Introduction

Cd is an environmental contaminant that has been recognized to be a risk factor in organism. It can enter human and animal bodies via different industrial products, environmental pollution, and different contaminated food. Several animal studies have revealed that Cd accumulates preferentially in the hepatic and renal tissue and causes distinct pathological changes to the liver and the kidney [1, 2] even in very low concentrations. Besides, Cd exposure can inhibit production of testosterone, reduce sperm motility, and cause testicular damage [3]. Haouem et al. have indicated that Cd has toxic effects on the testicular function of rats and leads to a significant decline in epididymal sperm count [4]. Cd has obvious toxic effects on estrogen synthesis, oocyte, ovary, and uterus of female animals; it can affect the expression of estrogen receptor [5]. Zhang et al. have injected different doses of Cd to Wistar rats for 6 weeks in order to find the effects of Cd on their reproductive system; they found that Cd could cause the decrease of progesterone and estradiol in the ovary of rats [6]. Höfer et al. have reported that an induction of estrogen-sensitive uterine gene expression was observed after treatment with Cd [7]. All these evidences showed that reproduction toxicity induced by Cd.

Autophagy is a conserved evolutionary lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelle [8]. LC3, mTOR, dynein, Beclin 1, and Atg5 are involved in autophagy. This process helps maintain cellular homeostasis. However, a persistent and massive autophagy can also result in tissue damage. Xue et al. have shown that in superior cervical ganglia neurons, HeLa or CHO cells treated with a cell death inducer in the presence of caspase inhibitors, the majority of the mitochondria were destroyed by autophagy, cells became smaller, and they were eventually irreversibly committed to death [9]. Several studies have reported that the toxic metals can cause autophagy including Cd [10, 11]. One of the major regulators of autophagy is the mTOR, which promotes autophagy due to poor nutrients [12]. Cd induces ROS and activates mTOR pathways, and then resulting in autophagy [13, 14]. Wang et al. have reported that the MES-13 mesangial cells were determined to have undergone autophagy after treatment with Cd [15]. Dong et al. have illustrated that low concentrations of Cd promoted autophagy and their results suggested that autophagy played a key role in Cd-induced endothelial dysfunction [16]. Besides, some studies have reported that autophagy was related to energy metabolism [17, 18]. Lock et al. demonstrated an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by mutationally active Ras [19].

Cd is harmful to organism, it is very important to prevent and/or attenuate Cd toxicity. One strategy to attenuate Cd exposure in animal is the use of Se. Se is an organism essential trace element, displaying an antioxidant effective oxygen free radical scavenging [20], protecting the organs and tissues from oxidative damage [21, 22]. Several studies have indicated that the treatment with Se protects various organs and tissues against harmful elements [23, 24] including Cd [25, 26]. Some studies found a protective effect of Se against the Cd toxicity in rodents and humans [27, 28]. Other studies have shown that organic Se-administered could protect broilers from Cd toxicity [29, 30]. Besides, Se is closely related to energy metabolism. Pinto et al. indicated that dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism of major insulin target tissues [31]. At the same time, Se can also exert some influences on autophagy. Liu et al. have indicated that autophagy was involved in the development of liver injury (pathological processes), which is induced by Se deficiency [32]. Frustaci et al. have shown that Se deficiency can cause decline of myocardial antioxidant reserve and enhance cell autophagy [33].

Although Cd toxicity and the antagonism of Se to Cd in mammals have been reported, the effects of Cd on the autophagy of avian ovarian tissue are not clear. In the study, we investigated the ovarian tissue autophagy in chickens after an exposure to Cd and the simultaneous administration of Cd and Se by a dietary route. We tested the ATPase activities, the mRNA expression of glucose metabolism-related genes, and the expression of autophagy-related genes in ovarian tissue of chickens. We evaluated the ovarian tissue autophagy caused by Cd exposure in chickens, as well as the ability of Se to reduce the toxicity of Cd. These results will not only add new and important data on Cd-induced autophagy but also enrich detoxification effect of Se on Cd in chicken ovary.

Material and Methods

Preparation of Animals

All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Forty-eight 50-day-old Isa Brown female chickens were divided randomly into four groups (n = 12 per group). The animal room was maintained at 18–26 °C; the chickens were kept under a 16-h light/8-h dark cycle and were given free access to standard food and water about 12 weeks. According to the LC50 of Cd (218.44 mg/kg diet CdCl2), the exposure doses of Cd (150 mg/kg diet CdCl2) were used in this study. In regard to Se doses, we added and supplied 2 mg/kg Se (super nutritional Se but not Se toxicity) with Na2SeO3 supplement. Control group was fed a basic diet containing 0.2 mg/kg Se; Se-treated group was fed with the basic diet supplemented with Na2SeO3 and the total Se content was 2 mg/kg; Se/Cd-treated group was fed with the basic diet supplemented with Na2SeO3; the total Se content was 2 mg/kg and supplemented with 150 mg/kg CdCl2. Cd-treated group was fed with the basic diet supplemented with 150 mg/kg CdCl2 [34]. The total rearing period was 12 weeks; in the end of experiment, ovarian tissues of chickens were collected.

ATPase Activity

The activities of Na+-K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase were determined according to the manufacturer’s instructions of the appropriate assay kits (Nanjing Jiancheng Bioengineering Institute, China). The activities of the Na+-K+-ATPase, the Ca2+-ATPase, and the Mg2+-ATPase were measured by quantifying the inorganic phosphorus (Pi) production from the conversion of ATP to ADP at 660 nm using the molybdenum blue spectrophotometric method, and were expressed as μmol/mg.pr. When one type of ATPase was tested, the inhibitors of other types of ATPase were added to depress the hydrolysis of phosphate radicals.

RNA Isolation and Quantitative Real-time Polymerase Chain Reaction (q-PCR)

Total RNA was dissociated from the ovarian tissue using Trizol reagent according to the manufacturer’s protocol. The concentration and purity of the total RNA were determined spectrophotometrically at 260/280 nm (Gene Quant 1300/100, General Electric Company, USA). First-strand cDNA was synthesized from 5 lg of total RNA using oligo dT primers and superscript II reverse transcriptase according to the manufacturer’s instructions (Roche, USA). Synthesized cDNA was diluted five times with sterile water and stored at − 80 °C before using.

All of the primers (Table 1) were designed by Premier Software (PREMIER Biosoft International, USA) for q-PCR. Detected via q-PCR, gene expression levels were performed on a LightCycler® 480 System (Roche, Basel, Switzerland) using fast Universal SYBR Green Master Mix (Roche, Basel, Switzerland). The reactions were performed in a 20 μL reaction mixture containing 10 μL of 2 × SYBR Green I PCR Master Mix (TaKaRa, China), 2 μL of either diluted cDNA, 0.4 μL of each primer (10 μM), 0.4 μL of 50 × ROX reference Dye II, and 6.8 μL of PCR-grade water. The PCR procedure for all of the primers consisted of heating the reaction mixture was 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 60 °C for 30 s. The melting curve analysis showed only one peak for each PCR product. Electrophoresis was performed with the PCR products to verify primer specificity and product purity. The relative mRNA abundance was calculated according to the method of 2-ΔΔCt, accounting for gene-specific efficiencies was normalized to the mean mRNA expressions of β-actin.

Table 1 Gene-special primers used in the present study
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Western Blot

Total protein was extracted from ovarian tissues of chickens after cell lyses. SDS-polyacrylamide gel electrophoresis was used to extract protein from 12% gel for western blot. Protein emergent was obtained on nitrocellulose membranes through transfer tank method. Membranes were blocked in 5% skim milk for 2 h and incubated with diluted primary chicken antibodies: autophagy LC3-II, dynein, Beclin 1, Atg5, and mTOR for 1 h at 37 °C, respectively. PBST was used to wash the membrane for four times, 5 min each. After that, membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit Ig G (1:500, Santa Cruz, CA, USA) for 1 h at 37 °C. After washing with PBST for four times, 5 min each, the signal was detected by Chemi Scope 5300 (Clinx Science Instruments, Shanghai, China). The optical density (OD) of each band was determined by Image VCD gel imaging system. Monoclonal β-actin antibody content was analyzed as a control by means of a rabbit polyclonal antibody (Sigma, USA).

