Involvement of CFTR in oviductal HCO3- secretion and its effect on soluble adenylate cyclase-dependent early embryo development.
BACKGROUND
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in electrolyte and fluid transport in epithelial cells, and women with cystic fibrosis (CF), caused by CFTR gene mutations, have a higher incidence of infertility.


METHODS
In the present study, we investigated the expression of CFTR in porcine oviduct and its functional role in oviductal HCO(3)(-) secretion and embryo development with RT-PCR, western blot, patch-clamp, short-circuit current (I(sc)), pH measurement and embryo culture.


RESULTS
RT-PCR and western blot analysis showed the expression of CFTR mRNA and protein in the oviduct with its localization demonstrated by immunohistochemistry. The whole-cell patch-clamp recording revealed a forskolin (FSK)-activated current with electrophysiological and pharmacological characteristics of CFTR. The I(sc) measurement showed that FSK-stimulated an increase in the I(sc), which could be significantly reduced by CFTR inhibitor or removal of both CO(2) and HCO(3)(-). pH measurement showed a FSK stimulated alkalization at the apical surface, which could be inhibited by CFTR inhibitor, indicating CFTR-mediated HCO(3)(-) secretion. Mouse embryo development from 2-cell to morula or blastocyst stage was significantly inhibited in the absence of HCO(3)(-) or when co-cultured with HCO(3)(-) secretion-deficient CFTR mutant cells as compared with the wild-type. RT-PCR, western blot and immunostaining showed the expression of soluble adenylate cyclase (sAC), the known HCO(3)(-) sensor, in embryos. Treatment with its inhibitors, 2-hydroxyestradiol and KH7, prevented the HCO(3)(-) dependent embryo development.


CONCLUSIONS
The present results suggest that CFTR-mediated oviductal HCO(3)(-) secretion may be vital for sAC-dependent early embryo development, a defect of which may contribute to the reduced fertility seen in women with CF.
Chen
Min Hui
MH
School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Chen
Hui
H
Zhou
Zhen
Z
Ruan
Ye Chun
YC
Wong
Hau Yan
HY
Lu
Yong Chao
YC
Guo
Jing Hui
JH
Chung
Yiu Wa
YW
Huang
Ping Bo
PB
Huang
He Feng
HF
Zhou
Wen Liang
WL
Chan
Hsiao Chang
HC
D000262:Adenylyl Cyclases / Q000502:physiology*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D015153:Blotting, Western; D005576:Colforsin / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004553:Electric Conductivity; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development; D005187:Fallopian Tubes / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D051379:Mice; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001639:Bicarbonates; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000262:Adenylyl Cyclases