Table 1.
Primers used for the study of Caspase3 and Caspase7 in Cynops orientalis.
Table 2.
Primers used for the study of p53 and Apaf1 in Cynops orientalis.
Figure 1.
Multiple alignment of Apaf1 in Cynops orientalis with its homolog from several vertebrates.
The protein alignment was created using Vector NTI10 (Invitrogen) with the following sequences: Cynops orientalis(JQ320086); Homo sapiens (O14727.2); Mus musculus(NP_033814.2); Xenopus laevis;(NP_001085834.1); Danio rerio(NP_571683.1). The identities of Apaf1 in Cynops orientalis with its homolog were 61.4% (Homo sapiens), 55.7% (Xenopus laevis), 51.2% (Danio rerio) and 59.9% (Mus musculus) respectively.
Figure 2.
Predicted protein structure of Apaf1 in Cynops orientalis.
(A) Cartoon representation of Apaf1 3-D structure. Yellow color stands for β-sheet and red color indicates the α-helix. The functional domains in the 3-D structures are indicated with arrows. The 3-D structure prediction was performed by I-TASSER server. (B) Schematic representation of Apaf1 subdomain structure. The subdomains are shown by the horizontal bar, with the numbers corresponding to the amino acid residues. The putative Apaf1 protein consists of three functional sudomains: the N-terminal caspase recruitment domain (CARD); the nucleotide-binding and oligomerization domain (NOD) and the C-terminal WD40 domain.
Figure 3.
Multiple alignment of p53 in Cynops orientalis with its homolog from several vertebrates.
The protein alignment was performed using Vector NTI10 (Invitrogen) with the following sequences: Cynops orientalis (HM627863)Homo sapiens (BAC16799.1); Mus musculus(BAA82343.1); Xenopus laevis(CAA54672.1); Danio rerio(NP_571402.1). The identities of p53 in Cynops orientalis with its homolog were 53.4% (Danio rerio), 49.6% (Homo sapiens), 48.5% (Mus musculus) and 52.8% (Xenopus laevis) respectively. Several amino acids that are essential for DNA binding are labeled with asterisks.
Figure 4.
Predicted protein structure of p53 in Cynops orientalis.
(A) Cartoon representation of p53 3-D structure. Yellow color stands for β-sheet and red color indicates the α-helix. The functional domains in the 3-D structure are indicated with arrows. The 3-D structure prediction was performed by I-TASSER server. (B) Schematic representation of p53 subdomain structure. The subdomains are shown by the horizontal bar, with the numbers corresponding to the amino acid residue. Several functional domains are in the p53: the transactivation domain, proline-rich domain, DNA binding domain, tetramerization domain and the C-terminal domain.
Figure 5.
Multiple alignments of Caspase3 and Caspase7 in Cynops orientalis with their homolog from several vertebrates.
The protein alignment was performed using Vector NTI10 (Invitrogen) with the following sequences: (A) Cynops orientalis(JQ320088); Danio rerio(CAX14649.1); Xenopus laevis (NP_001081225.1); Homo sapiens (P42574); Mus musculus (P70677); (B) Cynops orientalis (JQ320087); Homo sapiens (AAC50346.1); Mus musculus(BAA19730.1); Xenopus laevis (NP_001091272.1); Danio rerio (AAH95327.1). The identities of Caspase3 in Cynops orientalis with its homolog were 56.2% (Danio rerio), 54.4% (Xenopus laevis), 57.6%(Homo sapiens) and 56.3% (Mus musculus). The identities of Caspase7 in Cynops orientalis with its homolog were 67.6% (Danio rerio), 64.6% (Xenopus laevis), 62.6% (Homo sapiens) and 62.3% (Mus musculus). Putative cleavage sites at aspartic acid residues, which separate Caspase3 and Caspase7 into large subunits and small subunits, are labeled red arrowheads. Histines and Cystines that are essential for the catalytic centre of Caspase3 and Caspase7 are indicated by asterisk.
Figure 6.
Predicted protein structures of Caspase3 and Caspase7 in Cynops orientalis.
(A)(B) Schematic representation of the predicted subdomain structure of Caspase3 (A) and Caspase7 (B) in Cynops orientalis. The subdomains are shown by the horizontal bar, with the numbers corresponding to the amino acid residues. The cleavage active Aspadine are marked with green or red arrow head, which separate the Caspase3 or Caspase7 into large subunits and small subunits. The four surface loops (L1-L4) that shape the catalytic groove are also labeled with different colors. (C)(D) Cartoon representation of the 3-D structures of Caspase3 and Caspase7. The red color and green stands for the large subunit of Caspase3 and Caspase7, and the gray color indicates the small subunits of Caspase7 and Caspase3. Two cleavage active Asp residues are shown with the form of stick and ball in the cartoon. The 3-D structure prediction was performed by I-TASSER server.
Figure 7.
p53, apaf1, caspase3 and caspase7 gene expression in various tissues of Cynops orientalis.
