
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00719-3
10.1016/j.psj.2024.104140
104140
GENETICS AND MOLECULAR BIOLOGY
Role of PI3K/AKT signaling pathway involved in self-renewing and maintaining biological properties of chicken primordial germ cells
Liu Xin *†‡
Ye Liu *†‡
Ding Ying *†‡
Gong Wei *†‡
Qian Hongwu *†‡
Jin Kai *†‡
Niu Yingjie *†‡
Zuo Qisheng *†‡
Song Jiuzhou §
Han Wei #
Chen Guohong *†‡
Li Bichun yubcli@yzu.edu.cn
⁎†‡ǁ1
⁎ Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
† Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China
‡ Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
§ Animal & Avian Sciences, University of Maryland, College Park, MA 20742, USA
# Poultry Institute, Chinese Academy of Agricultural Sciences Poultry Institute of Jiangsu, Yangzhou 225003, China
ǁ College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
1 Corresponding author: yubcli@yzu.edu.cn
08 8 2024
11 2024
08 8 2024
103 11 10414017 1 2024
25 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

Key words

chicken primordial germ cell
PI3K/AKT
proliferation
biological property
==== Body
pmcINTRODUCTION

Primordial germ cells are important culture cells for the establishment of gene-modified chickens, transgenic animals, and conservation of avian genetic resources (Fujimoto, et al., 1976; van de Lavoir, et al., 2012; Schusser, et al., 2013). However, the number of PGCs isolated in vivo is far from sufficient for research purposes, and the tendency of these cells to lose their germline potential during culture poses a challenge for in vitro culturing. Separate PGC cultures have been assessed in poultry, such as turkeys and ducks, with the goal of achieving species preservation and recovery of endangered birds (Guan, et al., 2010; Hall, et al., 2020). However, the efficiency of PGC in vitro culture for lineage establishment is low because of the inter-species differences in traits, such as those of the reproductive cycle (Shi, et al., 2021).

Using Dulbecco's Modified Eagle Medium (DMEM) conditioned medium and STO feeder layer cells, van de Lavoir et al. succeeded for the first time in establishing chicken PGC lines that can grow indefinitely, transgenic, and germline-transmissible chicken PGC lines (van de Lavoir, et al., 2006), and subsequent researchers have developed different culture systems. For example, Choi developed a system containing a high-sugar DMEM medium and hESC-qualified Matrigel whereas Macdonald et al. successfully cultured PGCs using BRL as a feeder layer (Macdonald, et al., 2010; Miyahara, et al., 2014; Song, et al., 2014), and Naito et al. developed culture systems containing 2% chick embryo extract and fibroblast cells (Naito, et al., 2015). However, these culture systems were ill-defined and had uncertain nutrients.

A recent study used a specific culture system that excluded the effects of undefined molecules in serum or feeder layer and found that PGCs were unable to proliferate in the absence of insulin (Whyte, et al., 2015). The physiological functions of insulin are mediated primarily via activation of the PI3K/AKT signal transduction pathway (Saltiel, 2021). Insulin activates the PI3K/AKT pathway in hESCs, and constitutive activation of the AKT pathway ensures cell survival (Godoy-Parejo, et al., 2019). Moreover, AKT signaling plays a key role in self-renewal and cell proliferation in mouse and human embryonic stem cells (Jirmanova, et al., 2002; Paling, et al., 2004; Li, et al., 2007). In addition, the AKT signaling pathway has a significant influence on PGC development (De Felici and Klinger, 2021). Although PI3K inhibitors inhibit AKT phosphorylation in mouse PGCs (Farini et al., 2005; Rossi et al., 2000), induction of mutant PI3K and inhibition of AKT signaling in mouse PGCs using wortmannin and LY294002 have no appreciable effect on germ cell proliferation (De Miguel, et al., 2002). AKT regulates the expressions of CCND1 and Cyclin B1 and induces cell cycle arrest in tumor and mouse embryonic stem cells (Lee, et al., 2009; Li, et al., 2012a; Thanaussavadate, et al., 2020). These findings indicate a relationship between insulin, cell proliferation, and apoptosis, mediated via the PI3K/AKT pathway, which deserves further investigation.

To the best of our knowledge, this study is the first to investigate the effects of AKT signaling on the proliferation, biological properties, apoptosis, cell cycle, and ferroptosis of PGCs. Our findings provide insights into the self-renewal of vertebrate PGCs and the potential evolutionary changes in this unique cell population, and may serve as a theoretical foundation for the optimization and improvement of culture systems for chicken PGCs.

