Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride.
Three experiments determined first, the effect of increasing ammonium chloride (NH(4)Cl) concentrations on the growth and metabolism of bovine granulosa cells isolated from small and medium-sized bovine ovarian follicles; secondly, whether the changes in granulosa cell growth and metabolism induced by NH(4)Cl were reversible; and thirdly, whether granulosa cells, previously conditioned with NH(4)Cl, were able to support maturation of oocytes in vitro. In Experiment 1, using a 2 (follicle size class) x 5 (NH(4)Cl concentration) factorial design, granulosa cells from small or medium-sized ovarian follicles were incubated for 96 h with 0, 0.2, 0.4, 0.8 or 1.6 micromol NH(4)Cl/ml. Experiment 2 used a split plot factorial design where granulosa cells were incubated for 96 h in the presence or absence of 1 micromol/ml NH(4)Cl and then incubated in the absence or presence of 1 micromol/ml NH(4)Cl for a further 48 h. Finally in Experiment 3, ovine oocytes were matured on layers of bovine granulosa cells which had not been conditioned with NH(4)Cl or conditioned with 0.5 or 1.0 micromol/ml NH(4)Cl and development of embryos to the blastocyst stage followed and blastocyst quality assessed. In Experiment 1, incubation of granulosa cells in increasing concentrations of NH(4)Cl reduced cell growth, increased cell protein concentrations and increased the amounts of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) oxidised and oestradiol and progesterone produced per 10(5) cells. Cells from medium-sized follicles were more sensitive to NH(4)Cl concentration and oxidised more MTT and produced less progesterone at high NH(4)Cl concentrations than cells from small-sized follicles. When, in Experiment 2, NH(4)Cl was removed from cell culture after 96 h incubation, cells previously exposed to NH(4)Cl grew at a slower rate during the subsequent 48 h, contained more cellular protein, oxidised more MTT and produced more oestradiol and progesterone than cells not previously exposed to NH(4)Cl. Maturation of ovine oocytes in coculture with bovine granulosa cells not exposed to NH(4)Cl (Experiment 3) increased egg cleavage rate and the proportion of cleaved eggs which developed to the blastocyst stage. Conditioning of granulosa cells with NH(4)Cl supported egg cleavage and development to the blastocyst stage at rates similar to those observed in the absence of granulosa cells. In conclusion, these experiments showed that the in vitro growth and metabolism of granulosa cells were altered by concentrations of NH(4)Cl similar to ammonium ion concentrations measured in follicular fluid and that these effects were not immediately reversible. Furthermore, the ability of granulosa cells conditioned with NH(4)Cl to support in vitro maturation of oocytes was impaired.
Sustainable Livestock Systems, Scottish Agricultural College, Craibstone Estate, Aberdeen AB21 9YA, UK. jrooke@ab.sac.ac.uk
J A
JA
Rooke
M
M
Ewen
K
K
Mackie
M E
ME
Staines
T G
TG
McEvoy
K D
KD
Sinclair
D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D046508:Culture Techniques; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000502:physiology; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D006107:Granulosa Cells / Q000187:drug effects* / Q000502:physiology*; D009865:Oocytes / Q000254:growth & development*; D010084:Oxidation-Reduction; D011374:Progesterone / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles
D017077:Culture Media, Conditioned; D011506:Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D000643:Ammonium Chloride; D011374:Progesterone; D004958:Estradiol; D019289:Pyruvic Acid; C022616:thiazolyl blue