Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine.
The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.
Vacková
I
I
Department of Biology of Reproduction, Research Institute of Animal Production, Prátelství 815, P.O. Box 1, 104 01 Prague, Uhríneves, Czech Republic.
Engelová
M
M
Marinov
I
I
Tománek
M
M
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D016193:G1 Phase; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects; D007150:Immunohistochemistry; D008898:Mimosine / Q000008:administration & dosage / Q000494:pharmacology*; D013552:Swine; D013997:Time Factors
D008898:Mimosine; D004815:Epidermal Growth Factor; D004247:DNA