Electron Microscopy Detection

Cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4 °C and then post-fixed in 0.1 M phosphate buffer containing 1% osmium tetroxide for 1 h. Ultrathin sections were stained with uranyl acetate-lead citrate and examined by a transmission electron microscopy (TEM).

Statistical Analyses

Statistical analyses of all data were performed using GraphPad Prism (version 5.0, GraphPad Software Inc., San Diego, CA, USA). All the data were analyzed by Student’s t test or one-way ANOVA, had a normal distribution, and passed equal variance testing. Quantitative data are shown as the mean ± SD. Differences of P < 0.05 were considered to be significant.

Results

ATPase Activities in Chicken Ovarian Tissues

In order to explore the toxic mechanism of Cd on chicken ovary, we detected the Na+-K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase in ovarian tissue of chickens. We did not find the chickens died because of Cd poisoning or other infections. As shown in Fig. 1, there was little difference between control group and Se-treated group for the activity of Na+-K+-ATPase in ovarian tissue. Compared with the control group, the activity of Na+-K+-ATPase in corresponding Se/Cd-treated group and Cd-treated group was lower, but the activities of ATPase in Se/Cd-treated group were higher than Cd-treated group. For Ca2+-ATPase, we found that both Se/Cd-treated group and Cd-treated group were lower than corresponding control group; in addition, the activity of Ca2+-ATPase in Se/Cd-treated group was higher than that of Cd-treated group. There were significant differences between control group and Se/Cd-treated group or Cd-treated group (P < 0.05). The activity of Mg2+-ATPase in Se/Cd-treated group and Cd-treated group was significantly lower compared to corresponding control group. There was no significant difference between Se/Cd-treated group and Cd-treated group (P > 0.05). However, the activity of Mg2+-ATPase in Se/Cd-treated group was higher compared to that of Cd-treated group.

Fig. 1
figure 1

The activities of ATPase in ovarian tissue in ovarian tissue of chickens. Bars with different letters represented statistically significant difference (P < 0.05) between the different treated groups at the same ATPase. Each value represents the mean ± SD (n = 12)

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The mRNA Expression of Glucose Metabolism-Related Genes in Chicken Ovarian Tissues

As shown in Fig. 2, we found that the mRNA expression levels of HK2, PK, SDH, and PDHX for Se/Cd-treated group and Cd-treated group were lower than corresponding control group and Se-treated group. Compared with control group, the mRNA expression level of SDH for Cd-treated group decreased the most. For the Se/Cd-treated group, the mRNA expression level of HK2 decreased the least and the mRNA expression level of SDH decreased the most compared to the corresponding control group. Besides that, we found that the mRNA expression levels of HK2 and PDHK had almost no difference in Cd-treated group. Our results showed that the mRNA expression of all the glucose metabolism-related genes for Se/Cd-treated group and Cd-treated group was significantly higher than that of corresponding control group and Se-treated group (P < 0.05).

Fig. 2
figure 2

The mRNA expression of glucose metabolism-related genes in ovarian tissue of chickens. Different letters indicate significant differences (P < 0.05) in different treated groups at the same glucose metabolism-related genes expression. The data are shown as the means ± SD (n = 12)

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The mRNA Expression of Autophagy-Related Genes in Chicken Ovarian Tissues

As shown in Fig. 3, the mRNA expression levels of Beclin 1, dynein, Atg5, LC3-I, and LC3-II for Se/Cd-treated group and Cd-treated group were significantly higher than corresponding control group and Se-treated group. In the Se/Cd-treated group and Cd-treated group, the mRNA expression level of LC3-II increased the most and the mRNA expression level of LC3-I increased the least compared to that of the control group. Whereas the expression of mTOR in Cd-treated group was lower than other three groups. There was a significant difference between control group and Se/Cd-treated group or Cd-treated group (P < 0.05). Additionally, the ratio of LC3-I and LC3-II for Se/Cd-treated group was higher than that of the Cd-treated group.

Fig. 3
figure 3

The mRNA expression of autophagy-related genes in ovarian tissue of chickens. Different letters indicate significant differences (P < 0.05) at the same autophagy-related genes expression. Bars with different letters represented statistically significant difference (P < 0.05) in the different treated groups at the same autophagy-related gene. The data are shown as the means ± SD (n = 12)

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The Protein Expression of Autophagy-Related Genes in Chicken Ovarian Tissues

As shown in Fig. 4, the protein expression levels of Beclin 1, dynein, ATG5, LC3-I, and LC3-II for Se/Cd-treated group and Cd-treated group were significantly higher than corresponding control group and Se-treated group. The expression of LC3-II for Cd-treated group increased the most compared to control group. Whereas the protein expression of mTOR for Se/Cd-treated group and Cd-treated group was lower than corresponding control group and Se-treated group. Moreover, we could find there was a significant difference between control group and Se/Cd-treated group or Cd-treated group about the protein expression level of the autophagy-related genes (P < 0.05). Besides this, the ratio of LC3-I and LC3-II in Se/Cd-treated group was higher than that of Cd-treated group.

Fig. 4
figure 4

The protein expression of autophagy-related genes in ovarian tissue of chickens. a Different letters indicate significant differences (P < 0.05) in different treated groups at the same autophagy-related genes expression. The data are shown as the means ± SD (n = 12). b The immunoblotting of mTOR, Beclin 1, dynein, Atg5, LC3-I, LC3-II, and β-actin at various treated groups in chicken ovarian tissues

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Electron Microscopic Observation of Chicken Ovarian Tissues

To determine whether Se and Cd affect autophagy in the ovarian tissue, TEM was used to perform ultrastructural analysis. From the control group (Fig. 5a), we found the ovarian cell structure integrity, nuclear membrane was clear, and the structure of the mitochondria in the cytoplasm was integrated. From Cd-treated group (Fig. 5d), we found that Cd led to ultrastructure changes, including nuclear membrane shrinkage, variation of chondriosome’s structure. In addition, there were several double-membrane vesicles appeared in Cd-treated cells; similar vesicles were rarely seen in the normal cells. The result showed the formation of autophagosomes and autophagolysosomes in Cd-treated cells, suggesting that Cd promotes autophagy in ovarian cells. From the Se/Cd-treated group (Fig. 5c), we also found the ultrastructure changes like the Cd-treated group; however, the degree was smaller.

Fig. 5
figure 5

The results of the ovarian tissue ultrastructure observations by electron microscopy. In control, Se-treated, Se/Cd-treated, and Cd-treated groups are shown respectively in a, b, c, and d. Magnification was 30,000 for all parts. Red arrow represents mitochondria. Green arrow represents chromatin aggregation. Yellow arrow represents autophagy vacuole

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Discussion and Conclusion

From the experiment investigation, we found Cd exposure can cause mitochondrial damage. In eukaryotic cell, mitochondrion is the main site of energy metabolism, whereas mitochondrial damage can cause energy metabolism disorder [35]. We found that the activities of ATPase and the mRNA expression of glucose metabolism enzyme including HK2, PK, PDHX, and SDH decreased when the ovarian tissue of chickens were treated with Cd. Krstić et al. found some metals including Cd-inhibited Na+-K+-ATPase and Mg2+-ATPase activities [36]. Kim et al. found a significant increase in Na+-K+-ATPase activity after exposure to Cd and Pb in a concentration-dependent manner [37]. Both HK2 and PK are the important enzymes in the anaerobic glycolysis process. HK2 can catalyze the conversion of glucose to glucose-6-phosphate; it is a key enzyme in the process of glycolysis. ATP is formed in the payoff phase of glycolysis by two reactions, one of which promoted by PK. PDHX and SDH are all involved in aerobic glycolysis pathway in the mitochondrial inner membranes. PDHX can oxidize pyruvate to acetyl coenzyme A and SDH is an essential enzyme in the citric acid cycle. Canesi et al. found a significant decrease in HK2 activity in Cu-exposed mussels [38]. Pysher et al. suggested that alterations in renal HK2 expression may be involved in arsenic-induced pathological conditions involving the kidney [39]. Our results are similar to those reported above. It is suggested that Cd toxicity could cause mitochondrial damage and then cause the disorder of energy metabolism.

