247 GENE EXPRESSION OF MAMMALIAN RELATIVE OF DNAJ IN BOVINE ENDOMETRIUM DURING EARLY PREGNANCY

Abstract

The mammalian relative of DNAJ (MRJ), a member of the molecular chaperones that are known as heat shock proteins, plays an important role in the process of murine chorioallantoic fusion. The objective of this study was to determine the expression pattern of MRJ in the bovine endometrium during early and mid-pregnancy using quantitative RT-PCR. Twenty-eight Japanese Black cows, aged between 1.2 and 15.2 years, with normal estrous cycles, were used in this study. Twenty-one cows were used for study of the level of MRJ during pregnancy. They were artificially inseminated, and their endometrial tissues were collected on Days 16 to 21 (n = 7), 30 to 36 (n = 6), 48 to 49 (n = 4) and 74 to 140 (n = 4) of pregnancy. Seven cows were used as controls for the study of cyclic level of MRJ, and their endometrial tissues were collected on Days 13 to 14 (n = 4) and 17 to 20 (n = 3) of the estrous cycle. The caruncles and the intercaruncles were isolated from the endometrial tissues. All tissues were frozen immediately using liquid nitrogen. Total RNA from these samples was extracted from the tissue using Trizol (Invitrogen Corp., Carlsbad, CA, USA). The first strand of cDNA was synthesized from the total RNA by oligo (dT)_12–18 and SuperScript^™ II RNase H- Reverse Transcriptase (Invitrogen Corp.) according to the manufacturer’s instructions. Primers and the TaqMan probe for MRJ and glyceraldehyde-phosphate-dehydrogenase (GAPDH) were designed using the primer design software Primer Express^™ (Applied Biosystems, Foster City, CA, USA). Bovine GAPDH was used as an internal standard. All PCR reactions were performed using a TaqMan^™ PCR Reagent Kit and a MicroAmp Optical 96-Well Reaction Plate and Cap (Applied Biosystems). The assay used an ABI Prism 7700 Sequence Detector (Applied Biosystems). Signals were detected according to the manufacturer’s instructions. The relative level of MRJ expression was calculated on the basis of GAPDH quantity (the method of calculation: relative level = MRJ quantity/GAPDH quantity). Data were analyzed by one-way ANOVA, and means were conpared by Tukey-Kramer’s HSD test. Mammalian relative of DNAJ genes were expressed in all samples examined;; the levels in intercaruncle tended to be greater than those in caruncle. Although MRJ expression level at Days 16 to 21 of pregnancy was greater than at other days during pregnancy, there were no significant differences between the levels at Days 16 to 21 of pregnancy and those of the estrous cycle. These results suggest that MRJ is produced in the endometrium and may play a role in early and mid-pregnancy and the estrous cycle.