MATERIALS AND METHODS

Ethics Statement

The animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee of the Yangzhou University Animal Experiments Ethics Committee [approval number: SYXK (Su) 2021-0027]. All experimental procedures were performed with strict adherence to the Regulations of the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China.

PGC Culture Conditions

Fertilized breeder eggs (Rugao yellow chicken) were obtained from the Poultry Research Institute (Chinese Academy of Agricultural Sciences, Yangzhou, China). The eggs were incubated at 37°C and 60% relative humidity in an OVA-EASY 100 ADVANCE incubator (Brinsea, Titusville, USA), and were turned at 6-h intervals. Following incubation for 4.5 d, PGCs were isolated from the kidney mesenchyme of chick embryo. Isolated mesonephric kidneys were placed in 200 μL of BSA solution (0.1%, aseptic) (C1007; Beijing Biotech Co., Ltd., Beijing, China). The supernatant post-centrifugation at 1,000 rpm for 3 min using an eppendorf centrifuge (5424R; Eppendorf, Hamburg, Germany) was discarded, and the gonads were crushed with a pestle and digested with accutase enzyme (AT001; Hangzhou Lianke Biology Technology Co., Ltd, Hangzhou, China). The digestion was terminated by adding 200 μL of PGC complete medium (Table 1 culture system), followed by centrifugation at 1,000 rpm for 3 min. After discarding the supernatant, the pellet was resuspended in 200 µL of culture medium followed by incubation at 37°C and 5% CO2 in an incubator (51032872; Thermo Scientific, Waltham, MA). The cells were observed daily and half of the liquid was replaced every 3 d during the purification phase. After 60 d of purification and cultivation (20th passage), PGCs were identified based on morphology, periodic acid-Schiff (PAS) staining, quantitative real-time PCR (RT-qPCR), and pluripotent gene expressions.Table 1 Cell culture medium.

Table 1DMEM basal medium	
Composition	Source	Concentration	Volume	
DMEM	Meilunbio-PWL037, Dalian, China	75%	37.5 Ml	
Ultra-filtered Water	Sigma-W3500, Shanghai, China	24%	12 Ml	
CaCl2·2H2O	Sigma-C7902, Shanghai, China	0.15 mM	0.5 Ml	
DMEM basal medium	Discard 3.368ml		46.632 mL	
B-27TM supplement	Gibco-17504044, Shanghai, China	1×	1 Ml	
GlutaMax	Gibco-35050061, Shanghai, China	2 mM	0.5 Ml	
MEM NEAA	Gibco-11140050, Shanghai, China	1×	0.5 Ml	
2-Mercaptoethanol	Gibco-21985023, Shanghai, China	0.1 mM	91 µL	
Chicken serum	Gibco-16110082, Shanghai, China	0.2%	100 µL	
EmbryoMax Nucleosides	Sigma-ES-008-D, Shanghai, China	1×	0.5 Ml	
Sodium pyruvate	Gibco-11360070, Shanghai, China	1.2 mM	0.6 Ml	
Ovalbumln	Sigma-A5503, Shanghai, China	0.2%	0.1 g	
Sodium heparin	MCE-HY-17567A, Shanghai, China	0.01%	50 µL	
Basic fibroblast growth factor	MCE-HY-P70600, Shanghai, China	1×	20 µL	
Human Activin A	MCE-HY-P70311, Shanghai, China	1×	25 µL	
Pen Strep	Gibco-15070063, Shanghai, China	1×	0.5 Ml	

Drug Treatment

In this experiment, the B-27 supplement containing insulin used in the PGC culture system served as a control (Table 1 culture system). LY294002 (HY-10108; MedChemExpress, Shanghai, China) was dissolved in dimethyl sulfoxide and IGF-1 (HY-P7018; MedChemExpress) was dissolved in phosphate buffered saline (PBS). PGCs in the logarithmic growth phase were harvested and counted using a cell counting chamber (C10283; Invitrogen, Shanghai, China). Assays were performed in 24-well plates with 1 × 105 cells/well. Cells in the control group were left untreated, whereas in the activation experiment, the cells were assigned to control, 60 nM, and 120 nM IGF-1 groups, and those subjected to inhibition were exposed to 0, 10, 20, 30, 40, and 50 μM LY294002, followed by incubation for 72 h.

Periodic Acid-Schiff Stain

Identification of PGCs was performed using a PAS staining kit (G1281; Solarbio Life Science, Beijing, China), The cells were initially fixed in 4% paraformaldehyde, followed by oxidant treatment, rinsing, dropwise addition of Schiff's staining solution, washing, xylene clearing, and neutral gum sealing. The stained cells were observed under an Olympus BX41 microscope (Olympus, Tokyo, Japan).

