93 Efficient introduction of green fluorescent protein-9R, a protein with cell-penetrating peptides, into oocytes using intracytoplasmic sperm injection

The introduction of functional molecules such as oligonucleotides, nucleic acids, peptides, and antibodies into gametes has been beneficial not only for research purposes but is also expected to provide a treatment modality for failure of assisted reproductive technology (ART). The use of cell-penetrating peptides (CPPs) has been established as a method to introduce proteins and nucleic acids, which cannot normally pass through the cell membrane, into cells. Cell-penetrating peptides are short sequences of amino acids that facilitate the penetration of conjugated cargoes across mammalian cell membranes. To date, reports on the introduction of proteins via CPP into germ cells have been limited, and the method has not achieved success in preimplantation embryos. Simple polyarginine peptides have been known to induce higher cell penetration rates among CPPs. In this study, we examined whether green fluorescent protein-conjugated nine arginines (GFP-9R) can be effectively introduced into mouse oocytes and spermatozoa. Oocytes were collected from oviducts of 8- to 10-week-old female ICR mice after administration of equine chorionic gonadotropin and human chorionic gonadotropin. Spermatozoa were collected from epididymides of 8- to 10-week-old male mice. Some oocytes were used to perform IVF and subsequently cultured. These oocytes, spermatozoa, and embryos were cultured with GFP-9R or GFP for 30min to 1h and were then fixed with 4% paraformaldehyde and observed with a confocal laser microscope to check the introduction of GFP-9R into intact germ cells. In addition, we also attempted to introduce GFP-9R into oocytes by performing IVF or intracytoplasmic sperm injection (ICSI) using GFP-9R-introduced spermatozoa. In the case of ICSI, the tails of sperm were removed by sonication. After performing IVF or ICSI, embryos were cultured and observed with a fluorescence microscope. As a result, the GFP fluorescence signal was not detected in the ooplasm incubated with GFP-9R but was detected in the perivitelline space, the zona pellucida, and the collapsed first polar body. No GFP fluorescence signal was detected in the oocytes incubated with GFP. On the other hand, GFP fluorescence was detected in some spermatozoa that were incubated with GFP-9R (GFP-9R 43.3% (26 out of 60) but not with GFP alone 0% (0 out of 67)). However, when IVF was performed using these spermatozoa, the fluorescence signal was not detected in spermatozoa attached to the zona pellucida, but a strong signal was detected in dead spermatozoa, which are not involved in fertilization. Furthermore, we found that efficiency of introduction of GFP-9R into sperm heads increased after sonication (GFP-9R 93.1% (27 of 29) vs. GFP 0% (0 of 23)). Finally, we performed ICSI with GFP-9R-introduced sperm heads which were sonicated. These ICSI embryos developed to blastocysts normally (GFP-9R 46.7% (7 of 15) vs. non-GFP-9R 50.0% (8 of 16)). In this study, we demonstrated efficient CPP protein delivery of sonicated sperm heads into oocytes via ICSI in mice, even though the CPPs did not enter effectively into intact germ cells directly.