Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes.
The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the G(s)-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.
Mehlmann
Lisa M
LM
Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA. lmehlman@neuron.uchc.edu
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D008540:Meiosis / Q000502:physiology*; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006080:Ovarian Follicle / Q000166:cytology / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics
C494613:GPR3 protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP