Y Yu1, C M Saunders, F A Lai, K Swann. 1. Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK.
Abstract
BACKGROUND: A sperm-specific phospholipase C zeta (PLCzeta) has been shown to trigger Ca(2+) oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLCzeta into oocytes, but it was unclear how much PLCzeta protein is required for development. Here we have injected cRNA encoding luciferase-tagged human PLCzeta (hPLCzeta-luc) into mouse oocytes and established the relationship between hPLCzeta-luc expression, Ca(2+) oscillations and development. METHODS: Mouse oocytes were injected with hPLCzeta-luc cRNA and a fluorescent Ca(2+)dye to monitor hPLCzeta-luc expression and Ca(2+) oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLCzeta-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr(2+). RESULTS: Repetitive Ca(2+) oscillations and oocyte activation were triggered by hPLCzeta over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLCzeta-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLCzeta expression. CONCLUSIONS: Human PLCzeta can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLCzeta-luc protein expression levels.
BACKGROUND: A sperm-specific phospholipase C zeta (PLCzeta) has been shown to trigger Ca(2+) oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLCzeta into oocytes, but it was unclear how much PLCzeta protein is required for development. Here we have injected cRNA encoding luciferase-tagged humanPLCzeta (hPLCzeta-luc) into mouse oocytes and established the relationship between hPLCzeta-luc expression, Ca(2+) oscillations and development. METHODS:Mouse oocytes were injected with hPLCzeta-luc cRNA and a fluorescent Ca(2+)dye to monitor hPLCzeta-luc expression and Ca(2+) oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLCzeta-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr(2+). RESULTS: Repetitive Ca(2+) oscillations and oocyte activation were triggered by hPLCzeta over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLCzeta-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLCzeta expression. CONCLUSIONS:HumanPLCzeta can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLCzeta-luc protein expression levels.
Authors: Michail Nomikos; Yuansong Yu; Khalil Elgmati; Maria Theodoridou; Karen Campbell; Vyronia Vassilakopoulou; Christos Zikos; Evangelia Livaniou; Nazar Amso; George Nounesis; Karl Swann; F Anthony Lai Journal: Fertil Steril Date: 2012-09-21 Impact factor: 7.329
Authors: Yuansong Yu; Michail Nomikos; Maria Theodoridou; George Nounesis; F Anthony Lai; Karl Swann Journal: Mol Biol Cell Date: 2011-11-23 Impact factor: 4.138
Authors: Karl Swann; Shane Windsor; Karen Campbell; Khalil Elgmati; Michail Nomikos; Magdalena Zernicka-Goetz; Nazar Amso; F Anthony Lai; Adrian Thomas; Christopher Graham Journal: Fertil Steril Date: 2012-01-02 Impact factor: 7.329