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. 2022 Dec 16;12(24):3570.
doi: 10.3390/ani12243570.

The Photoperiod Regulates Granulosa Cell Apoptosis through the FSH-Nodal/ALK7 Signaling Pathway in Phodopus sungorus

Yan Qi  1 , Hui-Liang Xue  1 , Yun-Jiao Zheng  1 , Yun-Fei Yin  1 , Wen-Lei Xu  1 , Jin-Hui Xu  1 , Ming Wu  1 , Lei Chen  1 , Lai-Xiang Xu  1
Affiliations

The Photoperiod Regulates Granulosa Cell Apoptosis through the FSH-Nodal/ALK7 Signaling Pathway in Phodopus sungorus

Yan Qi et al. Animals (Basel). .

Abstract

The photoperiod regulates the seasonal reproduction of mammals by affecting the follicle development, for which the granulosa cells provide nutrition. However, the underlying mechanism remains unclear. Here, Djungarian hamsters (Phodopus sungorus) were raised under different photoperiods to study the ovarian status and explore the potential mechanism of the follicle development mediated by the FSH-Nodal/ALK7 signaling pathway. Compared with the moderate daylight (MD) group, the short daylight (SD) group exhibited a significant decrease in the ovarian weight and increase in the atretic follicle number and granulosa cell apoptosis, whereas the long daylight (LD) group showed an increase in the ovarian weight, the growing follicle number, and the antral follicle number, but a decrease in the granulosa cell apoptosis. Based on these findings, the key genes of the Nodal/ALK7 signaling pathway controlling the granulosa cell apoptosis were studied using the quantitative real-time polymerase chain reaction and western blotting. In the SD group, the follicle-stimulating hormone (FSH) concentration significantly decreased and the Nodal/ALK7/Smad signaling pathways were activated, while the phosphatidylinositol 3-kinase (PIK3)/Akt signaling pathway was inhibited. The BAX expression was significantly increased, while the Bcl-xL expression was significantly decreased, leading to an increase in the caspase-3 activity, the granulosa cell apoptosis, and ovarian degeneration. However, in the LD group, the FSH concentration significantly increased, the Nodal/ALK7/Smad signaling pathway was inhibited, and the PIK3/Akt signaling pathway was activated. Taken together, our results indicate that the photoperiod can regulate the apoptosis of the granulosa cells by regulating the concentration of FSH, activating or inhibiting the Nodal/ALK7 signaling pathway, thereby affecting the ovarian function. Our research provides an important theoretical basis for understanding the photoperiod-regulated mechanisms of the mammalian seasonal reproduction.