Immunohistochemistry

For immunofluorescence analysis, PGCs were initially fixed in 4% paraformaldehyde for 30 min at room temperature, after which they were permeabilized with 0.1% Triton X-100 in PBS (P1020; Solarbio Life Science, Beijing, China) followed by 3 washes with PBS containing 0.1% Tween-20 (PBST). Samples were incubated overnight at 4°C with either the primary antibody SSEA-1 (1:400 dilution: ab16285; Abcam, Shanghai, China) or anti-DDX4/MVH antibody (1:400: ab13840; Abcam) dissolved in NCM universal antibody diluent (WB100D; New Cell & Molecular Biotech Co., Ltd, Suzhou, China). The following day, after 3 washes with PBST, the cells were incubated with the corresponding secondary antibody (Alexa-Fluor 488 (1:400 dilution, ab150077, Abcam, Shanghai, China), goat anti-Rabbit IgG (H+L) (1:50: AS039; ABclonal, Wuhan, China)) for 2 h at room temperature. Thereafter, the samples were washed and stained with Hoechst (C1017; Beyotime, Shanghai, China) to visualize nuclei. After washing the stained cells 3 times with PBST, they were mounted under coverslips in Antifade Mounting Medium (P0126; Beyotime, Shanghai, China) and visualized using a Leica d-35578 Wetzlar microscope (Leica Microsystems Inc., Germany).

Cell Counting Kit-8 Assay

PGCs in the logarithmic growth phase were collected and treated with either LY294002 (10, 20, 30, 40, or 50 μM) or IGF-1 (60 or 120 nM), with insulin as control. Following incubation for 72 h, the cells were counted using a cell counting kit (CCK-8) (CK04; Dojindo, Shanghai, China). The treated cells were added to culture medium containing reagent at a 10:1 ratio, followed by incubation at 37°C in a 5% CO2 incubator for 2 h, after which the absorbances of the well contents were recorded at 450 nm using a microplate reader (30086376; SPARK, Austria). For each treatment group, reactions were performed in triplicate. The viability of cells was determined using the following formula:

Cell viability (%) = [(experimental group) - (blank)]/[(control group) - (blank)] ×100.

EdU Proliferation Assay

Proliferation assays were performed using a Cell-Light EdU Apollo567 in vitro Imaging Kit (C10310-1; Ribobio, Guangzhou, China) in accordance with the instructions of the manufacturer. At the end of culture, 1 × 105 cells were taken from each well and incubated in 50 μM EdU medium for 2 h. Thereafter, the culture medium was discarded, and following washing, the cells were fixed on slides using 10 μL of 4% paraformaldehyde for 30 min at room temperature. The fixed cells were then washed, permeabilized, stained with Apollo stain followed by DNA staining, and washed again. The stained cells were observed under a Leica d-35578 Wetzlar microscope (Leica Microsystems Inc.).

RNA Extraction and Real-Time Quantitative PCR

RT-qPCR was used to determine the mRNA levels of the genes of interest. Briefly, total RNA was extracted using 1 mL of TRIzol reagent (15596026; Thermo Fisher Scientific Inc., Shanghai, China) according to the protocol of the manufacturer, and the concentration of the isolated RNA was measured using a Nanodrop spectrophotometer (NanoDrop 1000; Shanghai, China). After determining the total RNA concentration, reverse transcription to cDNA was carried by utilizing 1,000 ng of total RNA with FastKing gDNA Dispelling RT SuperMix (KR118, TIANGEN, Beijing, China). A real-time PCR thermal cycler (CFX Connect real - Time system 1855201; Bio-RAD, CA) was used to perform RT-qPCR analysis. The PCR cycle conditions were set as follows: an initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. All reactions were performed in triplicate to ensure data accuracy. Relative mRNA expression levels were quantified using the 2−ΔΔCT method, with β-actin as the reference gene for normalization of the target gene's mRNA level. The primers employed in the amplification process were synthesized by Beijing Tsingke Biotech, with a concentration of 10 μM, and their sequences detailed in Table S1.

