Determination of glucose metabolism and TCA cycle activity of early antral and late antral feline cat follicles employing [13C6]glucose and mass spectrometry

Abstract Text:

Improving in vitro culture systems for follicles and oocytes requires knowledge of their metabolism. Thus, our objectives were to establish glycolytic and TCA cycle activity in feline follicles at two developmental stages, and after 13 d of in vitro culture. Paired feline ovaries from sexually mature cats (≥1 yr) were acquired from a clinic. Morphologically healthy early (<0.5 mm o.d.) and late (>2 mm o.d.) antral follicles with a visible antrum were isolated. Early (n=10 per cat, n=9 cats) and late (n=1 per cat, n=9 cats) antral follicles were placed into individual wells with culture media (0.5 mL, DMEM containing glutamine and pyruvate plus a 50:50 mix of unlabeled and [¹³C₆]glucose) and incubated for 24 h (5% CO₂) at 38.5°C. To determine whether in vitro culture of early antral follicles leads to acquisition of a late antral metabolism, a group of early antral follicles (n=10 per cat, n=9 cats) were encapsulated in 0.5% alginate hydrogel and cultured individually for 13 d with media containing [¹³C₆]glucose the last 24 h. Following incubation, in vivo derived and in vitro cultured early antral follicles were pooled separately by cat for analysis, while late antral follicles were analyzed individually. Metabolites from follicles were extracted, and ¹³C-isotopomer enrichments of metabolites determined by gas chromatography-mass spectrometry. The TCA cycle intermediate equilibrium partners alanine (pyruvate) and glutamate (α-ketoglutarate) were monitored for calculation of glycolytic and TCA cycle fluxes. Data were analyzed as a mixed model ANOVA with cat and cat age as blocking factors. A greater proportion of pyruvate flux derived from glucose metabolism in late (56%) compared to early (33%) antral follicles, indicating higher rates of glycolysis by late antral follicles. For both early and late antral follicles, only 2% of acetyl-CoA flux derived from glucose, indicating that TCA cycle oxidative metabolism relies on other substrates. Early antral follicles cultured in vitro for 13 d metabolized glucose and had TCA cycle flux activity similar to that of the early antral follicles cultured for 1 d. Thus, in vitro culture of early antral follicles for 13 d did not result in these follicles acquiring a similar metabolism as late antral follicles. The current research demonstrates a metabolic shift between early and late antral follicles derived in vivo, as well as a limited ability of early antral follicles to acquire a late antral metabolism after in vitro culture for 13 d.

Keywords:

feline, follicle, metabolism