274 LOCALIZATION OF MACROPHAGE COLONY STIMULATING FACTOR (M-CSF) GENE IN THE ENDOMETRIUM OF JAPANESE BLACK COW

Abstract

Macrophage colony stimulating factor (M-CSF), a member of the group of hemopoietic cytokines, plays an important role in placental physiology of humans and mice. The objective of this study was to investigate the localization of M-CSF gene in bovine endometrial tissues during pregnancy using in situ hybridization (ISH). Twelve Japanese Black cows aged between 1.4 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. Cows were artificially inseminated, and pregnancy was confirmed by presence of conceptus and/or corpus luteum using ultrasonography. They were euthanized and their uteri were collected on Days 20 to 21 (n = 2), 36 to 42 (n = 3), 59 to 64 (n = 3), 127 to 137 (n = 2), and 225 to 226 (n = 2) of pregnancy. Pregnancy was confirmed finally 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. After being fixed in 4% PFA solution for 20 to 24 h, tissue pieces were embedded in paraffin using routine procedures. Several tissue pieces were collected from each individual cow. Sections (6 mm thick) were cut and placed on MAS-coated glass slides (Matsunami Glass, Kishiwada, Japan). The sections were dried in an oven for 3 days at 40°C. Antisense and sense biotin-labeled oligonucleotide probes for bovine M-CSF were designed from the sequence information (GenBank accession number D87917). Sense probe was used as the negative control. ISH was carried out using the GenPoint System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. The sections were observed under an Eclipse 800 microscope (Nikon, Tokyo, Japan), and the positive signal for M-CSF gene was detected. Density analysis was performed by using 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. The M-CSF gene was expressed in the stromal and luminal epithelial cells of endometrium, stromal cells of the fetal cotyledonary villus and caruncular crypt, and mono- and multi-nuclear epithelium cells of the fetal cotyledonary villus and caruncular crypt. Intensities of M-CSF gene-positive signals in all positive cells at Days 225 to 226 of pregnancy were stronger than those at other periods (P < 0.05). These results suggest that M-CSF is produced in several cell types in the endometrium and placental tissues and may play important roles in bovine pregnancy.