Negative effects of heat stress on ovarian tissue in female rabbit

Abstract

Numerous studies have highlighted the role of miRNA in the deformation and necrosis of cells of ovarian tissue caused by heat stress (HS), which ultimately affects ovarian function. Although the role of small RNAs has been investigated in alterations in ovarian tissue functioning in response to HS, the expression profile of ovarian miRNA has been explored to a lesser extent. In this study, female rabbits were subject to HS treatment by using electrical heater. The current work demonstrated that HS could significantly change physiological performance of female rabbits including body weight, rectal temperature and relative ovary weight, and significantly reduce serum IL-2, IL-8, CAT, and GSH-Px concentrations by enzyme-linked immunosorbent assay (ELISA) technique. As a result, an increase in apoptosis in ovarian cells, as well as unhealthy follicles, were observed by Hematoxylin-eosin (HE) and TUNEL staining. Additionally, small RNA-seq revealed changes in the miRNA expression profile of rabbit ovaries under HS. Five hundred fourteen miRNAs were obtained including known miRNAs 442 and novel miRNAs 72. Among these miRNAs, 23 miRNAs were significantly expressed under HS. Eleven differentially expressed miRNAs (DE miRNAs) and 9 their predicted targets were confirmed by qPCR, which were expected miRNA-mRNA negative regulation pattern. Among the DE miRNAs and targets, miR-141-39 may target COQ6, miR-449a-5p and miR-34c-5p may control RFC5 and RTN2 together, miR-449a-5p may target ACADVL, miR-34c-5p potentially targets Bcl-2 and miR-196b-5p potentially regulates CASK and HOXB6. Thus, the current work suggested the negative effects of HS on the ovarian tissue of female rabbits, and in conclusion these changes could be caused by decreased serum IL-2, IL-8, CAT and GSH-Px levels, increased ovarian apoptosis, and changed the expression of miRNAs.

Keywords: gene expression; heat stress; microRNA; ovary; rabbit.

Figure 1

Rectal temperature of female rabbits (n = 23 per group) in HS group and CON group. CON indicates CON group and HS indicates HS group. ****indicated statistically significant difference.

Figure 2

(A–D) Serum oxidative and immune biomarkers levels (n = 3 per group) from the HS group and CON group. CAT, catalase; GSH-Px, glutathione peroxidase; IL-2, interleukin 2; IL-8, interleukin 8. *, **, *** and **** indicated statistically significant difference.

Figure 3

(A) Ovarian histomorphology (n = 3 per group) in HS group and CON group. Green arrows indicated normal primary follicles and red arrows indicated vacuolated primary follicles; (B) Apoptotic cell count and cells apoptosis rate in ovarian tissues (n = 3 per group) in HS group and CON group. Nuclei were stained with blue dye (DAPI). Furthermore, red arrows indicated TUNEL-positive cells (green, Bar = 20 μm). Cells apoptosis rate = TUNEL positive cells/DAPI cells. ** indicated statistically significant difference.

Figure 4

Analysis of DE miRNAs from rabbit ovary in CON group and HS group. (A) Venn diagram illustrating common and different DE miRNAs between HS and CON; (B) Analysis of DE miRNAs by volcano map; (C) Hierarchical cluster diagram of DE miRNAs; (D) KEGG pathway enrichment analysis of target genes in DE miRNAs; (E) GO enrichment analysis of target genes in DE miRNAs.

Figure 5

Validation of DE miRNAs and their targets was done by qPCR. (A) Validation of 11 DE miRNAs; (B,C) Validation of potential targets. COQ6, coenzyme Q6; monooxygenase; RFC5, replication factor C subunit 5; RTN2, reticulon 2; ACADVL, acyl-CoA dehydrogenase very long chain; Bcl-2, B-cell CLL/lymphoma 2 protein; CASK, calcium/calmodulin dependent serine protein kinase; HOXB6, homeobox B6; LOC100339409, ATPase inhibitor; mitochondrial-like; LOC100009591, sodium channel protein type 1 subunit alpha. * and ** indicated statistically significant difference.