–polymerase chain reaction
Total RNA (1.0 µg for testis or 0.5 µg for spermatozoa) was reverse-transcribed to complementary DNA using oligo dT primers and MMLV reverse transcriptase (Toyobo Company, Osaka, Japan). The complementary DNAs were further amplified by PCR using selected primers. To eliminate a possible contamination by genomic DNA, primers were chosen in different exons (Table I).


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  		Table I. Primers used for RT–PCR

 
Non-quantitative RT–PCR was performed to identify the presence of transcripts of CatSper1–4 in the human ejaculated spermatozoa. Complementary DNA corresponding to 0.1 µg of total sperm RNA was used as template. The PCR amplification was performed on a Perkin Elmer DNA thermal cycler 480 (Wellesley, USA) in a final volume of 25 µl PCR mixture consisting of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 µM dNTPs, 1.5 IU TaKaRa TaqTM DNA polymerase (TaKaRa Biotechnology, Dalian, China) and 0.4 µM of each primer. Human sperm RNA reverse-transcribed without MMLV reverse transcriptase was used as negative control, and human testis complementary DNA was used as positive control. An initial denaturation step of 95°C for 5 min was used for each amplification, followed by 40 cycles of 95°C for 30 s, annealing for 30 s, 72°C for 30 s and one cycle of 72°C for 5 min. The annealing temperature for each pair of primers was different and is shown in Table I. PCR products were run on a 1.5% agarose gel stained with ethidium bromide and visualized under ultraviolet transillumination.

Relative quantitative RT–PCR was performed to determine the mRNA levels of mouse testes ranging from 8 to 120 days as well as high- and low-motile spermatozoa. Real-time RT–PCR was performed with an Mx3000P thermocycler (Stratagene, CA, USA) using SYBR GREEN 1 fluorescence detection of amplified products (Lopez-Casas et al., 2003). ß-Actin was used in parallel for each run as internal control (Killian et al., 2003; Ellis et al., 2004). A 25 µl PCR reaction was used and included 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 µM dNTPs, 1.5 IU TaKaRa TaqTM DNA polymerase, 0.4 x SYBR GREEN 1 (Invitrogen, Basel, Switzerland) and 0.15 µM of each primer. A four-step experimental run protocol (Pfaffl, 2001) was used and the amplification conditions were as follows: 95°C for 10 min (initial denaturation); 35 cycles of 25 s at 95°C (denaturation); 30 s at annealing temperature (Table I), 30 s at 72°C (elongation), 8 s at fluorescence measurement temperature (Table I). A melting curve was generated at the end of every run to ensure product uniformity (Figure 1). PCR products were run on a 1.5% agarose gel (Figure 1) and further verified by nucleotide sequencing. For each sample, a replicate was run omitting the reverse transcription step and a template-negative control was run for each primer combination. Standard curves were constructed with serial dilutions of complementary DNA from adult mouse testis or human testis (Pfaffl et al., 2002).


Figure 1
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  		Figure 1. Real-time RT–PCR products from mouse testis (A–E) and spermatozoa (F–H) confirmed by a melting curve and running on a 1.5% agarose gel. Total RNA was extracted from the mouse testis during post-natal development and spermatozoa. Real-time RT–PCR was performed using SYBR GREEN 1 fluorescence detection of amplified products. The melting temperatures and sizes of products are as follows: ß-Actin (A and F): 89°C, 385 bp; CatSper1 (B): 91°C, 227 bp; CatSper2 (C and G): 86.5°C, 261 bp; CatSper3 (D and H): 86°C, 140 bp; CatSper4 (E): 88.5°C, 320 bp; M, DNA Marker; 21d, testis from 21 days old mice; 70d, testis from 70 days old mice; hT, human testis complementary DNA; SH, high-motile sperm; SL, low-motile sperm; NTC, negative control, in which adult mouse testis RNA (A–E) or human sperm RNA (F–H) reverse-transcribed without MMLV reverse transcriptase was used as template.

 
Statistical analysis
The data are presented as mean ± SD. Student's t-test was performed to analyse the changes of CatSpers expression during post-natal development. The paired samples t-test was used to compare the data between the high- and low-motile sperm fractions after checking for normal distribution by means of the Kolmogorov–Smirnov test. All statistical tests were two-tailed and a P-value <0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Morphology of mouse testes during post-natal development
The morphology of testes during post-natal development in the present study was consistent with the previous observations (McKinney and Desjardins, 1973; Vergouwen et al., 1993). Briefly, leptolene spermatocyte and pachytene spermatocyte could be seen on day 11 and 15, respectively. Few round spermatids appeared at day 18 and 21, and these cells were frequently seen on day 25. At day 28 elongated spermatids were found, and by day 35 spermatogenensis was complete.

