Melatonin attenuates cadmium-induced ovulatory dysfunction by suppressing endoplasmic reticulum stress and cell apoptosis

Female CD-1 mice (5-week-old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All the animals were kept under a temperature- and humidity-controlled condition on a 12- h light/dark cycle. They were allowed to acclimatize for a week before treatment and had ad libitum access to food and water. All the experiments have received the approval of institutional review board and the First Affiliated Hospital of Zhengzhou University.

Sixty CD-1 female mice were randomly assigned to four groups. The mice were administered intraperitoneal injections of the vehicle or, melatonin (25 mg/kg; Sigma, St. Louis, Mo, USA), cadmium chloride [8] (CdCl₂, 5 mg/kg; Sigma, St. Louis, Mo, USA) with or without melatonin [30] (25 mg/kg) daily for 2 weeks respectively. Melatonin stock solutions were dissolved in 100% ethanol and diluted in saline. The final ethanol concentration in the working solutions did not exceed 0.1%. CdCl₂ was dissolved in saline (5 mg/kg).

Ovaries were collected, fixed, and embedded in paraffin. Tissues were serially sectioned (5-um thickness) and stained with hematoxylin and eosin. Images were captured with the Nikon Ni-E microscope. To assess the ovulation function, pregnant mare serum gonadotropin (PMSG, 7.5 I.U.) was intraperitoneally injected to stimulate the ovaries of the mice, followed by injection of 7.5 I.U. human chorionic gonadotropin (hCG) after 48 h to induce super-ovulation. After 12–14 h, the mice were sacrificed and the oviductal ampullae were broken to obtain the cumulus-oocyte complexes (COCs). Subsequently, COCs were pipetted in the in-vitro fertilization (IVF) medium (Vitrolife Sweden AB) containing 5% human serum albumin (Irvine Scientific) and 0.1% hyaluronidase (Sigma) to remove the cumulus cells. The number of ovulated oocytes was counted under a stereoscopic microscope (Nikon SMZ800N, Tokyo, Japan).

At least three ovaries were homogenized in the lysis buffer from the protein extraction kit (Sangon Biotech, Shanghai, China). A protein quantitation kit (Bio-Rad) was used to measure the concentrations of the protein solutions. Briefly, the protein samples were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were blocked with 5% defatted milk in Tris-buffered saline containing Tween20 (TBST) for 1 h at room temperature. Next the proteins were incubated with primary antibodies- GRP78 (1:1000, Cell Signaling Technology, Inc.), ATF4 (1:1000, Cell Signaling Technology, Inc.), CHOP (1:1000, Cell Signaling Technology, Inc.), phosphor-JNK (1:1000, Cell Signaling Technology, Inc.), Bax (1:1000, Proteintech, Inc.), Bcl-2 (1:1000, Proteintech, Inc.), and cleaved caspase 3 (1:1000, Cell Signaling Technology, Inc.), overnight at 4 °C respectively. Anti-GAPDH monoclonal antibody (1:2000, Proteintech, Inc.) was used as a loading control. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, the proteins were detected by enhanced chemiluminescence detection system (Bio-Rad).

Total RNA was extracted by using the micropurification kit (Qiagen, Dusseldorf, Germany), and the synthesis of cDNA was performed with a reverse transcription kit (Takara, Kusatsu, Japan) according to the manufacturer’s protocol. Real-time PCR was performed using the SYBR Green PCR kit (Qiagen, Dusseldorf, Germany) and Quantstudio 12 K Flex (Applied Biosystems). The primers sequences of GRP78, ATF4, s-XBP1, CHOP, GAPDH are shown in Table 1. Relative gene expression was calculated basing on the 2^-ΔΔCT method. Each sample was measured in triplicate in each experiment.

To detect the apoptotic rates of the ovarian sections, In Situ Cell Death Detection Kit (Roche, Penzberg, Germany) was purchased to carry out TUNEL analysis based on the manufacturer’s instructions. After incubation in TUNEL reaction mixture for 1 h at 37 °C, the sections were washed twice and counterstained with Hoechst 33342 (10 μg/mL) for 10 min to stain the nuclei. Finally, the sections were mounted on slides and the images were captured under a fluorescence microscope (Nikon Ni-E, Tokyo, Japan).

