Honggang Li, Ph.D., a Xiaofang Ding, Ph.D., b Huangtao Guan, B.A., a and Chengliang Xiong, M.D a

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1 Inhibition of human sperm function and mouse fertilization in vitro by an antibody against cation channel of sperm 1: the contraceptive potential of its transmembrane domains and pore region Honggang Li, Ph.D., a Xiaofang Ding, Ph.D., b Huangtao Guan, B.A., a and Chengliang Xiong, M.D a a Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People s Republic of China; and b Centre of Reproductive Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People s Republic of China Objective: To explore the contraceptive potential of the CatSper1 transmembrane domains and pore region in vitro. Design: In vitro study with human sperm and mouse fertilization. Setting: Andrology laboratory of an academic research center. Patient(s) and Animal(s): Normozoospermia and viripotent BALB/c mice. Intervention(s): The specific binding of an anti-catsper1 IgG antibody (H-300) to CatSper1 was confirmed by Western blot and immunofluorescence. Sperm from humans and mice were incubated with H-300. Main Outcome Measure(s): The effects of H-300 on human sperm progressive motility, abnormal acrosome, hyperactivated motility, and mouse in vitro fertilization rates were analyzed. Result(s): A significant decline in sperm progressive motility was observed after 1, 2, and 4 hours of incubation with H-300; the change was mainly ascribed to the decline of fast progressive motility. Significant inhibition of the hyperactivated motility was observed after 5 hours of incubation with H-300. The incubation of mouse sperm with H-300 before insemination reduced the in vitro fertilization rate to 28% of control levels (72% inhibition). Conclusion(s): CatSper1 may be a potential target for immunocontraception, and the antibody may be a tool to study the function of ion channels in sperm in which relatively fewer methods can be applied. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Immunocontraception, CatSper1, sperm, ion channel There is an obvious need for new types of safe, sustained, effective contraceptives, especially for males. The world population continues to explode, even though the available contraceptives have contributed to a slowing of the growth. It is predicted that the total world population will reach 8 billion by 2020 and 8.9 billion by 2050 (1). The currently available contraceptives cannot provide the wide ranges of choices by people at different stages in their lives. In particular, the male contraceptive options are limited to the condom and vasectomy, wheras many men wish to carry contraceptive responsibility (2). The lack of availability of effective and satisfactory contraceptive methods for many couples and adolescents caused millions of unintended pregnancies annually. More than one-half of these unwanted pregnancies are terminated by elective abortion, and >700,000 women died Received May 6, 2008; revised July 6, 2008; accepted July 9, 2008; published online October 31, H.G.L. has nothing to disclose. X.F.D. has nothing to disclose. H.T.G. has nothing to disclose. C.L.X. has nothing to disclose. The first two authors contributed equally to this article. Supported by grant 2004BA720A33-01 from the Science and Technology Department of China to CLX, and grant JX2B03 from the Health Department of Hubei Province to H.G.L. Reprint requests: Chengliang Xiong, M.D., Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, HangKong Road 13, Wuhan , P.R. China (FAX: ; clxiongcn@yahoo.com.cn). between 1995 and 2000 worldwide, as a result of the complications related to unintended pregnancies (3). Therefore, developing a new approach for contraception is urgently needed. Immunocontraception may provide viable and valuable alternatives to the currently few options for male contraception. The rationale and feasibility of immunocontraception is clearly supported by the large number of overtly healthy men and women in whom infertility is caused by antisperm antibodies (4). There are immunocontraceptive vaccines that have entered clinical trials. Immunocontraception has many features. It is long acting, reversible, and easy to administer. The execution of this approach should be facilitated in most countries by the service infrastructure for the delivery of vaccines