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Genes to Cells (2008) 13, 851-861. doi:10.1111/j.1365-2443.2008.01210.x
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Cd52, known as a major maturation-associated sperm membrane antigen secreted from the epididymis, is not required for fertilization in the mouse

 Ryo Yamaguchi1,2, Kazuo Yamagata3, Hidetoshi Hasuwa1, Emiko Inano1, Masahito Ikawa1 and Masaru Okabe1,2,*

1 Research Institute for Microbial Diseases, and
2 Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
3 Laboratory for Genomic Reprogramming, RIKEN, Center for Developmental Biology, Kobe 650-0047, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
CD52 is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes and in epididymal epithelial cells. CD52 is also known as "maturation-associated sperm antigen" but its function is unknown. We therefore generated Cd52 disrupted mice. The resulting Cd52 null mice were healthy, even though Cd52 is expressed on cells of the immune system. We then examined a possible role for CD52 in reproduction. Sperm from Cd52-deficient males were investigated and the viability, motility, morphology, and incidence of spontaneous acrosome reactions were found to be all similar to values for wild-type sperm. In in vitro fertilization system, the sperm showed normal fertilizing ability. As CD52 was found to be transferred onto sperm only after they had migrated into the vas deferens, we examined the behavior of sperm from Cd52-deficient mice in vivo. The mice mated naturally and we observed that a normal number of sperm passed through the uterotubal junction, known to the crucial hurdle for various gene knockouts resulting in infertile sperm. As a consequence, there was no difference in the litter size from the wild-type and Cd52-null males. Our results therefore indicate CD52 is not required for fertilization in the mouse either in vivo or in vitro.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Sperm are produced from spermatogonial cells as a highly differentiated cell to fertilize eggs but they have no fertilizing ability when they leave testis. Sperm require a maturation process during passage through epididymis. For example, swimming ability and egg interacting ability of sperm are reported to be acquired during the maturation step (Cooper 1995; Toshimori 2003). The ability of sperm to undergo the acrosome reaction, a prerequisite for sperm to fertilize eggs, is also known to increase during the epididymal maturation (Williams et al. 1991). This sperm maturation process is associated with reorganization of membrane structures such as processing of membrane proteins on sperm surface (Kim et al. 2006), changes in plasma membrane phospholipids composition (Sullivan et al. 2005), and the accumulation or modification of glycoproteins on sperm surface (Kirchhoff 1996; Tulsiani 2006). Various glycoproteins are known to be secreted from the epithelial cells of male reproductive tract and transferred on sperm during epididymal passage (Cornwall et al. 1990; Vreeburg et al. 1990; Sullivan et al. 2005; Busso et al. 2007). Thus these glycoproteins may function as a primary interface between sperm and female reproductive tract and eggs.

A highly sialylated glycosylphosphatidylinositol (GPI)-anchored protein CD52 initially found on lymphocytes is also known as SAGA-1, GP20, Cambridge pathology 1 antigen and epididymal secretory protein E5. It is one of the few well-defined antigens secreted from the epididymal cells, is transferred to the sperm plasma membrane during epididymal passage, and alters the characteristics of sperm surface. CD52 is exposed in the equatorial region of the sperm head at the end of the capacitation process in human (Yeung et al. 2001). Both CD52 on sperm and lymphocyte share the same peptide sequence but carbohydrate moiety is different in these two cases. Thus, CD52 on sperm could be antigenic to females. Actually, anti-CD52 mab (MAb H6-3C4) is generated from an infertile woman's peripheral blood lymphocytes. The antibody was shown to have a strong complement-dependent, sperm-immobilizing activity, which indicates the possibility of CD52 being a candidate contraceptive target molecule. Although a clear implication of CD52 in fertilization mechanism is not reported, the covering of sperm membrane with CD52 might have an effect on the sperm-egg, or sperm-female reproductive tract interactions (such as storage of spermatozoa in the caudal part of the isthmus, in tight contact with the epithelium cells lining the oviduct (Topfer-Petersen 1999)).

