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Construction of sperm-specific lactate dehydrogenase DNA vaccine and experimental study of its immunocontraceptive effect on mice CHEN Yong*, ZHANG Duo*, XIN Na, XIONG YongZhong, CHEN Ping, LI Bo, TU XiangDong & LAN FengHua† Research Laboratory of Medical Genetics, PLA Institute of Technology for Birth Control, Fetal Health and Child Growth, Fuzhou General Hospital of Nanjing Military Region, Fuzhou 350025, China

Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic expression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vaccinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vaccinated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive DNA vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future. sperm-specific lactate dehydrogenase, DNA vaccine, contraceptive vaccine, mucosal immunization

Sperm-specific lactate dehydrogenase or lactate dehydrogenase C4 (LDHC4) exists only in testis and spermatozoa of mammalian and avian species, the function of which relates to energy metabolism and capacitation of the sperm. Because of its specific distribution, LDHC4 is regarded as one of the candidates of immunocontraceptive vaccine[1]. It has been reported that purified LDHC4 or chemically modified LDHC4 peptides could elicit an immune response in many animal species and the production of LDHC4 specific antibodies could be induced in the sera or the reproductive fluids. The birthrate of the treated animals decreased markedly and the ― contraception effect was found reversible[2 7].

Following the attenuated vaccine and genetically engineered vaccine, nucleic acid vaccine belongs to the third-generation vaccine. It is a recombinant plasmid which contains exogenous genes encoding one or more proteins. When such a plasmid is introduced into the body, the exogenous antigens are synthesized with the help of the body’s expression system, which can in turn Received July 18, 2007; accepted September 27, 2007 doi: 10.1007/s11427-008-0035-7 † Corresponding author (email: [email protected]) *Contributed equally to this work Supported by Grants from the Key Research Projects of Science and Technology of Fujian Province, China (Grant No. 2002Y032) and Chinese PLA Foundation of Medical Sciences during the Tenth Five–Year Period (Grant No. 01MA032)

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elicit immune responses. Compared with the previous two generations of vaccine, the nucleic acid vaccine has many obvious advantages. It can induce both humoral and cellular immune responses, the responses can last for a long time, the conformation and antigenicity of the antigen protein is identical to native antigen, and most importantly, it has no potential pathogenicity. The nucleic acid vaccine includes both DNA and RNA vaccines. At present, DNA vaccine is studied most extensively. Shen et al.[8] constructed an LDHC4 DNA vaccine by inserting the gene of LDHC4 into an expression vector. After vaginal DNA immunization using a controlled delivery matrix, this DNA vaccine could induce the production of specific antibodies in mice. Shi et al.[9] and Chang et al.[10] also constructed the LDHC4 DNA vaccine and studied its effect of immunocontraception. According to the sequences of the cDNA of human and mouse LDHC4 in GenBank, we designed PCR primers and amplified the coding sequences of the genes in vitro. Two DNA vaccines were obtained by inserting the amplified products into the eukaryotic expression vector pVAX1. To explore their contraception effects as well as the underlying mechanism, we immunized both male and female BALB/c mice by mucosal surface instillation.

1 Materials and methods 1.1 Materials The prokaryotic expression vector pET-28a(+) was a product of Novagen Company and the eukaryotic expression vector pVAX1 was purchased from Invitrogen Corporation. Both the Endofree Plasmid Maxi Kit and Ni-NTA Matrix Kit were purchased from Qiagen Company. The BALB/c mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences with the number of qualification being SCXK (Hu) 2003-003. Breeding and all animal experiments were carried out at Department of Comparative Medicine, Fuzhou General Hospital, Nanjing Military Region. Goat anti-mouse IgG-HRP was a product of KPL Company (USA, Maryland) and Goat anti-mouse IgA-HRP was purchased from the Southern Biotech Company (USA, Birmingham, Alabama). 1.2 Methods 1.2.1 Construction of pVAX1-hLDHC and pVAX1mLDHC. Based on the human LDHC4 mRNA (GI:

