A transcriptomal analysis of bovine oviductal epithelial cells collected during the follicular phase versus the luteal phase of the estrous cycle

Cerny et al. Reproductive Biology and Endocrinology (2015) 13:84 DOI 10.1186/s12958-015-0077-1 RESEARCH Open Access A transcriptomal analysis of bovine oviductal epithelial cells collected during the follicular phase versus the luteal phase of the estrous cycle K. L. Cerny, E. Garrett, A.J. Walton, L.H. Anderson and P. J. Bridges* Abstract Background: Reproductive success depends on a functional oviduct for gamete storage, maturation, fertilization, and early embryonic development. The ovarian-derived steroids estrogen and progesterone are key regulators of oviductal function. The objective of this study was to investigate luteal and follicular phase-specific oviductal epithelial cell function by using microarray-based transcriptional profiling, to increase our understanding of mRNAs regulating epithelial cell processes, and to identify novel genes and biochemical pathways that may be found to affect fertility in the future. Methods: Six normally cycling Angus heifers were assigned to either luteal phase (LP, n = 3) or follicular phase (FP, n = 3) treatment groups. Heifers in the LP group were killed between day 11 and 12 after estrus. Heifers in the FP group were treated with 25 mg PGF2α (Lutalyse, Pfizer, NY) at 8 pm on day 6 after estrus and killed 36 h later. Transcriptional profiling by microarray and confirmation of selected mRNAs by real-time RT-PCR analyses was performed using total RNA from epithelial cells isolated from sections of the ampulla and isthmus collected from LP and FP treatment groups. Differentially expressed genes were subjected to gene ontology classification and bioinformatic pathway analyses. Results: Statistical one-way ANOVA using Benjamini-hochberg multiple testing correction for false discovery rate (FDR) and pairwise comparison of epithelial cells in the ampulla of FP versus LP groups revealed 972 and 597 transcripts up- and down-regulated, respectively (P < 0.05). Within epithelial cells of the isthmus in FP versus LP groups, 946 and 817 transcripts were up- and down-regulated, respectively (P < 0.05). Up-regulated genes from both ampulla and isthmus were found to be largely involved in cholesterol biosynthesis and cell cycle pathways, while down-regulated genes were found in numerous inflammatory response pathways. Conclusions: Microarray-based transcriptional profiling revealed phase of the cycle-dependent changes in the expression of mRNA within the epithelium of the oviducts’ ampulla and isthmus. Keywords: Heifer, Oviduct epithelial cells, Ampulla, Isthmus, Estradiol, Progesterone Background Reproductive success depends on a functional oviduct for gamete storage and maturation, fertilization, and early embryonic development. Ovarian-derived steroids are well known regulators of oviductal function. Both estrogen and the progesterone receptors are abundant in the bovine oviducts’ mucosal epithelium [1–6], yet our understanding of how the steroidal environment affects * Correspondence: Department of Animal and Food Sciences, University of Kentucky, Lexington 40546KY, USA the ability of the oviduct to function remains only partially understood. Maturation of gametes and breeding will occur in an estrogen dominant environment, fertilization and early cleavage after the steroidogenic shift and later stages of cleavage and formation of the morula occur within an oviduct exposed to increasing concentrations of circulating progesterone. Increasing our understanding of the steroidal control of oviductal function is critical to the design and implementation of interventions used to manage breeding and the establishment of a pregnancy. © 2015 Cerny et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cerny et al. Reproductive Biology and Endocrinology (2015) 13:84 Functionally, the oviduct is divided into two distinct segments; the upper ampulla situated immediately below the ovarian bursa and infundibulum, and the lower isthmus which spans the interval from the ampulla to the uterus. The epithelial mucosa within these two sections consists of ciliated and secretory simple columnar epithelial cells [7], the ratio of which is regulated by steroid hormones [8, 9]. Steroidal regulation of processes that facilitate sperm binding [10], sperm release [11], capacitation [12] and hyperactivation [13] are all established in the literature. Epithelial cells are also an active site of biosynthesis and secretion. A 3- to 5-fold increase in the rate of oviductal secretions can be expected around the time of estrus [14] and amino acids including glycine, glutamate, aspartate, alanine and lysine are all found in higher concentrations in oviductal fluid than in peripheral plasma [15]. Overall, steroid-regulated epithelial cell secretions can be considered an important mediator of the microenvironment that facilitates gamete and zygote health and early development. While major efforts have been directed at investigating the ovary and uterus at the transcriptional level [16–18], less extensive investigation has been directed towards the oviduct. Suppressive subtractive hybridization was used in the detailed study of Bauersachs et al., [19], in which changes in gene expression within the oviductal epithelium were determined in heifers killed on the morning of estrus or 12 days thereafter; our report expanding on their analysis with the use of more current transcriptomal profiling technologies and the determination of spatial differences between the ampulla and the isthmus. The objective of this descriptive study was therefore to determine global oviductal epithelial cell gene expression profiles during the follicular and luteal phases of the estrous cycle. Specifically, microarray-based transcriptional profiling was used to identify spatial and phase of the cycle-dependent changes in mRNA expression in epithelial cells isolated from the ampulla and isthmus, with the overall goals of increasing our understanding of epithelial processes and identifying novel genes that may be identified as key regulators of fertility in the future. Our results must be interpreted, however, with the knowledge that this analysis does not extract potential spatial (isthmus to ampulla and visa versa) signaling mechanisms that could affect oviductal epithelial cell gene expression profiles independent to phase of the estrous cycle. Given the size of the dataset generated by this analysis, our approach to this descriptive study is not to provide a detailed discussion of genes or processes affected by phase of the estrous cycle, but to summarize the results generated, pro (...truncated)