Developmental expression of transcripts of CatSper family members
The expected products were amplified in mouse testis mRNA by real-time RT–PCR (Figure 1). Primer dimers and unspecific amplicons were absent.

The expressions of CatSper family members were developmentally regulated during the mouse sexual maturation, but the initiation and the expression pattern of these four members were considerably different (Figure 2).


Figure 2
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  		Figure 2. Temporal expressions of CatSper1–4 mRNAs normalized to ß-Actin in the mouse testis during post-natal development through to adulthood as measured by real-time RT–PCR. The levels of CatSper1, 2 (A) and CatSper3, 4 (B) mRNAs are expressed as ratios relative to adult (120 days old) mouse testis values. Results are averaged from three independent experiments in all stages. The data are presented as mean ± SD. The up-regulations with significance were marked (compared with their former time point, *P < 0.001, **P < 0.005, ***P < 0.05).

 
Very low levels of CatSper1 mRNA were detected in the mouse testes on day 18 and 21 (after meiosis). The significant up-regulation of its mRNA expression was observed at day 25, 28 and 42 (compared with their former time point, P < 0.001, P < 0.005 and P < 0.05, respectively). Then, CatSper1 mRNA increased gradually from day 42 onwards, such that the amount of CatSper1 mRNA was maximal in the adult testis stage.

A weak expression of CatSper2 mRNA was found as early as day 8 (before meiosis). With further development, the sharp up-regulations of CatSper2 mRNA occurred at day 18 and 35 (compared with their former time point, P < 0.001 both), and then the expression of CatSper2 mRNA decreased to adult levels, thus during testis post-natal development CatSper2 mRNA peaks around day 35 and 42.

CatSper3 and CatSper4 mRNAs have similar expression profiles during mouse post-natal development, initiated on day 15 (after meiosis), increased significantly at 21, 25 and 35 (compared with their former time point, P < 0.005, P < 0.05 and P < 0.001, respectively), and then increased gradually as the animals aged till maximal levels in the adult testis.

Characterization of the purified spermatozoa as well as the high- and low-motile sperm fractions
Round cells and cytoplasmic droplets were absent in the purified spermatozoa as well as the high- and low-motile sperm fractions isolated by Percoll gradients. Moreover, the CatSper family members are testified to express solely in testis, and the absence of C-Kit mRNA discards the possible contamination of testicular germ cells (Figure 3D).


Figure 3
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  		Figure 3. The presence of CatSper2 (B) and CatSper3 (C) transcripts in human ejaculated spermatozoa identified by RT–PCR. ß-Actin was used as a loading control (A). C-Kit (D) was included to control the quality of sperm purification as well as high- and low-motile spermatozoa isolation. The sizes of products were as follows: ß-Actin (A): 385 bp; CatSper2 (B): 261 bp; CatSper3 (C): 140 bp; C-Kit (D): 781 bp. M, DNA Marker; hT, human testis complementary DNA; SP, purified spermatozoa; SH, high-motile sperm; SL, low-motile sperm; NTC, negative control, human sperm RNA reverse-transcribed without MMLV reverse transcriptase was used as template.

 
Ten sperm samples were used to compare the levels of CatSper mRNAs between the high- and low-motile sperm fractions. In each sample, the motility of the high motile sperm fractions was >90% and that of the low-motile sperm fraction was <30% (Table II). Two sperm samples were excluded because the high-motile sperm fraction showed motility <90%.


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  		Table II. Motility, viability and ß-Actin, CatSper2, CatSper3 mRNA expression in the high- and low-motile sperm fractions from each sample

 
The viability of the high- and low-motile spermatozoa was determined by eosin vital stain. The percentage of live cells was not different between the high-motile spermatozoa and low-motile spermatozoa (Table II, P = 0.121, n = 10).

The presence of mRNA of CatSper family members in the purified spermatozoa
The transcripts of CatSper2 and CatSper3 were found in purified spermatozoa from all five semen samples by RT–PCR (Figure 3B and C), and CatSper1 mRNA was detected in two samples, whereas there was no expression of CatSper4 mRNA in these purified spermatozoa even after increasing the PCR cycle number to 45.

Expression of CatSper2 and CatSper3 mRNAs in the high- and low-motile sperm fractions
The quantification of CatSper2 and CatSper3 mRNA in the high- and low-motile sperm fractions isolated from 10 semen samples individually was performed by real-time RT–PCR, by taking ß-Actin as internal control. The threshold cycle of ß-Actin varied from 14.23 to 15.16 and mRNA levels of ß-Actin in the high- and low-motile sperm fractions were similar (Table II, P = 0.932, n = 10).