187065) sequence in GenBank, PCR primers were designed. The sequence of the upstream primer was: 5′CCGAAGCTTGCACCATGTCAACTGTCAAGGAGCAGC-3′, with a Hind III restriction site and the downstream primer was: 5′-CCGCTCGAGTTAAAATATTAGATCCTTTTGAATATTCC-3′, with an Xhol I restriction site. With the human testis λTripIEx cDNA library as templates, the human LDHC4 coding sequence was amplified by PCR. The expected size was 1016 bp. Based on the mouse LDHC4 mRNA (GI: 52885) sequence in GenBank, the sequence of the upstream primer was: 5′-CCGGATCCCTCGCCACCATGGCCACCGTCAAGGAG-3′, with a BamH I restriction site and the downstream primer was: 5′-CGGAATTCGCGAGTTTATAACTGCAGATCCTTC-3′, with an EcoR I restriction site. The mouse LDHC4 coding sequence was obtained by RT-PCR. The expected product size was 1028 bp. The recombinant pVAX1-hLDHC and pVAX1mLDHC expression vectors were constructed by inserting these amplification products into pVAX1 and amplified by transformation of E. coli DH5α. 1.2.2 Preparation of DNA vaccines and immunization of BALB/c mice. The plasmids used for immunization were purified using Qiagen’s Endofree Plasmid Maxi Kit and suspended in sterile saline at a concentration of 2 mg/mL. Sixty four female BALB/c mice which were 6―8 week old were randomly divided into 4 groups. Group 1 consisting of 8 female mice received sterile saline as control. The mice were immunized in two manners: simple vaginal instillation or combined vaginal and nasal instillation, with 4 mice for each manner. The volume of saline for each mouse was 50 μL. Group 2 received void pVAX1 plasmid, both the number of mice and the immunization manner being the same as Group 1. Group 3 were immunized with pVAX1-mLDHC recombinant plasmid. The immunization manner was the same as previous two groups, but three dosages were included: 50, 100 and 150 μg per mouse, with 4 mice for each dosage. Group 4 received the pVAX1-hLDHC recombinant plasmid, and the dosages, the number of mice and the immunization manner were the same as Group 3. These mice were subsequently given booster doses twice at 2-week intervals. 1.2.3 ELISA of LDHC4 antibody. One week after the first immunization, 0.1―0.3 mL of blood from retroorbital bleeding was collected. The serum was separated

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by centrifugation at 8000 r/min for 10 min after overnight coagulation at 4℃ and stored at −20℃. Vaginal secretion was collected by washing the vaginal lumen with 100 μL PBS. The supernatant was separated by centrifugation at 7000 r/min for 10 min and stored at −20℃. The serum and vaginal secretion were collected at 2-week intervals. The coding sequence of human LDHC4 was obtained by PCR, using the human testis λTripIEx cDNA library as templates. The recombinant plasmid pET-28a (+)hLDHC was constructed by inserting this PCR product into pET-28a (+) vector. E. coli. BL21(DE3)was transformed with pET-28a(+)-hLDHC. Soluble recombinant protein was induced by IPTG, and protein purification was carried out with Ni+-NTA agarose. The purity of the recombinant human LDHC4 (hLDHC4) was confirmed by 10% SDS-PAGE. Mouse LDHC4 coding sequence was obtained by RT-PCR, with total mRNA of testis as templates. The purified recombinant mLDHC4 was prepared in the same way as recombinant hLDHC4. Purified recombinant hLDHC4 and mLDHC4 were diluted in bicarbonate buffer to 1 μg/mL, applied to ELISA plates with 50 μL per well and incubated overnight at 4℃. Wells were blocked with PBS containing 4% bovine serum albumin and incubated at 37℃ for 2 h. Sera (100 μL) at dilution 1:100 or vaginal secretions (100 μL) at dilution 1:10 were added to each well. After incubation at 37℃ for 1 h, wells were washed 6 times with PBS containing 0.05% Tween-20, and 100 µL goat anti-mouse IgG-HRP diluted 1:4000 (for sera) or goat anti-mouse IgA-HRP diluted 1:4000 (for vaginal secretions) was added. Again the wells were incubated for 1 h at 37℃ and then washed six times. Coloration started when 100 μL 3,3′,5,5′-Tetramethylbenzidine (TMB) was added. After incubation at 37℃ for 20 min, the reaction was stopped by addition of 50 μL H2SO4 (2 mol/L). The plate was measured with a plate reader (Rayto) at 450 nm. 1.2.4 Western blot analysis of LDHC4 antibody. The purified recombinant mLDHC4 and hLDHC4 proteins were separated by 10% SDS-PAGE and transferred onto the nitrocellulose membrane. The membrane was blocked with 3% BSA overnight at 4℃ and incubated with 1:50 sera or 1:10 vaginal fluids from immunized mice at room temperature for 2 h. After being washed three times with PBS containing 0.05% Tween-20, the 310