Western Blotting

Harvested PGCs were lysed by the addition of radio-immunoprecipitation assay (RIPA; R0010; Attachment Phenylmethanesulfonyl fluoride (PMSF) Solarbio Life Science, Beijing, China) in the presence of a phosphatase inhibitor cocktail (B15001; Selleck, Shanghai, China). Total protein was extracted by lysis on ice, with the concentration determined using a BCA Protein Assay Kit (CW0014, CWBIO; Taizhou, China). Lysates were separated on 4–20% GenScript SurePAGE gels (M00655; GenScript, Nanjing, China) and subsequently transferred to hydrophobic PVDF transfer membranes (ISEQ00010; Merck, Shanghai, China). The membranes were initially blocked in 5% skimmed milk for 2 h at ambient temperature, followed by washing in 1× TBST and overnight incubation at 4°C with the following antibodies diluted with NCM universal antibody diluent: pan-AKT1/2/3 antibody – internal (1:1000: AF6261; Affinity, Changzhou, China), JAM-A/CD321/F11R Rabbit pAb and SLC7A11/xCT rabbit pAb (1:500: A1241; 1:1000: A13685; Abclonal, Shanghai, China), ZO-1 polyclonal antibody (1:500: 617300; Thermo Fisher Scientific Inc., Shanghai, China), GPX4 monoclonal antibody and GAPDH polyclonal antibody (1:1000: 67763-1-Ig; 1:5000: 10494-1-AP; Proteintech, Wuhan, China), mouse anti-PIK3R1 monoclonal antibody, rabbit anti-phospho-PI3 kinase p110 beta (Ser1070) antibody, and rabbit anti-phospho-AKT (Ser473) antibody (1:1000: bsm-33219M, bs-6417R, bs-0876R; Bioss, Beijing, China). Thereafter, the membranes were washed 3 times with 1× TBST and incubated with a suitable secondary antibody [goat anti-mouse IgG (1:5,000: CW0102 CWBIO; Taizhou, China) or goat anti-rabbit IgG (1:5,000: CW0103, CWBIO; Taizhou, China)] at room temperature for 2 h. Finally, the expression of the target proteins was monitored using an enhanced ECL chemiluminescence detection kit (E411-04; Vazyme, Nanjing, China) in a chemiluminescent system (ChemiDocImagingSystems, BIO-RAD, Singapore). Grayscale values were obtained using ImageJ software (version 1.8, National Institutes of Health, Bethesda, USA), which was also used to perform quantitative analysis.

Detection of Apoptosis Using the FITC/PI Double-Staining Method

PGCs seeded in 6-well plates were treated with LY294002 as described in a previous section to assess the levels of cell apoptosis. Following a 72-h incubation, the cells were treated using an Annexin V-FITC Apoptosis Detection Kit (40302ES60; Yeasen Biotechnology, Shanghai, China), with apoptosis determined based on flow cytometric analysis (BD LSRFortessa, Biosciences, Shanghai, China), with reference to the manual of the manufacturer for specific steps. Experimental data were processed using FlowJo software (FlowJo_v10.8.1; BD, Ashland, USA).

Cell Cycle Analysis

PGCs were collected and centrifuged at 1,200 rpm for 5 min, with removal of the resulting supernatant. Following the addition of pre-cooled PBS, the cells were re-centrifuged, after which they were gently mixed with pre-cooled 70% ethanol and left to fix overnight. The following day, a mixture of 0.5 mL of staining buffer, 10 μL of propidium iodide, and 10 μL of RNase A was added to the cells followed by incubation for 30 min at 37°C, ensuring protection from light. Thereafter, the cells were subjected to a flow-through assay using cell cycle and apoptotic analysis kit (40301ES60; Yeasen Biotechnology, Shanghai, China) in accordance with the instructions of the manufacturer.

Reactive Oxygen Species Assay

Cells were collected by centrifugation, and following the addition of 10 μM DCFH-DA, they were incubated at 37°C for 2 h, with mixing at 10-min intervals. The cells were then washed with serum-free cell culture medium to remove any DCFH-DA that had not entered the cells and were subsequently subjected to flow cytometry. Assays were performed using a Reactive Oxygen Species Assay Kit (50101ES01; Yeasen Biotechnology, Shanghai, China) in accordance with the instructions of the manufacturer.

Assays for GSH/GSSG/MDA and Fe2+

The reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) contents of cells was assayed using a GSH and GSSG assay kit (S0053; Beyotime, Shanghai, China) in accordance with the instructions of the manufacturer. The GSH content of the test samples was calculated as: total glutathione GSSG × 2. The content of malondialdehyde (MDA) in cells was measured using a lipid peroxidation MDA assay kit (KGT004-1; KeyGEN BioTECH, Nanjing, China) according to the instructions of the manufacturer. Cells were centrifuged for 10 min at 12,000 × g and 4°C and the resulting supernatants were collected for ELISA assays.

Ferrous ion detection was performed using a cell ferrous iron colorimetric assay kit (E-BC-K881-M; Elabscience, Wuhan, China). PGCs were lysed on ice for 10 min following the addition of 0.2 mL of reagent I to approximately 1 × 106 cells. After centrifuging the lysates at 15,000 × g for 10 min, 80 μL of Reagents II and III each were added to 80 μL of supernatant, followed by incubation for 10 min at 37°C and optical density (OD) determination at 593 nm.