against diseases. There has also been considerable interest in the development of immunocontraceptive vaccines for limiting fertility in animal species (5). In contrast, improvements for immunocontraception are still needed, mainly to increase its efficacy, and diminish its harmful side effects. Indeed, many researchers are dedicated to these goals, such as developing vaccines against B-cell epitopes or multiantigens, changing immunization routes, and upgrading vaccine delivery vectors. Meanwhile, it is also essential to find new targets for immunocontraception. Cation channel of sperm (CatSper) is a unique sperm cation channel protein family exclusively expressed in the testis /09/$36.00 Fertility and Sterility â Vol. 92, No. 3, September doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 All four members of the CatSper family (CatSper1 4) are located primarily in the principal piece of the sperm tail, and are required for sperm hyperactivation and male fertility (6 9). Mice deficient in any one of the CatSper members are completely infertile as a result of an impairment in sperm mobility and an inability to fertilize intact oocytes (6, 9). Based on its restricted pattern of expression and the important role in sperm mobility and male fertility, CatSper family is predicted to be an ideal target with high performance and low side effects for contraception (6, 10). However, there is no report documenting the contraceptive potential of the CatSper family thus far, and no specific antagonist has been found. It has been demonstrated that the calcium current through CatSper1 is not sensitive to the L-type Ca v channel blockers, including nifedipine and nimodipine (10). We suppose that CatSper family is an ideal sperm specific antigen for immunocontraception. First, CatSper members meet the important criteria for the potential immunocontraceptive target antigen (11). They are exclusively expressed by spermatogenic cells, and specifically localized to the membrane of spermatozoa. Therefore, the antibody can contact and bind with them in the live cell. They are essential for sperm function and male fertility. Second, some autoimmune diseases (12) caused by autoantibodies to ion channels has shown that it is possible to block the function of an ion channel by antibody, although there is no ion channel that has been tested as a target for immunocontraception. Moreover, current progress in the elucidation of roles of ion channels in sperm biology lends belief to developing the next generation of contraceptives targeting sperm ion channels (13). To validate our hypothesis, in the present study, an IgG antibody against the transmembrane domains and pore region of human CatSper1 was used to evaluate the immunocontraceptive potential of CatSper1. MATERIALS AND METHODS Patient Recruitment and Semen Sample Collection After 3 to 5 days of abstinence, human semen samples (n ¼ 9) were obtained from healthy donors by masturbation who met all the World Health Organization (WHO) criteria (14) for normozoospermia. The study was approved by the institutional review board, and informed patient consent was obtained for the use of sperm samples. Animals and Housing Twelve 90-day-old male (body weight, g) and day-old female (body weight, g) BALB/c mice were used. Animals were maintained in a temperature- and humidity-controlled animal facility with a 12-hour light and 12-hour dark cycle. Mice were humanely killed by cervical dislocation after intraperitoneal injection of 0.2 ml of 10% chloral hydrate. All experimental procedures with the animals in this study were performed in accordance with the NIH Guiding Principles in the Care and Use of Animals. Antibody The rabbit anti-catsper1 IgG (clone number, H- 300) and normal rabbit IgG (SC-2027) used in the present study were obtained from Santa Cruz Biotechnology Incorporation (Cants Cruz, CA). H-300 is a rabbit polyclonal antibody raised against human CatSper1 amino acids at the C-terminus, and it is recommended by the manufacturer for detection of CatSper1 of mouse, rat, and human origin. The amino acids of human CatSper1 cover five of six transmembrane domains and the pore region. The transmembrane domains share 81% identity/93% similarity, and the pore region shares 89% identity/100% similarity between human and mouse (6). Experimental Design Immunoblotting and