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, 22 (1), 9-16

The Effect of Melatonin on Mitochondrial Function and Autophagy in In Vitro Matured Oocytes of Aged Mice

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The Effect of Melatonin on Mitochondrial Function and Autophagy in In Vitro Matured Oocytes of Aged Mice

Zahraa Nasheed Hamad Almohammed et al. Cell J.

Abstract

Objective: This study examined the in vitro effect of melatonin on the protein synthesis of mitochondria, as well as autophagy in matured oocytes of aged mice.

Materials and methods: In this experimental study, germinal vesicles (GV) oocytes were collected from aged (with the age of six-months-old) and young mice (with age range of 6-8 weeks old) and then cultured in the in vitro culture medium (IVM) for 24 hours to each metaphase II (MII) oocytes and then supplemented with melatonin at a concentration of 10 μM. The culture medium of MII oocytes was devoid of melatonin. Afterward, the expression of the SIRT-1 and LC3 was assessed by immunocytochemistry. ATP-dependent luciferin-luciferase bioluminescence assay was employed for the measurement of the ATP contents. Intracellular reactive oxygen specious (ROS) was detected by DCFH-DA, and the total antioxidant capacity (TAC) level was determined by TAC assay.

Results: The expression of SIRT-1 and LC3, as well as the measurement of the ATP content, was significantly increased in oocytes treated with melatonin compared with the oocytes receiving no treatment. Moreover, TAC was considerably higher in melatonin-treated oocytes than oocytes receiving no treatment. On the other hand, the level of ROS was significantly decreased in oocytes treated with melatonin in comparison with the untreated oocytes. The results indicated that melatonin considerably improved the development of oocytes as well.

Conclusion: According to the data, melatonin increased mitochondrial function and autophagy via an increase in the expression of SIRT1 and LC3, as well as the ATP contents while it decreased the levels of ROS and increased TAC in oocytes derived from aged mice.

Keywords: Aged Mice; Autophagy; Melatonin; Mitochondria.

Conflict of interest statement

There is no conflict of interest in this study.

Figures

Fig.1
Fig.1
The expression of SIRT-1 at the MII stage of in vitro matured oocytes, isolated from young and aged mice was evaluated usingimmunofluorescence staining. A. The micrograph represents the intensity of the SIRT-1 expression among the young MII oocyte, young MII oocyte+melatonin, aged MII oocyte+melatonin, and aged MII oocyte groups. The nuclei were stained by DAPI. The secondary antibody was conjugated with FITC and B. The expression of SIRT-1 in the aged MII oocyte+melatonin group was significantly higher than the aged MII oocyte (P<0.01). Accordingly, the SIRT-1 expression was elevated in the young MII oocyte+melatonin group compared with the young MII oocyte group (P<0.05) (magnification × 400, scale bars: 20 µm). Y+M; Young MII oocyte+melatonin and O+M; Aged MII oocyte+melatonin.
Fig.2
Fig.2
The expression of the LC3 protein in in vitro matured MII oocytes, isolated from aged and young mice was determined by the Immunofluorescence staining. A. The micrograph represents a significant difference in intensity of the LC3 expression between the young MII oocyte, young MII oocyte+melatonin, aged MII oocyte+melatonin, and aged MII oocyte groups. The nuclei were stained by DAPI. The secondary antibody was conjugated with FITC (magnification ×400, scale bars: 20 µm) and B. Significantly higher levels of LC3 were found in the aged MII oocyte+melatonin compared with the aged MII oocyte groups (P<0.01). The expression of the LC3 was significantly higher in the young MII oocyte+melatonin than the young MII oocyte groups (P<0.01). Y+M; Young MII oocyte+melatonin and O+M; Aged MII oocyte+melatonin.
Fig.3
Fig.3
The levels of the ATP contents of in vitro matured MII oocytes in all experimental groups, namely aged MII oocyte, young MII oocyte, aged MII oocyte+10 µM melatonin, and young MII oocyte+10 µM melatonin. Each group consisted of 35-50 MII oocytes. The obtained data were represented as mean ± SD. ¥; P<0.001 vs. aged group, ß; P<0.05 vs. young group, a; P<0.01 vs. young group, µ; P. 0.001 vs. young group, Y+M; Young+melatonin, and O+M; Aged+melatonin.
Fig.4
Fig.4
The total antioxidant capacity (TAC) of MII stage in vitro matured mouse oocytes in four groups: old and young or old and young supplemented by 10 µM melatonin. 50 µL of culture media of each group were used for TAC content measurement. The data was represented based on mean ± SD. Although TAC level increased in aged MII oocyte+melatonin in comparison to the aged MII oocyte group, (0.35 ± 0.06 vs. 0.11 ± 0.05 mM) but there is no significant difference between them (P=0.07). The result shows that there is no significant difference between aged MII oocyte+melatonin and Young groups as well (P=0.31). It also shows a significant difference between young MII oocyte+melatonin vs. young MII oocyte group. µ; P<0.01, ß; P<0.05, Y+M; Young MII oocyte+melatonin, and O+M; Aged MII oocyte+melatonin.
Fig.5
Fig.5
Intracellular reactive oxygen specious (ROS) levels of MII in vitro matured oocytes were measured by immunofluorescence dye (DCFHDA) in all experimental groups, namely aged MII oocyte, young MIIoocyte, aged MII oocyte+10 µM melatonin, and young MII oocyte+10µM melatonin and they were quantified by the ImageJ software. Eachgroup consisted of 40-50 MII oocytes. The results were expressed asmean ± SD. The different symbols represent a significant differencebetween the two experimental groups. Although the ROS level wasdecreased in young MII oocyte+melatonin group compared with theyoung MII oocyte group, the difference was not statistically significant(4 ± 0.81 vs. 17 ± 3.09, P=0.71). The results also showed that there wasno significant difference between the aged MII oocytes+melatoninand young MII oocytes groups (P=0.10). ¥; P. 0.05 vs. aged group, µ; P<0.001 vs. young group, Y+M; Young+melatonin, and O+M; Aged+melatonin.
Fig.6
Fig.6
The levels of DCFH-DA representing the reactive oxygen specious (ROS) production in MII in vitro matured oocytes, isolated from young and aged mice. The micrograph depicts the different intensity of ROS among the young MII oocytes, young MII oocytes+10 µM melatonin, aged MII oocytes, and aged MII oocytes+10 µM melatonin groups. The phase contrast of each group shows the morphology of oocytes. The fluorescence intensity of DCFH-DA was applied to probe ROS within the cytoplasm of oocytes (magnification: ×200, scale bars: 100 µm).

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