membrane was incubated with 1:4000 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (for sera) or 1:4000 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA (for vaginal secretion) for 2 h at room temperature. Following washing six times with PBS, the membrane was developed with 4-chlorine-1naphthol. For detection of LDHC4 antibody with sperm crude protein, normal human sperm suspension was centrifuged at 1500 r/min for 15 min after liquefaction. The sediment was washed twice with saline and resuspended in 0.2% Triton X-100 and swelled at room temperature for 60 min. The supernatant was collected after centrifugation at 3000 r/min for 20 min. Western blot analysis was performed as described above. 1.2.5 Mating test. Mice (9 female, 5 male) were immunized with pVAX1-hLDHC or pVAX1-mLDHC DNA vaccines three times at 2-week intervals. The dosage for each mouse each time was 150 µg. For female mice, combined vaginal and nasal instillations were used and for male mice, combined oral and nasal instillations were used. Five days after the third immunization, the female mice were paired with mature normal male mice at a ratio of 3:1 and the male mice were paired with normal female mice at estrus at a ratio of 1:3. The female mice in this experiment were checked daily for whether there was a vaginal plug as an indication of mating. The pregnant female mice were bred individually. Groups of void plasmid-treated mice, saline-treated mice and nonimmunized mice were also set as controls, each group containing six female mice and two male mice. 1.2.6 Sperm agglutination and immunohistochemmistry assays. Normal sperm suspension was prepared as described above and resuspended in PBS. The sera and vaginal secretions from various groups of female mice were mixed with sperm suspension (1:1, v/v) and incubated at 37℃ for 1 h. The mixture was checked under a microscope. For immunohistochemistry, 10 μL fresh normal sperm suspension was dropped onto paraform-coated cover slips, air-dried and then fixed with methanol for 10 min at room temperature. The slides were washed three times with PBS and blocked with 4% BSA for 30 min. After being washed three times with PBS, sperms on the slide were incubated with 10 μL first antibodies, which was

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the serum or vaginal fluid from immunized mice, at 37℃ for 3 h. The slides were washed three times with PBS and sperms were incubated with secondary antibodies (1:500 diluted goat anti-mouse IgG-HRP for sera, 1:1000 diluted goat anti-mouse IgA-HRP for vaginal

used as vaccines were characterized spectrometrically, with the A260/A280 ratio being about 1.8, and they produced right fragments when digested with the abovementioned restriction enzymes.

secretions) at 37℃ for 2 h. Slides were washed 3 times again with PBS and were developed with the addition of DAB solution.

Compared with control mice, anti-LDHC4 antibody could be easily detected by ELISA in sera and vaginal fluids from vaccinated mice one week after the first immunization. The titer was highest at the fifth week and declined gradually after that. The antibody was still detectable at the 13th week and the titer of antibodies from the mice immunized by combined vaginal and nasal instillation was higher than that from mice immunized by simple vaginal instillation (Figure 2).

1.2.7 Other methods. Five days after the third immunization, 3 female and 3 male mice from each group of mating test were killed by cervical dislocation (2 male mice for 3 control groups). The testis, vagina, nasal mucosa and other tissues were fixed in 10% formaldehyde and conventional pathological examination was carried out. Statistic analysis was done by using analysis of variance based on data of randomized complete block design.

2 Results 2.1 Construction of DNA vaccines The recombinant plasmids pVAX1-hLDHC and pVAX1mLDHC were introduced into E. coli DH5α and amplified in it. The plasmids were purified using Qiagen’s Endo-free Plasmid Maxi Kit and confirmed by restriction enzyme digestion (the pVAX1-mLDHC plasmid digested with BamH I and EcoR I and the pVAX1hLDHC plas mid digested with Hind III and

Figure 1 Agarose gel electrophoresis of recombinant plasmids pVAX1-hLDHC and pVAX1- mLDHC. Lane M, DNA Markers; lane 1, the void pVAX1 plasmid before restriction digestion; lane 2, the void pVAX1 plasmid after restriction digestion; lane 3, the pVAX1-mLDHC plasmid before restriction digestion; lane 4, the pVAX1-mLDHC plasmid after restriction digestion; lane 5, the pVAX1-hLDHC plasmid before restriction digestion; lane 6, the pVAX1-hLDHC plasmid after restriction digestion.

