Nucleoside reverse transcriptase inhibitor-induced rat oocyte dysfunction and low fertility mediated by autophagy

Abstract

Low fertility is one of the most common side effects caused by nucleoside reverse transcriptase inhibitors (NRTIs), whereas the molecular mechanism underlying this process were largely unclear. This study was conducted to investigate whether autophagy plays a role in NRTIs-induced oocyte dysfunction and low fertility in female rat. Both in vivo and in vitro experiments were conducted. For the in vivo experiment, female adult Sprague-Dawley rats were subjected to zidovudine (AZT) and lamivudine (3TC) intragastric treatment for 3, 6, 9, and 12 weeks; a control was also set. Oocytes were collected for maturation evaluation, in vitro fertilization and mitochondrial function assays, and apoptosis and autophagy analysis. For the in vitro experiment, oocytes were collected and assigned to the control, 3-methyladenine (3-MA, an effective autophagy inhibitor), AZT, AZT+3-MA, 3TC, and 3TC+3-MA groups. The oocytes were cultured with the abovementioned drugs for 24, 48, and 72 h and then, subjected to the same assays as in the in vivo study. The results showed a significant time-dependent decrease in oocyte maturation-related maker levels, oocyte cleavage rate, blastocyst formation rate, mitochondrial DNA copy number and adenosine triphosphate level, and apoptosis, and a significant increase in the reactive oxygen species levels (all P-values < 0.05), in both the in vivo and the in vitro experiments. These changes, except for the changes in the oocyte maturation-related markers, were partially attenuated by 3-MA. In conclusion, we demonstrated that NRTIs can cause rat oocyte dysfunction and low fertility, and this damage was, at least partially, mediated by autophagy.

Keywords: NRTIs; low fertility; mitochondrial toxicity, autophagy.

Figure 1

Maturation of rat oocytes was evaluated by measuring the levels of MPF, GDF-9, TFG-β, IGF-1, KL, and PDK1 (A–F) with enzyme-linked immunosorbent assay (ELISA), for the in vivo experiment. The rats were treated with AZT and 3TC for 3, 6, 9, and 12 weeks, and then, their oocytes were isolated for measuring the aforementioned biomarkers. Here, a represents the statistically significant differences with the control group (P < 0.05), and b denotes the statistically significant differences between the AZT and the 3TC groups (P < 0.05).

Figure 2

Maturation of rat oocytes was evaluated by measuring the levels of MPF, GDF-9, TFG-β, IGF-1, KL, and PDK1 (A–F) with ELISA, for the in vitro experiment. The oocytes were treated with AZT and 3TC for 24, 48, and 72 h. Here, a represents the statistically significant differences with the control groups (P < 0.05), and b denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).

Figure 3

Oocyte cleavage rate and blastocyst formation rate in different treatment groups, for the in vivo experiment (A and B) and the in vitro experiment (C and D). The rates in the AZT and 3TC groups were all significantly lower than those in the controls, at different time points, for the in vivo experiment (all P-values < 0.05). Here, a represents the statistically significant differences with the control group (P < 0.05), and b denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).

Figure 4. Effects of AZT and 3TC on the mtDNA copy number, and the mitochondrial function-related gene expression of mice oocytes, for the in vivo experiment

The rats in the AZT and 3TC groups were suffering from AZT and 3TC for 3, 6, 9, and 12 weeks. The mtDNA copy number was evaluated by measuring the ND2 gene content (A). mtTFA (B) and NRF-1 (C) were measured to evaluate the mitochondrial function. Here, a represents the statistically significant differences with the control group at P < 0.05, and b denotes the statistically significant differences between the AZT and the 3TC groups at P < 0.05.

Figure 5. Effects of AZT and 3TC on the mtDNA copy number, and the mitochondrial function-related gene expression of mice oocytes, for the in vitro experiment

The mtDNA copy number was evaluated by measuring the ND2 gene content (A). mtTFA (B) and NRF-1 (C) were measured to evaluate the mitochondrial function. Here, a represents the statistically significant differences with the control group (P < 0.05), and b denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).

Figure 6. Effects of AZT and 3TC on the ATP, cAMP, and ROS levels of mice oocytes, for the in vivo experiment

Rats were treated with AZT and 3TC for 3, 6, 9, and 12 weeks. The ATP level was measured with the ATP Assay KiT (A), and the level of cAMP was detected using luciferase (B). Further, the ROS level in the mitochondria was detected using dichloro-dihydro-fluorescein diacetate (C). The fluorescence intensity was in line with the ROS level (D). Here, a represents the statistically significant differences between the AZT, 3TC, and control groups at P < 0.05, and b denotes the statistically significant differences between the AZT and the 3TC groups at P < 0.05.

Figure 7. Effects of AZT and 3TC on the ATP, cAMP, and ROS levels of rat oocytes, for the in vitro experiment

The ATP level was measured with the ATP Assay KiT (A), and the level of cAMP was detected with luciferase (B); the ROS level in the mitochondria was detected using dichloro-dihydro-fluorescein diacetate (C). The fluorescence intensity was in line with the ROS level (D). Here, a represents the statistically significant differences with the control group (P < 0.05), and b denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).

Figure 8. Effects of AZT and 3TC on the Bax and Bcl-2 mRNA expressions, for the in vivo experiment

Here, (A) represents the statistically significant differences between the AZT, 3TC, and control groups at P < 0.05, and (B) denotes the significant differences between the AZT and the 3TC groups at P < 0.05.

Figure 9. Effects of AZT and 3TC on the Bax and Bcl-2 mRNA expressions, for the in vitro experiment

Here, (A) represents the statistically significant differences with the control group (P < 0.05), and (B) denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).

Figure 10. AZT- and 3TC-induced oocyte autophagy, for the in vivo experiment

The ATG5 (A), ATG7 (B), and Beclin 1 (C) mRNA levels were assayed using qRT-PCR. Here, a represents the statistically significant differences between the AZT, 3TC, and control groups at P < 0.05, and b denotes the statistically significant differences between the AZT and the 3TC groups at P < 0.05.

Figure 11. AZT- and 3TC-induced oocyte autophagy, for the in vitro experiment

The ATG5 (A), ATG7 (B), and Beclin 1 (C) mRNA levels were assayed using qRT-PCR. LC3-II and mTOR were also analyzed in the different groups by using a Western blot (D). Here, a represents the statistically significant differences with the control group (P < 0.05), and b denotes the statistically significant differences between the AZT and the AZT+3-MA groups, and between the 3TC and the 3TC+3-MA groups (P < 0.05).