326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

Abstract

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca²⁺) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.