Growth hormone ameliorates the age-associated depletion of ovarian reserve and decline of oocyte quality via inhibiting the activation of Fos and Jun signaling

Abstract

Oocyte quality typically begins to decline with aging, which contributes to subfertility and infertility. However, there is still no effective treatment to restore the ovarian reserve and improve aged-oocyte quality. According to the present study, growth hormone (GH) secretion changes with maternal age in female mice. After intraperitoneal injection with GH (1 mg/kg body weight) every two days for two months, the 10-month-old mice showed a better ovarian reserve and oocyte quality than control mice. GH treatment decreased the occurrence rate of aneuploidy caused by spindle/chromosome defects. Additionally, the single oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. It was also observed that GH treatment reduced the expression of γH2AX and apoptosis of aged oocytes via decreasing the activation of Fos and Jun. Collectively, our results indicate that GH treatment is an effective way to reverse the age-associated depletion of ovarian reserve and the decline of oocyte quality by decreasing apoptosis.

Keywords: aging; apoptosis; growth hormone; meiosis; oocyte quality.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1

The GH levels and oocyte quality were declined in aged mice. (A) The GH levels in the peripheral blood were measured in young (n = 6) and aged (n = 6) mice. (B) Micrographs of young and aged WT mouse ovaries (Scale bar, 1 mm) and HE staining of these ovaries (Scale bar, 100μm, 500 μm). (C) Follicle counts in 6-week (n = 3) and 10-month (n = 3) old WT mice. (D) Chromosomes misalignment and spindle defects (arrowheads) in aged oocytes. The oocytes were stained with α-tubulin (green) and propidium iodide (PI) (red) respectively. Scale bar, 50 μm. (E) Left: The rate of GVBD and Pb1 extrusion were recorded after 4 h and 14 h of culture in M2 medium respectively. Right: Percentages of oocytes with spindle defects in young (n = 97) and aged mice (n = 104). (F) Representative images of MII oocytes collected from young (n = 76) and aged (n = 19) mice and IVF outcomes from these two groups. Black arrowheads point to abnormal oocytes. Scale bar, 100 μm, 400 μm. (G) Quantification of MII oocytes, 2-cell embryo and blastocyst from young and aged mice. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0005.

Figure 2

Effects of GH treatment in vitro on the meiotic progress of aged oocytes. (A) Representative images of oocytes after 14 hours cultured in M2 medium (n = 87) and GH treated medium (n = 88). Black arrowheads point to oocytes that fail to extrude a polar body or fail to survive. Scale bar, 100 μm. (B) The rate of GVBD and Pb1 extrusion in these two groups respectively. (C) Left: Spindle morphologies in control and GH group. Oocytes were stained with α-tubulin (green) and hoechst (blue). Scale bar, 50 μm. Right: Percentages of oocytes with spindle defects in control (n = 51) and GH group (n = 68). Data are presented as mean ± SD. **P < 0.01.

Figure 3

Effects of GH administration in vivo on the ovarian reserve. (A) Schematic illustration for the NS and GH-treated mice. (B) The GH levels in the peripheral blood was measured in GH (n = 6) and vehicle (n = 7) group. (C) The changes of body weight and ovary index after GH administration. NS, no significance. (D) Micrographs of NS-treated and GH-treated mouse ovaries. Scale bar, 1 mm. (E) Left: Estrous cycle in representative females. Right: Average numbers of cycles in 8 days in two groups. P, proestrus; E, estrus; M, metestrus; D, diestrus. (F) HE-stained of NS-treated and GH-treated mouse ovaries. Scale bar, 400 μm, 200 μm. (G) Follicle counts and the number of corpus luteum in NS-treated (n = 3) and GH-treated (n = 5) mice. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Figure 4

Effects of GH treatment in vivo on the quality and meiotic progress of aged oocytes. (A) Left: Representative images of MII oocytes, morula and blastocysts from NS-treated and GH-treated mice. Black arrows point to fragmented MII oocytes. Scale bar, 200 μm, 100 μm. Right: Number of MII oocytes and percentage of 2-cell embryos and blastocysts in NS-treated and GH-treated mice. (B) After cultured in M2 medium, the rate of GVBD and Pb1 extrusion were recorded. (C) Left: The MII oocytes from NS-treated (n = 37) and GH-treated (n = 42) mice were stained with α-tubulin (green) and propidium iodide (PI) (red). Scale bar, 50 μm. Right: Quantification of NS-treated and GH-treated oocytes with abnormal spindle/chromosomes. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

ScRNA-seq of NS-treated and GH-treated MII oocytes. (A) Left: Mapping rate of NS-treated oocytes (n = 4) and GH-treated (n = 4) oocytes. Right: The number of detected genes (FPKM > 1) in vehicle and GH group oocytes. NS, no significance. (B) Left: The gene expression heatmap showed the differentially expressed genes (DEGs) in these two groups. Right: Principal components analysis (PCA) of eight samples. (C) The volcano map showed the DEGs between NS-treated and GH-treated mice. (D) The top 30 KEGG pathways involved in the down-regulated genes. The red box encloses the apoptosis pathway. (E) The top 22 KEGG pathways involved in the up-regulated genes. The red box encloses the GnRH signaling, cAMP signaling, calcium signaling and Rap1 signaling pathway. (F) 2 DEGs were selected for QPCR validation (n ≥ 10). Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Figure 6

GH treatment decreased oocyte apoptosis and DNA damage. (A) Left: Immunostaining for Ki-67 in ovaries from the NS and GH groups. Brown represents positive staining. Scale bar, 400, 100, 50 μm. Right: The mean density levels of Ki-67 in ovarian. (B) The ovaries from NS-treated and GH-treated mice were stained with γH2AX (red) and DAPI (blue). Scale bar, 50 μm. (C) Left: The MII oocytes from NS-treated (n = 7) and GH-treated (n=7) mice were stained with γH2AX (red) and hoechst (blue). Scale bar, 50 μm. Right: Mean density levels of NS-treated and GH-treated oocytes. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Figure 7

Effect of GH treatment on the FOS/JUN pathway. (A) The expression of GHR in GH-treated and NS-treated oocytes. (B) The expression of Fos, Fosb, Jun, Junb, Jund, Lmnb2 and Ctsb in GH-treated and NS-treated oocytes according to the sequencing data. (C) Results of single oocyte QPCR in the two groups (n ≥ 10). (D) Immunostaining for Fos, Jun, Fosb and Junb in ovaries from the NS and GH groups. Brown represents positive staining. Data are presented as mean ± SD. *P < 0.05.

Figure 8

Schematic diagram regarding how GH reverses the age-associated depletion of ovarian reserve and declining quality of oocytes. In old mice, the ovarian reserve and the levels of GH are declined. The decreased GH enhances the expression levels of Fos and Jun family which increase the oocyte apoptosis and DNA damage. Administration of GH restores the depletion of ovarian reserve and induces the increment of GHRs. Additionally, GH supplementation exerts an influence on reversing apoptosis via lowering the expression of Fos and Jun which improves the oocyte quality.