Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization.
BACKGROUND
Granulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP3Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins.


METHODS
Ovarian tissue and granulosa cells were analyzed by multiphotonic and confocal microscopy to determine the intracellular presence of IP3R types 1, 2 and 3, RyR, thapsigargin-sensitive Ca2+-ATPase, and endomembranes. Cellular fractionation and Western blot assays were also used to further confirm the nuclear occurrence of the three IP3R isoforms. Free nuclear and cytosolic Ca2+ concentrations were measured using Fluo-4 AM by confocal microscopy.


RESULTS
By using antibodies and specific fluorophores, was shown that granulosa cells endomembranes contain three isoforms of IP3R, the RyR, and the thapsigargin-sensitive Ca2+-ATPase (SERCA). Interestingly, all these proteins were also detected in the nuclear envelope and in well-defined intranuclear structures. Microsomal membranes depicted characteristic bands of the 3 types of IP3R, but also variants of lower molecular weight. Analysis of nuclear membranes and nucleoplasmic fraction confirmed the nuclear localization of the IP3R types 1, 2 and 3. We demonstrated ATP-induced Ca2+ transients in the nuclear and cytoplasmic compartments. Remarkably, the inhibitory effect on ATP-induced Ca2+ mobilization of brefeldin A was more accentuated in the cytoplasm than in the nucleus.


CONCLUSIONS
These findings provide evidence that granulosa cells, including nuclei, express the Ca2+-handling proteins that allow Ca2+ mobilization. All three IP3R were also detected in ovarian slices, including the nuclei of granulosa cells, suggesting that these cells use the three IP3R in situ to achieve their physiological responses.
Díaz-Muñoz
Mauricio
M
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus UNAM-Juriquilla, Querétaro, QRO, México. mdiaz@inb.unam.mx
de la Rosa Santander
Patricia
P
Juárez-Espinosa
Anna Berenice
AB
Arellano
Rogelio O
RO
Morales-Tlalpan
Verónica
V
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010053:Ovary / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism
D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D020126:Brefeldin A; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium