Abstract
This study aims to explore the role of PERK‐eIF2α signaling pathway in fetal male rats with hypospadias induced by maternal exposure to di‐n‐butyl phthalate (DBP). DBP was used to treat pregnant SD rats by gastric intubation from gestation day (GD) 14–18 to construct a hypospadias rat model. The amount, weight, anogenital distance (AGD), and hypospadias incidence of rats were recorded and the genital tubercle (GT) of fetal male rats was collected on GD 19. Western blotting was performed to detect the expressions of PERK‐eIF2α pathway‐ and autophagy‐related proteins, and cell apoptosis was detected using TUNEL method. Then, GT fibroblasts of fetal rats were obtained and transfected with PERK‐siRNA to detect cell apoptosis and autophagy in each transfected group. The incidence of hypospadias was 43.49% in fetal male rats induced by DBP. The fetal rats in DBP group presented the decreased birth weight and anogenital distance (AGD)/body weight ratio than the Control group (all P < 0.05). Further, p‐PERK, p‐eIF2α and ATF4 protein expressions and the ratio of LC3‐II/LC3‐I were greatly increased in the GTs of fetal rats, while apoptosis index (AI) and P62 protein expression were evidently decreased (all P < 0.05). In addition, the apoptosis rate was increased in GT fibroblasts after transfection of PERK‐siRNA with the increased P62 and reduced LC3‐II/LC3‐I ratio (all P < 0.05). Activation of PERK‐eIF2α signaling pathway can influence the GT development of fetal male rats with hypospadias induced by DBP through activation of autophagy and inhibition of apoptosis.
Keywords: PERK‐eIF2α, Hypospadias, Dibutyl phthalate, Autophagy, Apoptosis
Introduction
Hypospadias, a congenital malformations in males, is characterized by an aberrant opening of the urethra on the ventral aspect of the glans penis or, on the shaft or scrotum, owing to the incomplete fusion of urethral folds [1]. As the second‐most common birth abnormality of the male reproductive system after cryptorchidism, hypospadias would occur in nearly 1 in 125–300 individuals at birth, with an increasing incidence during the past decade [[2], [3]]. However, hypospadias is so complex that environmental, genetic, and other‐related factors are believed to contribute to its pathogenesis [4]. Nowadays, dibutyl phthalate (DBP), as one of common environmental endocrine disruptors (EEDs) [5], is known to cause a variety of serious diseases, especially affecting the male reproductive system in embryonic development, thereby resulting in hypospadias, cryptorchidism or other congenital malformations [6]. But the specific mechanism of DBP leading to hypospadias of fetal male rat remains unclear.
To our knowledge, cell apoptosis is essential for the normal development of the genital tubercle (GT) [7]. For example, the apoptosis of fetal mouse GT cells has been found to be significantly reduced after the mice were exposed to di‐(2‐ethylhexyl) phthalate (DEHP) [8] In addition, during embryonic development, autophagy is also thought to be an important dynamic catabolic process to maintain the normal cell differentiation and development [9]. Moreover, recent studies about male prostate disease have reported that androgen deprivation (one of leading causes of hypospadias) could induce cell autophagy enhancement and apoptosis change [[10], [11]].
Protein kinase R‐like ER kinase (PERK), an endoplasmic reticulum (ER)‐located type I transmembrane protein, is generally credited as an important component of the unfolded protein response (UPR), as well as a key regulator of protein synthesis [12]. To be specific, PERK activated by unfolded proteins in the ER could inhibit protein synthesis through phosphorylation of the ‐subunit of the eukaryotic initiation factor 2a (elF2a) in response to ER stress and regulation of cell apoptosis and autophagy [[13], [14]], thereby participating in the occurrence and development of various diseases, such as diabetes [15], tumor [16], neuron‐degeneration [17] and myocardial ischemia‐reperfusion injury [18]. For this reason, we hypothesized that PERK‐eIF2α signaling pathway may also be a critical signal molecule involved in autophagy and apoptosis of GTs development of congenital hypospadias.