Autophagy is an evolutionarily conserved lysosomal pathway of degrading cytoplasmic proteins and organelles. There is a close relationship between autophagy and energy metabolism. mTOR, a receptor of amino acids and ATP, is a key component that coordinately regulates the balance between growth and autophagy in response to cellular physiological conditions and environmental stress. The expression of mTOR is inhibited under nutrient starvation, which has been known as a crucial step for autophagy induction in eukaryotes. Lin et al. authenticated that during autophagy induced in cancer cells by (i) starvation through serum and amino acid deprivation or (ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism, would be affected [40]. Liu et al. found that the autosis (a unique type of autophagic cell death) is inhibited both in vitro and in vivo by cardiac glycosides, which are Na+-K+-ATPase antagonists used in clinical medicine [41]. The regulation of autophagy is a very complex process, including mTOR, LC3-I, LC3-II, Beclin 1, Atg5, and dynein. LC3 are considered important markers of autophagy; the amount of LC3-II or the LC3-II/LC3-I ratio correlates with the number of autophagosomes. Beclin 1 is a critical component of mammalian autophagy [42]; it is engaged in the initial stages of autophagosome formation and promotes the nucleation of the autophagosome. Dynein also has an essential role in the process of autophagy, because it brings autophagosomes closer to lysosomes for subsequent fusion [43]. Increased levels of ATG can maintain stressful conditions during the autophagic process. If Atg5 gene was knocked out, autophagy would be blocked. Any changes in these genes can cause changes in autophagy. Some studies have indicated that Cd can induce autophagy in different organs and tissues of animals. Chargui et al. have suggested that rats’ kidney cortex adapts to subtoxic Cd dose by activating autophagy [44]. Zou et al. found that autophagy can occur in rat liver cells treated with Cd; the mRNA expression of autophagy-related genes Atg5, Beclin 1, and LC3 increased [45]. Our results were consistent with the above reports; the expression of all autophagy-related genes increased except mTOR after Cd exposure in ovarian tissue of chickens. Therefore, we inferred Cd exposure can cause energy metabolism disorder by damaging mitochondria, at the same time, the expression of autophagy-related genes changed. This finally led to autophagy in ovarian tissue of chickens. The results of electron microscopy proved our conjecture. Taken together, we suggested that autophagy was involved in the damage process of chicken ovarian tissue induced by Cd.

Se can antagonize many heavy metals including Cd to reduce their toxic effects on organs and tissues of animals and alleviate the damage of mitochondria. Several studies have reported that Se can antagonize Cd, which reduce the toxicity of Cd to organs or tissues of animals. Banni et al. have suggested that the well-established protective effect Se against Cd-induced toxicity in rat liver passes through non-MT gene expression mechanisms [46]. Taskin et al. have indicated that Se could restore the functions of liver mitochondria after dysfunction induced by adriamycin in rats [47]. Our results showed that both the activities of ATPase and the mRNA expression of glucose metabolism enzyme in the Se-treated group were higher than that of the Cd-treated group. This indicated that Se improved the energy metabolism disorder caused by Cd. Becker et al. found that the mRNA levels of the PK increased in diabetic rats after Se treatment [48]. Besides, results from our experiment found that the expression of autophagy-related genes (LC3-I, LC3-II, Beclin 1, Atg5, and dynein) was lower in Se antagonistic group than in Cd-treated group while mTOR increased significantly in the Cd-treated group. Therefore, we think that Se reduces the damage caused by Cd-induced autophagy in ovarian tissue of chickens. Wang et al. found that the expression of autophagy-related genes (Atg5, LC3-I, LC3-II, Beclin1, and dynein) increased when Se deficiency happened [49]. Santosh et al. found that Se could prevent glutamate-activating autophagy markers Beclin 1 and LC3-II [50]. As mentioned above, our study also demonstrated that Se can reduce the toxicity of Cd to the ovarian tissue of chickens by reducing autophagy.

In conclusion, our results emphasized that Cd exposure can cause energy metabolism disorder by mitochondrial injury and then induce autophagy, which cause damage to ovarian tissue of chickens. Whereas Se had an ability to inhibit Cd-induced autophagy in ovarian cells of chickens and provided mechanistic evidence that cytoprotective effects may mediate through improving energy metabolism and decreasing autophagy.