Effect Of PCXN2-mIZUMO On The Fertility Of C57BL/6 Mice

[BACKGROUND & AIM] It is a very interesting project for scientists to explore a method ofconvenience and availability for human family planning and pest animal birth control. Theirwork focuses on application of immunocontraception vaccine in many countries, especiallydeveloping ones. In the developed world, steroid contraceptives for humans are both widelyused and efficacious. Elsewhere they are too costly. The development of less expensive methodsis considered necessary. One such method is immunocontraception, i.e. the vaccination againstsperm, egg, or reproductive hormones to prevent either fertilization or the production of gametes.With the development of immunology and gene engineering, gene immunization, also known asDNA or polynucleotide immunization, is becoming a novel strategy for vaccine development inwhich plasmid DNA encoding either individual or a collection of antigens is directlyadministered to a host. Such immunization leads to host expression of the delivered foreign gene,resulting in the induction of a specific immune response against the in vivo produced antigen. InMarch 2005 Naokazu Inoue et al firstly reported a novel protein named Izumo which belongs toimmunoglobulin superfamily. Izumo only detected on the inner acrosomal membrane is a testis(sperm)-specific protein. To address the physiological role of Izumo in vivo they generatedIzumo-deficient mice by homologous recombination. Izumo was confirmed to be the keymolecule which impact on the sperm-egg fusion in the process of fertilization. Due to anabsence of Izumo, Izumo-deficient mice were healthy but males were sterile. In our study, weare intended to elucidate the expression of IZUMO gene in the muscle and the effect on thefertility of C57BL/6 mouse by means of gene immunization with pCXN2-mIZUMO plasmid.The results will provide useful experimental data for the immunocontraceptive research. [MATERIALS AND METHODS]Materials:①eukaryotic expressed plasmid pCXN2-mIZUMO;②control plasmid pCXN2as the control;③male and female C57BL/6 mice, 6-8weeks of age;④anti-serum was collectedfrom the immunized C57BL/6 mice;⑤spermatozoa were collected from the epididymides ofmature C57BL/6 male mice;⑥oocytes were collected from the oviduct ampulla of matureC57BL/6 female mice by superovulationMethod:①pCXN2 plasmid was constructed and identified by enzyme cut.②Extractionand purification of plasmid DNAs of pCXN2-mIZUMO and pCXN2 were performed.③Pretreatment of C57BL/6 mice before inoculation: all experimental C57BL/6 mice receivedvaccine i.m. by multispot injection in the leg muscles along with 100μL of 0.25％bupivacaine-HCL. Three days later, pCXN2-mIZUMO (100μg/mouse) was inoculated in thesame fashion, and the mice received the second and third injection every two weeks.④Immninization: all the animals were divided into two groups randomly and were immunizedwith pCXN2-mIZUMO and pCXN2 plasmids respectively.⑤Serum preparation: the bloodsample was taken from the tail vein of the animals for serum preparation before everyimmunization and two weeks after the third immunization.⑥RT—PCR: total RNA was isolatedfrom intramuscular injection sites six week postinoculation and RT-PCR was performed usingmouse IZUMO upstream and downstream primers to examine the level of in vivo expression,and then its product was confirmed by sequencing.⑦ELISA assay: sera titers of antibodiesagainst mIzumo were measured using ELISA.⑧Fertilization in vitro : detect the effect of theanti-serum on fertilization rate in vitro.⑨Antifertility affect: two week after the lastimmunization, all the animals were introduced to mate with normal mice at a female/male ratioof 2:1 to determine the effect of inoculation on the fertility of C57BL/6 mice. The presence ofvaginal plug meant successful mating on the subsequent day. The newborns of experimentalgroups were compared with the controls.[RESULTS]①enzyme cut: pCXN2-mIZUMO and pCXN2 plasmids were identified usingEcoR1 digestion and double digestion of HindⅢand XbaⅠ, respectively. The special positivefragments confirmed that the pCXN2-mIZUMO plasmid is correct and the control plasmid isconstructed successfully;②RT-PCR: The product amplified by RT-PCR is a 468 bp fragment,which was derived from mIZUMO mRNA confirmed by sequencing and BLAST analysis. ③ELISA assay: the ELISA result shows that specific antibodies were detected 2 week afterthe first immunization. The mice immunized by pCXN2-mlZUMO plasmid maintained arelatively high level of antibodies after two times enhanced immunization.④the fertilizationrate in vitro was decreased in the presence of anti-serum.⑤the analysis of the litter size afterdelivery reveals the fertility of the female mice immtmized with pCXN2-mlZUMO plasmid wasimpaired compared with the female control group.[CONCLUSION]①The plasmid of pCXN2 was successfully constructed.②ThepCXN2-mIZUMO plasmid is able to express its function in the muscle cell in vivo.③Thespecific response of the immune system of C57BL/6 mice to mlzumo antigen was induced byimmunization with pCXN2-mIZUMO plasmid.④Anti-serum from mice immunized withpCXN2-mIZUMO plasmid reduced the in vitro fertilization rate.⑤The fertility of female miceinoculated with pCXN2-mIZUMO plasmid was impaired.⑥The results provide the newexperimental evidences for Izumo gene as a targeted-antigen of immunocontraception.