Master of Science / Department of Clinical Sciences / James W. Carpenter / 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.
| Identifer | oai:union.ndltd.org:KSU/oai:krex.k-state.edu:2097/770 |
| Date | January 1900 |
| Creators | Wesselowski, Sonya |
| Publisher | Kansas State University |
| Source Sets | K-State Research Exchange |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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