Keywords: FSH; Nodal/ALK7 signaling pathway; apoptosis; granulosa cell; ovarian function; photoperiods.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Ovarian experiment design. Note, the SD group was taken as an example.
Figure 2
Figure 2
Differential analysis of the serum hormone concentrations in Phodopus sungorus under different photoperiod conditions. (A) IGF-1; (B) GnRH; (C) FSH; and (D) LH. Values are the means ± SEM. n = 11. SD, short daylight; MD, moderate daylight; LD, long daylight. ** p < 0.01.
Figure 3
Figure 3
Differences in the number of follicles by the H&E staining of the ovaries of Phodopus sungorus under different photoperiods. (A) Ovarian section from the SD individuals. (B) Ovarian section from the MD individuals. (C) Ovarian section from the LD individuals. (D) Differences in the number of growing follicles across the different photoperiods. (E) Differences in the number of antral follicles across the different photoperiods. (F) Differences in the number of atretic follicles across the different photoperiods. (G) Indices of the growing follicles, antral and atretic follicles under the different photoperiods. g, growing follicle; a, antral follicle; *, atretic follicle. Values are means ± SEM. Bar = 200 μm; n = 3. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
TUNEL detection results of the ovary tissue in Phodopus sungorus under three photoperiod conditions. (A) Fluorescent TUNEL staining of the ovaries of Phodopus sungorus under three photoperiod conditions. Blue, nuclei stained with 4′, 6-diamino-2-phenylindole (DAPI); green, TUNEL results of the FITC. The area indicated by the red arrow is an enlarged image of Merge. (B) Apoptosis activity of the granulosa cells in Phodopus sungorus under three photoperiod conditions. Bar = 100 μm; n = 3. Oo, oocyte; Gr, granulosa cells. SD, short daylight; MD, moderate daylight; LD, long daylight, ** p < 0.01.
Figure 5
Figure 5
Co-localization of Nodal and ACVR1C proteins in the ovaries of Phodopus sungorus under three photoperiod conditions. Blue, nuclei stained with 4′, 6-diamino-2-phenylindole (DAPI); red, expression and distribution of Nodal protein; green, expression and distribution of ACVR1C protein. Oo, oocyte; Gr, granulosa cells; arrow shows theca cells. Bar = 20 μm; n = 3. SD, short daylight; MD, moderate daylight; LD, long daylight.
Figure 6
Figure 6
Key gene expression levels in the Nodal/ALK7 signaling pathway in the ovaries of Phodopus sungorus under three photoperiod conditions. (A) Nodal, (B) ALK7, (C) SMAD4, (D) XIAP, (E) Akt, (F) HtrA2, (G) DIABLO, (H) Bcl-xL, (I) Bcl2, and (J) BAX. Values are means ± SEM. n = 6. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
Levels of the Nodal and ACVR1C protein expression in the ovaries of Phodopus sungorus under three photoperiod conditions. (A,B) Representative immunoblots of the key Nodal and ACVR1C proteins and internal reference proteins (α-tubulin and β-actin) in three different photoperiod groups. (C,D) Relative protein expression of Nodal and ACVR1C. Values are means ± SEM. n = 8. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05.
Figure 8
Figure 8
Levels of the Smad signaling pathway protein expression in the ovaries of Phodopus sungorus under three photoperiod conditions. (A,B) Representative immunoblots of the key SMAD4 and XIAP proteins and internal reference proteins (β-actin and GAPDH). (C,D) Relative protein expression of SMAD4 and XIAP. Values are means ± SEM. n = 8. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05, ** p < 0.01.
Figure 9
Figure 9
Levels of the PI3K/Akt signal pathway protein expression in the ovaries of Phodopus sungorus under three photoperiod conditions. (A,E,F) Representative immunoblots of the key P-Akt, Akt, HtrA2, and DIABLO proteins and internal reference proteins (β-actin and α-tubulin). (B,C,G,H) Relative protein expression of P-Akt, Akt, HtrA2, and DIABLO. (D) Ratio of P-Akt to Akt. Values are means ± SEM. n = 8. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05.
Figure 10
Figure 10
Levels of the B-cell lymphoma-2 (BCL-2) family protein expression in the ovaries of Phodopus sungorus under three photoperiod conditions. (A–C) Representative immunoblots of the key Bcl-xL, Bcl2, and BAX proteins and internal reference proteins (α-tubulin and β-actin). (D–F) Relative protein expression of Bcl-xL, Bcl2, and BAX. Values are means ± SEM. n = 8. SD, short daylight; MD, moderate daylight; LD, long daylight. * p < 0.05.
Figure 11
Figure 11
Detection of the caspase-3 activity in Phodopus sungorus under three different photoperiods. Values are means ± SEM. n = 6. SD, short daylight; MD, moderate daylight; LD, long daylight, ** p < 0.01.
Figure 12
Figure 12
Molecular mechanism diagram of the photoperiod regulating the seasonal reproduction in female Phodopus sungorus. (A) Regulation mechanism of FSH-Nodal/ALK7 signal pathway under short daylight condition. (B) Regulation mechanism of FSH-Nodal/ALK7 signal pathway under long daylight condition. IGF-1: insulin growth factor 1; GnRH: gonadotropin-releasing hormone; FSH: follicle-stimulating hormone; LH: luteinizing hormone; ALK7: activin receptor-like kinase 7; BAX: bcl-2-associated X protein; Bcl-2: B-cell lymphoma-2; P-Akt: phosphate-Akt; HtrA2: High temperature requirement protein a2.
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