Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season

Abstract

Background: Seasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season.

Results: A total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified.

Conclusions: The miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.

Figure 1

Serum concentration of progesterone, estrogen, FSH and LH in different reproductive stages of Tan and Small Tail Han ewes. Four hormones were measured in ng/mL, pg/mL, ng/mL and ng/mL, respectively. P: progesterone; E: estrogen. TSA: anestrous Tan ewes in spring; TAL: Tan ewes in luteal phase in autumn; TAP: proestrous Tan ewes in autumn; TAE: estrous Tan ewes in autumn; HSL: Small Tail Han ewes in luteal phase in spring; HSP: proestrous Small Tail Han ewes in spring; HSE: estrous Small Tail Han ewes in spring. Each Column represented mean value of each stage, and the par represented standard deviation. The different letters meant the significant difference among stages (P < 0.01). The triangles stood for the hormone concentration of samples used to perform RNA-seq.

Figure 2

Sequence distribution of mapped reads. Frequency distribution of sequence length of STH sheep (A) and Tan sheep (B) based on the abundance of mapped reads. The composition of the RNA classes in each library was shown for STH sheep (C) and Tan sheep (D).

Figure 3

Expressed miRNAs and miRNA families. (A) The number of expressed miRNAs, including known, conserved and predicated novel miRNAs; (B) Distribution of miRNA family size; (C) The top ranked 20 expressed miRNA families in each sample.

Figure 4

Comparison of the miRNA expression profiles between anestrus and the other three stages. (A) Expressed miRNAs for each sample; (B) The top expressed miRNAs in each sample.

Figure 5

Differentially expressed miRNAs between anestrus and the other stages during estrus cycle. (A) The number of differentially expressed miRNAs. (B) The number of down-regulated miRNAs (indicates in D) and up-regulated (indicates in U) genes in anestrus when compared to the other three stages during estrus cycle. A-L: anestrus vs. luteal phase; A-P: anestrus vs. proestrus; A-E: anestrus vs. estrus. “U/U” and “D/D”: the expression patterns (up-regulation or down-regulation) of miRNAs between anestrus of Tan and the other stages of Tan were basically consistent with those between anestrus of Tan and the other stages of STH sheep; “D/U” means differentially expressed miRNAs are inconsistent between above two comparisons. (C) Venn diagram showed the 25 overlapped differentially expressed miRNAs between anestrus and the other three stages.

Figure 6

Hirpin structure and enriched KEGG pathways of significantly down-regulated miRNAs in anestrus.

Figure 7

Network consisting of differentially expressed miRNAs, their target genes and enriched KEGG pathways.

Figure 8

Predicated piRNAs and piRNA clusters. (A) Statistics of predicated piRNAs (B) chromosome distribution of predicated piRNAs with reads numbers >5 in at least one sample.