Therefore, on the basis of DBP‐induced hypospadias male rats, this study aims to explore the regulatory effect of PERK‐eIF2α signaling pathway on the autophagy and apoptosis in the GTs of fetal male rats with hypospadias, and to explain the molecular mechanism of DBP‐induced hypospadias, thus providing the guidance for clinical treatments.
Materials and methods
Ethics statement
This study has been approved by the laboratory animal ethics committee of Guangzhou Women and Children's Medical Center. All treatments about the experimental animals were in accordance with guidelines for laboratory animal care and use published by National Institutes of Health (NIH) [19].
Animals and treatments
The normal Sprague–Dawley (SD) rats of specific pathogen‐free (SPF) grade (aged 7–8 weeks) were purchased from Shanghai Laboratory Animal Center of Chinese Academy of Sciences, including 30 male rats with the mean weight of 307 ± 18 g and 60 female rats with the mean weight of 265 ± 13 g. After 1‐week adaptation feeding, male and female rats were kept in one cage with the ratio of 1: 2. Vaginal secretion was examined daily using microscope, and the gestation Day 0 (GD 0) was designated as the day that copulatory plug and vaginal sperm was firstly detected. Then, the pregnant female rats (GD 0) were randomly divided into DBP group and Control group, with 20 rats in each group. The pregnant rats from GD 14 to GD 18 were given daily intra‐gastric administration at fixed time, in which DBP group was administrated at a dosage of 750 mg/kg/d DBP (Sigma–Aldrich Corporate, Louis, Missouri, USA) dissolved in corn oil [20] and Control group was administrated in a dosage of 1.5 ml/kg corn oil. Caesarean sections were performed on GD 19, and the gonads and penises were carefully examined to identify fetal male rats, as well as the amount, weight, and anogenital distance (AGD) of fetal male rats was collected. Under dissecting microscope, the internal and external genitalia were observed to identify male fetal rats with hypospadias in DBP group and Control group respectively, thereby dividing into Hypospadias group and Normal group. Genital tubercle (GT) tissues in Hypospadias group and Normal group were collected and stored at −80 °C used for further analysis.
Western blotting
GT tissues were homogenized in ice‐cold lysis buffer, moved into 1.5 ml EP (Eppendorf) tube, incubated on ice for 30 min, and centrifuged at 4 °C in 14,000 rpm for 30 min. The total protein concentration of supernatant was determined by Bradford method. Next the protein was separated by polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane using semi‐dry transfer apparatus (Bio‐Rad, USA). The membrane strips were hybridized with rabbit anti‐mouse PERK, p‐PERK, eIF2α, p‐eIF2α, activating transcription factor‐4 (ATF4), LC3, P62 and GAPDH (monoclonal antibodies were purchased from Abcam Inc., Cambridge, UK) for 1 h and then hybridized with horse radish peroxidase labeled goat anti‐rabbit IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h, followed by poly (butylene succinate‐co‐terephthalate)s (PBST) washing 5 times × 3 min. Horseradish peroxidase (HRP) substrate (Bio‐Rad, USA) was used to develop the target protein. The ratio of gray value of target protein versus GAPDH is the relative content of the target protein.
TUNEL (terminal deoxyribonucleotidyl transferse(TdT)‐mediated biotin‐16‐dUTP nick‐end labelling) staining
GT paraffin sections were dewaxed in xylene and dehydrated by graded ethanol. TUNEL staining was then performed using One Step TUNEL Apoptosis Assay Kit (Beyotime Institute, China) according to the manufacturer's instructions. The number of apoptotic cells was counted in 5 randomly selected high power ( × 400) fields with at least 1000 cells per field. Apoptosis index (AI) was calculated as the percentage of positive cells in total cell number.
GT fibroblast isolation and culture
The GT was taken out and put into dulbecco's modified eagle medium (DMEM) (Sigma–Aldrich, USA) containing 20% fetal bovine serum (FBS), 1% penicillin and streptomycin for 15 min. Then, ophthalmic scissors were used to cut the GT tissue into 1 mm × 1 mm tissue blocks, which was incubated in a culture dish (60 mm diameter). When primary fibroblasts covered 70% of culture dish, fibroblasts were digested, passaged, and purified using differential passage digestion and differential adherence methods. The purified fibroblasts were observed under reversed microscope, and vimentin expression of the third generation of fibroblasts was detected using immunofluorescence staining to identify cell types.
