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 PGF (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.

Document Type

Article

Publication Date

8-5-2015

Notes/Citation Information

Published in Reproductive Biology and Endocrinology, v. 13, article 84, p. 1-13.

© 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.

Digital Object Identifier (DOI)

http://dx.doi.org/10.1186/s12958-015-0077-1

Funding Information

This work was supported by National Institutes of Health Grant P01 HD071875, the University of Kentucky, and Kentucky Agricultural Experiment Station. The information reported in this paper (publication no. 15-07-075 ) is part of a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.

s12958-015-0077-1-s1.pdf (49 kB)
Supplementary Figure 1. Box plot of the log2 expression signal for each sample (microarray chip).

s12958-015-0077-1-s2.pdf (128 kB)
Supplementary Figure 2. Overlapping sample signal intensity histogram indicating the frequency of transcripts at specific signal intensity values.

s12958-015-0077-1-s3.pdf (186 kB)
Supplementary Table 1. Differentially expressed transcripts between follicular and luteal phases of the estrous cycle in epithelial cells exclusive to the ampulla and isthmus (FDR adjusted P < 0.05).

s12958-015-0077-1-s4.pdf (201 kB)
Supplementary Table 2. Differentially expressed transcripts in common between follicular and luteal phases of the estrous cycle in epithelial cells of the ampulla and isthmus (FDR adjusted P < 0.05).

s12958-015-0077-1-s5.pdf (58 kB)
Supplementary Table 3. Enriched gene ontology classifications from up-regulated DEGS within epithelial cells of the ampulla in follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s6.pdf (39 kB)
Supplementary Table 4. Enriched gene ontology classifications from down-regulated DEGS within epithelial cells of the ampulla in follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s7.pdf (71 kB)
Supplementary Table 5. Canonical pathway analysis of up-regulated DEGS within epithelial cells of the ampulla in the follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s8.pdf (86 kB)
Supplementary Figure 3. Top 6 Canonical pathways from up- and down-regulated differentially expressed genes within epithelial cells of the ampulla in the follicular versus luteal phase. Ingenuity Pathway Analysis software was used to determine significant pathways based on the number of significant genes expressed within the pathway using Fischer’s Exact test (P <0.05). A) Ampulla: Up-regulated pathways in the follicular versus luteal phases. B) Ampulla: Down-regulated pathways in the follicular versus luteal phases.

s12958-015-0077-1-s9.pdf (69 kB)
Supplementary Table 6. Canonical pathway analysis of down-regulated DEGS within epithelial cells of the ampulla in the follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s10.pdf (56 kB)
Supplementary Table 7. Enriched gene ontology classifications from up-regulated DEGS within epithelial cells of the isthmus in follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s11.pdf (53 kB)
Supplementary Table 8. Enriched gene ontology classifications from down-regulated DEGS within epithelial cells of the isthmus in follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s12.pdf (44 kB)
Supplementary Table 9. Canonical pathway analysis of up-regulated DEGS within epithelial cells of the isthmus in the follicular versus luteal phase groups (P < 0.05).

s12958-015-0077-1-s13.pdf (62 kB)
Supplementary Figure 4. Top 6 Canonical pathways from up- and down-regulated differentially expressed genes within epithelial cells of the isthmus in the follicular versus luteal phase. Ingenuity Pathway Analysis software was used to determine significant pathways based on the number of significant genes expressed within the pathway using Fischer’s Exact test (P <0.05). A) Isthmus: Up-regulated pathways in the follicular versus luteal phases. B) Isthmus: Down-regulated pathways in the follicular versus luteal phases.

s12958-015-0077-1-s14.pdf (85 kB)
Supplementary Table 10. Canonical pathways of down-regulated DEGs within epithelial cells of the isthmus in the follicular versus luteal phase groups (P < 0.05).

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