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. 2023 Jan 16:11:e14612.
doi: 10.7717/peerj.14612. eCollection 2023.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin

Wenju Liu  1   2 , Zhihao Chen  3 , Rui Li  3 , Menghao Zheng  3 , Xunsheng Pang  3 , Aiyou Wen  3 , Bing Yang  3 , Shujuan Wang  3   4
Affiliations

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin

Wenju Liu et al. PeerJ. .

Abstract

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-β superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole.

Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-β superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA.

Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin.

Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Keywords: 4-P-PDOT; Bovine; Gene regulation; Granulosa cell; Hormonal synthesis; Luzindole; Melatonin.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Effects of high dose (10−5 M) and low dose (10−9 M) melatonin and melatonin receptor antagonist supplementation on endocrine related gene expression (StAR, RUNX2, CYP11A1, and CYP19A1).
The mRNA levels of StAR (A), RUNX2 (B), CYP11A1 (C), and CYP19A1 (D) were examined by real-time PCR in granulosa cells 48 h after melatonin supplementation in the absence/presence of luzindole or 4-P-PDOT. (E) Protein abundance was detected by Western blot. The quantity of mRNA was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. P < 0.05 was considered significant difference. The experiment was repeated three times independently.
Figure 2
Figure 2. Effects of high dose (10−5 M) and low dose (10−9 M) melatonin and melatonin receptor antagonist supplementation on TGF-β superfamily related gene expression, including (BMP6, TGFBR3, INHA, INHBA, and INHBB).
The mRNA abundance of BMP6 (A), TGFBR3 (B), INHA (C), INHBA (D), and INHBB (E) were examined by real-time PCR at 48 h after melatonin supplementation in the absence/presence of luzindole or 4-P-PDOT. (F) Protein abundance was detected by Western blot. mRNA abundance was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. P < 0.05 was considered significant difference. The experiment was repeated three times independently.
Figure 3
Figure 3. Effects of high dose (10−5 M) and low dose (10−9 M) melatonin and melatonin receptor antagonist supplementation on development related genes expression (DNMT1A, EGFR, and FSHR).
The mRNA abundance of DNMT1A (A), EGFR (B), and FSHR were examined by real-time PCR 48 h after melatonin supplementation in the absence/presence of luzindole or 4-P-PDOT. mRNA abundance was normalized to that of β-actin. The statistical differences were performed using one-way ANOVA. P < 0.05 was considered significant difference. The experiment was repeated three times independently.
Figure 4
Figure 4. Effects of high dose (10−5 M) and low dose (10−9 M) melatonin and melatonin receptor antagonist supplementation on hormone secretions (estradiol, progesterone, Inhibin A, Inhibin B, and Activin B).
Abundance of estradiol (A), progesterone (B), inhibin A (C), inhibin B (D) and activin B (E) were measured 48 h after melatonin supplementation in granulosa cell medium in the absence/presence of luzindole or 4-P-PDOT. The statistical differences were performed using one-way ANOVA. P < 0.05 was considered significant difference. The experiment was repeated three times independently.
Figure 5
Figure 5. Melatonin and melatonin combined with its receptor antagonist mediated effects on bovine granulosa cells.
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