Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

Sperm-specific post-acrosomal WW-domain binding protein (PAWP) does not cause Ca2+ release in mouse oocytes.

Mature mammalian oocytes undergo a prolonged series of cytoplasmic calcium (Ca(2+)) oscillations at fertilization that are the cause of oocyte activation. The Ca(2+) oscillations in mammalian oocytes are driven via inositol 1,4,5-trisphosphate (IP3) generation. Microinjection of the sperm-derived phospholipase C-zeta (PLCζ), which generates IP3, causes the same pattern of Ca(2+) oscillations as observed at mammalian fertilization and it is thought to be the physiological agent that triggers oocyte activation. However, another sperm-specific protein, 'post-acrosomal WW-domain binding protein' (PAWP), has also been reported to elicit activation when injected into mammalian oocytes, and to produce a Ca(2+) increase in frog oocytes. Here we have investigated whether PAWP can induce fertilization-like Ca(2+) oscillations in mouse oocytes. Recombinant mouse PAWP protein was found to be unable to hydrolyse phosphatidylinositol 4,5-bisphosphate in vitro and did not cause any detectable Ca(2+) release when microinjected into mouse oocytes. Microinjection with cRNA encoding either the untagged PAWP, or yellow fluorescent protein (YFP)-PAWP, or luciferase-PAWP fusion proteins all failed to trigger Ca(2+) increases in mouse oocytes. The lack of response in mouse oocytes was despite PAWP being robustly expressed at similar or higher concentrations than PLCζ, which successfully initiated Ca(2+) oscillations in every parallel control experiment. These data suggest that sperm-derived PAWP is not involved in triggering Ca(2+) oscillations at fertilization in mammalian oocytes.