Ovarian Response And Recovery To Repeated Ovarian Stimulations And Shank3 Gene Mutant In Embryos In Cynomolgus Monkeys

Autism and autism spectrum disorders (ASDs) are neurodevelopmental disorders diagnosed based on a triad of criteria:deficits in communication, impaired social interaction, and repetitive or restricted interests and behaviors. Mutations in shank3 have been reported also in schizophrenia, suggesting that shank3 deficiency may contribute to both neurobehavioral disorders. In this study, explore the new gene editing technique-CRISPR/Cas9 combined with efficient cynomolgus monkey assisted reproductive technologies generate the genetic mutation-related autism gene modification of cynomolgus monkeys.This research investigated the effects of repeated ovarian stimulation using recombinant human follicle-stimulating hormone (rhFSH) for 2 years on cynomolgus monkeys. A total of 128 adult female cynomolgus macaques were given with 1.375 μg of rhFSH twice daily for 8 d, followed by 83.3μg of recombinant human chorionic gonadotropin. After the first ovarian stimulation, ovarian responses and oocyte recovery did not differ among the four seasons throughout the year. The duration of the third and fourth menstruation cycle (MC) was consistent with that of the MC before ovarian stimulation but differed from that of the first and second MC. Thus, monkeys were restimulated at intervals of two, three, and four MCs. The proportion of responders and the number of oocytes retrieved after repeated stimulation were similar to those of the first cycle. The number of oocytes produced in response to the first (39.8±24.3), second (33.8±11.9), third (35.4±26.9), and fourth (37.4±20.4) stimulation did not differ between the cycles. However, the outcome of the fifth repeated stimulation significantly decreased compared with that of the four previous stimulation cycles. The poor outcome of the first stimulation indicated the low efficiency of repeated ovarian stimulation. In conclusion, cynomolgus monkeys can respond to finite repeated ovarian stimulation for an entire year. This study provided a basis for ART application in biotechnology and biomedicine research on human diseases.Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, A total of 3 embryos with normal development to morula or blastocyst stages were collected and examined for the presence of site-specific genome modification analysis by PCR, T7EN1 cleavage assay, and sequencing. It demonstrated that the CRISPR/Cas9 system functioned well in genetically mutation of shank3 in cynomolgus monkey’s embryos, provide availability for creating shank3 knockout cynomolgus monkeys.