Statistical Analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS (version 25.0; IBM Corp, Armonk, NY, USA) and GraphPad Prism softwares (version 8; GraphPad Software, California, USA) and respective histograms were drawn. Statistical significance was set at P < 0.05.

RESULTS

Culture and Identification of Chicken Primordial Germ Cells

To investigate the effects of the AKT pathway on the biological characteristics of chicken PGCs, these cells were initially cultured using the method described by Collarini. Morphological observations revealed that the cultured PGCs grew in a circular, single-dispersed suspension (Collarini, et al., 2019) (Figure 1A). PAS staining revealed that these cells were PAS positive, indicating that large amounts of lipid droplet-like material is attached to the cell surface (Figure 1B). RT-qPCR results revealed a significantly higher expression of the totipotency genes NANOG, Oct4, and SOX2 and germline marker genes DAZL, CVH, and C-kit than those of CEF (P < 0.05, Figures 1C–I). Combined with the positive immunofluorescence results obtained for SSEA-1 and CVH (Figure 1J), these findings indicated that the chicken PGCs were successfully cultured.Figure 1 . In vitro cultivation and identification of primordial germ cells. (A) Typical primordial germ cell (PGC) morphology, Scale bars indicate 50 μm. (B) Periodic Acid Schiff stain, Scale bars indicate 10 μm. (C–I) Expression of pluripotency and marker genes determind by RT-qPCR. Data are expressed as the mean ± SD of 3 independent experiments (*p < 0.05, **P < 0.01, ***P < 0.001). (J) Immunocytochemical detection of SSEA-1/CVH in cultured chicken PGCs. Scale bars indicate 100 μm.

Figure 1

AKT Signaling Pathway Plays a Key Role in PGCs

AKT signaling plays an essential role in the in vitro culture of PGCs (Matsui, et al., 2014). To further clarify the effects of the AKT signaling pathway on PGC culture in vitro, we assessed the effects of 0, 60, and 120 nM IGF-1 (an activator of the AKT signaling pathway) on PGC cultures. Observations after 3 d revealed a significant aggregation of cells with increasing activator concentration (Figure 2A). Contrastingly, activation of the AKT pathway had no significant effect on the proliferation, pluripotency, or marker genes of PGCs (Figures 2B–2C). However, in response to high IGF-1 concentration, we detected increases in the levels of AKT phosphorylation and cell adhesion factors (Figures 2D–2F). These observations indicate that the selected concentrations of IGF-1 do not promote PGC proliferation in the culture system assessed in this study, and thus do not optimize the culture system.Figure 2 . Activation of the AKT signaling pathway in PGCs during in vitro culture. (A) Morphological observation of PGCs after 3 d of IGF-1 culture. (B) Cell proliferation rate of PGCs determined using a CCK-8 assay. (C) Pluripotency and marker gene expression of PGCs. (D–F) Effects of IGF-1 on the PI3K/AKT signaling pathway and cell adhesion factors. Data are expressed as the mean ± SD of 3 independent experiments. Values within a column followed by different superscript letters differ significantly (P < 0.05).

Figure 2

Previous studies conducted in our laboratory have revealed that upon removal of insulin from B27, PGCs show limited proliferation and AKT phosphorylation is inhibited (Ye, et al., 2023). In a subsequent experiment, we examined the effects of the AKT pathway inhibitor LY294002 at concentrations of 0, 10, 20, 30, 40, and 50 μM for 3 d. The results revealed a gradual increase in cell fragmentation with increasing inhibitor concentration (Figure 3A). In addition, the findings of a CCK8 assay indicated that inhibition of the AKT pathway led to a reduction in PGC proliferation (Figure 3B), with the degree of inhibition increasing with increasing concentration of LY294002, and complete inhibition of the PI3K/AKT pathway was detected at a concentration of 40 μM (P < 0.05; Figures 3C–E). Consequently, this concentration was selected for subsequent experiments.Figure 3 Effects of LY294002 on the PI3K/AKT signaling pathway. (A) Morphology and proliferation of PGCs after incubation with LY294002 for 3 d. (B) Cell proliferation of PGCs after incubation with LY294002 determined using a CCK-8 assay. (C–E) Effects of LY294002 on the PI3K/AKT pathway determined by western blotting. (F and G) EdU proliferation detection and statistical analysis (H) Effects of LY294002 on the cell cycle of PGCs determined by flow cytometry. (I) The expression of cell cycle genes in PGCs. Data are expressed as the mean ± SD of 3 independent experiments. The same letter indicates no significant difference between the 2 groups of data, whereas different letters indicate that the difference is statistically significant (P < 0.05).