Total RNA was extracted from fresh testis, muscle, liver, intestine and heart, and then transcribed into cDNA for RT-PCR analysis. β-actin served as an internal control. p53, apaf1, caspase3 and caspase7 are expressed in all the examined tissues. In the newt testis, Apaf1 and Caspase3 were expressed at higher level than p53 and Caspase7. The intensity of the signal was analyzed by Tanon-Gis system and the data were processed with SPSS 9.0. The gene expression level in testis were presented as their percentage of β-actin. Significance of difference was assessed by SPSS9.0. P<0.05. n = 3.
Figure 8.
p53, Apaf1, Caspase3 and Caspase7 gene expression in the testis of Cynops orientalis after starvation and cadmium exposure.
(A)(B) gene expression level after starvation (one month).(C)(D) gene expression level after cadmium exposure determined by real-time reverse transcription-PCR. Messenger RNA levels in experimental group were presented as the folds of gene expression level in control group, which was set as 1.0. All data were expressed as Means±SD, and T-test in SPSS9.0 applied to analyze the significance of differences between experimental group and control group. * P<0.05 when compared with control. n = 3.
Figure 9.
p53, Apaf1, Caspase3 and Caspase7 gene expression after stress treatment.
The gene express pattern in the testis of Cynops orientalis after cold exposure at (4°C 4 h and 12 h) and heat exposure (38°C 2 h, 12 h and 24 h; 40°C 2 h and 4 h) determined by real-time reverse transcription-PCR. Messenger RNA levels in experimental group were presented as the folds of gene expression level in control group, which was set as 1.0. All data were expressed as Means±SD, and T-test in SPSS9.0 applied to analyze the significance of differences between experimental group and control group. * P<0.05 when compared with control. n = 3.
Figure 10.
Immunofluorescence analysis of spontaneous apoptosis and stress induced apoptosis in newt early spermatids.
The apoptotic germ cells were determined by TUNEL kit (red signal) (Beyotime, China) and the nucleus were dyed with DAPI (Blue signal). Apoptotic sperms were found in normal newt testis (A1-3), and severer mature sperm apoptosis detected under various stress including starvation (B1-3), cold exposure(4°C for 12 h) (C1-3), cadmium exposure (5 mg/Kg body weight for 36 h) (D1-3) and heat exposure (40°C for 2 h) (E1-3). The scale bar is 10 µm.
Figure 11.
Immunofluorescence analysis of spontaneous apoptosis and stress induced apoptosis in newt mature sperms.
The apoptotic germ cells were determined by TUNEL kit (Beyotime, China) and the nucleus were dyed with DAPI. Apoptotic sperms were found in normal newt testis (A1-3), and severer mature sperm apoptosis detected under various stress including starvation (B1-3), cold exposure(4°C for 12 h) (C1-3), cadmium exposure (5 mg/Kg body weight for 36 h) (D1-3) and heat exposure (40°C for 2 h) (E1-3). The scale bar is 10 µm.
Figure 12.
Immunofluorescence analysis on apoptosis.
No apoptosis occurred to spermatogonia in normal newt testis and stress treated newt. The apoptotic germ cells were determined by TUNEL kit (red signal) (Beyotime, China) and the nucleus were dyed with DAPI (Blue signal). No apoptotic spermatogonia were found in normal newt testis (A) and stress treated newts (B) while severe apoptotic signal was in spermatogonia in the testis treated with DNase I in the TUNEL positive preparation Kit (Beyotime, China). The scale bar is 10 µm.
Figure 13.
Caspase-3, -8, -9 activities in the testis of newts treated with various stress.
The Caspase activities in the testis of newts were analyzed after stress treatment including starvation (one month), cadmium injection (36 h), cold exposure (4°C, 12 h) and heat exposure (40°C, 2 h). Caspase-3, -8, -9 activity was measured by colorimetric assays based on caspase-3, -8, -9 to change Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA into a yellow formazan product (pNA), respectively. The caspase activities in stress exposure groups were presented as the percentage of enzyme activity in the control group. Value represents the Mean±SD, and T-test in SPSS9.0 applied to analyze the significance of differences between stress treated group and control group. * P<0.05 when compared with control. n = 3.
Figure 14.
(A) Longitudinal section of the mature sperm. The nucleus is exceedingly long and thin (white arrow). Scale bar:1 µm (B) Cross section of the mature sperm nucleus. A large number of mitochondria were arranged around the nucleus (white arrow). Scale bar: 0.5 µm.
Figure 15.
Schematic representation of possible mechanism for the stress triggered germ cell apoptosis in Chinese fire-belly newt, Cynops orientalis.
(A) Potential pathways initiated by various stress in germ cell apoptosis. Cold exposure, cadmium exposure and heat exposure probably induced germ cell apoptosis via both extrinsic pathway and intrinsic pathway, but starvation likely only via extrinsic pathway. (B) Hypothesized sperm features and unique reproduction policy responsible for high occurrence of spermatid and sperm apoptosis. The nuclear morphology and mitochondria arrangement may be the physiological basis for observed high frequency of spermatid and sperm apoptosis, and a unique reproduction policy of newts may also account for this phenomenon.