Xhol I). The result is shown in Figure 1. Plasmids to be

2.2 ELISA analysis of LDHC4 antibody

2.3 Western blot analysis of LDHC4 antibody The sera and vaginal fluids from the mice immunized with pVAX1-hLDHC and pVAX1-mLDHC DNA vaccines were probed with purified recombinant hLDHC and mLDHC antigens, respectively. It was showed that specific protein bands appeared on the nitrocellulose membrane. Furthermore, sera from the immunized mice also were probed with native sperm crude proteins and a specific protein band was visualized, the molecular weight of which was approximately 35 kD (Figure 3). 2.4 Mating test of immunized mice The mice in each group of mating test were mating on day 5 after the third immunization and checked daily for mating. The number of newborns in each group was recorded and the difference was analyzed by t-test. It was showed that there was no significant difference among saline-treated group, the void pVAX1 plasmid-treated group and nonimmunized group in terms of the number of newborns, but the number of newborns of DNA vaccine-treated mice decreased obviously, showing significant difference from that of control mice (Table 1). It was also indicated that the immunocontraceptive effect of pVAX1-mLDHC DNA vaccine was better than that of pVAX1-hLDHC DNA vaccine and the immunocontraceptive effect on male mice might be better than that on female mice. 2.5 Sperm agglutination and immunohistochemistry Both sera and vaginal secretions from pVAX1-mLDHC and pVAX1-hLDHC immunized mice could restrain sperm motility and induce sperm agglutination. These

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Figure 2 LDHC4 antibody levels assayed by ELISA. (a) Sera from the groups immunized in the simple vaginal instillation manner; (b) vaginal secretions from the groups immunized in the simple vaginal instillation manner; (c) sera from the groups immunized in the combined vaginal and nasal instillation manner; (d) vaginal secretions from the groups immunized in the combined vaginal and nasal instillation manner.

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Figure 3 Specificity of LDHC4 antibody by Western blot analysis. (a) Sera from the immunized mice were probed with purified recombinant protein. Lane 1, sera from pVAX1-mLDHC immunized mice probed with purified recombinant mLDHC4 protein; lane 2, sera from pVAX1-hLDHC immunized mice probed with purified recombinant hLDHC4 protein. (b) Vaginal fluids from immunized mice were probed with purified recombinant protein. Lane 1, vaginal fluids from pVAX1-mLDHC immunized mice probed with purified recombinant mLDHC4 protein; lane 2, vaginal fluids from pVAX1-hLDHC immunized mice probed with purified recombinant hLDHC4 protein. (c) Sera from the immunized mice were probed with sperm crude protein. Lane 1, sera from pVAX1-mLDHC immunized mice probed with sperm crude protein; lane 2, sera from pVAX1-hLDHC immunized mice probed with sperm crude protein. Lane M: protein markers. The sera and vaginal secretions were all obtained from the immunized female mice (dosage of 150 µg), which were immunized by combined vaginal and nasal instillation, at the fifth week after the first immunization. Table 1

Comparison of the number of newborns of each group Vaccinated mice Number of pregnant S P Number of newborns of each litter Total number Group x (number) female mice 1 Female (3) 3 8 9 9 26 8.67 0.58 2 Female (3) 3 8 7 8 23 7.67 0.58 >0.10 3 Female (3) 3 7 6 8 21 7.00 1.00 >0.05 4 Female (6) 6 2 2 1 0 0 2 7 1.17 0.98 <0.001 5 Female (6) 6 2 4 3 3 2 1 15 2.50 1.05 <0.001 6 Male (2) 6 0 1 2 0 3 1 7 1.17 1.17 <0.001 7 Male (2) 6 1 1 3 2 1 3 11 1.83 0.98 <0.001 a) Group 1: nonimmunized mice; group 2: saline-treated mice; group 3: void pVAX1 plasmid-treated mice; group 4: pVAX1-mLDHC DNA vaccine-treated female mice; group 5: pVAX1-hLDHC DNA vaccine- treated female mice; group 6: pVAX1-mLDHC DNA vaccine-treated male mice; Group 7: pVAX1-hLDHC DNA vaccine-treated male mice. Because of incorrect performance, one of male mice in group 6 and 7 died during experiment. Other treated male mice were healthy during the experiment as compared with preimmunization and control male mice. a)