Figure 3

Inhibition of AKT Signaling Reduces PGC Proliferation

To further verify the effects of the AKT signaling pathway on the proliferative capacity of PGCs, we examined the effects of inhibitor treatment on PGC proliferation using an EdU assay and compared with the control. We observed that LY294002 induced a significant inhibition of PGC proliferation (P < 0.05; Figures 3F and 3G). Abnormal cell cycle progression can lead to a dysregulation of cell proliferation (Otto and Sicinski, 2017). To examine the cell cycle status in LY294002-inhibited PGCs, we performed flow cytometric analysis based on PI labeling. Compared with the control group, we found that LY294002 treatment of PGCs resulted in a reduction of cells in the G0/G1 phase, an increase in the proportion of cells in the S phase, and no significant change in the percentage of cells in the G2 phase (Figure 3H). These findings indicate that inhibition of AKT signaling can contribute to inducing G1/S cell cycle arrest and thereby inhibit the proliferation of PGCs.

To examine the potential mechanism whereby the inhibition of AKT signaling leads to G1/S phase block in PGCs, we assessed the effect of LY294002 on the expression of the cell cycle-related genes CCND1 and E2F1. The results revealed that LY294002 significantly inhibited the expression of these 2 genes (P < 0.05; Figure 3I), thereby indicating that inhibition of the AKT pathway down-regulates expression of the CCND1 and E2F1 genes, and consequently causes cell cycle arrest in PGCs.

Inhibition of the AKT Signaling Pathway Reduces PGC Adhesion

Cell proliferation is associated with the activity of cell adhesion molecules (Chen, et al., 2021), and to investigate the effects of LY294002 on the adhesion of PGCs, we performed RT-qPCR to analyze the expression of cell adhesion-related genes. The results revealed that LY294002 significantly reduced the expression of the ZO-1, JAM, and β-catenin genes (Figure 4A), and the expression of cell adhesion-related proteins was further assessed by performing western blot analysis (P < 0.05; Figures 4B–4C). These findings accordingly indicated that treatment with LY294002 reduced the adhesive capacity of PGCs.Figure 4 Inhibition of the AKT signaling pathway reduces PGC adhesion. (A) Relative expression of cell adhesion markers. (B and C) The expression and statistics of ZO-1 and JAM in PGCs following LY294002 inhibition. (D) The expression of pluripotency and marker genes. (n = 3). Data are expressed as the mean ± SD of 3 independent experiments (*p < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Inhibition of the AKT Signaling Pathway Reduces the Reproductive Potential of PGCs

To assess the effects of AKT signaling on the reproductive potential of PGCs, we examined gene expression in these cells 72 h after treatment with LY294002. RT-qPCR analysis revealed that the expression of PGC pluripotency and that of marker genes, including NANOG, Oct-4, SOX2, CVH, C-kit, and DAZL, was markedly down-regulated in the LY294002-treated group (P < 0.05, Figure 4D), thereby indicating that inhibition of the AKT signaling pathway results in a significant reduction in the reproductive potential of PGCs.

Inhibition of the AKT Signaling Pathway Promotes Apoptosis in PGCs

To investigate the effects of AKT signaling on apoptosis in PGCs, we initially determined the expression of apoptosis-related genes by RT-qPCR. The results indicated that in response to the inhibition of AKT pathway, the expression levels of apoptosis-related marker genes caspase-3, caspase-6, caspase-8, caspase-9, C-myc, and BAX were significantly up-regulated in PGCs (P < 0.05), whereas the anti-apoptotic marker BCL-2 was significantly down-regulated (P < 0.05) (Figures 5A–5G). The combination of BCL-2 down-regulation and BAX up-regulation is an important marker for the occurrence of apoptosis in cells (AlBasher et al., 2018), and thus our observations provide evidence for the occurrence of PGC apoptosis following inhibition of the AKT pathway. To further clarify the effects of AKT pathway inhibition on the levels of apoptosis in PGCs, we performed flow cytometric analysis based on FITC/PI labeling and accordingly detected a significant increase in the apoptosis of cells in the LY294002 group (P < 0.05), which was consistent with the results of our gene expression analysis (Figures 5H–5J). On the basis of these findings, we thus concluded that inhibition of the AKT pathway promotes apoptosis in PGCs.Figure 5 Inhibition of the AKT signaling pathway promotes the apoptosis of PGCs. (A–G) Expression of apoptosis related genes at 3 d after LY294002 treatment. (H–J) Flow cytometry analysis and statistics of PGC apoptosis after inhibition by LY294002. (n = 3). Data are expressed as the mean ± SD of 3 independent experiments (*p < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Inhibition of the AKT Signaling Pathway Induces Ferroptosis in PGCs