The body weights of the mice were recorded every other day. After treatment for 14 days, the ovaries of each group were harvested, and weighed with an electronic weighing balance. Relative ovary weight was calculated as a proportion of the final body weight before euthanasia, expressed as mg/g body weight. The results showed that the Cd-treated group had significantly less body weight than the control group from the 4th day of the treatment. Melatonin ameliorated the effect of Cd on body weights (Fig. 1a). However, there were no significant differences in the relative ovary weights between the groups (Fig. 1b).

To determine the effects of Cd on ovarian function, we observed the ovarian morphological changes in each group. The ovary consists of follicles at different developmental stages. The formation of antral follicles determined the ovulation in individuals. According to a previous study, the antral follicles had independent antral spaces containing follicular fluid, and the antral follicles were defined as atretic if they contained at least 20 apoptotic granular cells, and a degenerating or apoptotic oocyte [34]. After the injection of the Cd-treated mice with ovulatory gonadotropins, the ovarian histology of the mice showed less antral and more atretic follicles compared to other groups (Fig. 1c), which indicated the ovulatory dysfunction in Cd-exposed ovaries. The addition of melatonin could partially attenuate the degeneration of oocytes. To further evaluate the variation of ovulatory function, we measured the number of ovulated oocytes after the stimulation of super-ovulation. As expected, the number of ovulated oocytes in the oviduct was significantly reduced after Cd treatment, and melatonin considerably restored the impaired oocyte release in the Cd-treated mice (Fig. 1d). These results indicate that intraperitoneal injection of Cd induces ovulatory dysfunction and melatonin could alleviate the damage.

ER stress has been demonstrated to be a key mechanism underlying Cd-induced cell death. To elucidate the molecular mechanisms underlying the alleviation of ovulation disorders in mice by melatonin, we carried out the western blot assay, and the results showed that the expression of the proteins— GRP78 and ATF4 was significantly upregulated in ovaries of Cd-exposed mice. However, melatonin notably attenuated the Cd-evoked upregulation of GRP78 and ATF4 (Fig. 2a and b). CHOP, a downstream target of ATF4, was analyzed. The results showed that melatonin significantly inhibited Cd-induced upregulation of CHOP (Fig. 2a and b). Phosphorylated JNK (p-JNK), downstream of sXBP-1 also exhibited the similar variation tendency as CHOP (Fig. 2a and b). We also examined the expression of ER stress-related genes (GRP78, ATF4, spliced XBP-1 transcript, CHOP) in the ovaries of different groups (Fig. 3c). The results revealed that Cd exposure significantly increased the mRNA levels of GRP78, ATF4, CHOP comparing to the control ovaries, and tended to have higher expression of sXBP-1. Notably, administration of melatonin was able to reduce the ER stress in Cd-treated mice comparable to the controls (Fig. 3a). These results suggest that administration of melatonin markedly inhibited ER-stress and its downstream targets, and hence prevented cell apoptosis in the Cd-exposed ovaries. In melatonin-treated group, the ER pathway was not be inhibited, we speculate that the oxidation of tissues is at low levels in normal circumstances, therefore, melatonin does not influence the ER pathway when it was given alone.

Increased ER stress induced cell apoptosis by activating the caspase pathway; hence, we examined the protein expression of cleaved caspase-3, Bax and Bcl-2 in the ovary by western blotting. Results showed that the protein expression of cleaved caspase-3 indeed substantially increased in Cd-exposed ovaries. Meanwhile, administration of melatonin attenuated the cleaved caspase-3 level indistinguishable from the controls. The Cd group showed a higher level of Bax and a lower level of Bcl-2 protein than the control. Melatonin reversed the negative effects of Cd treatment on the expression of Bax and Bcl-2 proteins. We assessed the Bax/Bcl-2 ratio; results showed that the ratio of Bax/Bcl-2 was considerably higher in the Cd-exposed ovaries than in the ovaried of the mice in the control group. The elevated ratio was reversed by melatonin (Fig. 3a and b). Next, we performed the TUNEL staining to determine apoptosis levels in the ovaries of the mice in the Cd with and the Cd without melatonin groups. TUNEL assay exhibited more positive signals after Cd exposure, and there were more apoptotic granular cells in the developing follicles; the addition of melatonin reduced the number of TUNEL-positive cells in the ovary sections (Fig. 3c), indicating that Cd induced cell apoptosis which could be attenuated by the administration of melatonin.