GT fibroblast grouping and transfection
GT fibroblasts were incubated in 6‐well plate. When cells were fused till 70%–80%, cell transfection were performed and divided into Mock group (no transfection), PERK‐siRNA group (transfected with PERK‐siRNA) and NC‐siRNA group (transfected with NC (Negative control)‐siRNA). The sequence of the siRNA for PERK (NM_031599.1) was obtained from NCBI database, which was synthesized by Shanghai Gene Pharma Co., Ltd. In addition, PERK‐siRNA and NC siRNA were transfected following the Lipofectamine 2000 (Invitrogen Inc., California, USA). Here, the stock and working concentrations of siRNA were 20 mM and 50 nM respectively. After 48 h transfection, the expressions of PERK‐eIF2αpathway‐ and autophagy‐related proteins in each group were detected using western blot, and cell apoptosis was determined by flow cytometry (BD Pharmingen, San Diego, CA, USA).
Statistical analysis
All data were analyzed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Measurement data were presented as mean ± standard deviation, and normality test was performed. The comparison of measurement data between each two groups was analyzed using t‐test, while comparison among multiple groups was analyzed by Analysis of Variance (ANOVA). Enumeration data was expressed by percentage and analyzed using χ2 test. The P value of <0.05 was considered significantly different.
Results
Effects of DBP exposure on fetal male rats
The hypospadias rat model was successfully constructed. As demonstrated in Table 1, no hypospadias was presented in Control group, while the incidence of hypospadias in fetal male rats induced by DBP was 43.48% (30/69). Compared with Control group, the birth weight and AGD were obviously decreased, as well as the ratio of AGD/weight in fetal male rats in DBP group (all P < 0.05).
Table 1.
Effects of DBP exposure on related parameters of the fetal male rats.
| DBP group | Control group | |
|---|---|---|
| Pregnant rats (n) | 15 | 15 |
| Fetal male rats (n) | 69 | 82 |
| Birth weight of fetal male rats (g) | 5.03 ± 0.22 | 6.12 ± 0.36∗ |
| Birth weight of AGD (mm) | 2.08 ± 0.17 | 3.27 ± 0.21∗ |
| AGD/weight (mm/g) | 0.41 ± 0.04 | 0.54 ± 0.05∗ |
| Fetal male rats with hypospadias (with/without) | 30/39 | 0/82 |
| Hypospadias incidence (%) | 43.49∗ | 0 |
Notes: ∗ P < 0.05, compared with Control group.
Expressions of PERK‐eIF2 pathway‐related proteins in the GT of fetal male rats in Hypospadias group and Normal group
As shown in Fig. 1, no significant differences were found in the expressions of PERK and eIF2α protein between Hypospadias group and Normal group (P > 0.05). However, the protein levels of p‐PERK, p‐eIF2α and ATF were higher in the Hypospadias group than the Normal group (all P < 0.05).
Figure 1.
Expressions of PERK‐eIF2 pathway‐related proteins in the GT of fetal male rats in Hypospadias group and Normal group. Notes: *P < 0.05, compared with Normal group.
Comparison of the apoptosis and autophagy in the GT of fetal male rats in Hypospadias group and Normal group
TUNEL staining was used to observe the number of apoptotic cells in the GT of fetal male rats in two groups. As displayed in Fig. 2A, AI was significantly lower in Hypospadias group than Normal group (P < 0.05). The autophagy‐related protein detected by western blot presented that the expression of LC3‐II was enhanced, together with the ratio of LC3‐II/LC3‐I, while P62 protein was greatly decreased in the GT of fetal male rats in Hypospadias group, as compared with Normal group (P < 0.05, Fig. 2B).
Figure 2.