In the study of mechanism of fertilization, many factors are designated as "important" factors from the experiments in which antibodies and ligands added to in vitro fertilization system showed inhibitory activities. However, many such discovered factors turned out to be "not essential" by gene disruption, suggesting that conclusive demonstrations of the protein functions become more reliable from the observation of the gene disrupted animals. Therefore, we tried to disrupt CD52 gene to examine its role in fertilization.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Expression of Cd52 in various organs (Northern and Western blotting)

Cd52 is known to be expressed in thymus and in spleen (Kubota et al. 1990). The anti-CD52 monoclonal antibodies are used as one of the treatments to prevent the rejection of transplanted organs (Calne et al. 1999; Hale et al. 2000). However, the expression levels of Cd52 were much higher in epididymal tissues than in those immunity-related organs (Fig. 1A). It has been reported that the human CD52 is secreted from the male reproductive tract and is bound to the sperm surface (Kirchhoff 1996). When we examined the presence of CD52 in the mouse using Western blotting, CD52 was not found in the caput epididymis but substantial amounts of CD52 were found starting from the corpus epididymal section to vas deferens. Interestingly, CD52-positive sperm (examined by Western blotting) were only found in the vas deferens (Fig. 1B).


Figure 1
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  		Figure 1  Tissue distribution of Cd52 mRNA and protein. (A) Northern blot containing equal amounts of total RNA (10 µg) was hybridized with 32P-dCTP labeled cDNA fragments of Cd52 and Gapdh. (B) Testes, male reproductive ducts and sperm protein were extracted with lysis buffer containing Triton X-100 and subjected to immunoblot analysis. Western blots containing equal amounts of tissue proteins (30 µg) and sperm protein (10 µg) were hybridized with anti-CD52 polyclonal antibody. For control sperm protein, mouse IZUMO1 was detected with anti-IZUMO1 monoclonal antibody (#125).

 
Immunohistochemistry

The localization of CD52 was examined by immunostaining. We detected almost no staining in caput epididymal sections, but the epithelial cells of the cauda epididymal sections showed an intense staining (Fig. 2). In the vas deferens, an equally strong staining was observed in the epithelial cells, but different from epididymis, the staining was spread to the ductal area of the vas deferens. This staining was not a nonspecific binding, because no staining was observed in Cd52–/– mice.


Figure 2
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  		Figure 2  Expression of CD52 in epididymis and vas deferens. The sections of caput epididymis (A, B), cauda epididymis (C, D), and vas deferens (E, F) were subjected to indirect immunofluorescence employing anti-CD52 monoclonal antibody followed by Alexa Flour 488-conjugated anti-rat second antibody. Immunoreactivity was visualized with fluorescent microscopy. CD52 protein was only detected in the cauda epididymis and vas deferens from heterozygous mouse (C, E, and G). No fluorescence was detected in the tissues from Cd52-deficient mice (B, D, and F).

 
While sperm from cauda epididymis showed no reactivity to anti-CD52 antibody, approximately 25% of sperm population from vas deferens was stained by anti-CD52 antibody (Fig. 3C). The staining pattern of CD52 was spotty and was basically localized in the midpiece area and sometimes extended to the head regions. Ejaculated sperm recovered from the uterus within 1 h after plug formation showed a similar spotty staining pattern as sperm from vas deferens shown in Fig. 3E, indicating that the encounter of sperm with accessory gland secretions at ejaculation seems not to affect the localization of CD52 on sperm. Together with the result of Western and Northern blotting experiments, it was indicated that CD52 is secreted from the epithelial wall and binds to the sperm in the vas deferens in mouse.


Figure 3
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  		Figure 3  Immunolocalization of CD52 protein on mouse sperm. Sperm from cauda epididymis (A, B), vas deferens (C, D) and recovered from uterus (E, F) were immunostained with anti-CD52 monoclonal antibody combined with Alexa Flour 488-conjugated second antibody. Immunoreactivity was visualized with fluorescent microscopy (x400). No fluorescence was detected on sperm from the cauda epididymis (A) and on Cd52-deficient sperm (B, D, and F).

