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. 2019 May;157(5):475-484.
doi: 10.1530/REP-18-0466.

VIP activates primordial follicles of rat through ERK-mTOR pathway in tissue culture

Song Li  1 , Qi Fan  1 , Yanqiu Xie  2 , Haiyan Lin  1 , Qi Qiu  1 , Yihua Liang  1 , Qingxue Zhang  1   3
Affiliations

VIP activates primordial follicles of rat through ERK-mTOR pathway in tissue culture

Song Li et al. Reproduction. 2019 May.

Abstract

In vitro activation of primordial follicles is becoming more essential in assisted reproductive technologies. Vasoactive intestinal peptide (VIP) is one of the members of the neurotrophin family which has demonstrated to have an impact on follicle development in recent years. This study aims to investigate the effect of VIP on the activation of primordial follicles in neonatal rat in an in vitro culture system and to determine the relevant molecular mechanism of their activation. Ovaries of 4-day-old rats were examined for the expression of VIP receptors and were cultured in mediums containing VIP with or without inhibitors of the ERK-mTOR signalling pathway. They were then collected for histological analysis or measurement of the molecular expression of this pathway. The receptors of VIP were found in granular cells and oocytes of primordial and early-growing follicles in neonatal ovary. The ratio of growing follicle increased in the presence VIP at different concentrations, with the highest level of increase being observed in the 10-7 mol/L VIP-treated group. The ratio of PCNA-positive granular cells was also increased, while that of the apoptotic oocytes were decreased, and protein analysis showed increased phosphorylation of ERK1/2, mTOR and RPS6 in the VIP-treated group. However, the effect of VIP on the activation of primordial follicle became insignificant with the addition of MEK inhibitor (U0126) or mTORC1 inhibitor (rapamycin). This study indicated that VIP could activate neonatal rat primordial follicle through the ERK-mTOR signalling pathway, suggesting a strategy for in vitro primordial follicle recruitment.

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Conflict of interest statement

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1
Figure 1
The expression of vasoactive intestinal peptide (VIP) and its receptors in 4-day-old rat ovary. Four-day-old rat ovaries were freshly collected and cut into 5 μm sections, followed by immunohistochemistry for detecting VIP and its receptors – VPAC1 and VPAC2 (arrowheads, positive primordial follicle; arrows, positive early growing follicles). Scale bar: 50 μm.
Figure 2
Figure 2
VIP promoted the transformation from primordial follicles to early growing follicles in neonatal rat ovary. Four-day-old rat ovaries were cultured in basal medium without VIP (control group) or with VIP in different concentrations for three days. (A) The representative photos of all groups stained with hematoxylin and eosin: control (a), 10−6 mol/L (b), 10−7 mol/L (c), 10−8 mol/L (d). (B) and (C) The numbers of early-growing follicles and primordial follicles were counted, and their ratio was calculated. Scale bar: 50 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP−6 mol/L group, & P < 0.05 compared with the VIP−7 mol/L group, # P < 0.05 compared with the VIP−8 mol/L group).
Figure 3
Figure 3
VIP6–28 inhibits the activation of neonatal rat ovary by VIP. Four-day-old rat ovaries were cultured in basal medium without VIP (control group) or with 10−7 mol/L VIP or VIP + VIP6–28 (5 × 10−6 mol/L) for 3 days. (A) The representative photos of all groups stained with hematoxylin and eosin: control (a), VIP (b), VIP+VIP6–28 (c). (B and C) The numbers of early-growing follicles and primordial follicles were counted, and their ratio was calculated. Scale bar: 50 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP-treated group, & P < 0.05 compared with the VIP+VIP6–28-treated group).
Figure 4
Figure 4
VIP promotes the proliferation of granular cells and inhibits the apoptosis of oocytes. Four-day-old ovaries were cultured with 10−7 mol/L VIP or VIP + VIP6–28 (5 × 10−6 mol/L) for 3 days. (A and D) PCNA staining and TUNEL assay were conducted in the three groups mentioned above for proliferation and apoptosis observation. (B) Western blot was conducted to quantify the expression of PCNA. (C and E) The percentage of primordial follicles or oocytes with PCNA-positive or TUNEL-positive section. Scale bar: 100 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP-treated group, & P < 0.05 compared with the VIP + VIP6–28-treated group).
Figure 5
Figure 5
(A and B) The mRNA expression of Sohlh1 and Amh in neonatal ovary after treatment with10−7 mol/LVIP with or without VIP6–28 (5 × 10−6 mol/L) for 3 days. Values are mean ± s.d. of at least three experiments. (Gene expression was normalized to Gapdh. *P < 0.05 compared with the VIP group, & P < 0.05 compared with the VIP + VIP6–28-treated group).
Figure 6
Figure 6
Effect of U0126 or rapamycin on VIP-induced follicle development. Four-day-old rat ovaries were cultured with (i) 10−7 mol/L VIP, (ii)VIP plus 10−8 mol/L U0126, or (iii) VIP + 8.75×10−8 mol/L rapamycin for three days. (A) HE stained ovaries of different groups: control (a), VIP (b), VIP + U0126 (c), VIP + Rapa (d). (B and C) The numbers of early-growing follicles and primordial follicles were counted, and their ratio was calculated. Scale bar: 50 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP group, & P < 0.05 compared with the VIP + U0126 group, # P < 0.05 compared with the VIP + Rapa group).
Figure 7
Figure 7
U0126 or rapamycin inhibits the VIP-induced proliferation of granular cells and promotes oocyte apoptosis. Ovaries were cultured with (i) 10−7 mol/L VIP, (ii) VIP + 10−8 mol/L U0126 or (iii) VIP plus 8.75 × 10−8 mol/L rapamycin for 3 days. (A and D) PCNA staining and TUNEL assay were conducted for proliferation and apoptosis observation. (B) Western blot was conducted to quantify the expression of PCNA. (C and E) The numbers of primordial follicles with positive PCNA staining and oocytes with positive TUNEL staining were counted. Scale bar: 100 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP group, & P < 0.05 compared with the VIP + U0126 group, # P < 0.05 compared with the VIP + Rapa group).
Figure 8
Figure 8
VIP activated primordial follicle through ERK-mTOR signalling way. Ovaries were cultured with 10−7 mol/L VIP + 10−8 mol/L U0126 or 8.75 × 10−8 mol/L rapamycin for 3 days. (A and B) Western blot was conducted to investigate the level of protein phosphorylation in the ERK-mTOR signalling pathway, as well as AKT and FOXO3A. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP group. & P < 0.05 compared with the VIP + U0126 group. # P < 0.05 compared with the VIP + Rapa group).
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