259 LOCALIZATION OF LEUKEMIA INHIBITORY FACTOR (LIF) GENE IN BOVINE PLACENTA

Abstract

Leukemia inhibitory factor (LIF), a member of the group of hemopoietic cytokines, plays a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. The objective of this study was to investigate the localization of LIF gene in bovine placenta tissues during pregnancy using in situ hybridization (ISH). Eleven Japanese Black cows aged between 1.8 and 14.5 years, with normal estrous cycles, were used in this study. They were observed daily for estrous behavior, and the day of estrus was considered as Day 0. They were artificially inseminated, and their uteri were collected on Days 61 to 63 (n = 3), 127 to 142 (n = 4), and 225 to 232 (n = 4) of pregnancy. Pregnancy was confirmed by the presence of a conceptus in the uterus, and the uterus was isolated by dissection, avoiding damage of the uterine artery. The uterus was perfused with 4% paraformaldehyde (PFA) solution using catheters inserted into the uterine artery, and placental tissues were isolated by dissection. Each tissue was cut into small pieces (5 mm thick) and fixed in 4% PFA solution for 20-24 h, after which they were embedded in paraffin using routine procedures. Several tissue pieces were collected from each individual cow. Six micrometer-thick sections were cut and placed on MAS coated glass slides (Matsunami Glass, Kishiwada, Japan). The sections were dried in an oven for three days at 40°C. Anti-sense and sense biotin-labeled oligonucleotide probes for bovine LIF were designed from the sequence information (GenBank accession number D50337). The sense probe was used as the negative control. ISH was carried out using GenPoint System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. The sections were observed under an Eclipse 800 microscope (Nikon, Tokyo, Japan) to detect the positive signal for LIF gene. Density analysis was performed with Scion Image (Scion Corporation, Frederick, MD, USA). Data were analyzed by one-way ANOVA and Tukey-Kramer's HSD, using 0.05 as a significant level. Leukemia inhibitory factor gene was expressed in stromal cells of the fetal cotyledonary villus and caruncular crypt and in mono- and multi-nuclear epithelium cells of the fetal cotyledonary villus and caruncular crypt. Intensities of LIF gene positive signals in all positive cells at Days 127 to 142 tended to be weak compared with those at other periods. Furthermore, intensities of LIF gene positive signals of multinuclear cells of the villus and crypt demonstrated a tendency to be strong compared with those of other cells. These results suggest that LIF is produced in several cell types in the placenta and may play important roles in pregnancy.