DOI: https://doi.org/10.21203/rs.3.rs-2202541/v1
Mercury (Hg) is a toxic heavy metal that is widely present in various feeds and showed the serious risk to animal reproductive performance [1]. Normal ovarian follicular development is important for reproductive performance in laying hens. Objective Previous studies have demonstrated that Hg exposure could cause ovarian pathological changes and follicular atresia in mammals [2,3]. During follicular development, apoptosis of granulosa cells in mammalian follicles is the fundamental reason of follicular atresia [4]. Ma et al. found that Hg could not only cause the nephrotoxicity, but also reach the reproductive system inducing reproductive toxicity in mammals [5-7]. Therefore, the investigation of Hg-induced apoptosis follicular granulosa cell apoptosis will be the crucial in revealing the molecular mechanism of reproductive performance in laying hens.
A close relationship was occurred between unfold protein response (UPR) and apoptosis [8]. Once the cell is in an unstable state, various of signaling pathways were activated by UPR to correct the deposition of faulty or unfolded proteins in the endoplasmic reticulum (ER) lumen [8,9]. When the endoplasmic reticulum stress (ERS) is activated, unfold protein deposition separates glucose regulated protein 78 from the three transmembrane receptors, which include glucose activating protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6) [10,11]. ATF6 is a transmembrane type of protein in the ER. Once apoptosis is triggered, ATF6 is dissociated from the Bip protein and transferred to the Golgi apparatus, which is cleaved by S1P and S2P, which were two kinds of Golgi proteases. Cleaved ATF6 penetrates the nucleus, activating the CHOP gene expression and induces the apoptosis progress [10,11]. Therefore, studying the apoptosis in follicular granulosa cells mediated by ERS will provide a basis for revealing the mechanism of reproduction in laying hens.
Selenium (Se), an essential trace element in animals, is vital for sustaining reproductive performance success [12]. Previous studies proved that appropriate selenium levels could promote the development of reproductive organs and sexual maturity in animals [13,14]. So far, it is not clear whether Se could effectively alleviate the apoptosis in follicular granulosa cells and whether ATF6/CHOP pathway is involved is also unknown. Therefore, we investigated the impact of mercury on apoptosis of follicular granulosa cells in laying hens, investigated the protective effect of selenium on apoptosis and further studied the ATF6/CHOP pathway to reveal the molecular mechanism. The findings provided some theoretical basis for confirming the toxicological effect of Hg and the protective effect of selenium on reproductive performance at the molecular level in laying hens.
Cell Culture and Reagents
The follicular granulosa cells in Hyline-Brown laying hens aged two years were isolated and cultured according to the preliminary research method [15]. In brief, the ovaries of non-gestating laying hens were procured from a local slaughter house and immediately brought to the 4°C refrigerator in the laboratory. The follicular liquid in ovary was extracted by a syringe needle and immediately centrifuged at 800g for 5min to isolate the granulosa cells. Cells were cultured with 0.2% collagenase (abs47048001, Absin, Shanghai, China) in a water bath at 37°C for 6 min. Cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (abs9483, DMEM, Absin, Shanghai, China) containing 14% fetal bovine serum (abs972, Absin, Shanghai, China) at a humidified 5% CO2 incubator (Ou Meng Industrial Co. Ltd., Shanghai, China) at 37°C.
Hg (B8945, purity > 99.0%, Baiaolaibo Bio-Tech, Beijing, China) dissolving in 0.1 M sterile phosphate buffered saline (PBS) was added to the medium to provide 15 μM Hg. Se (sodium selenite, X0367, purity > 99.0%) was obtained from Xinxinjiali Bio-Tech Co. (Wuhan, China) and 4-phenyl butyric acid (B3859, 4-PBA, an inhibitor of ATF6/CHOP pathway) was purchased from Baiaolaibo Bio-Tech Co. (Beijing, China). The Se dissolving in sterile PBS was prepared to the corresponding concentration for the further experiment. Antibodies of CHOP, ATF6 and β-actin were obtained from Abcam (Cambridge, England).
Experimental Design
For detection of the function of ERS in the protection of Se on apoptosis, granulosa cells (GCs) were cultured, exposed with 15μM Hg (15Hg), 12μM Se (12Se) and 0.1μM 4-PBA alone or in combination. The 5 groups were as followed: Control, 15Hg, 12Se, 15Hg +12Se, 15Hg+4-PBA and 15Hg +12Se+4-PBA. The representative pictures of follicular granulosa cells isolated successfully were shown in Supplement Figure 1.
Cell Viability Assay
Cells viability assay was performed according to a cholecystokinin-8 (CCK-8) kit detection. After 24 hours of treatment, the follicular granulosa cells were incubated in 96 well-plates with 10 μL CCK-8 reagent (Absin, Shanghai, China) for 2 hours at 37°C. The absorbance of cells was then measured using a microplate reader (Absin, Shanghai, China) at 450 nm. The cell activity was calculated according to a previous methodology [16].
Nuclear Condensation Assays
When the cells were treated for 24 hours, the follicular granulosa cells fixed in 4% paraformaldehyde were rinsed 3 times with sterile PBS. The cells were incubated with 4',6-diamidino-2-phenylindole (DAPI, Absin, Shanghai, China) to examine the nuclear morphology below a fluorescence microscope (Ou Meng Industrial Co. Ltd., Shanghai, China). The degree of nuclear condensation was qualitatively determined according to fluorescence brightness.
Annexin V/Propidium Iodide Staining Assay
When the cells were treated for 24 hours, the follicular granulosa cells were digested by 0.2% trypsin, the cells suspensions were obtained by means of washing the cells with PBS and centrifugating suspension at 1200g for 5min. The cells were stained with Annexin V-FITC binding and propidum iodide (Absin, Shanghai, China) and quantitated by a flow cytometer (Ou Meng Industrial Co. Ltd., Shanghai, China). The apoptosis rate was calculated as followed: Apoptosis rate (%) = The numbers of early apoptotic and late apoptotic cells / The total number of cells × 100%.
Mitochondrial Membrane Potential Assay
When the cells were treated for 24 hours, the follicular granulosa cells were digested by 0.2% trypsin, the cells suspensions were obtained by means of washing the cells with PBS and centrifugating suspension at 1200g for 5min. Cells were treated with the JC-1 fluorescent probe for 30 minutes at 37°C in a dark environment. The follicular granulosa cells’ fluorescent intensities were determined by a flow cytometer (Ou Meng Industrial Co. Ltd., Shanghai, China). The calculation method of mitochondrial membrane potential reduction is based on a previous study [17].
Quantitative Reverse Transcription-polymerase Chain Reaction
After 24 hours of treatment, the follicular granulosa cells were lysed with cell lysis fluid (Absin, Shanghai, China). The intracellular total RNA was extracted using a TRIzol reagent (Absin, Shanghai, China) and a reverse transcription kit (Absin, Shanghai, China) was used to reverse-transcribed to the cDNA. The PCR assays were carried out in a LightCycler96 (Roche, Basel, Switzerland). The PCR reaction system as as follows: 5 μL of 2 × SYBR Premix Ex TaqTM (Absin, Shanghai, China), 0.5 μL of diluted cDNA template, 4.1 μL of RNase-free water, and forward and reverse primers. The 5’ to 3’ oligonucleotide primer sequences were referenced with a previous literature, which was presented in Supplement Table 1. The PCR amplification was as followed: initial denaturation at 94.5°C for 29 s, followed by 41 cycles of 94.5°C for 5 s, 59.5°C for 29 s, and 71.5°C for 31 s. The 2−ΔΔCt method was used to analyze the relative quantitative data of each gene by the according to a previous study [18].
Western-blot analysis
The expressions of ATF6 and CHOP proteins were determined by the western-blot analysis. Intracellular total proteins were extracted according to the radioimmunoprecipitation method. The total proteins were separated using 10% polyacrylamide gels electrophoresis and then transferred to the polyvinylidene difluoride membranes (Beyotime Co. Ltd, Wuhan, China). The membranes were blocked with 5.0% skim milk for 20 h and incubated overnight with ATF6 (1:500, Beyotime Co. Ltd, Wuhan, China), CHOP (1:500, Beyotime Co. Ltd, Wuhan, China) and β-actin (1:3000, Beyotime Co. LTD, Wuhan, China) antibodies, and then were incubated with the secondary antibody against rabbit IgG. The protein signals were detected using a western blot imaging system (JP-K300, Jinpeng science technology Co. Ltd, Shanghai, China) and relative abundance of each protein was expressed as optical density ratios.
Statistical Analysis
All variance statistical methods in this study were based on one-way variance analysis and all experimental results were expressed as mean ± standard error. Tukey multiple range test was used for the multiple comparison by SPSS 21.0 (SPSS Inc., Chicago, IL, USA) when there was any significant difference among any groups (P < 0.05).
The effect of Se on cell viability in follicular granulosa cells
In comparison to the control group, 15 μmol/L Hg significantly decreased cell viability (P < 0.05), while 12 μmol/L Se addition totally reverse this reduction (P < 0.05). Meanwhile, after pretreated with the 4-PBA, the cell viability was sharply increased compared with the 15Hg group (P < 0.05; Figure 1).
The effect of Se on nuclear condensation in follicular granulosa cells
The picture in the control group presented that the nuclear morphology in follicular granulosa cells was similar to the round shapes under a fluorescence microscope. However, the nuclear in follicular granulosa cells became condensate after 15 μmol/L Hg addition. However, the degree of nuclear concentration was reduced after pretreated with the 4-PBA (Figure 2).
The effect of Se on apoptosis rate and apoptosis-related gene expression in follicular granulosa cells
The apoptosis rate was sharply increased in 15 μmol/L Hg group compared to the control group (P < 0.05), but 12 μmol/L Se addition partly reverse this enhancement (P < 0.05). Meanwhile, after pretreated with the 4-PBA, the apoptosis rate was sharply decreased compared with the 15Hg group (P < 0.05; Figure 3A, B).
Gene expressions of Bax/Bcl-2, Caspase-3 and Caspase-9 were sharply increased in 15 μmol/L Hg group compared with the control group (P < 0.05), while 12 μmol/L Se addition partly reverse these increases (P < 0.05). Meanwhile, when pretreated with the 4-PBA, gene expressions of Bax/Bcl-2, Caspase-3 and Caspase-9 were sharply decreased compared with the 15Hg group (P < 0.05; Figure 3C).
The effect of Se on mitochondrial membrane potential in follicular granulosa cells
When compared to the control group, the percentage of mitochondrial potential decrease was significantly higher in 15 μmol/L Hg group (P < 0.05), while 12 μmol/L Se addition partly reverse this enhancement (P < 0.05). Meanwhile, compared with the 15Hg group, the percentage of mitochondrial potential decrease was significantly increased after 4-PBA treatment (P < 0.05; Figure 4).
The effect of Se on ERS gene expressions in follicular granulosa cells
The gene expressions of PERK, ATF4, CHOP, IRE1α, TRAF2α, ATF6 and ASK1 were sharply increased in 15 μmol/L Hg group compared with the control group(P < 0.05), while 12 μmol/L Se addition partly reverse these increases (P < 0.05). Meanwhile, after pretreated with the 4-PBA, the gene expressions of PERK, ATF4, CHOP, IRE1α, TRAF2α, ATF6 and ASK1 were sharply decreased compared with the 15Hg group (P < 0.05; Figure 5).
The effect of Se on expressions of ATF6 and CHOP proteins in follicular granulosa cells
When compared to the control group, the expression of ATF6 and CHOP proteins was significantly higher in the 15 mol/L Hg group (P < 0.05), while 12 μmol/L Se addition partly reverse these increases (P < 0.05). Meanwhile, when ATF6 and CHOP proteins were pretreated with 4-PBA, their expression levels were considerably lower than in the 15Hg group (P < 0.05; Figure 6).
Evidence has accumulated that many factors, including abrupt changes in the breeding environment and hazardous feed additives, can cause aberrant follicular development and follicular atresia in laying hens[19-21]. Hg is the most toxic in various feeds and showed the serious risk to animal reproductive performance. In mammals, follicular development is crucial to the reproductive performance [22]. The underlying cause of follicular atresia, which plays a crucial role in reproductive ability, is the apoptosis of follicular granulosa cells[23,24]. Apoptosis is a normal component in the development of multicellular organisms and is essential for follicular atresia processes [25]. Therefore, the investigation of Hg-induced apoptosis in follicular granulosa cells will be crucial in determining the molecular mechanism of reproductive efficiency in laying hens. In this investigation, we discovered Hg expose could significantly reduce the cell viability and induced the nuclear condensate, which meant that apoptosis progress was might be triggered in follicular granulosa cells. Meanwhile, the Flow cytometry showed that Hg could induce the apoptosis and reduction of mitochondrial membrane potential in follicular granulosa cells. According to this study, Hg could induced the cytotoxicity and apoptosis in PC12 cells [26]. The Caspases family, which include caspase-3 and caspase-9, have been proven in studies to play a crucial role in cell apoptosis [27,28]. Caspase is called cysteinyl aspartate specific proteinase. Caspases are a group of proteases with similar structures that exist in the cytoplasm, including caspase-3 and caspase-9. Besides, Bcl-2 (b-cell lymphoma/leukemia-2 gene) is an oncogene, which has a significant inhibitory effect on apoptosis [29]. In this research, Hg-induced apoptosis in follicular granulosa cells was accompanied by significantly reduced gene expressions of caspase-3 and caspase-9, indicating that the caspase family was involved. Previous research found that Hg exposure significantly reduced mitochondrial membrane potential and induced the changes of gene expressions of caspases and Bcl-2 families in vertebrate, which were accordance with this study [30].
Selenium (Se), as the essential trace mineral, is fundamental importance to animal health [31]. Therefore, we explored selenium's protective mechanism against Hg-induced apoptosis in laying hen follicular granulosa cells. In this study, our study elucidated Se could significantly alleviated the decrease of cell viability induced by the Hg exposure in follicular granulosa cells. The nuclear of follicular granulosa cells became condensate after induction by the Hg, while Se addition alleviated the nuclear condensation to some extent, implying that the follicular granulosa cells were in the apoptosis program following the Hg exposure and Se addition alleviated this program. Han et al. found that selenium ameliorated Hg-induced the reduction of cell viability in hepG2 cells, which was accordance with this study [32]. In addition, the Flow cytometry showed that Se supplementation alleviated the Hg-induced apoptosis and reduction of mitochondrial membrane potential in follicular granulosa cells in the present study. Accumulation evidences showed that Se could protect various organs from Hg-induced apoptosis in vertebrate, which were been proved in this study [5]. In this study, the enhancements of gene expressions of caspase-3, caspase-9 and Bax/Bcl-2 were alleviated by Se addition, implying that nano-selenium could alleviate the toxic effect of Hg by regulating the levels of apoptosis genes.
Accumulation previous investigations reported that ERS played a key role in apoptosis induced by various of toxic substances, including heavy metals [33-35]. When Hg activities the apoptosis in cells, intracellular unfold proteins are accumulated and induce endoplasmic reticulum chaperone (glucose regulated protein 78) overloading in cells [36]. Three transmembrane receptors were separated from the glucose regulated protein 78 and inducing the CHOP factor to trigger the cell death, including PERK, IRE1 and ATF6 [10,11]. ATF6 is a kind of transmembrane protein located in the ER. Once apoptosis is triggered, ATF6 is dissociated from the Bip protein and transferred to the Golgi apparatus, which is cleaved by S1P and S2P, which were two kinds of Golgi proteases. Cleaved ATF6 enters the nucleus to activates the CHOP gene expression and induces the apoptosis progress [10,11]. In this study, 4-PBA is a chemical ERS inhibitor in apoptosis progress [37]. In this research, Hg-induced apoptosis was decreased by 4-PBA pretreatment, indicating that Hg-induced apoptosis was related to ERS in follicular granulosa cells. Further, our study elucidated that Hg exposure substantially increased the gene expressions of PERK, ATF4, CHOP, IRE1α, TRAF2α, ATF6 and ASK1 in the present study. However, after 4-PBA pretreatment, the gene expressions of them were considerably down-regulated in follicular granulosa cells. Hg exposure sharply enhanced the expressions of ATF6 and CHOP proteins, while 4-PBA pretreatment partly reverse these increases. These results suggested that the Hg exposure caused apoptosis via ATF6/CHOP pathway in follicular granulosa cells. Meanwhile, Se addition sharply decreased the gene expressions of PERK, ATF4, CHOP, IRE1α, TRAF2α, ATF6 and ASK1, and the expressions of ATF6 and CHOP proteins, implying that Se alleviated Hg cytotoxicity by inhibiting ATF6/CHOP pathway. The possible mechanism for this is that ATF6 could not dissociated from the Bip protein and transferred to the Golgi apparatus leading to the ATF6 could not be cleaved in follicular granulosa cells in laying hens.
Hg exposure caused apoptosis, lowered mitochondrial membrane potential, and dramatically decreased cell viability in follicular granulosa cells of laying hens. Meanwhile, Se addition significantly alleviated the Hg-induced apoptosis in follicular granulosa cells. Furthermore, The apoptosis caused by Hg and the protective impact of Se on the follicular granulosa cells in laying hens were both significantly influenced by the ATF6/CHOP pathway. This study provided the theoretical basis for revealing the effect of Hg on reproductive performance and the protective effect of Se in laying hens.
Compliance with Ethical Standards
The experiments were carried out in accordance with the International Animal Care standards. All experiments were carried out in accordance with the animal use guidelines established by Henan University of Science and Technology in China (approve number: 099-2022).
Conflict of Interest
The authors declare that no commercial or financial relationships that could be construed as a potential conflict of interest existed during the research.