144 EXPRESSION OF ACETYL COENZYME A CARBOXYLASE α (ACCα) IN FELINE, CANINE AND PORCINE OOCYTES

Abstract

High lipid content in in vitro–derived embryos of several species is associated with poor developmental potential and cryosurvival of oocytes. Lipid content of oocytes varies among species and embryonic lipid content is further influenced by the culture environment. In spite of the variation among species, the relative contribution of de novo lipid synthesis during oocyte maturation or subsequent embryonic development is unknown. In the present study, we evaluated the expression of acetyl coenzyme A carboxylase (ACCα), the key rate-limiting enzyme of lipogenesis, in oocytes of 3 species with high lipid content. Ovaries of dogs and cats were collected from a local veterinary clinic and those of pigs from an abattoir. Cat and dog oocytes were recovered by the slicing method of the ovaries. Porcine oocytes were recovered by aspiration of ovarian follicles. Immediately after collection, oocytes were fixed for 30 min using a solution of 10% neutral buffered formalin. The ACCα enzyme expression was evaluated in porcine, cat and dog oocytes by immunofluorescence using a goat anti-human ACCα primary antibody followed by fluorescein isothiocyanate-conjugated anti-goat secondary antibody. The study was replicated, with negative controls, 3 times using ≥30 oocytes per species. Messenger RNA expression of ACCα gene was also evaluated in pig oocytes. The RNA was isolated from fat tissue, pooled good-quality oocytes (n = 30), pooled poor-quality oocytes (n = 30) and cumulus cells using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Complementary DNA was synthesised from 200 ng of RNA using Quantitect reverse-transcription kit (Qiagen) according to the manufacturer's instructions. Real-time PCR assays were carried out in duplicate and expression of ACCα mRNA levels relative to fat was determined. Oocytes and cumulus investment in cats, dogs and pigs strongly expressed ACCα. Expression of the protein was uniformly distributed through the entire ooplasm. The mRNA expression of ACCα in good- and poor-quality oocytes and cumulus cells relative to fat tissue was 11.5, 1.4 and 40.1%, respectively. Further studies are warranted on the dynamics of expression of ACC during in vitro maturation and the functional activity of the enzyme.

We extend our appreciation to the Alabama Animal Alliance Inc. (Montgomery) for providing us with dog and cat ovaries; the Lambert-Powell Meats Laboratory of Auburn University for donating pig ovaries; and Dr. James Webster, Dr. Tsegaye Habtemariam and Dr. Abdalla Eljack for administrative support.