title: Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.
abstract: BACKGROUND
  Oocyte activation is a crucial step that comprises the release of the oocyte from meiotic arrest, pronuclear formation and subsequent embryo development. Oocytes are activated by repetitive increases in the intracellular concentration of free Ca(2+), [Ca(2+)](i) oscillations, which are triggered during fertilization by the introduction of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca(2+)](i) oscillations or oocyte activation. We first purified recombinant human PLCZ1 (hPLCZ1) protein and evaluated its [Ca(2+)](i) oscillation activity in mouse and human oocytes with the view to investigate its application in the clinic for assisted oocytes activation in lieu of chemical agents.
  
  
  METHODS
  Recombinant hPLCZ1 was synthesized using the Escherichia coli system, and subjected to immunoblot analysis with anti-PLCZ1 and anti-His tag antibodies. [Ca(2+)](i) oscillations by microinjection of recombinant hPLCZ1 into mouse or human oocytes were examined by [Ca(2+)](i) monitoring with Fluo 4. Ploidy of the oocytes with recombinant hPLCZ1 injection was confirmed with fluorescence in situ hybridization.
  
  
  RESULTS
  A band of 68 kDa on recombinant protein was detected with both antibodies. Injection of recombinant hPLCZ1 induced [Ca(2+)](i) oscillations in a dose-dependent manner in both mouse and human oocytes. These oscillations, which closely resembled those initiated by the sperm upon fertilization, triggered activation and cleavage in oocytes of both species, although further development of the mice embryos was low. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. Microinjection of recombinant hPLCZ1 into ICSI-failed human oocytes rescued fertilization failure in five of eight attempts.
  
  
  CONCLUSIONS
  Repeated [Ca(2+)](i) oscillations and oocyte activation were induced in mouse and human oocytes by microinjection of recombinant hPLCZ1 synthesized in E. Coli. Injection of recombinant protein could thus provide a biological solution for inducing artificial activation of oocytes.
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, CHA University, Gangnam-gu, Seoul, Republic of Korea.
authors.forename: Sook-Young
authors.initials: SY
authors.lastname: Yoon
authors.forename: Jin Hee
authors.initials: JH
authors.lastname: Eum
authors.forename: Jeoung Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Hoi Chang
authors.initials: HC
authors.lastname: Lee
authors.forename: You Shin
authors.initials: YS
authors.lastname: Kim
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Han
authors.forename: Hyung Jae
authors.initials: HJ
authors.lastname: Won
authors.forename: Sang Hee
authors.initials: SH
authors.lastname: Park
authors.forename: Sung Han
authors.initials: SH
authors.lastname: Shim
authors.forename: Woo Sik
authors.initials: WS
authors.lastname: Lee
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Fissore
authors.forename: Dong Ryul
authors.initials: DR
authors.lastname: Lee
authors.forename: Tae Ki
authors.initials: TK
authors.lastname: Yoon
mesh_terms: D000328:Adult; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D008297:Male; D051379:Mice; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology*; D054801:Phosphoinositide Phospholipase C / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology*
chemical_list: D011994:Recombinant Proteins; C499235:PLCZ1 protein, human; D054801:Phosphoinositide Phospholipase C; D002118:Calcium