Ferroptosis is a novel mode of iron-dependent cell death discovered in recent years, characterized by the iron-dependent accumulation of reactive oxygen species (ROS) and lipid peroxides (MDA) and depletion of GSH (Mou, et al., 2019; Yan and Zhang, 2019). To investigate the effects of the AKT pathway on ferroptosis in PGCs, we assessed the effects of pathway inhibition on the levels of Fe2+, ROS, MDA, and GSH. Compared with the control group, those treated with LY294002 were found to be characterized by significantly higher levels of Fe2+, ROS, MDA, and GSSG (P < 0.05), and a significant reduction in the levels of GSH (P < 0.05), thereby providing evidence for the occurrence of ferroptosis in PGCs (Figures 6A–6G). SLC7A11 and glutathione peroxidase 4 (GPX4) play key roles in the regulation of ferroptosis (Bridges, et al., 2012; Wang, et al., 2022a), and in the present study, we detected a significant reduction in the expression of both GPX4 and SLC7A11, as well as that of the TFR1, ACSL4, and NOX4 genes (P < 0.05; Figures 6H–6L). Corresponding western blot analysis revealed reductions in the levels of the SLC7A11/GPX4 proteins in LY294002-treated cells (P < 0.05; Figures 6M–6O). On the basis of these findings, we thus speculate that AKT signaling may induce ferroptosis in PGCs by activating SLC7A11/GPX4.Figure 6 Inhibition of the AKT signaling pathway induces ferroptosis in PGCs. (A) Effects of LY294002 treatment on the Fe2+ content of PGCs. (B and C) Flow cytometry analysis of the changes in reactive oxygen species (ROS) content in PGCs and statistical chart of mean fluorescence intensity of ROS in each group. (D) Changes in malondialdehyde (MDA) in PGCs following LY294002 treatment. (E–G) Changes in the reduced glutathione (GSH)/oxidized glutathione disulfide (GSSG) content in PGCs treated with LY294002 for 3 d. (H–L) Expression of ferroptosis-related genes determined by RT-qPCR. (M–O) The expression and statistics of SLC7A11 and GPX4 in PGCs following LY294002 inhibition. Data are expressed as the mean ± SD of 3 independent experiments (*p < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

DISCUSSION

Although chicken PGCs can be cultured in vitro for long periods of time, prolonged culturing results in a gradual loss of germline competence and retarded proliferation (Raucci, et al., 2015). Consequently, to facilitate the effective self-renewal of PGCs, optimising the culture conditions and detemining the specific effects of each of the different culture medium constituents is important. The proliferation of avian PGCs is associted with the activity of multiple signaling pathways, such as Hedgehog (Chen, et al., 2016) and Wnt (Lee, et al., 2016). Activation of the PI3K/AKT pathway promotes cell proliferation (Díaz-Muñoz, et al., 2013), and cell adhesion is involved in cell proliferation (Achiha, et al., 2020). Furthermore, integrin and E-cadherin promote cell survival and proliferation via the PI3K/AKT pathway (Crossland, et al., 2013) and PI3K/AKT signaling enhances cell adhesion (Panebianco, et al., 2017; Ding, et al., 2021; Hsu, et al., 2022), whereas disruption of the integrin or E-cadherin pathway typically leads to cell death and differentiation (Shim, et al., 2001; Li, et al., 2012b). In undifferentiated hESCs, E-cadherin is co-expressed with the pluripotency factors Oct4, Nanog, and Sox2 (D'Amour., et al., 2005). In addition, the expression of the pluripotency genes Oct-4 and Nanog depends on the activation of the AKT signaling pathway (Almozyan, et al., 2017). In the present study, we found that although treatment of PGCs with the PI3K/AKT activator IGF-1 promoted the expression of PI3K/AKT and adhesion factor genes and proteins, it had no similar effect on the PGC proliferation, and had no significant effects on the expression of germ cell characterization genes, such as Oct-4 and NANOG. Contrary to the findings of previous studies, we speculate that IGF-1 supplementation under these culture conditions does not contribute to their optimization and that an appropriate level of AKT activation is essential for germ cell proliferation.