phenomena did not appear in sperm samples when they were incubated with the sera and vaginal secretions from nonimmunized mice and void pVAX1 plasmidtreated mice (Figure 4). The immunohistochemical analysis was performed on normal sperm samples which were incubated with the sera or vaginal secretions from pVAX1-mLDHC or pVAX1-hLDHC DNA vaccine-treated mice. It was showed that specific coloration appeared at the cytoplasm, acrosome membrana externa and acrosome capsule of the sperms, whereas no staining appeared in the sperm samples incubated with the sera or vaginal secretions from void pVAX1-treated mice and nonimmunized mice (Figure 5). 2.6 Histopathological check Conventional histopathological test was carried out on testis of male mice and nasal mucosa, vaginal mucosa

and ovaries of female mice and other related tissues. In male mice, the testis structure was normal and no sign of inflammatory cell infiltration was seen. In female mice, the morphology, thickness and number of cells of vaginal mucosa, nasal mucosa and ovaries were normal (results not shown).

3 Discussion LDHC4 is a key enzyme for sperm energy metabolism. It is distributed largely in the cytoplasm and mitochondrial matrix of the sperm. A small proportion of LDHC4 locates in the acrosome membrana externa and acrosome capsule, while a few LDHC4 sits in the chief piece membrane of the sperm[11]. The expression of LDHC4 is initially activated in the mid-pachytene of primary spermatocyte of puberty mammals and its activity accounts for about 46.3% of the total lactate dehydro-

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Figure 4 Sperm agglutination analysis. (a) Normal sperm suspension mixed with vaginal secretions from nonimmunized mice; (b) normal sperm suspension mixed with sera from nonimmunized mice; (c) normal sperm suspension mixed with vaginal secretions from void pVAX1 plasmid-treated mice; (d) normal sperm suspension mixed with sera from void pVAX1 plasmid-treated mice; (e) normal sperm suspension mixed with vaginal secretions from pVAX1-mLDHC DNA vaccine-treated mice; (f) normal sperm suspension mixed with sera from pVAX1-mLDHC DNA vaccine-treated mice; (g) normal sperm suspension mixed with vaginal secretions from pVAX1-hLDHC DNA vaccines-treated mice; (h) normal sperm suspension mixed with sera from pVAX1-hLDHC DNA vaccines-treated mice. In this experiment, sera and vaginal secretions from female mice immunized with 100 µg plasmid by combined vaginal and nasal instillations (5 weeks after the first immunization) were used. Arrows indicate the agglutination site. Magnification: ×100.

Figure 5 Immunohistochemistry analysis. (a) normal sperm reacted with vaginal fluids from nonimmunized mice; (b) normal sperm reacted with sera from nonimmunized mice; (c) normal sperm reacted with vaginal fluids from void pVAX1 plasmid-treated mice; (d) normal sperm reacted with sera from void pVAX1 plasmid-treated mice; (e) normal sperm reacted with vaginal fluids from pVAX1-mLDHC plasmid-treated mice; (f) normal sperm reacted with sera from pVAX1-mLDHC plasmid-treated mice; (g) normal sperm reacted with vaginal fluids from pVAX1-hLDHC plasmid-treated mice; (h) normal sperm reacted with sera from pVAX1-hLDHC plasmid-treated mice. In this experiment, sera and vaginal secretions from the female mice immunized with 100 µg plasmid by combined vaginal and nasal instillations (5 weeks after the first immunization) were used. Magnification: ×1000.

genase activity of normal sperm cell. The amino acid composition of LDHC4 is highly conserved in different species. For example, the homology of LDHC4 between human and mouse is 74%, while that between human and fox is 86%[12]. Owing to its strong immunogenicity, when immunized, LDHC4 can induce specific immune responses in many experimental animals, which in turn results in reduced birthrate significantly. In this study, BALB/c mice were immunized via mucosal immunization with recombinant plasmids pVAX1314

mLDHC and pVAX1-hLDHC, whose ability to direct the synthesis of LDHC4 protein was verified in advance by transfection and in vitro expression experiments (results not shown). The two contraceptive vaccines were shown to stimulate the local and systemic humoral immune responses in mice obviously. The induced antibodies could not only react with the purified recombinant LDHC4, but also recognize native mouse LDHC4 protein from testis. The antibody titer increased with the times of immunization. It was observed that the titer