Comparison of the apoptosis and autophagy in the GT of fetal male rats in Hypospadias group and Normal group. Notes: A, The cell apoptosis in the GT of fetal male rats in Hypospadias group and Normal group detected by TUNEL staining, *P < 0.05, compared with Normal group; B, The relative expressions of autophagy‐related proteins (LC3‐II/LC3‐I and P62) in Hypospadias group and Normal group detected by western blot, *P < 0.05, compared with Normal group.
Identification of fibroblasts from the GT of fetal male rats and comparison of expressions of pathway‐related proteins in GT fibroblasts
Under inverted microscope, cells were in spindle or polygonal shape with regular oval nucleus and obviously large nucleolus, and most cells had long and thin filopodia. Vimentin was a marker of fibroblasts, and immunoflurescence results showed that all of the isolated fibroblasts were vimentin‐positive, which confirming the fibroblast phenotype (Fig. 3A). In addition, the expressions of pathway‐related proteins of GT fibroblasts in each group were detected by western blot. Specifically, the relative protein expressions of PERK, p‐PERK, p‐eIF2α and ATF4 were significantly decreased in PERK‐siRNA group when compared to Mock group (all P < 0.05), but no alterations were found with respect to the total protein expression of eIF2α (all P > 0.05). Moreover, there were no significant differences in protein expressions between Mock group and NC‐siRNA group (all P > 0.05, Fig. 3B).
Figure 3.
Identification of fibroblasts from the GT of fetal male rats and comparison of expressions of signaling pathway‐related proteins in GT fibroblasts. Notes: A, The identification of fibroblasts from the GT of fetal male rats, a, Morphological observation of the fibroblast under inverted microscope, b, The immunofluorescence results of vimentin in fibroblast cells (40 × ); B, Expressions of PERK‐eIF2α pathway‐related proteins in GT fibroblasts of fetal male rats in each group, *P < 0.05, compared with Mock group.
Comparison of the apoptosis and autophagy in the GT fibroblast of fetal male rats in each group
As shown in Fig. 4, NC‐siRNA group was not different from Mock group regarding the cell apoptosis ratio (P > 0.05), but an evident increased apoptosis ratio was presented in PERK‐siRNA group by comparison with the Mock group (P < 0.05). In terms of autophagy‐related proteins in each group, the decreased LC3‐II/LC3‐I but the elevated P62 were discovered in ERK‐siRNA group in contrast to Mock group, no significant changes of these proteins were shown between Mock and NC‐siRNA groups (all P > 0.05).
Figure 4.
Comparison of the apoptosis and autophagy in the GT fibroblast of fetal male rats in each group. Notes: A, The cell apoptosis in the GT fibroblast of fetal male rats in each group detected using flow cytometry, B, The relative expression of autophagy‐related proteins (LC3‐II/LC3‐I and P62) in the GT fibroblast of fetal male rats in each group detected by western blot, *P < 0.05, compared with Mock group.