Converse to the relatively stable and constant ß-Actin mRNA levels in different samples as well as between the high- and the low-motile sperm fractions, CatSper2 and CatSper3 transcript amounts varied in different samples with normal distributions. In 9 of the 10 semen samples, higher CatSper2 and CatSper3 transcript amounts normalized by ß-Actin in high-motile sperm fractions than in low-motile sperm fractions were observed, respectively (Table II). Only in one sample, the ratio of CatSper3 mRNA to ß-Actin mRNA was 0.88 in the low-motile sperm fraction and was higher than 0.82 in the high-motile sperm fraction. The ratios of both CatSper2 and CatSper3 mRNA to ß-Actin mRNA were statistically different between the high-motile and low-motile fraction (P = 0.008, P = 0.004-respectively, n = 10; Table II).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
The recent cloning and characterization of the CatSper family is a matter of significance to the study of male reproduction and infertility (Carlson et al., 2003; Felix, 2005). The four members of this family were identified to be exclusively expressed in the testis and differentially localized in the sperm. They play an important role in sperm motility and male fertility. Among several candidate calcium channels in sperm, only CatSper1 and CatSper2 were identified to be required for male fertility so far. Based on their restricted expression patterns and their vital roles in sperm functions, CatSper family members are predicted to be potential targets for male infertility screening and ideal targets for contraception (Ren et al., 2001; Quill et al., 2001; Avidan et al., 2003; Nikpoor et al., 2004).

Considering the important roles of CatSper family members in sperm functions and male fertility, and the considerable potential of the sperm RNA as a tool for study, we have focused on the temporal expression profiles of the CatSper transcripts in post-natal mouse testis development, and their presence in human ejaculated spermatozoa as well as their relationship to sperm motility.

We used a quantitatively rigorous approach based on reverse transcription followed by real-time PCR amplification to provide valuable new information on the temporal profiles of CatSper family members expression during mouse testis development. Furthermore, the high-temperature fluorescence measurement point set at the end of the fourth segment can improve SYBR Green I quantification, because it melts the unspecific PCR products below the chosen temperature and ensures accurate quantification of the desired product.

The differences between the temporal expression profiles of the CatSper transcripts in post-natal mouse testis development suggest different regulations to their transcriptions, although the induction or repression may be the indirect result of changes in the cell populations of the testis (Shima et al., 2004).

The different expression profile of CatSper transcripts shed light on the possibility of forming heteromeric channels among these four CatSper family members. Distinct localization patterns (Jin et al., 2005) between CatSper1/2 and CatSper3/4 have overthrown the hypothesis that CatSper1/2 form heteromeric channels with CatSper3/4. Moreover, they have different functions. CatSper1/2 have been shown to be required for sperm motility and hyperactivated movement, whereas CatSper3/4 are supposed to have roles in the acrosome reaction (Jin et al., 2005). Our results also show distinct expression patterns between CatSper1/2 and CatSper3/4 transcripts during mouse testis development.

It is possible that CatSper1 and CatSper3 form different heteromeric channels with CatSper2 and CatSper4, respectively. Identical phenotypes of CatSper1 and CatSper2 null sperm and the reciprocal requirement of CatSper1 and CatSper2 to stabilize both proteins in sperm strongly suggested that these two proteins are components of a heteromeric channel (Carlson et al., 2005). But the different initiation and distinct temporal expression pattern between CatSper1 and CatSper2 transcripts found in a previous study (Schultz et al., 2003) and in the present study suggest that CatSper1 and CatSper2 were co-dependently expressed at other levels distinct from transcription. More recently, the physical association of Cav3.3 with CatSper1 and CatSper2 has been identified, but these three proteins do not form a triple complex. It was postulated that CatSper1 and CatSper2 bind competitively to Cav3.3 (Zhang et al., 2006). Therefore, it is possible that CatSper1 and CatSper2 fulfill their functions at different stages during testis development, through binding to Cav3.3. As to the possibility of CatSper3 forming a heteromeric channel with CatSper4, it was postulated that the two proteins may form a heterodimer based upon their identical patterns of protein expression and localization (Jin et al., 2005). The similar initiations and temporal expression patterns of their transcripts observed in the present study also support this hypothesis. A weakness of the present study is the lack of data on temporal protein expression profiles of CatSper members in post-natal mouse testis development, primarily due to lack of availablity of antibodies against them.

After the determination of the changes of mRNAs of CatSper family members in post-natal mouse testis development, their presence in human ejaculated spermatozoa was examined in the present study. We found that transcripts of CatSper2 and CatSper3 existed in purified human ejaculated spermatozoa of all samples, whereas transcript of CatSper4 did not. As to CatSper1, we detected its transcript in all unpurified semen samples in a previous study (Li et al., 2006). Its presence in purified human ejaculated spermatozoa has also been reported recently (Gur and Breitbart, 2006), but in the present study, its transcript was only detected in some samples after purification. It is possible that sperm purification decreases the level of its transcript through RNA degradation and/or removing round cells and cytoplasmic droplets.