 
Generation of Cd52-deficient mice

In order to examine the roles of CD52, we disrupted the Cd52 gene by homologous recombination. The mouse Cd52 gene consists of two exons and is mapped to chromosome 4 (Tone et al. 1999). The targeting vector was designed to remove both exons of Cd52 (Fig. 4A) and was electroporated into D3 ES cells after linearization. Potentially targeted ES cell clones were separated by positive–negative selection with G418 and acyclovir. Correct targeting of the Cd52 allele in ES cell clones was determined by PCR for homologous recombination on both ends (Fig. 4B). Mating between heterozygous mutant mice yielded the expected Mendelian ratios: Cd52+/+, 26%; Cd52+/–, 52%; Cd52–/–, 22% of offspring (n = 116). Northern and Western blot analysis showed that Cd52 mRNA expression was undetectable in the Cd52–/– epididymis (Fig. 4C). CD52 protein was also not detected in sperm from the vas deferens in Cd52–/– mice (Fig. 4D). Cd52–/– mice exhibited normal development and grew up as healthy adults with normal CD4/CD8 positive cell ratios (Fig. S1 in Supplementary Material). We do not exclude the possibility that we overlooked the phenotype, but the chance is high that the role of CD52 in immune system is masked by some compensating factors or the role is not essential in lymphocytes and in splenocytes.


Figure 4
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  		Figure 4  Production of Cd52-deficient mice. For the targeted disruption of mouse Cd52 allele, all two exons encoding mouse CD52 protein (closed boxes) was replaced by the neomycin-resistant-cassette (Neor). A herpes simplex virus thymidine kinase gene (tk) was introduced into the targeting construct for negative selection. (B) Genotyping of tail tip DNA by PCR amplification with primers indicated in the figure. (C) Northern blot analysis of total RNA from Cd52+/+ (+/+), Cd52+/– (+/–), and Cd52–/– (–/–) cauda epididymis. Blots were hybridized with 32P-dCTP labeled cDNA fragments of Cd52 and Gapdh. (D) Western blot analysis of cauda epididymal lysate and sperm lysate in vas deferens from Cd52+/+ (+/+), Cd52+/– (+/–), and Cd52–/– (–/–) mice.

 
Fertility of sperm in CD52 null epididymis

CD52 expressed in epididymis may contribute to supporting epididymal function per se in nursing sperm. We collected and observed the motility of epididymal sperm from Cd52–/– male mice using automated sperm analyzer SMAS. No difference in the motility or the swimming pattern was found compared to sperm from wild-type mice (Table 1 & Fig. 5). Using the double transgenic mouse line, CD52 KO bearing Acr-EGFP transgene, we examined the effect of CD52 disruption on the spontaneous acrosome reaction of sperm from cauda epididymis and from vas deferens by a flow cytometer. Sperm from Cd52–/– male showed no significant difference from those from control male, even sperm from the cauda epididymis and vas deferens (Fig. 6). The ionophore induced acrosome reaction which was caused by the addition of A23187 [GenBank] was also found not to be affected by the CD52 disruption (data not shown). To confirm the fact that sperm maturation in epididymis was normal in CD52 null mice, in vitro fertilization assay was performed using sperm from CD52 –/–, +/–, and +/+ mice and it resulted in a similar fertilization ratio as expected (Table 2).


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  		Table 1  Comparison of sperm motility prepared from CD52 +/+, +/–, and –/– mice
 

Figure 5
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  		Figure 5  A change in swimming patterns of sperm during incubation in TYH medium. Sperm from CD52 +/– and –/– mice were incubated in TYH medium and analyzed by the computer-aided Sperm Motility Analysis System (SMAS). Almost all the sperm showed straightforward movement at 15 min of incubation (arrows), but a comparable number of characteristic hyperactivated movements (arrowheads) appeared in the sperm population after 120 min of incubation both in sperm from CD52 +/– and –/– mouse lines.