Discussion
In this study, DBP was used to treat pregnant SD rats to induce hypospadias, and we observed the decreased birth weight, shortened AGD, as well as the reduced ratio of AGD/weight, meanwhile, the incidence of hypospadias was 43.49% in the fetal male rats induced by DBP, suggesting that the hypospadias model was successfully constructed via prenatal exposure to DBP in rats, thereby verifying the toxicity of DBP to offspring development, which was in accordance with previous studies [[20], [21]]. In addition, the apoptosis in the GT of fetal male rats with hypospadias by TUNEL method were also found to be significantly decreased than normal fetal rats. To the best of our knowledge, cell apoptosis plays an important role in the embryonic development of the male anterior urethra, especially the proper apoptosis in the distal urethral plate, as such, the abnormal decreased apoptosis was supposed to be closely related to hypospadias [[22], [23]]. Consistent with the study by Liu et al., the apoptosis of fetal rats GT cells induced by DEHP presented a significant decrease, along with a decrease of pro‐apoptotic protein P53 [8]. Besides, in Hoxa13‐mutant mice with hypospadias, the defects of the distal urethra and glans were possibly attributed to the loss of apoptosis in the urethra, which was an important cause of hypospadias formation [24]. On the other hand, the enhanced ratio of LC3II/LC3I and a decrease P62 protein were also displayed in the GT of fetal rats with hypospadias in our research. As shown previously by accumulated evidence, autophagy acted as a key element to affect the cell death procedure, since it could respond to a wide range of stresses [25]. Of note, LC3 was regarded as a unique autophagic marker protein with a molecular mass of 651,765 kDa in mammalian cells [26]. During the process of autophagy, LC3‐I, a cytosolic form of LC3, was modified to LC3‐II, the membrane‐bound form, and thus, the ratio of LC3‐II/LC3‐I protein levels was used as an indicator of the extent of autophagy [[25], [26]]. Another autophagic induction marker P62, a multifunctional ubiquitin‐binding protein, was selectively degraded by autophagy, and its expression was negatively correlated with the autophagy activity [[27], [28]]. Similarly, Li et al. also observed that the LC3II/LC3I ratio was significantly increased, and the expression of polyubiquitin‐binding protein p62 was significantly decreased in the GT from the hypospadias male fetuses [29], indicating that exposure to DBP during pregnancy inhibited cell apoptosis and promoted cell autophagy in the GT of fetal male rats, thereby resulting in the formation of hypospadias.
Subsequently, expressions of PERK‐eIF2α pathway‐related proteins were examined in the GT of hypospadias fetal male rats, presenting an increased expression pattern of p‐PERK, p‐eIF2α and ATF4. It has been well‐documented that ER stress could trigger the disassociation of GRP78 from the transmembrane ER‐stress receptor (PERK). As a result, eIF2a at serine 51 (S51) was phosphorylated by activated PERK to inhibit unfolded protein translation, but at the same time it induced translation of specific mRNAs encoding proteins, such as ATF4, to alleviate ER stress [[30], [31]], indicating that activation of ER stress triggers PERK‐eIF2α signaling pathway under the pathological condition of hypospadias. As recorded in the study by Kouroku et al., in rat embryonic cancer cells and rat embryonic fibroblasts, activation of PERK‐elF2a signaling pathway can up‐regulate Atg12 expression and induce autophagy, thereby involving in ERS caused by ectopic expression of polyQ72 [32]. Meanwhile, both Back et al. [33] and Boyce et al. [34] found that PERK mediated the persistent phosphorylation of its downstream effector molecule eIF2α to weaken the cell apoptosis at ERS. Hence, it was reasonably to predict that activation of PERK‐e IF2α signaling pathway regulated cell autophagy and apoptosis in GT development of fetal male rats.
Furthermore, the GT fibroblast of fetal rat was isolated to perform in‐vitro transfection experiments, and the downregulated PERK‐eIF2α signaling pathway, eIF2α phosphorylation and ATF4 expression, and increased cell apoptosis, as well as reduced ratio of LC3‐II/LC3‐I and increased P62 were detected after PERK silencing, implying that cell autophagy was restricted but apoptosis was enhanced through inhibition of PERK‐eIF2α signaling pathway. In radiation myelitis induced by Iodine‐125, the expressions of LC3II and ATG12 were also significantly declined with increased cell apoptosis in neural cells when transfected with a PERK siRNA [35], which was in agreement with our findings. Therefore, PERK silencing reduced the conversion of LC3‐I to LC3‐II and inhibited cell autophagy via alleviation of eIF2α phosphorylation and ATF4 expression, thereby facilitating the cell survival and influencing the development of hypospadias.
Taken together, our findings illustrated that activation of PERK‐eIF2αpathway can promote cell autophagy and inhibit cell apoptosis to affect the GT development of DBP‐induced hypospadias from fetal male rats, which also provides a new approach for elucidating the pathogenesis of hypospadias.
Acknowledgements
The authors appreciate the reviewers for their useful comments in this study.
Supporting information
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Supplementary data
Supplementary data related to this article can be found at https://doi.org/10.1016/j.kjms.2018.04.002.
Conflicts of interest: All authors declare no conflicts of interests.
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