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Metabolic analysis of glucose, pyruvate, and glutamine in dog oocytes collected from different sized follicles and matured in vitro

by Wesselowski, Sonya

Abstract (Summary)
Current in vitro maturation (IVM) systems for domestic dog oocytes are

inefficient, largely due to the species' unique reproductive physiology. The size

of donor follicle influences developmental competence of dog ovarian oocytes.

Specifically, oocytes from follicles > 2 mm in diameter complete in vitro nuclear

maturation at a higher rate than those from smaller follicles. The objective was to

determine the influences of follicular size, maturation time, and meiotic status on

oocyte metabolism. We hypothesized that metabolic patterns differed between

oocytes from small versus large follicles. Oocytes (n = 531) from adult ovaries

were collected and grouped based on follicular size (small, < 1 mm, n = 252;

medium, 1 to 2 mm, n = 231; and large, > 2 mm, n = 48). Oocytes were cultured

for 0, 24, or 48 hours at 38.5°C in 5% CO[subscript]2 in 80 [Mu]L of TCM 199 + 25[Mu]M [Beta]-

mercaptoethanol + 10 ng/ml epidermal growth factor + 0.25 mM pyruvate + 2.0

mM glutamine + 0.1% polyvinyl alcohol + 0.03 mg/ml streptomycin + 0.03 mg/ml

penicillin G sodium (IVM medium), assessed for metabolism and evaluated for

nuclear status. For metabolic assessments, oocytes were incubated for 3 h in 3

[Mu]l of IVM medium containing (1) 0.005 mM [0.064 [Mu]Ci/[Mu]l] D-53H-glucose

(glycolysis) + 1 mM D-614C [0.053 [Mu]Ci/[Mu]l] glucose (glucose oxidation) or (2) 0.001

mM [0.041 [Mu]Ci/[Mu]l] L-G-3H-glutamine + 1 mM [0.027 [Mu]Ci/[Mu}l] 1-14C pyruvate,

placed on the lid of a centrifuge tube containing 25 mM NaHCO3 and trapped

radioactivity was measured using a [Beta]-counter. Only oocytes at an appropriate

meiotic stage for each culture period (n = 380) were included in data analysis

(e.g., germinal vesicle stage at 24 and 48 h culture were excluded). Differences

in metabolism among groups were analyzed by ANOVA (main effects being

follicular class, culture interval, and meiotic status). Oocytes recovered from

large follicles metabolized significantly more pyruvate, glutamine, and glucose

(via glycolysis) than those from small ones (p < 0.05). Across meiotic stages and

follicular sizes, glycolytic rate was lowest in oocytes cultured for 24 hours (p <

0.05) compared to 0 or 48 hours. Metaphase II oocytes had a significantly higher

glycolytic rate than those at other meiotic stages (p < 0.05). At culture onset (0

h), oocytes from small follicles predominately used pyruvate (p < 0.05), while

oocytes from larger follicles (p < 0.05) predominately metabolized glucose. The

present data suggests that dog oocytes preferentially use glucose as an energy

substrate and that increasing glycolytic rate correlates with meiotic maturation.

In addition, oocytes collected from large follicles exhibit increased metabolic

capabilities that may be responsible for their increased developmental

competence during IVM.

Bibliographical Information:

Advisor:

School:Kansas State University

School Location:USA - Kansas

Source Type:Master's Thesis

Keywords:canine oocyte nuclear maturation energy substrate metabolism biology molecular 0307

ISBN:

Date of Publication:01/01/2008

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