EFFECT OF MICROINJECTION OF MESSENGER RNA OF PLCZ INTO ZYGOTE IN MOUSE EMBRYO DEVELOPMENT IN VITRO

Abstract

Sperm are special cells with minimal cytoplasm to ensure motility and protect DNA during sperm formation and migration to the egg. Therefore, it is known that a series of gene expression mechanisms such as DNA replication-RNA transcription-translation do not occur in mature sperm. And it is known that most of the cytoplasmic components required for fertilization and embryonic development are derived from the egg, and sperm have only paternal gene transfer function. However, spermatozoa RNAs are also delivered to the oocyte contributing to early embryo development. Mature spermatozoa, in spite of being transcriptionally and translationally inactive, possess several types of RNAs that accumulate in the sperm cell. The study of sperm RNAs has been challenging because of the difficulty associated with sperm RNA isolation. The sperm-specific phospholipase C zeta (PLCZ) is identified as candidate sperm-borne oocyte-activating factor that triggers a cytoplasmic Ca2+ oscillations during fertilization. Such oscillations are known to be important for optimal embryonic development. In this study, we investigated the purifying of messenger RNA of PLCZ from mouse sperm, and produced cDNA of PLCZ. Also we investigated the role of cDNA of PLCZ in embryonic development. cDNA library originated from mouse testis. cDNA was produced by in vitro transcription with or without GFP tagging. Total mRNA from mouse testes and sperm were isolated by Dynabeads™ mRNA Purification Kit. To evaluate the [Ca2+]i oscillatory activity, we performed microinjection of cDNA into mouse mature eggs and [Ca2+]i monitoring. To investigate the effect of microinjection of cDNA of PLCZ on mouse embryo development, we produces artificially activated zygote with 10 mM strontium, and examined microinjection of cDNA PLCZ into zygote stage. Further embryonic development were observed blastocyst formation for 5 days culture in KSOM. mRNA of PLCZ were purified from mouse sperm and testes, but not in mouse granulosa cells. Microinjection of cDNA of PLCZ into zygote stage induced higher blastocyst formation than control eggs significantly (P<0.02). The ability of PLCZ to increase [Ca2+]i appears to have influenced intracellular signaling and induced embryogenesis in vitro. SUPPORT: This research was supported by a grant from Republic of Korea, NRF-2018R1D1A1B07043250.