Adiponectin modulates steroid hormone secretion, granulosa cell proliferation and apoptosis via binding its receptors during hens' high laying period

Abstract

Adiponectin is an important adipocytokine and plays the roles in multiple metabolic processes via binding its receptors - AdipoR1 and AdipoR2, which has also been found to participate in the regulation of the reproductive system of animals, in particular by influencing the secretion of ovarian steroid hormones. To further investigate the expression of adiponectin and its receptors in follicles after in vitro incubation, and their role in the steroid synthesis of laying hens' ovaries, we performed qRT-PCR and ELISA to detect the expressions of AdipoQ, AdipoR1, and AidpoR2, and determined the key genes involved in steroidogenesis and the secretion of estradiol (E2) and progesterone (P4) through the in vitro activation of adiponectin (AipoRon) and overexpression or knockdown of AdipoR1 and AdipoR2. Our results revealed that adiponectin and its receptors wildly exist in follicles and granulosa cells, and AdipoRon (5 and 10 µg/mL) had no effect on granulosa cell proliferation and apoptosis but significantly stimulated the secretion of adiponectin and its receptors in granulosa cells after incubation for 24 h. Furthermore, AdipoRon could significantly stimulate the secretion of P4 and inhibit E2 level compared to those of the control group through modulating the key genes expression of steroidogenesis (CYP19A1, StAR, CYP11A1, FSHR, and LHR). The secretion of E2 was also decreased in granulosa cells by the treatments of overexpression and knockdown of AdipoR1/2, however, there was no difference in terms of the level of P4 and StAR expression between them if there was overexpression or knockdown of AdipoR1/2. In addition, it was shown that the secretion of E2 only exhibits a marked drop if co-processing 10 µg/mL AdipoRon and pGMLV AdipoR2 compared to single treatments. Taken together, the study highlighted the role of adiponectin and its receptors in the regulation of steroid synthesis and secretion in ovarian granulosa cells in laying hens.

Keywords: AdipoR1/2; adiponectin; laying hen; reproduction; steroid hormones.

Figure 1

The mRNA expression levels of AdipoQ, AdipoR1 and AdipoR2 in follicles of different sizes (A) and granulosa cell (B). Different letters above each bar represent significant difference (P < 0.05) and all the values in the bars with the vertical lines represent mean ± SD, which are the same as the following figures.

Figure 2

Effect on granulosa cell proliferation and apoptosis of the supplement of AdipoRon. (A) The granulosa cell vitality of the supplements of AdipoRon at different levels and incubation times. (B) The proportion of apoptotic cells in the total granulosa cells. (C) Annexin-V/PI analysis of granulosa cells treated with 0, 5, 10, 20, 40 µg/mL AdipoRon, respectively.

Figure 3

The secretion of Adiponectin (A), AdipoR1 (B), AdipoR2 (C), P4 (D) and E2 (E) in granulosa cells treated with 0, 5, 10 µg/mL AdipoRon, respectively.

Figure 4

Effect of overexpression or knockdown of AdipoR1 and AdipoR2 on the expressions of AdipoR1 and AdipoR2. AdipoR1 overexpression (A) and knockdown (B) in granulosa cells enhances or suppresses the mRNA expression levels of AdipoR1. AdipoR2 overexpression (C) and knockdown (D) in granulosa cells enhances or suppresses the mRNA expression levels of AdipoR2. AdipoR1 (E) and AdipoR2 (F) overexpression and knockdown in granulosa cells led to a higher or lower protein expression of AdipoR1 or AdipoR2. * means P < 0.05, ^⁎⁎ means P < 0.01 and different letters above each bar means P < 0.05.

Figure 5

Effect of AdipoRon and overexpression or knockdown of AdipoR1/2 on the mRNA expressions of steroidogenesis related genes in granulosa cells. The mRNA expressions of CYP19A1, StAR, CYP11A1, FSHR, and LHR in granulosa cells treated with overexpression AdipoR1 (A), overexpression AdipoR2 (B), knockdown AdipoR1(C) and knockdown AdipoR2(D) individual or co-processing with AdipoRon at different concentrations, respectively.

Figure 6

Effect of AdipoRon and overexpression or knockdown AdipoR1/2 on the secretion of steroid hormones in granulosa cells. The secretions of E2 and P4 in granulosa cells treated with overexpression AdipoR1 (A), overexpression AdipoR2 (B), knockdown AdipoR1 (C) and knockdown AdipoR2 (D) individual or co-processing with AdipoRon at different concentrations, respectively.