 

Figure 6
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  		Figure 6  Time course of the acrosome reaction in Cd52–/– mice. Mice with the Cd52–/– allele were bred with a green sperm transgenic mouse line and a double-transgenic mouse line, Cd52–/– mice with green sperm was obtained. Sperm from cauda epididymis (A) and vas deferens (B) from this double-transgenic mouse line were incubated in TYH medium and analyzed for their acrosomal integrity by flow cytometry. Sperm from Cd52+/– (closed column) and Cd52–/– (open column) littermate mice were compared. Error bars represent mean ± SDs from seven (cauda epididymis) or four (vas deferens) independent experiments.

 

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  		Table 2  Comparison of fertilizing ability of sperm prepared from CD52 +/+, +/– and –/– mice in vitro
 
Fertility of ejaculated sperm in CD52 null mice

Usually, cauda epididymal sperm are used for in vitro fertilization experiments. However, this sperm population is not yet covered with CD52 (Fig. 3), indicating that mouse epididymal sperm are competent to bind and penetrate eggs without having CD52 on their surface. Therefore, if CD52 functions in fertilization, it might be through a characteristic step involved in in vivo fertilization processes, such as ejaculated sperm moving up the female reproductive tract to where fertilization takes place.

To clarify the role of CD52 in fertilization in vivo, we mated Cd52–/– male mice with superovulated wild-type female mice. Two hours after coitus, whole mount sections of oviducts were made and serial sections were observed by bright field microscopy. Close examination of sections of the uterotubal junction revealed that sperm derived from both CD52 +/+ and –/– sperm had migrated through the ostium of the colliculus tubarius, and were found inside the oviduct (Fig. 7).


Figure 7
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  		Figure 7  Migration of Cd52–/– sperm into oviduct. Sperm transits from uterus to oviduct were observed by removing the uterotubal junction 2 h after coitus and making frozen sections stained with hematoxylin. (A, C) heterozygous type: (B, D) Cd52–/–. Boxed areas in (A) and (B) were magnified in (C) and (D). Arrow heads indicate sperm present in the uterus (u) and colliculus (c).

 
There was no difference in the mating ratio and fertility compared to wild-type mice (Table 3). In natural mating, the number of pups sired by wild-type and Cd52–/– male mice were similar (control, 9.55 ± 1.70, n = 18; homozygous, 9.14 ± 1.02, n = 14), coinciding with the result of migrating equal number of sperm into the oviduct. Thus the disruption experiment revealed that CD52 is "dispensable" in fertilization.


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  		Table 3  Fertilized eggs recovered from oviducts from copulated females
 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the process of studying fertilization, various antibodies to sperm were produced in the past. The clarification of the antigens and subsequent disruptions of the corresponding genes indicated various antigens to be essential (ADAM2, ADAM1a, ADAM3, ACE, and IZUMO1). On the other hand, many factors turned out to be "not essential" (acrosin, CD46, PH20, ADAM1b, GalTase etc.) for fertilization (see review, Okabe & Cummins (2007)). These data suggest that we have to be careful when we examine the roles of a certain factor in fertilization using antibodies and/or ligands. The gene disruption experiment is a time-consuming effort but could often provide clear understanding which is not possible to obtain with normal biochemical and/or physiological analysis.

CD52 is a GPI-anchored membrane protein found in mouse, rat, monkey, dog and human (Hale 2001). Although the lengths of the mature peptides are different among species, all of them have a single potential site for the N-linked glycosylation. The rat CD52 antigen has been characterized for many years as the "major maturation-associated antigen" of sperm (Kirchhoff 1996; Yeung et al. 2001). It is the most abundant antigen among the sperm glycoproteins and its acquisition during epididymal transit explains much of the remarkable change in surface charge and lectin-binding characteristics which occurs during sperm maturation (Kirchhoff & Schroter 2001). In view of the accumulation of CD52 in rat and human sperm, it is natural to expect an important role of CD52 in sperm maturation in various species. Therefore, we chose CD52 as a candidate gene to disrupt to elucidate the mechanism of epididymal maturation process toward fertilization.

