title: Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes.
abstract: The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.
authors.affiliation: Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Chunmin
authors.initials: C
authors.lastname: Wang
authors.forename: Kiho
authors.initials: K
authors.lastname: Lee
authors.forename: Erzsébet
authors.initials: E
authors.lastname: Gajdócsi
authors.forename: Agnes Bali
authors.initials: AB
authors.lastname: Papp
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Machaty
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D005306:Fertilization; D005307:Fertilization in Vitro; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D013552:Swine
chemical_list: D015220:Calcium Channels; D002118:Calcium