Amelia Verhoeven 
Marquette University
Milwaukee, WI 
Faculty Mentor: Dr. Stephen Downs 

Previous studies have indicated that altering the composition of media used to culture oocytes has a significant effect on meiotic regulation. I have examined the possibility that the different amounts of glutamine (gln) in minimal essential medium (MEM) and M16 medium modify the ability of the meiotic inhibitors, dbcAMP and hypoxanthine, to maintain mouse oocytes in meiotic arrest in vitro by carrying out extensive gln dose response experiments. M16 is a simple buffered salt solution while MEM is much more complex, containing additional vitamins and amino acids. The amino acid, gln a common supplement to many media, is present in MEM but is absent in M16 medium. Denuded oocytes (DO) and cumulus cell-enclosed oocytes (CEO) from hormonally primed immature mice were cultured 17-18h at 37šC in medium containing either 300µM dbcAMP or 4mM hypoxanthine plus increasing concentrations of glutamine. Oocytes were also cultured in the presence or absence of glucose, a hexose that can profoundly affect meiosis. Maturation was assessed by examining oocytes at the end of culture for germinal vesicle breakdown (GVB). In hypoxanthine-supplemented M16, 50-60% of DO underwent GVB in control Gln-free medium, and increasing concentrations of the amino acid had no effect. On the other hand, nearly all CEO cultured in control conditions resumed maturation (92-100%), but GVB was profoundly suppressed by gln in dose-dependent fashion (22-32% GVB). Glucose had little effect in hypoxanthine-supplemented M16. In dbcAMP containing M16 medium, far fewer DO underwent GVB, in the absence of gln (0-6%), and no significant effect of gln was observed. Again, the majority of CEO resumed maturation under gln-free conditions, and the suppressive effect of glutamine was even more dramatic (only 2-6% GVB at 2mM). Inhibition of maturation in CEO was consistently greater in the absence of glucose. I have initiated similar experiments in MEM, and while results are preliminary, a similar pattern is emerging. However, the differences in maturation between DO and CEO are much more compressed. This suggests that other amino acids and factors in this more complex media may also play a role in meiotic maturation. My results support a role for glutamine in contributing to meiotic suppression due to augmented de novo purine synthesis, since purines contribute to a decrease in meiotic maturation. There may also be an interaction between glutamine and glucose under certain culture conditions, as glucose can provide substrate for purine synthesis after transit through the pentose phosphate pathway. My evidence indicates that the principal site for this metabolism of glutamine and glucose is the cumulus oophorus, the somatic cells that surround the oocyte and provide regulatory input. In future experiments I will test whether the gap juctional pathway that couples these two cell types is required for transmission of the inhibitory signal.