Although CD52 was highly expressed in the epididymal tissues, the disruption of CD52 caused no apparent effect on the epididymal functions per se. As an alternative role, we could speculate that CD52, as one of the membrane proteins after transition to sperm, is functioning to interact with the female reproductive tract. However, the sperm from Cd52-deficient mice could successfully migrate into the oviduct and fertilize the eggs and Cd52-deficient males sired similar numbers of pups. The fertilizing ability of Cd52-deficient males remained normal even at 50 weeks of age (data not shown), indicating that no immunological disorders in the reproductive tract took place, differing from the case of CD59-disrupted mouse line, in which a progressive loss of fertility associated with immobile dysmorphic and fewer sperm cells after 5 months of age was observed (Qin et al. 2003; Qin et al. 2005). Thus with respect to all these aspects, CD52 turned out to be not essential in the fertilization system. Approximately 25% of the sperm population had detectable CD52. If CD52 has an inhibitory or stimulatory activity on sperm function when attached to the sperm surface, the remaining CD52-free population might mask the effect of CD52 in our experimental system. Although, no apparent phenotype of CD52 disruption was observed in the present experiment, the fact that the CD52 has been retained as an active gene in epididymis and is found on mouse, rat, monkey and human sperm suggests that CD52 has unknown but important roles in fertilization.

In human, all sperm are reported to possess CD52 on their surface, while in mouse, approximately a quarter of the population was found to react to anti-CD52 antibody (Fig. 3). The localization of CD52 on mouse sperm turned out to be different from human (midpiece in mouse versus whole sperm in human) and the site of CD52 transition from epididymal tissue to sperm seemed to be different between mouse and human (vas deferens in mouse versus in epididymis in human). It is not clear how CD52 is transferred from epididymal epithelium to sperm membrane. As a similar mechanism of transfer of membrane proteins to sperm, prostasomes are known in prostate and are speculated to transport membrane proteins to the sperm membrane (Ronquist & Brody 1985). Recently, exosome (Leblanc et al. 2006) in the epididymis, termed epididymosome, was reported to serve in epididymal maturation of sperm (Rejraji et al. 2006). CD52 might be transported through this secretion system and epididymosomes might be the origin of the speckled stainings observed in immunofluorescent analysis. Further investigation is awaited to learn if CD52 is transferred from epididymal epithelium to sperm plasma membrane or CD52-containing exosomes are only attaching on sperm membrane.

Although CD52 was found to be dispensable in fertilization, it does not mean that CD52 has no function in the system in wild-type mice. In the present paper, we could not find any apparent phenotype derived from the disruption of CD52 in immune system or in fertilization in mice kept under normal conditions. However, with combination to other gene disrupted mice, a severe phenotype might be expressed as in the case shown in Hox gene disruptions (Davis et al. 1995). In other words, various factors are known to be compensated by other factors, but their role becomes evident if the disruption is overlaid on another genetic background in which some other gene was disrupted (Nef et al. 2003). The CD52-disrupted mouse line may have such a characteristic. In order to make it possible to pursue further analysis of the role of CD52 in vivo, the CD52 disrupted mouse line was submitted to Riken BioResource center and is available to the scientific community.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Northern blot analysis

Northern hybridization was performed using 10 µg of total RNA extracted from various tissues of adult ICR mice. RNAs were separated by electrophoresis on agarose gels, transferred to Hybond-N+ membranes (GE Healthcare Bio-Sciences Corp, Piscataway, NJ), and hybridized to 32P-labeled probes at 60 °C overnight. Mouse Cd52 and glyceraldehydes-3-phosphatate dehydrogenase (Gapdh) cDNAs were used as probes. The Cd52 probe consisted of a cDNA fragment amplified from mouse epididymal total RNA by RT-PCR using 5'-TGAATTCTTCAAAGTGGCCTGCA GACTGTC-3' and 5'-TGAATTCGCCATTGGCTGTCAAC TTTAGCC-3' as primers.

