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Jian-Jun Chang, Jing-Pian Peng, Ying Yang, Jing-Ling Wang, and Li Xu

Partial cDNA sequence coding for Microtus brandti radde (Brandt’s vole) testes-specific lactate dehydrogenase (brLDH-C4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR). By inserting the product into the eukaryotic expression vector pCR3.1, pCR3.1-brLDH-C4′ was obtained as the prototype of contraceptive DNA vaccine. Immunization with pCR3.1-brLDH-C4′ in BALB/c mice generated antibodies specific to purified brLDH-C4′ and native mouse LDH-C4 protein. The birth rate of the pCR3.1-brLDH-C4′ immunized mice was found to be decreased significantly (80% lower than that of those immunized with pCR3.1). Functions of the elicited antibodies in sera from pCR3.1-brLDH-C4′ inoculated mice were further explored. The results indicated that the antibodies from the mice injected with pCR3.1-brLDH-C4′ could cause the agglutination of normal sperm suspension, while the ovarian structure and the development of ovarian follicles of these mice were not impaired, which gives a possible explanation for the immunocontraceptive effects of the pCR3.1-brLDH-C4′ DNA vaccine.

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Jinxiu Dong, Siqi Liu, Ziming Wang, Kai Zheng, Mengli Yang, Jianjun Liu, and Liuwang Nie

The specificity of sperm–egg recognition is crucial to species independence, and two proteins (Izumo1 and JUNO) are essential for gamete adhesion/fusion in mammals. However, hybridization, which is very common in turtles, also requires specific recognition of sperm–egg binding proteins. In this study, we discovered that natural selection plays an important role in the codon usage bias of Tu-Izumo1 and Tu-JUNO. Positively selected sites and co-evolutionary analyses between Tu-Izumo1 and Tu-JUNO have been previously reported, and we confirm these results in a larger analysis containing 25 turtle species. We also showed that Tu-JUNO is expressed on the oocyte surface and that Tu-Izumo1 and Tu-JUNO interact with each other directly in different species hybridization combinations. Co-immunization assays revealed that this interaction is evolutionarily conserved in turtles. The results of avidity-based extracellular interaction screening between Tu-Izumo1 and Tu-JUNO for sperm–oocyte binding pairs (both within and across species) likely suggest that the interaction force between Izumo1 and JUNO has a certain correlation in whether the turtles can hybridize. Our results lay a theoretical foundation for the subsequent development of techniques to detect whether different turtle species can interbreed, which would provide the molecular basis for breeding management and species protection of turtles.