The inhibitor LY294002 has previously been used to study the PI3K/AKT signaling pathway (Zhuang, et al., 2011), which mediates cell proliferation based on the degree of phosphorylation of signaling molecules (Horn, et al., 2017; Shin, et al., 2019; Brand, et al., 2022). Treatment of mouse H9C2 cardiomyocytes with LY294002 reduces cell proliferation and the expression of P-PI3K and P-Akt, although had no significant effect on the expression of PI3K and AKT (Xing, et al., 2022). Additionally, inhibition of PI3K/AKT signaling significantly suppressed cell adhesion in gastric cancer and leukemia cells (Peng, et al., 2020). Our observations in the present study are consistent with these findings. Additionally, LY294002 can down-regulate the expression of cell cycle proteins B1 and CCND1, thereby inducing G2/M blockade and inhibiting the proliferation of renal carcinoma and progenitor Leydig cells (Barrett, et al., 2019; Li, et al., 2023). Again, our findings in the present study are consistent with those repoted previously, as treatment of PGCs with LY294002 resulted in reduced levels of cell cycle genes. These findings thus provide evidence to indicate that inhibition of PI3K/AKT alters cell cycle progression and suppresses PGC proliferation.

The BAX/Bcl-2 pathway can induce the apoptosis of Huh7 cells in response to the inhibition of PI3K/AKT in Huh7 cells (Sui, et al., 2015). In the present study, we thus investigated the extent of apoptosis following the inhibition of PI3K/AKT signaling, as an increased BAX/Bcl-2 ratio can lead to the release of cytochrome c from mitochondria and activation of caspase-9 and caspase-3 (Czabotar, et al., 2014). We found that LY294002 treatment promoted an increase in the expression of the pro-apoptotic factor BAX, whilst causing a reduction in the expression of Bcl-2, thereby resulting in an increase in the BAX/Bcl-2 ratio. We also evaluated the expression of other apoptosis-related genes, namely, caspase-3, -6, -8, and -9 and accordingly found that LY294002 treatment promotes an up-regulation of all 4 genes. The findings of flow cytometric analyses confirmed that LY294002 promotes apoptosis in PGCs, which is consistent with previous results. Thus, our findings indicate that inhibition of the PI3K/AKT pathway promotes apoptosis in PGCs.

Previous research has indicated that inhibition of the AKT/mTORC1 pathway can lead to the suppression of GPX4 synthesis (Cai, et al., 2023). Additionally, the process of ferroptosis is characterized by reductions in the expression of GPX4 and SLC7A11 and a corresponding increase in the expression of NOX4 (Li and Leung, 2020; Wu, et al., 2021). TFR1 is a specific marker of ferroptosis, the high levels of which contribute to the deposition of iron in mouse liver (Feng, et al., 2020; Song, et al., 2022). Similarly, high levels of ACSL4 can promote ferroptosis in mouse kidneys (Wang, et al., 2022b). Elevated levels of Fe2+ in organisms are associated with disturbance of the oxidative/antioxidant system, biochemical manifestations of which include a depletion of oxidized GSH, an accumulation of ROS, and excessive levels of the toxic lipid peroxidation product MDA (Shah, et al., 2018; Seibt, et al., 2019; Ursini and Maiorino, 2020). We found that LY294002 inhibits the expression of GPX4, promotes significant increase in the intracellular levels of Fe2+, GSSG, and MDA, and induces the occurrence of ferroptosis in PGCs, which is consistent with the findings of previous studies. Our findings thus indicate that inhibition of PI3K/AKT signaling induces ferroptosis in PGCs.

Collectively, our observations indicate that an elevation in AKT signaling levels does not result in an acceleration of PGC proliferation. Conversely, the inhibition of AKT signaling was found to have a pronounced effect on PGC proliferation, characterized by the induction of cell apoptosis. These findings indicate that the maintenance of appropriate AKT levels is conducive to PGC self-renewal.

CONCLUSIONS

In this study, we established that PI3K/AKT plays important role in the proliferation, reproductive potential, cell cycle, and apoptosis of chicken PGCs. These findings provide a theoretical basis for further optimizing the system used to culture chicken PGCs and will provide a valuable reference for the development of stable and effective in vitro culture media for PGCs. Moreover, we believe our findings will contribute to promoting the application of this cellular tool in developmental biology and transgenic chicken production.

DISCLOSURES

The authors declare no conflicts of interest.

Appendix Supplementary materials

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ACKNOWLEDGMENTS

This study was supported by STI 2030-Major Projects (2023ZD0407503 ), the National Key Research and Development Program of China (2021YFD1200301 ), National Natural Science Foundation of China (32272858, 32172718, 32372861 , and 32202655 ), Excellent Youth Foundation of Jiangsu (BK20220117 ), China Postdoctoral Science Foundation (2022M722697 ), International Science and Technology Cooperation Projects of Yangzhou (YZ2022206 ), JBGS Project of Seed Industry Revitalization in Jiangsu Province (JBGS (2021) 029 ), and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104140.
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