immunofluorescence were performed to confirm the specific binding of H-300 to CatSper1 of human and mouse sperm. Then human sperm were swum up and incubated with H-300. At different time points during incubation, human sperm motility, acrosomal abnormalities, and hyperactivited sperm motility were determined. Mouse sperm and eggs were used to study the inhibitory effect of H-300 on in vitro fertilization. Immunoblotting and Immunofluorescence The immunoblotting was performed as previously described (15). Briefly, plasma membrane proteins of sperm samples were obtained by using a plasma membrane protein extraction kit (BioVision Inc., Mountainview, CA) and subjected to 10% SDS-PAGE, transferred to a nitrocellulose filter, and incubated with H-300 at a 1:200 dilution. Immunoblots were developed with a secondary antibody conjugated with alkaline phosphatase and the WesternBreeze Chromogenic Immunodetection Kit (Invitrogen, San Diego, CA) according to the manufacturer s instructions. Immunohistochemistry was performed as previously described (16). H-300 at a 1:50 dilution was used as the primary antibody, and fluorescein isothiocyanate-labeled goat antirabbit antibody (Santa Cruz Biotechnology, Inc.) was used as the secondary antibody at a 1:2000 dilution. In both immunoblotting and immunofluorescence, normal rabbit IgG instead of primary antibody was used as the negative control. In immunoblotting, mouse liver was also used as a negative control. Human Sperm Swim-up and Incubation with H-300 Human semen samples were allowed to liquefy for 30 to 60 minutes at room temperature, and then were swum up in SpermRinse (Vitrolife AB, Billdal, Sweden) for 1 hour, as described in the WHO protocol (14). The swim-up sperm were suspended in SpermRinse, and the motility was determined using a computer-assisted motion analyzer (Weili, Beijing, China), according to the appropriate WHO guidelines (14). A minimum motility of 70% of was required for the following incubation with H-300. Concentrations of the swim-up sperm were adjusted to /ml. Aliquots (60 ml) of the sperm suspension were incubated with H-300 or normal rabbit IgG at a final concentration of 50 mg/ml. The sperm were incubated in an incubator at 37 C with 5% CO 2 for 1142 Li et al. Immunocontraceptive potential of CatSper1 Vol. 92, No. 3, September 2009

3 up to 5 hours. At different time points during incubation, sperm functions were analyzed as follows. Determination of Human Sperm Functions After 1, 2, and 4 hours of incubation, an 8-mL sperm suspension was loaded on a clear slide and covered with a coverslip. Progressive motility (WHO motility class A þ B) and fast progressive motility (WHO motility class A) was measured using the computer-assisted motion analyzer at 37 C. At least 200 spermatozoa in randomly chosen fields of vision were analyzed. After 2 hours of incubation, an 8-mL sperm suspension was loaded on a clear slide and observed under a light microscope to examine sperm agglutination. After 4 hours of incubation, a 10-mL sperm suspension was fixed with 4% paraformaldehyde solution (in 0.01 mol/l PBS [ph 7.4]) for 10 minutes at room temperature, then centrifuged and smeared on a clear glass slide. Sperm acrosomal abnormalities were determined using a Coomassie Blue G-250 staining procedure as reported earlier (17). After 5 hours of incubation, analysis of the hyperactivated sperm motility was performed, as previously described (18). Briefly, 2 ml of sperm suspension was diluted with 8 ml of SpermRinse. Eight milliliters of diluted suspension were loaded on a clear slide and covered with a coverslip. The parameters of hyperactivated motility (curvilinear velocity [VCL], straight progressive velocity [VSL], and average path velocity [VAP]) of single sperm were obtained under the 40 objective of the computer-assisted motion analyzer. Straightness of trajectory (STR) was caculated as follws: (STR) ¼ VSL/VAP 100. In each sample, at least 20 randomly chosen sperm with progressive motility were analyzed. VCL and STR of hyperactivated sperm were delimited by the 90th percentile of VCL and the 10th percentile of STR values from noncapacitated sperm (Fig. 1), which were immediately suspended in SpermRinse after semen liquefaction. In Vitro Fertilization Mouse sperm were collected from the cauda epididymis and incubated in SpermRinse in vitro for 60 minutes with 20 mg/ ml H-300 or normal rabbit IgG (negative control). Before insemination, sperm were centrifuged and washed once by SpermRinse to remove the unbound antibody, and were resuspended in SpermRinse. Eggs were collected from female mice synchronized with 10 units of pregnant mare serum gonadotropin and 10 units of hcg 48 hours and 16 hours before collection, respectively. Eggs were incubated with roughly sperm/egg for 4 hours at 37 C with 5% CO 2.Successful fertilization was scored as two-cell embryos 25 to 28 hours after insemination. for normal distribution by means of the Kolmogorov-Smirnov test. The chi-squared test was used to compare the number of hyperactivated sperm and in vitro fertilization rates. All statistical tests were two tailed, and a P value <.05 was considered as statistically significant. RESULTS To confirm the quality and the specificity of the anti-catsper1 IgG (H-300), immunoblotting and indirect immunofluorescence were performed. In immunoblotting, a clean single band at approximately 80 kda was detected in the 40-kDa to 120-kDa protein range of human and mouse sperm, whereas no band was detected in the negative control. The results of immunofluorescence also showed that H-300 bound specifically with the principal piece of human and mouse sperm, which is the localization of CatSper1 (6, 16). No staining was observed in the head of the sperm or the negative control. After the quality and specificity of H-300 were confirmed, H-300 was used to block the function of sperm in vitro. After 1, 2, and 4 hours incubation with H-300, the progressive motility of the sperm declined significantly. Moreover, similar changes in the fast progressive motility were observed (Table 1). The decline in progressive motility resulting from incubation with H-300 was mainly ascribed to the change in fast progressive motility. Furthermore, the inhibitory effect of H-300 on the sperm motility was not caused by sperm agglutination, because 2 hours after incubation with H-300, no sperm agglutination was observed under the microscope. After 4 hours of incubation with H-300, the rate of abnormal acrosomes was similar with the control (Table 1; P¼.61). As for the hyperactivated sperm motility, to delimit the parameters of the hyperactivated sperm, the VCL and STR of 185 randomly chosen noncapacitated sperm with progressive motility from the nine semen samples were obtained after liquefaction. The 90th percentile VCL and 10th percentile STR values of these cells were mm/sec and 81.02, respectively. After a 5-hour incubation with H-300, significant inhibition of the hyperactivated motility was observed (Fig. 1): only 1 of 203 randomly chosen sperm with progressive motility from the nien semen samples treated with H-300 was classified as a hyperactivated sperm. In the control group incubated with normal rabbit IgG, 12 of 173 sperm from the nine semen samples were defined as hyperactivated sperm. The effect of H-300 on mouse fertilization was further examined. Significant inhibition of fertilization in vitro was achieved by incubating the mouse sperm with H-300. The fertilization rate in the presence of 20 mg/ml H-300 was reduced to 28% of the control levels (24.0% vs. 85.1%; 72% inhibition; Fig. 2). Statistical Analysis Comparisons of the sperm motility and acrosomal abnormalities were performed by paired sample t tests after checking DISCUSSION The targets for immunocontraception can be divided into three main categories: targeting gamete production, gamete Fertility and Sterility â 1143

4 TABLE 1 Sperm function analysis results in H-300 incubation. Incubation time Parameters H-300 Normal IgG control Negative control 1 hour Motility AþB (%) a Motility A (%) b hours Motility AþB (%) a Motility A (%) a hours Motility AþB(%) b Motility A (%) a Abnormal acrosome (%) Note: Values are means SD. Motility A and B are defined according with WHO guidelines (14). Statistically significantly different from the normal IgG control group ( a P<.005, b P<.01). Li. Immunocontraceptive potential of CatSper1. Fertil Steril function, and gamete outcome. Sperm antigens belong to the antigen targeting gamete function and constitute the most promising and exciting targets for immunocontraception because they are feasible and effective targets for causing infertility, while avoiding potentially pathogenic autoimmune