Delivery of Morpholino Antisense Oligos into Mouse and Monkey Ovarian Follicles

To test the feasibility of in vivo gene knockdown in the mammalian ovary, we performed in vitro delivery of Morpholino antisense oligos into intact secondary and antral follicles in mice and rhesus macaques (Macaca mulatta). The Morpholino antisense oligo is a powerful tool for modification of gene expression. Usually 25 bp in length, Morpholinos are synthesized nucleic acid analogs that can downregulate target proteins by blocking translation or modifying pre-mRNA splicing. Endo-Porter is a novel peptide that delivers substances into the cytosol of cells by an endocytosis-mediated process. Through co-incubation of intact mouse or monkey ovarian follicles with a fluorescein-labeled Morpholino and Endo-Porter reagent, we observed an even distribution of green fluorescence within the entire follicles derived from both species. There is no noticeable difference in the intensity of green fluorescence between the cumulus and mural granulosa cells. However, no green fluorescence was visible in the oocytes. The fluorescence within the follicles lasted for approximately 72 hours. Incubation of ovarian follicles with fluorescein-labeled Morpholino or Endo-Porter reagent alone showed no green fluorescence in the follicles. Mammalian follicular fluid contains many negatively charged molecules that can easily block the access of cationized delivery reagents to follicular cells. Our data show that Endo-Porter is an effective delivery reagent for non-anionized molecules, such as Morpholinos and antibodies, in cultured intact mouse and monkey follicles. While this approach is very useful for specific gene/protein knockdowns in granulosa cells within an ovarian follicle in mice and nonhuman primates, the Endo-Porter reagent is not applicable to target oocyte-expressed genes. Experiments designed to determine the efficacy of local and systematic delivery of vivo-Morpholino in the ovary is underway.