Synthesis and messenger ribonucleic acid expression of apolipoproteins E and A-I by the bovine corpus luteum during the estrous cycle and pregnancy.
In an attempt to characterize proteins secreted by the corpus luteum, explant cultures of luteal slices from cows taken on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle, and Days 17, 88, 180, and > 240 of pregnancy were incubated with H-leucine for 24 h. Proteins in luteal-conditioned medium were separated by two-dimensional PAGE, transferred to polyvinylidene fluoride membrane, and subjected to N-terminal amino acid microsequencing. Microsequence analysis revealed that the bovine corpus luteum synthesized and released de novo synthesized apolipoproteins (Apo) E and A-I in culture during the estrous cycle and pregnancy. Release of Apo E was observed only on Day 3 of the estrous cycle. Release of Apo A-I was observed on Days 11, 14, 17, and 19 of the estrous cycle, and on all days of pregnancy examined. To demonstrate the presence of the appropriate mRNA and characterize the temporal relationship for these identified proteins, total RNA was isolated from corpora lutea on Days 2, 3, 7, 16, 17, and 20 of the estrous cycle, and on Days 17, 90, 170, 180, and 272 of pregnancy, and submitted to Northern and dot blot analysis. Apo E mRNA was expressed only on Days 2-3 of the estrous cycle and was not expressed on the other days of the cycle or during pregnancy. A single Apo E mRNA transcript about 1.0 kilobase (kb) in size was observed. Expression of Apo A-I mRNA was detected on all days of the estrous cycle and pregnancy examined. Apo A-I cDNA hybridized with a single mRNA transcript about 1.0 kb in size. Apo A-I mRNA levels did not differ among days of the estrous cycle, although higher levels of Apo A-I mRNA were observed during later stages of pregnancy. Serum concentrations of Apo A-I and progesterone were correlated across the estrous cycle but not during the prepartum period or after parturition. This study demonstrates for the first time that the corpus luteum synthesizes Apo E and Apo A-I and expresses their respective mRNAs. The pattern of expression of Apo E and Apo A-I mRNAs paralleled that of de novo synthesis of their respective proteins after incubation of luteal tissue with [H]leucine. The role of luteal apolipoproteins may involve an autocrine/paracrine function influencing luteal development, tissue remodeling, and steroidogenesis.
Ndikum-Moffor
F M
FM
Animal Science Department, University of Florida, Gainesville 32611-0910, USA.
Simmen
R C
RC
Fields
P A
PA
Katoh
N
N
Oikawa
S
S
Buhi
W C
WC
Rollyson
M K
MK
Chang
S M
SM
Fields
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis*; D001057:Apolipoproteins E / Q000096:biosynthesis*; D015152:Blotting, Northern; D002417:Cattle; D002784:Cholesterol / Q000096:biosynthesis / Q000097:blood; D003338:Corpus Luteum / Q000378:metabolism* / Q000648:ultrastructure; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004971:Estrus / Q000378:metabolism*; D005260:Female; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008079:Lipoproteins, VLDL / Q000096:biosynthesis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*
D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D003470:Culture Media; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D004247:DNA; D002784:Cholesterol