responses (19). Some sperm-specific antigens have been delineated and are being explored for contraceptive vaccines development. Notable among these are PH-20, PH-30, fertilization antigen, sperm protein-10, sperm protein-17, lactate dehydrogenase-c 4, contraceptive vaccinogen, testis-specific antigen-1, rsmp-b, protein A-kinase anchoring protein, SAMP32, SAMP14, epididymal protease inhibitor, and sperm-associated antigen-9. As the first member of the CatSper family, CatSper1 was cloned in 2001 (6). In mice, CatSper1 protein is expressed exclusively in the testis and not in other tissues. Moreover, the transcription of CatSper1 is initiated after meiosis (20). In FIGURE 1 The hyperactivated sperm of the blank control (B), normal rabbit IgG control (C), and H-300-treated group (D). Each data point represents an individual sperm cell. The boxes define regions containing hyperactivated sperm delimited by the 90th percentile VCL and 10th percentile STR values from the noncapacitated sperm (A). Li. Immunocontraceptive potential of CatSper1. Fertil Steril Li et al. Immunocontraceptive potential of CatSper1 Vol. 92, No. 3, September 2009

5 FIGURE 2 In vitro fertilization with mouse sperm from cauda epididymis pretreated with H-300 (B) or normal rabbit IgG control, (A). Scale bar represents 100 mm. Li. Immunocontraceptive potential of CatSper1. Fertil Steril mouse sperm, CatSper1 is localized primarily in the tail s principal piece, and has been identified as a component of the key flagellar calcium channel required for hyperactivated sperm mobility and male fertility (10). In human testis and sperm, the same expression and localization of CatSper1 as in mice has been observed (16), indicating a similar role of CatSper1 in human male fertility. Based on its restricted expression pattern and important role in sperm mobility and male fertility, CatSper1 is predicted to be an ideal target for contraception (6, 10). We propose that CatSper1 is an ideal sperm-specific antigen for immunocontraception. Herein, the immunocontraceptive potential of the transmembrane domains and pore region of CatSper1 was evaluated in vitro, by observing the influence of an anti-catsper1 IgG antibody targeting these regions upon human sperm function and mouse sperm fertilization. The inhibitory effects of H-300 upon the mobility of human sperm were observed. The depression of motility and hyperactivated mobility of human sperm was inferred as the specific action of H-300. First, the binding of H- 300 with human sperm is specific. Nonspecific staining was not observed by immunocytochemistry. Second, the inhibitive effects of H-300 upon the mobility of human sperm are similar to the phenotype reported for the CatSper1-null sperm, which displayed less directed movements (6), and were defective in their ability to hyperactive motility after capacitating incubation (21). Furthermore, sperm agglutination was not observed, and the rate of abnormal acrosome was not affected by treated with H-300. Third, the functional parts of ion channels are the transmembrane domains and pore region that have been demonstrated previously as the target of antibody in some autoimmune diseases (22, 23). Therefore, it is reasonable to conclude that the antibody targeting these areas could block the function of ion channels. Considering the essential role of CatSper1 in sperm function and fertilization (6), and with the high blocking efficacy of H-300, we expected a high inhibitory efficacy of this antibody on mouse fertilization. In our preliminary experiments, although the incubation of mouse sperm with 50 mg/ml H-300 significantly reduced the fertilization rates, it remained 57% of the control level. We then reduced the amount of mouse sperm from sperm/egg to sperm/ egg, and higher inhibition efficacy (72% inhibition) of H-300 (20 mg/ml) on mouse fertilization was observed. Although H-300 was raised against the transmembrane domains and pore region of human CatSper1, these areas of mouse CatSper1 exhibit a high degree of homology with its human counterparts. The transmembrane domains share 81% Fertility and Sterility â 1145

6 identity/93% similarity, and the pore region shares 89% identity/100% similarity (6). H-300 is also recommended for detecting mouse CatSper1 in the instruction of this product. The specific binding of H-300 with mouse sperm was observed, which fits the description. Therefore, the blocking of mouse fertilization in vitro should be the specific action of H-300. There are no reports documenting the immunocontraceptive potential of an ion channel on sperm membranes. This may be because of the rare ion channel that has been identified being essential for sperm function and fertility. The present study provides evidence that anti-catsper1 IgG antibody targeting its transmembrane domains and pore region can block the function of CatSper1. Our data indicated that CatSper members could be the effective and feasible targets for immunocontraception. It is also presumed that CatSper1 antibody ascribe to male infertility. Finally, antibodies against functional domains of ion channels might be a tool to study functions of sperm ion channels, which are difficult to study by using conventional electrophysiologic methods, because of their smaller size, complex geometry, and motile nature. Acknowledgments: The authors thank Professor Changhong Zhu for good advice in the design of the in vitro fertilization. REFERENCES 1. Nieschlaq E, Henke A. Hopes for male contraception. Lancet 2005;365: Hoesl CE, Saad F, P oppel M, Altwein JE. Reversible, non-barrier male contraception: status and prospects. Euro Urol 2005;48: Nass S, Strauss J, eds. New frontiers in contraceptive research: a blueprint for action. Washington, DC: Institute of Medicine National Academy Press, Bronson RG, Cooper G, Rosenfield D. Sperm antibodies: their role in infertility. Fertil Steril 1984;42: Cooper DW, Larson E. Immunocontraception of mammalian wildlife: ecological and immunogenetic issues. Reproduction 2006;132: Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, et al. A sperm ion channel required for sperm motility and male fertility. Nature 2001;413: Quill TA, Ren D, Clapham DE, Garber DL. A voltage-gated ion expressed specifically in spermatozoa. Proc Natl Acad Sci USA 2001;98: Lobley A, Pierron V, Reynolds L, Allen L, Michalovich D. Identification of human and mouse CatSper3 and CatSper4 genes: characterisation of a common interaction domain and evidence for expression in testis. Reprod Biol Endocrinol 2003;1: Qi H, Moran MM, Navarro B, Chong JA, Krapivinsky G, Krapivinsky L, et al. All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility. Proc Natl Acad Sci USA 2007;104: Kirichok Y, Navarro B, Clapham DE. Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2þ channel. Nature 2006;439: Aitken RJ. Immunocontraceptive vaccines for human use. J Reprod Immunol 2002;57: Lang B, Vincent A. Autoantibodies to ion channels at the neromuscular junction. Autoimmu Rev 2003;2: Zhang D, Gopalakrishnan M. Sperm ion channels: molecular targets for the next generation of contraceptive medicines. J Androl 2005;26: World Health Organisation. Laboratory manual for the examination of human semen and sperm cervical mucus interaction. 4th ed. New York: Cambridge University Press, Kong XB, Ma HG, Li HG, Xiong CL. Blockade of epithelial sodium channels improves sperm motility in asthenospermia patients. Int J Androl Feb 20 [Epub ahead of print]. 16. Li HG, Liao AH, Ding XF, Zhou H, Xiong CL. The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa. Asian J Androl 2006;8: Larson JL, Miller DJ. Simple histochemical stain for acrosomes on the sperm from several species. Mol Reprod Dev 1999;52: Quill TA, Sugden SA, Rossi KL, Doolittle LK, Hammer RE, Garbers DL. Hyperactivated sperm motility driven by CatSper2 is required for fertilization. Proc Natl Acad Sci USA 2003;100: Frayne J, Hall L. The potential use of sperm antigens as targets for immunocontraception; past, present and future. J Reprod Immunol 1999;43: Li HG, Ding XF, Liao AH, Kong XB, Xiong CL. Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility. Mol Hum Reprod 2007;3: Carlson AE, Westenbroek RE, Quill TA, Ren D, Clapham DE, Hille B, et al. CatSper1 required for evoked Ca2þ entry and control of flagellar function in sperm. Proc Natl Acad Sci USA 2003;100: Takamori M. An autoimmune channelopathy associated with cancer: Lambert-Eaton myasthenic syndrome. Intern Med 1999;38: Parsons KT, Kwok WW. Linear B-cell epitopes in Lambert-Eaton myasthenic syndrome defined by cell-free synthetic peptide binding. J Neuroimmunol 2002;126: Li et al. Immunocontraceptive potential of CatSper1 Vol. 92, No. 3, September 2009