Antibodies

Rabbit anti-mouse CD52 polyclonal antiserum was produced by immunization with mouse CD52 polypeptide (AASGTNKNSTSTKKTPLKSG). Rat monoclonal antibody against mouse CD52 was a kind gift from Dr Nagahiro Minato (Kyoto University, Kyoto, Japan) (Kubota et al. 1990). A new monoclonal antibody against mouse IZUMO1 (Inoue et al. 2005) was produced by screening after immunization of whole mouse sperm to rat and termed Mab #125. Monoclonal antibodies against mouse ADAM2 (fertilin β 9D2.2) were purchased from Chemicon International, Inc. (Temecula, CA).

Immunohistochemistry

Epididymis and vas deferens were collected from adult Cd52+/– and Cd52–/– mice and embedded in a TissueTek O.C.T. compound (Sakura Finetechnical Co., Tokyo, Japan). Frozen sections (16 µm) prepared from these tissues were mounted on APS (aminosilane) coated glass slides. Sperm from cauda epididymis or vas deferens were swum up in TYH medium and resuspended in PBS (Toyoda et al. 1971). Ejaculated sperm were collected from the uterus just after mating and resuspended in PBS. Sperm suspensions were mounted on glass slides and dried up. All samples were fixed in 4% paraformaldehyde/PBS for 30 min. After washing with PBS, slides were blocked with 10% New Born Calf Serum (NBCS)/PBS for 1 h and incubated with rat anti-mouse CD52 monoclonal antibody in 10% NBCS/PBS at 4 °C overnight. After washing with 10% NBCS/PBS containing 0.05% Tween-20, the slides were incubated with anti-rat IgG labeled with Alexa Fluor 488 (Invitrogen) in 10% NBCS/PBS for 1 h. After washing with PBS containing 0.05% Tween-20, the slides were observed under an Olympus IX-70 fluorescence microscope.

Immunoblot analysis

Immunoblot analysis was performed as described previously (Yamaguchi et al. 2006). Briefly, sperm from the epididymis and vas deferens were collected and incubated in lysis buffer containing 1% TritonX-100 for 20 min on ice. The testis, epididymis, and vas deferens were excised, minced, and homogenized in lysis buffer, and then placed on ice for 1 h. The sperm and tissue extracts were centrifuged, and the supernatants were collected. Proteins were separated by SDS-PAGE under reducing conditions and transferred electrophoretically to PVDF membranes. After blocking, blots were incubated with primary antibody overnight at 4 °C, and then incubated with horseradish-peroxidase conjugated goat anti-rabbit IgG, goat anti-mouse IgG and goat anti-rat IgG (GE Healthcare Bio-Sciences Corp.). The detection was performed using an ECL Western blotting detection kit (GE Healthcare).

Construction of the Cd52 gene disruption vector

A targeting vector was constructed using pPNT containing the Neo-resistance gene (Neor) as a positive selection marker and a herpes simplex virus thymidine kinase (tk) as a negative selection marker (Tybulewicz et al. 1991). A 2.1-kb NotI-SalI fragment as a short arm and a 6.0-kb SpeI-KpnI fragment as a long arm were obtained by PCR using genomic DNA in D3 embryonic stem (ES) cells as a template. The PCR primers used were as follows: 5'-GCGGCCGCAGTTAAAAGCACTTGTTGCAAGCCGGGC AG-3' and 5'-TTTGTCGACGTGCGGCAGTATTAGGAGT GAACCCAGTAC-3' for the short arm, 5'-GGACTAGT GGCCACTTTGAACCTGGCTGCTTTTTCTGC-3' and 5'-TGGTACCAGAGGTCTCAACCTGTGGCTTGTGACCCAG-3' for the long arm.

