Impact of in Vitro Heat Shock (42.50c) on Prostaglandins, Ionic and Metabolic Contents in Sheep Endometrial Epithelial Cells
Sukanta Mondal*, Avantika Mor, Sumanta Nandi and Ippala Janardhan Reddy
ICAR-National Institute of Animal Nutrition and Physiology, India
Received: March 30, 2017; Published: April 18, 2017
*Corresponding author: Sukanta Mondal, ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore-560 030, India, Tel: x002B;9180-25711164/25711304; Fax: x002B;9180-25711420; Email: sukanta781@gmail.com
How to cite this article: Sukanta M, Avantika M, Sumanta N, Ippala J R. Impact of in Vitro Heat Shock (42.50c) on Prostaglandins, Ionic and Metabolic Contents in Sheep Endometrial Epithelial Cells. Curr Trends Biomedical Eng & Biosci. 2017; 3(1): 555604. DOI: 10.19080/CTBEB.2017.03.555604
Abstract
Elevated temperature is one of the major factors responsible for reduced fertility in domestic ruminants including sheep. The aim of the study was to delineate the effect of heat shock on prostaglandins, ionic and metabolic contents of sheep endometrial epithelial cell in vitro. Endometrial epithelial cells were cultured in RPMI 1640 medium at 38.5 °C in CO2 incubator for 24hr (5% CO2 in air, 90-95% relative humidity; Control culture; n=6). Heat stressed cultures (n=6) were acclimated at 38.50C for 6 hr and then placed at 42.5 °C for 18 hr. In vitro heat stress (42.5 °C) significantly (P<0.05) increased calcium, glucose and PGE2 levels and increased (P>0.05) protein, phosphorus, urea, SOD, PGF2a levels in epithelial cells as compared to those exposed to 38.5 °C. It is concluded that heat shock altered prostaglandins, ionic and metabolic contents of endometrial epithelial cells in vitro.
Keywords: Heat stress; Endometrial epithelial cells; Ionic content; Metabolic content; Sheep
Introduction
Higher environmental temperature is one of the major factors responsible for reduced fertility in farm animals. Heat stress causes a great suppression in endometrial function [1] besides compromised follicular growth [2], embryonic function [3] and oocyte developmental potential [4]. Heat stress also increases the production of PGF2a in the endometrium, leading to the early regression of CL or the death of embryos [5]. Heat stress-induced hyperthermia has been found to decrease the pregnancy rate in cattle during the summer in regions associated with elevated ambient temperatures [6]. Prostaglandins (PGs) produced by endometrium serves as a crucial mediators in maternal recognition of pregnancy, implantation and parturition [7,8]. The endometrial epithelial cells preferentially produce PGF2a whereas stromal cells produce mainly PGE2. PGF2a acts as the luteolytic agent to control the estrous cycle in ruminants. Endometrial secretion of PGF2a by pregnant uterus has been found to increase in response to heat stress and decrease the embryonic survivality by altering the signals required for maintenance of corpus luteum function during early pregnancy. Increased PGF2a synthetic capacity of endometrium exposed to heat stress may be due to heat-induced alterations in endometrial cellular membranes resulting in increased mobilization of substrate for prostaglandin biosynthesis. Similar increases in uterine PGF2a secretion in response to heat stress in vitro by endometrial expiants from cows at Day 17 of pregnancy have been reported [5]. No reports are available on impact of in vitro heat stress on endometrial epithelial prostaglandins, ionic and metabolic contents in sheep. The present study was undertaken to investigate the effect of heat shock on prostaglandins, ionic and metabolic contents of sheep endometrial epithelial cells in vitro.
Materials and Methods
Uteri were collected from the local abattoir immediately after slaughter and transported to the laboratory on ice. The epithelial cells from the sheep endometrium were separated by the method as previously described [7]. Following isolation of epithelial cells, the cells were cultured in RPMI 1640 medium at 38.5 °C in presence of 5% CO2. Control cultures (n=6) were maintained at 38.50C for 24hr. Control cultures were incubated under conditions representing normal body temperature of normal sheep in a thermo-neutral environment. Heat stressed cultures (n=6) were acclimated at 38.5 °C for 6 hr and then placed at 42.5 °C for 18hr. The culture medium was collected after 24hr and stored at-70 °C until analysis. The concentrations of metabolites (glucose, total protein and urea) and ions (calcium, chloride, phosphorus) were analyzed by using Biochrom Libra S32 UV/Vis Spectrophotometer. The intra and inter assay coefficients of variation for all analyses were below 7%. The concentrations of PGF2a and PGE2 were determined in 50 |il aliquots of culture medium after 10 fold dilution with extraction buffer using ELISA kits supplied by Neogen, USA. The sensitivity of the PGF2a and PGE2 assays were 0.002 and 0.1ng/ml, respectively. The intra-and inter-assay coefficients of variation of PGF2a assay were less than 19%. The intra- and inter-assay coefficients of variation of PGE2 assay were less than 14%. Results are expressed as mean±SEM. Concentrations of each factor in control and heat stressed culture medium were analyzed by 't'-test using GraphPad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA). Differences between mean values were considered significant when the probability values were < 0.05.
Results and Discussion
The effect of heat shock on prostaglandins, ionic and metabolic contents of sheep endometrial epithelial cells were presented in Table 1. The elevated temperature (42.5 °C) significantly (P<0.05) increased calcium, glucose and PGE2 contents of epithelial cells. However, exposure of epithelial cells to 42.50C did not significantly (P>0.05) increase protein, phosphorus, urea, PGF2a and SOD contents in epithelial cells compared to those exposed to 38.5 °C. Chloride contents decreased (P>0.05) in epithelial cells followi