379 EFFECT OF SUPPLEMENTARY INOSITOL 1,4,5-TRIPHOSPHATE INJECTION ON FERTILIZATION AND EMBRYO DEVELOPMENT AFTER BOVINE ICSI

Abstract

During fertilization in mammals, sperm membrane-bound phospholipase C zeta induces breakdown of ooplasmic membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2), which leads to the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 induces intracellular Ca²⁺ oscillations ([Ca²⁺]_i), which trigger inactivation of maturation-promoting factor (MPF) and mitogen-activated kinase (MAPK), resulting in decondensation of the sperm head, resumption of meiosis, extrusion of a second polar body, cortical granule exocytosis, formation of pronuclei (PN), and entry into the first cell cycle. In bovine ICSI, injection of a single spermatozoon into an oocyte does not consistently induce [Ca²⁺]_i oscillations, as was observed in IVF, and this may, at least in part, explain the low rates of fertilization and embryo development. Although IP3 has been used as a powerful activator for nuclear transferred zygotes or parthenogenetic oocytes, few studies have evaluated the effect of IP3 injection on normal fertilization and embryo development after ICSI. The objective of this study was to determine the effect of injecting IP3 during bovine ICSI on fertilization and embryo development in vitro. Chemically defined media (CDM) were used throughout (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585–596). The injection volume of IP3, which was dissolved in calcium- and magnesium-free PBS, was approximately 0.01 nL, and the total dose for injection along with a spermatozoon was about 250 µM, which was determined by the 2PN formation rate in preliminary experiments. Semen from 3 bulls was used to produce embryos in 5 replicates. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22–23 h under 5% CO₂ in air at 38.5°C, and oocytes with a first polar body were used for ICSI. Motile sperm from frozen–thawed semen were used for sperm injection, with or without IP3 in a 50-µL drop of GMOPS medium, with a piezo-driven pipet of 7–8 µm inner diameter. After ICSI, presumptive zygotes were cultured in CDM-1 for 3 days, and then in CDM-2 for 5 days at 39°C under 5% CO₂/5% O₂/90% N₂. Cleavage and blastocyst development were evaluated at the end of each culture period. Data were subjected to Fisher's exact test. Cleavage in control and IP3 groups was 36.4 and 50.0%, respectively (P < 0.05). Respective blastocyst rates per oocyte were 5.5 and 13.0% (P > 0.05). This study showed that injection of IP3 during the sperm injection process improved cleavage of bovine oocytes after ICSI.