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reached the highest level at the first week after the third immunization with a gradual decline thereafter. The mating test was carried out when the antibody reached the highest level. The number of offsprings of the treated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings. T-test showed that in the first mating experiment, the difference of the number of newborns between vaccinated mice and control mice was significant and the effect of pVAX1-mLDHC immunization was better than that of pVAX1-hLDHC immunization for the average number of newborns of pVAX1- mLDHC immunized male and female mice was 1.17, whereas that of pVAX1-hLDHC immunized male and female mice was 1.83 and 2.5, respectively. The difference might result from the discrepancy of LDHC4 between species. It seemed that the contraceptive effect on male mice was possibly better than that on female mice. Although the same phenomenon had even been reported by others[3], further research is needed to get a clearer conclusion. In the second mating experiment which was carried out eight weeks after the third immunization, there was no difference in the number of offsprings between immunized and control mice, which indicated that the contraceptive effect of constructed DNA vaccines in this study was reversible. These results were similar to that of Goldberg et al.[13] Considering that the relationship between cell-mediated immunity and the reproductive behavior such as spermegg interaction is not closely related, changes in the cellular immunity induced by these DNA vaccines were not detected in the present study. The mechanism of antifertility of LDHC4 DNA vaccine might be that the induced antibodies inhibited the enzyme activity of LDHC4 in sperm cells, interfered the energy metabolism and restrained sperm motility. In this study, when incubated with normal sperm suspension, sera and vaginal secretions from vaccinated mice could reduce the swimming ability of sperms and induced sperm agglutination, suggesting that specific antibodies in sera and vaginal secretions could bind to the LDHC4 of sperms and the energy metabolism of sperms would be interfered. As the immunohistochemical results showed, the antibodies reacted mainly with LDHC4 antigen located in the cytoplasm, acrosome membrana externa and acrosome capsule of the sperm, due to the higher content of LDHC4 in these regions and caused head to head agglutination, thus generating strong staining in immunohistochemistry.

In earlier studies, purified native LDHC4 or chemically modified LDHC4 peptides were used to immunize mice, rabbits, baboons and other animals, which could reduce the fertility of these animals and induce the production of specific antibodies in sera, cervical mucus, oviducal fluids and vaginal fluids. But such protein vaccines had many disadvantages, such as short duration of their immunologic effects, complexity of their preparation, poor stability and difficulty in transportation. With the development of DNA vaccine, researchers began to construct LDHC4 DNA vaccine by inserting the gene of LDHC4 into various expression vectors. In 2006, Chang et al.[10] inserted the partial cDNA sequence of the Microtus brandti radde LDHC4 (brLDHC4) into the expression vector pCR3.1 and obtained the pCR3.1-brLDHC4′-DNA vaccine. Immunization of female BALB/c mice with this DNA vaccine by multi-spot injections at muscles induced the generation of specific antibodies in the sera and the birthrate of treated mice decreased significantly. The antibodies in the sera from vaccinated mice could cause the agglutination of normal sperms[10], but the secretary IgA-type antibody in reproductive tracts was not detected in their study. For the reproduction activities, the local humoral immune response might have a more direct effect in lowing the rate of pregnancy. Shi et al. immunized mice by oral feeding and nasal instillation with LDHC4 DNA vaccine and the effect was also significant[9]. Mucosal immunization could induce both local mucosal and systemic immune responses. Therefore, there were more and more reports of successful immunization with DNA vaccine by mu― cosal immunization[14 20]. The two LDHC4 DNA vaccines constructed in our laboratory could also reduce fertility of mice by muli-spot injections at muscles (results not shown), although the effect was not as good as that of mucosal immunization. Immunocontraception has been regarded as an ideal method for population control.When antisperm antibody is present in the sera, seminal plasma, and/or cervical mucus of anyone of a couple without any other abnormalies, immunological infertility will result definitely. Therefore, we propose that contraception can possibly be achieved by inducing the generation of antisperm antibody in normal male and female individuals through vaccines. The evident contraceptive effect of the two LDHC4 DNA vaccines in male and female BALB/c mice showed in this study has given support to our pro-

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