title: Small-conductance calcium-activated K(+) channels 3 (SK3) regulate blastocyst hatching by control of intracellular calcium concentration.
abstract: BACKGROUND
  The present study was designed to investigate the expression of small-conductance calcium-activated K(+) channels 3 (SK3) in preimplantation embryos and to explore their role in the underlying mechanism of blastocyst hatching.
  
  
  METHODS
  Human preimplantation embryos were donated by patients who achieved successful pregnancy with in vitro fertilization. Mouse preimplantation embryos in different stages were collected and cultured with or without siRNA cell injection. The expression of SK3 was examined by RT-PCR, quantitative real-time PCR, western blot and immunofluorescence. Functional expression of SK3 was investigated using the patch-clamp technique. [Ca(2+)]i was measured by fluorescent imaging. Embryos were cultured in vitro to investigate the effect of SK3 knockdown or apamin, an SK3 inhibitor, on blastocyst hatching and F-actin formation.
  
  
  RESULTS
  In human blastocysts, the level of SK3 expression was significantly lower in blastocysts that failed to hatch than in blastocysts that hatched successfully. In mouse embryos, SK3 mRNA and protein were not found in zygotes, but were detected from the 2-cell stage onward, with the highest levels observed in blastocysts. SK3 was predominately located in the trophectoderm cell membrane of expanded blastocysts. SK3 knockdown in trophectoderm cells not only suppressed the SK3 current, but also reduced [Ca(2+)]i elevation and membrane potential hyperpolarization induced by thapsigargin. Although the formation of expanded blastocysts was not affected, blastocyst hatching and F-actin formation were significantly inhibited after SK3 knockdown in trophectoderm cells.
  
  
  CONCLUSIONS
  SK3-mediated [Ca(2+)]i elevation and membrane potential hyperpolarization in trophectoderm cells are important for blastocyst hatching, and defects in SK3 expression may contribute to infertility.
authors.affiliation: Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China.
authors.forename: Yong-Chao
authors.initials: YC
authors.lastname: Lu
authors.forename: Guo-Lian
authors.initials: GL
authors.lastname: Ding
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Shuai
authors.initials: S
authors.lastname: Shi
authors.forename: Run-Ju
authors.initials: RJ
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Jie-Xue
authors.initials: JX
authors.lastname: Pan
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Sheng
authors.forename: He-Feng
authors.initials: HF
authors.lastname: Huang
mesh_terms: D000818:Animals; D001030:Apamin / Q000494:pharmacology; D001755:Blastocyst / Q000378:metabolism* / Q000502:physiology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D046149:Embryo Culture Techniques; D005455:Fluorescent Antibody Technique; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D008813:Mice, Inbred ICR; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*
chemical_list: C406328:KCNN3 protein, human; D051640:Small-Conductance Calcium-Activated Potassium Channels; D001030:Apamin; D002118:Calcium