Expression profile analysis of sheep ovary after superovulation and estrus synchronisation treatment

Abstract

Superovulation is a widely used reproductive technique in livestock production, but the mechanism of sheep's superovulation is not yet clear. Here, a method of superovulation and estrus synchronisation was used to treat female Duolang sheep. After treatment, there were significant differences in serum FSH and LH levels and the number of dominant follicles between the two groups of sheep. We identified a total of 5021 differentially expressed genes (11, 13 and 15 days after treatment) and performed RT-qPCR analysis to identify several mRNA expression levels. GO and KEGG enrichment analysis revealed that differentially expressed genes were involved in the regulation of signalling pathways of follicular development, cell cycle, material synthesis, energy metabolism, such as COL3A1, RPS8, ACTA2, RPL7 RPS6 and TNFAIP6 may play a key role in regulating the development of follicles. Our results show a comprehensive expression profile after superovulation and estrus synchronisation treatment. We provide the basis for further research on breeding techniques to improve the ovulation rate and birth rate of livestock.

Keywords: estrus synchronisation; ovary; sheep; superovulation; transcriptome.

FIGURE 1

Ovarian follicular status in sheep at different time periods after estrus synchronisation and superovulation. DT is estrus synchronisation, DC is superovulation, and above the picture is the time after processing

FIGURE 2

Identification of genes expression in sheep ovary treatment by RNA‐seq. (a) Flow chart of genes analysis. (b) A statistical chart of source information for all sample genes. Grey on behalf of intergenic, light red on behalf of intron and light blue on behalf of intron

FIGURE 3

The information of genes in sheep at different time points after estrus synchronisation and superovulation. (a) Under different experimental conditions, the horizontal box plot is expressed, where the abscissa is the sample (group) name, the ordinate is log2 (FPKM+1), and the box plot of each region is for the five statistics (top to bottom: value, upper quartile, median, lower quartile and minimum, respectively). (b) Sample correlation heat map. (c) Differential gene Venn diagram. The sum of all the numbers in the circle represents the total number of differential genes in the comparison combination, and the overlapping regions represent the differential genes shared between the combinations. Red representative DCA vs. DTA, green representative DCB vs. DTB and blue representative DCC vs. DTC (p < 0.05)

FIGURE 4

Validation of genes and their differential expression in sheep ovary. (a) RT‐PCR amplification of genes with specific primes. The order of electrophoresis are Marker (Takara DL500 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp and 50 bp), N is negative control, lane 1 is β‐actin, lanes 2–22 represent SLC27A2, AMIGO2, ADAM9, LDLR, EIF4EBP1, MGARP, COBLL1, PDE1C, CYP11A1, CAPN6, RRM2, KPNA2, LMNB1, PPIA, TNNI3, BIRC5, GTSE1, AURKB, TNFAIP6, TNFAIP6, KLF2 and APLP2, respectively. (b) Expression of 23 differentially expressed genes by RT‐qPCR. Blue is the result of qPCR, and red is the result of RNA‐seq (p < 0.05). The red gene name represents the DCA vs. DTA group; the black gene name represents the DCB vs. DTB group and the blue gene name represents the DCB vs. DTB group. The ordinate is the ratio of the relative expression levels of the gene in the treatment group and the estrus synchronisation (DTN/DCN, N stands for a, b and c)

FIGURE 5

Enrichment analysis of differentially expressed genes sheep at different time periods after estrus synchronisation and superovulation. GO enrichment analysis of differentially expressed genes at DCA vs. DTA (a) and DCB vs. DTB (b) groups (p < 0.05). The abscissa is the GO macromolecules and GO term at the next level, and the ordinate is the number and proportion of genes annotated to the term. KEGG enrichment analysis was performed on the hosting genes of differentially expressed genes in DCA vs. DTA (c), DCB vs. DTB (d) and DCC vs. DTC (e) groups. The vertical axis and the horizontal axis represent the name of the access and the access factor, respectively. The size of the dots represents the number of genes that are enriched in the access, and the colours correspond to different q‐values

FIGURE 6

Partial network diagram of PPI analysis. (a) Partial network diagram of DCA vs. DTA differentially expressed genes. (b) Partial network diagram of DCB vs. DTB differentially expressed genes. The red squares represent TNFAIP6 and CYP11A1