Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?

Abstract

This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.

Keywords: RNA-Seq; bovine; embryo-maternal interaction; microarray; oviduct.

Fig. 1

Experimental design. The estrous cycles of crossbred beef heifers were synchronized using a 7-day progesterone (P4)-releasing intravaginal device (CIDR; 1.38 g of P4) with administration of a prostaglandin F2α analog (PG) the day before CIDR removal. A) Experiment 1. Heifers (n = 19) were allocated to one of two groups: cyclic (nonbred) or pregnant (artificially inseminated at standing estrus). All heifers were slaughtered on Day 3 after estrus. B) Experiment 2. To ensure the ovulation, 0.02 mg of a GnRH agonist (buserelin) was administered 32 h after CIDR removal. Heifers (n = 17) were allocated to one of two groups: cyclic, sham transfer in both oviducts or pregnant with 50 in vitro-produced presumptive zygotes transferred in the ipsilateral oviduct and a sham transfer carried out in the contralateral oviduct. All transfers were carried out endoscopically on Day 1.5, and all animals were slaughtered on Day 3 after estrus. ET, endoscopic transfer.

Fig. 2

Correspondence analysis demonstrating the source of greatest variation in the oviduct transcriptional profile in experiment 1. Each dot represents all the transcripts expressed on one microarray representing one tissue site from one animal.

Fig. 3

Correspondence analysis demonstrating the source of greatest variation in the oviduct transcriptional profile in experiment 2. Each dot represents all the transcripts expressed on one RNA-Seq representing one tissue site from one animal.

Fig. 4

Illustration of DEGs between pregnant and cyclic heifers showing the top-10 up- and down-regulated genes in pregnant animals (123 and 155 genes, respectively) (P < 0.05, fold-change > 2). Values in parentheses indicate the fold-change for each gene. Complete gene list can be found in Supplemental Table S1.

Fig. 5

Quantitative real-time PCR analysis of selected genes for RNA-Seq validation (experiment 2). Across 20 comparisons (10 genes × 2 heifer groups [pregnant vs. cyclic]), the expression pattern of the selected genes obtained by qPCR was consistent with the results from the RNA-Seq analysis in all but two cases (QRFPR and CCL20). For each transcript, bars with different superscripts differ significantly (P < 0.05). PII, pregnant ipsilateral isthmus; CII, cyclic ipsilateral isthmus.

Fig. 6

Quantitative real-time PCR analysis of differentially expressed transcripts from experiment 2 in tissues from experiment 1. Across 20 comparisons (10 genes × 2 heifer groups [pregnant vs. cyclic]), the expression pattern of the selected genes was as expected, with no differences between groups in all but one case (KERA). For each transcript, bars with different superscripts differ significantly (P < 0.01). PII, pregnant ipsilateral isthmus; CII, cyclic ipsilateral isthmus.