The presence of CatSper1–3 mRNAs in human ejaculated spermatozoa could be a considerable tool for male infertility screening and further study. Members of the CatSper family are predicted to be potential targets for male infertility screening and contraception because of their restricted expression patterns and important roles in male fertility. Recently, the profiles of CatSper1 mRNA expression in testis biopsy of subfertile patients were investigated, and the data linked to CatSper1 gene expression to sperm motility and male fertility (Avidan et al., 2003; Nikpoor et al., 2004). However, testicular puncture is invasive, inconvenient and may not be accepted by patients because of the ethical considerations. The study of mRNAs in sperm could reflect past events of spermatogenesis and/or spermiogenesis, and could be used as a non-invasive proxy for investigations of testis-specific infertility (Aoki et al., 2006; Martins and Krawetz, 2005; Miller et al., 2005; Miller and Ostermeier, 2006; Ostermeier et al., 2002; Zhao et al., 2006). Therefore, if CatSper family members could be identified to be a target for male infertility screening and treatment, it is apparently more feasible to study its mRNA in ejaculated spermatozoa than in testicular biopsy. Moreover, taking into account the relatively fewer methods that can be used to study sperm ion channels, the CatSper1–3 transcripts present in human ejaculated spermatozoa could be good tools for further study.

The presence of CatSper1–3 mRNAs also implies an important role of CatSper family members in sperm function and fertilization. Considering that translation may occur in sperm to replace and supply the proteins they could be essential for fertilization (Gur and Breitbart, 2006). In fact, it has been shown that CatSper1 is translated in sperm during capacitation to replace the degraded protein (Gur and Breitbart, 2006).

We further compared the levels of CatSper2 and CatSper3 mRNAs in high- and low-motile spermatozoa isolated from the same sample of normospermic patients. In almost all semen samples, higher normalized CatSper2 and CatSper3 transcript amounts were observed in high-motile fractions than in low-motile fractions. Therefore, the ratios of CatSper2 and CatSper3 mRNA to ß-Actin mRNA were statistically different between the two populations of sperm. Differential distribution of some mRNA between high- and low-motile sperm, such as AROMATASE, PROTAMINE-1, eNOS and nNOS, have been reported recently (Lambard et al., 2003, 2004). The discrepancies suggest that low motility may be the result of defective transcription of some genes coding molecules involved in sperm motility and other functions. One or some of these genes may be defectively transcribed in some spermatozoa of the low-motile fraction, and in different spermatozoa the genes which were defectively transcripted may be different. The low-motile fraction may be composed of heterogeneous subsets in which different genes were defectively transcripted.

Another explanation for the differential distribution of mRNAs between high- and low-motile sperm might lie in the translation occurring in sperm (Gur and Breitbart, 2006). Low motility may be the result of low-level translation in sperm due to insufficient transcripts.

On the other hand, the discrepancies between high- and low-motile spermatozoa also suggest that CatSper2 and CatSper3 may be concerned with the acquisition of sperm motility. The loss of the ability to swim forward and the failure of hyperactivation of CatSper2 null sperm have verified that CatSper2 is responsible for the sperm forward velocities and is essential for the generation of a hyperactivated form of motility (Quill et al., 2001, 2003; Carlson et al., 2005). The function of CatSper3 remained obscure and may also be related to sperm motility, although the localization of CatSper3 in the acrosome of sperm imply its potential role in the acrosome reaction (Jin et al., 2005).

Taken together, the present study used real-time RT–PCR to provide valuable new information on the temporal profiles of CatSper family members expression in the mouse testis during post-natal development through to adulthood. CatSper1–3 transcripts were identified to be present in human ejaculated spermatozoa and may be good tools for further study. The significant higher expressions of CatSper2 and CatSper3 mRNA levels observed in the high-motile spermatozoa than in the low-motile fraction also suggest that CatSper2 and CatSper3 transcripts in human ejaculated spermatozoa be potential targets for male infertility screening.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We are grateful to Prof. Ming-Da Zuo and Ms Xue-Bing Pang for their excellent technical assistance and advice. The investigation was partly supported by grants from National ‘Ten times Five Years’ Key Technologies R&D Programme, China (No. 2004BA720A33-01) and the Hubei province health department project (No. JX2B03).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 12, 2006; resubmitted on January 22, 2007; accepted on January 24, 2007.


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Control of hyperactivation in sperm
Hum. Reprod. Update, November 1, 2008; 14(6): 647 - 657.
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