The Effect of Ghrelin on the Maturation of Sheep Oocytes and Early Embryonic Development In Vitro

Abstract

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Keywords: RNA-seq; cell cycle; early embryo; ghrelin; sheep oocyte.

Figure 1

Changes in oocyte nuclear morphology over time in response to ghrelin. (A) Diagram of different stages of IVM oocytes (GV, GVBD, MI, MII). (B) Control group: (0 ng/mL) oocyte nuclear maturation cell morphology (I–IV) and Hoechst 33342 stained cell morphology (V–VIII) (scale bar: 20 μm) (C) Experimental group: (200 ng/mL) oocyte nuclear maturation cell morphology (I–IV) and Hoechst 33342 stained cell morphology (V–VIII) (scale bar: 20 μm).

Figure 2

Relative expression of GADD45 and CCNB1 at different stages of IVM oocyte. Error bars indicate three independent biological replicates (mean ± standard deviation). * p < 0.05, ** p < 0.01.

Figure 3

Volcano plot of differentially expressed genes at different time points. Red represents genes that are significantly different and upregulated; blue represents genes that are significantly different and downregulated; gray represents genes that are not significantly different.

Figure 4

Gene set enrichment analysis (GSEA) of different time points. The green line shows the enrichment profile. NES: normalized enrichment score, FDR: false discovery rate.

Figure 5

Heatmap showing the expression comparison of markers at different time points. Ribosome synthesis, generation of oxidative phosphorylation, cell cycle, RNA transport, metabolism of inositol phosphate, meiosis of oocytes, PI3K-AKT signaling pathway, and other differentially significant genes.

Figure 6

Effect of ghrelin addition on early embryonic development in sheep. (A) Diagram of fertilization and early embryonic development in vitro. (B) Cell morphology during fertilization and early embryonic development in vitro. I. Fertilization, II. Fertilization finish, III. 2 cell, IV. 4 cell, V. 8 cell, VI. 16 cell, VII. Morula, VIII. Blastocyst. Scale bar: 20 μm.

Figure 7

qPCR analysis of GLUT1 in different development stages. Error bars indicate three independent biological replicates (mean ± standard deviation). * p < 0.05, ** p < 0.01.