G. W. Smith

Two forms of the prolactin receptor messenger ribonucleic acid are Present in ovine fetal liver and adult ovary

Previous binding studies indicated that there is little to no specific prolactin binding in ovine fetal liver and adult ovary. Therefore, we sought to determine if ovine prolactin receptor (PRLR) mRNA is present in those tissues. Primers were designed from the bovine PRLR cDNA sequence for use in reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR analysis of ovine fetal liver total cellular RNA (tcRNA) isolated from days 60, 90, 105, 120 and 135 of gestation, and luteal tcRNA isolated from days 3, 7, 10, 13 and 16 of the estrous cycle revealed that PRLR mRNA was present in these tissues. However, two RT-PCR products were generated from both tissues. The two RT-PCR products did not differ between the two tissue sources in sequence, and were designated oPRLR-1 and oPRLR-2. Ovine PRLR-1 is 513 bp in length and is 96.4% identical to the bovine cDNA. Ovine PRLR-2 is identical to oPRLR-1 until nucleotide (nt) 420 at which point a 39 bp insertion occurs. This insertion occurs between Homology Boxes 1 and 2 within the cytoplasmic domain of the receptor, resulting in an 11 amino acid divergent sequence, followed by two stop codons. Ribonuclease-protection assay revealed that oPRLR-1 mRNA is the most abundant in these tissues. Our data indicate that two forms of oPRLR mRNA are Present in fetal liver and adult ovary, and that one form (oPRLR-2) is predicted to encode a truncated PRLR.

Ontogenies of messenger RNA encoding tissue inhibitor of metalloproteinases 1 and 2 within bovine periovulatory follicles and luteal tissue

Tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) are important regulators of extracellular matrix remodeling and also possess growth factor activity. The objective of these studies was to characterize TIMP-1 and TIMP-2 mRNA expression by bovine periovulatory follicles/ corpora hemorrhagica (Experiment 1) and luteal tissue (Experiment 2). In Experiment 1, beef heifers (n = 27) were ovariectomized at-16 (n = 6), 0 (n = 5), 8 (n = 3), 16 (n = 4), 24 (n = 4), or 48 (n = 5) hr relative to a gonadotropin-releasing hormone induced gonadotropin surge (40 hr after prostaglandin F2 alpha-induced luteolysis). Total cellular RNA was isolated from the large steroidogenically active follicle or corpus hemorrhagicum obtained from each animal, and the expression of TIMP-1 and TIMP-2 mRNA was subsequently examined by northern and dot blot analysis. The expression of TIMP-1 or TIMP-2 mRNa did not differ in preovulatory follicles collected at -16 vs. 0 hr. Concentrations of both TIMP-1 and TIMP-2 mRNA (picograms per microgram of tissue DNA) were increased (P < 0.05) at 8 hr postgonadotropin surge, had declined to presurge levels by 24 hr (P < 0.05), and were increased (P < 0.05) in corpora hemorrhagica collected at 48 hr after a gonadotropin surge. In Experiment 2, corpora lutea were collected from beef heifers on Days 4, 10, 15 (n = 4 each), or 19 (n = 3) postestrus (Day 0 = estrus). Concentrations of TIMP-1 mRNA (picograms per microgram of tissue DNA) were greater in corpora lutea collected on Day 4 (P < 0.05) vs. Day 10, 15, or 19. Concentrations of TIMP-2 mRNA increased (P < 0.05) from Day 4 to 15 and decreased (P < 0.05) by Day 19. We conclude that: 1) during the periovulatory period, the ontogenies of TIMP-1 and TIMP-2 mRNA expression are similar, whereas 2) during luteal phase, TIMP-1 mRNA expression is maximal during the early luteal phase, whereas concentrations of TIMP-2 mRNA peak during the midluteal phase. TIMP-1 and TIMP-2 may play important roles in the regulation of extracellular matrix remodeling during the periovulatory period and the subsequent luteal phase.

Matrix Metalloproteinase (2, 9, and 14) Expression, Localization, and Activity in Ovine Corpora Lutea Throughout the Estrous Cycle

Abstract Members of the matrix metalloproteinase (MMP) family collectively degrade extracellular matrix (ECM) and help regulate luteal function. The objectives of these experiments were to characterize the mRNA expression, localization, and activity of MMPs 2, 9, and 14 in ovine corpora lutea (CL). Ovine CL were collected on Days 2, 4, 10, and 15 of the estrous cycle (Day 0 = estrus). Messenger RNA transcripts for MMPs 2 and 14 were detected using Northern analysis; however, expression of MMP-9 was undetectable. Expression of MMP-14 mRNA (membrane type-1 MMP) was increased (P < 0.05) on Day 4; whereas, expression of MMP-2 mRNA was highest (P < 0.05) on Day 10, which corresponded to the observed increases in gelatinolytic activity in luteal homogenates as measured by a fluroscein-labeled gelatin substrate assay. MMP 2 and 9 proteins were localized predominantly to large luteal cells (LLCs), whereas MMP-14 was localized primarily to cells other than LLCs as demonstrated by immunohistochemistry. Immunolocalization of MMP-2 to putative endothelial cells was also observed on Day 15. Localization of MMP activity was determined using in situ zymography. Luteal tissues contained gelatinolytic activity primarily localized pericellularly to various cell types, including LLCs. These results support the hypothesis that ECM remodeling occurs throughout the luteal phase and may help potentiate cellular migration, differentiation, angiogenesis, and growth factor bioavailability.