Differential gene expression profiling of endometrium during the mid-luteal phase of the estrous cycle between a repeat breeder (RB) and non-RB cows

Abstract

Background: Repeat breeding directly affects reproductive efficiency in cattle due to an increase in services per conception and calving interval. This study aimed to investigate whether changes in endometrial gene expression profile are involved in repeat breeding in cows. Differential gene expression profiles of the endometrium were investigated during the mid-luteal phase of the estrous cycle between repeat breeder (RB) and non-RB cows using microarray analysis.

Methods: The caruncular (CAR) and intercaruncular (ICAR) endometrium of both ipsilateral and contralateral uterine horns to the corpus luteum were collected from RB (inseminated at least three times but not pregnant) and non-RB cows on Day 15 of the estrous cycle (4 cows/group). Global gene expression profiles of these endometrial samples were analyzed with a 15 K custom-made oligo-microarray for cattle. Immunohistochemistry was performed to investigate the cellular localization of proteins of three identified transcripts in the endometrium.

Results: Microarray analysis revealed that 405 and 397 genes were differentially expressed in the CAR and ICAR of the ipsilateral uterine horn of RB, respectively when compared with non-RB cows. In the contralateral uterine horn, 443 and 257 differentially expressed genes were identified in the CAR and ICAR of RB, respectively when compared with non-RB cows. Gene ontology analysis revealed that genes involved in development and morphogenesis were mainly up-regulated in the CAR of RB cows. In the ICAR of both the ipsilateral and contralateral uterine horns, genes related to the metabolic process were predominantly enriched in the RB cows when compared with non-RB cows. In the analysis of the whole uterus (combining the data above four endometrial compartments), RB cows showed up-regulation of 37 genes including PRSS2, GSTA3 and PIPOX and down-regulation of 39 genes including CHGA, KRT35 and THBS4 when compared with non-RB cows. Immunohistochemistry revealed that CHGA, GSTA3 and PRSS2 proteins were localized in luminal and glandular epithelial cells and stroma of the endometrium.

Conclusion: The present study showed that endometrial gene expression profiles are different between RB and non-RB cows. The identified candidate endometrial genes and functions in each endometrial compartment may contribute to bovine reproductive performance.

Keywords: Caruncle; Cow; Endometrium; Intercaruncle; Microarray; Repeat breeder.

Fig. 1

qPCR analysis of top two up- and down-regulated known genes in ipsilateral uterine horns between RB and non-RB cows for validation of the gene expression changes obtained from microarray analysis. a, b, c and d CAR and e, f, g and h ICAR. a, b, e and f up-regulated known genes in RB cows when compared with non-RB cows. c, d, g and h) down-regulated known genes in RB cows when compared with non-RB cows. The expression of mRNA was normalized to the expression of SUZ12 measured in the same RNA preparation. Data are shown as the mean ± SEM. Asterisks show significant differences (P < 0.05)

Fig. 2

qPCR analysis of top two up- and down-regulated known genes in contralateral uterine horns between RB and non-RB cows for validation of the gene expression changes obtained from microarray analysis. a, b, c and d CAR and e, f, g and h ICAR. a, b, e and f up-regulated known genes in RB cows when compared with non-RB cows. c, d, g and h down-regulated known genes in RB cows when compared with non-RB cows. The expression of mRNA was normalized to the expression of SUZ12 measured in the same RNA preparation. Data are shown as the mean ± SEM. Asterisks show significant differences (P < 0.05)

Fig. 3

qPCR analysis of top five up- and down-regulated known genes in whole uterus between RB and non-RB cows for validation of the gene expression changes obtained from microarray analysis. a, b, c, d, e up-regulated known genes in RB cows when compared with non-RB cows. f, g, h, i, j down-regulated known genes in RB cows when compared with non-RB cows. The expression of mRNA was normalized to the expression of SUZ12 measured in the same RNA preparation. Data are shown as the mean ± SEM. Asterisks show significant differences (P < 0.05)

Fig. 4

Representative photomicrographs of protein localization of CHGA, GSTA3 and PRSS2 in endometrial tissue from RB and non-RB cows on Day 15 of estrous cycle. Protein localization of (a, b, c and d) CHGA, (e, f, g and h) GSTA3 and (i, j, k and l) PRSS2 in endometrial tissue from RB (a, b, e, f, i and j) and non-RB (c, d, g, h, k and l) cows was detected by immunohistochemistry. Seven-micrometer sections of bovine endometrial tissues of ipsilateral uterine horns on Day 15 of estrous cycle were immunostained with anti-human CHGA, anti-human GSTA3 and anti-bovine PRSS2 polyclonal antibodies. Positive staining of CHGA and PRSS2 were found in the uterine luminal epithelium, uterine stroma and glandular epithelium of both RB and non-RB cows. GSTA3 was detected in the uterine luminal, uterine stroma and glandular epithelium in RB cows, whereas positive staining was not observed in non-RB cows. No signal was detected in the negative control sections using normal rabbit IgG (inserted panels). LE, luminal epithelium; US, uterine stroma; GE, glandular epithelium. Scale bars = 50 μm