These two fragments were inserted into a pPNT vector and the targeting construct was linearized with NotI digestion. ES cells were electroporated and colonies were screened.

Generation of Cd52 mutant mice

G418-resistant colonies were amplified, and genomic DNA was prepared from them and screened by PCR analysis. Several of the recombinant ES cell lines carrying the disrupted Cd52 allele were identified and subsequently used to generate chimeras by injection into blastocysts from C57BL/6 Cr mice (> 2 months old; Japan SLC, Inc., Shizuoka, Japan). Injected blastocysts were transferred to ICR pseudopregnant foster mothers, resulting in the birth of male chimeric mice. These mice were crossed with C57BL/6 to obtain F1 heterozygous offspring. Cd52-deficient mice were generated by the intercrossing F1 offspring mice. Mice used in this study were of B6; 129 mixed background.

All experiments were performed with the consent of the Animal Care and Use Committee of Osaka University.

Analysis of acrosome reaction and sperm motility

To investigate the influence of Cd52 disruption on the sperm acrosome reaction, females from a transgenic mouse line which have enhanced green fluorescent protein in sperm acrosome (Acr-EGFP) were crossed with Cd52–/– males (Nakanishi et al. 1999). Double transgenic F1 offspring were intercrossed to generate Cd52–/– and Acr-EGFP+/–. Sperm from double-transgenic mice were assayed as described in our previous paper (Inoue et al. 2003). Briefly, sperm were squeezed out from the incisions made in cauda epididymis or vas deferens and were suspended and incubated in TYH medium to induce a spontaneous acrosome reaction. Acrosomal statuses were analyzed from the acrosomal fluorescence by flow cytometer at 0, 30, 60, 120 and 180 min after insemination. Sperm motility was measured using epididymal sperm and automated Sperm Motility Analysis System (SMAS, Kaga Electronics Co. Ltd, Tokyo, Japan).

Sperm migration analysis

B6D2F1 females were superovulated by intraperitoneal injection of 5 units of equine chorionic gonadotropin followed 48 h later by 5 units of human chorionic gonadotropin (HCG). Superovulated females were caged together with test males 12 h after HCG injection, and the formation of vaginal plug was observed every 30 min. About 2 h after copulation, oviducts were excised together with the connective part of the uterus. To detect sperm in the uterotubal junction, the oviducts with attached uterus were fixed in 4% paraformaldehyde–PBS for 6 h, followed by washing with PBS, and were then prepared for frozen sections. Total of three females were examined using three males of each genotype.

Assessment of the fertilizing ability of Cd52-deficient mice

Female B6D2F1 mice (older than 8 weeks; Clea Japan Inc., Tokyo, Japan) were superovulated following intraperitoneal injections of eCG (Teikoku Zoki, Co. Ltd, Kanagawa, Japan) and hCG (Teikoku Zoki) at 48 h intervals. Cd52+/– or Cd52–/– males were mated with superovulated females 7 h after hCG injection. Eggs were recovered from females at their pronuclear stage and placed in a KSOM medium (Ho et al. 1995). Fertilization rates were assessed by pronuclear formation and subsequent 2-cell formation, observed by Olympus IX-70 microscope. In vitro fertilization was performed as previously described (Ikawa et al. 1997).


   Acknowledgements
 
Authors thank Dr Nagahiro Minato (Kyoto University) for providing the antibody against CD52. Authors also thank Y. Maruyama, A. Kawai and Y. Koreeda for technical assistance with gene disruption. This work was supported in part by grants from the Ministry of Education, Science, Sports, Culture, and Technology, and the 21st Century 200 COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Takeo Kishimoto

* Correspondence: okabe{at}gen-info.osaka-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Ho, Y., Wigglesworth, K., Eppig, J.J. & Schultz, R.M. (1995) Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol. Reprod. Dev. 41, 232–238.[CrossRef][Medline]

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Accepted: 13 May 2008




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