The expression of cytosolic and mitochondrial type II acyl-CoA thioesterases is upregulated in the porcine corpus luteum during pregnancy.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to free fatty acids and CoASH, thereby regulating fatty acid metabolism. This activity is catalyzed by numerous structurally related and unrelated enzymes, of which several acyl-CoA thioesterases have been shown to be regulated via the peroxisome proliferator-activated receptor alpha, strongly linking them to fatty acid metabolism. Two protein families have recently been characterized, the type I acyl-CoA thioesterase gene family and the type II protein family, which are expressed in cytosol, mitochondria and peroxisomes. Still, only little is known about regulation of their expression and precise functions in vivo. In the present study, we have investigated the activity and expression of acyl-CoA thioesterase in the porcine ovary during different phases of the estrus cycle. The activity was low in homogenates obtained during the immature and follicular phases, increasing nearly 4-fold during the luteal phase, with the highest activity being found in the pregnant corpus luteum (about 7-fold higher than in immature follicles). The increase in homogenate activity in corpus luteum from pregnant pigs was due to a moderate increase in the cytosolic activity, and an approximately 20-25-fold increase in the mitochondrial fraction. Western blot analysis showed no detectable expression of the type I acyl-CoA thioesterases (CTE-I and MTE-I) and revealed that the increased activity in cytosol and mitochondria is due to increased expression of the type II acyl-CoA thioesterases (CTE-II and MTE-II). This apparent hormonal regulation of expression of the type II acyl-CoA thioesterase may provide new insights into the functions of these enzymes in the mammalian ovary.

Detailed analytical subcellular fractionation of non-pregnant porcine corpus luteum reveals peroxisomes of normal size and significant UDP-glucuronosyltransferase activity in the high-speed supernatant.

A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies. In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000 x g(av) for 10 min) and not in the post-mitochondrial fraction prepared at 30,000 x g(av) for 20 min by Peterson and Stevensson. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ. Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000 x g(av) for 1h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat.