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Jun-Xue Jin
Northeast Agricultural University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0932-2105
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Background: The importance of the processes of lipogenesis and lipolysis in providing an essential energy source during oocyte maturation is increasingly being recognized. Recent our studies have demonstrated that melatonin up-regulated lipid metabolism during oocyte maturation. Nevertheless, there is still limited information regarding the underlying molecular mechanisms of action of melatonin on lipid metabolism in porcine cumulus-oocyte complexes (COCs). Here, our aim was to investigate the effect of melatonin on COCs, and the melatonin receptor-mediated lipid metabolism signaling pathway.
Materials/methods: To determine the melatonin-mediated lipolysis pathway in cumulus cells, COCs were treated with melatonin and the correlated metabolic responses were assessed using melatonin receptor-mediated signaling.
Results: The results showed that exposure of COCs to melatonin during in vitro maturation significantly increased cumulus expansion index, blastocyst formation rate and total cell numbers/blastocyst, although nuclear maturation was no significant difference. The levels of proteins MT1, MT2, Gsα, PKA, and lipolysis-related factors (AGTL, HSL, PLIN A+B) were significantly increased by melatonin supplementation, and this effect was inhibited by simultaneous treatment with melatonin antagonists (luzindole or 4P-PDOT), although 4P-PDOT treatment did not completely block the effect of melatonin on MT1. Further, the gene expression patterns reflected their relevant protein levels in cumulus cells. Melatonin-mediated lipolysis could significantly reduce lipid droplets (LDs) numbers and increase fatty acid (FA) production and ATP levels by increasing the β-oxidation-related gene expression in cumulus cells. Simultaneously, melatonin significantly increased the amount of LDs, FAs, ATP, and enhanced the lipid metabolism-related gene expression in oocytes. Finally, the oocyte quality was improved by increasing GDF9, BMP15 and GSH and decreasing ROS levels.
Conclusion: These findings revealed that the MT2-mediated cAMP/PKA signaling pathway promotes intracellular lipolysis and FA production in cumulus cells, which provided an essential energy source for COCs development.
Keywords
Melatonin, MT2 receptor, Protein kinase A, Lipolysis, Cumulus cells, Oocytes
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.jpg
Figure S1. Expression of mRNA and proteins in cumulus cells by Real-time PCR and Western blot analysis, respectively. (A) Relative protein expression and (B) mRNA expression. Melatonin/MTn, 10-9 mol/L melatonin; Luzindole/Lu, 10-9 mol/L Luzindole; 4P-PDOT/4P, 10-9 mol/L 4P-PDOT. Different letters denote significant difference (P < 0.05).
- SupplementaryFigure2.jpg
Figure S2. Expression of oocyte developmental indicators and lipid metabolism-related genes in oocytes. (A and D) GDF9, (B and E) BMP15, (C, F and G) GSH and ROS, and (H) mRNA expression related to lipid metabolism. Melatonin/MTn, 10-9 mol/L melatonin; Luzindole/Lu, 10-9 mol/L Luzindole; 4P-PDOT/4P, 10-9 mol/L 4P-PDOT. Scale bar = 25 μm. Different letters denote significant difference (P < 0.05).
- SupplementaryTable1.docx
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Jun-Xue Jin
Northeast Agricultural University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0932-2105
Badges
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This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
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History
CURRENT STATUS: Posted
Version 1
Posted 01 Jun, 2021
No community comments so far
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Comments: 0
PDF Downloads: ...
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The importance of the processes of lipogenesis and lipolysis in providing an essential energy source during oocyte maturation is increasingly being recognized. Recent our studies have demonstrated that melatonin up-regulated lipid metabolism during oocyte maturation. Nevertheless, there is still limited information regarding the underlying molecular mechanisms of action of melatonin on lipid metabolism in porcine cumulus-oocyte complexes (COCs). Here, our aim was to investigate the effect of melatonin on COCs, and the melatonin receptor-mediated lipid metabolism signaling pathway.
Materials/methods: To determine the melatonin-mediated lipolysis pathway in cumulus cells, COCs were treated with melatonin and the correlated metabolic responses were assessed using melatonin receptor-mediated signaling.
Results: The results showed that exposure of COCs to melatonin during in vitro maturation significantly increased cumulus expansion index, blastocyst formation rate and total cell numbers/blastocyst, although nuclear maturation was no significant difference. The levels of proteins MT1, MT2, Gsα, PKA, and lipolysis-related factors (AGTL, HSL, PLIN A+B) were significantly increased by melatonin supplementation, and this effect was inhibited by simultaneous treatment with melatonin antagonists (luzindole or 4P-PDOT), although 4P-PDOT treatment did not completely block the effect of melatonin on MT1. Further, the gene expression patterns reflected their relevant protein levels in cumulus cells. Melatonin-mediated lipolysis could significantly reduce lipid droplets (LDs) numbers and increase fatty acid (FA) production and ATP levels by increasing the β-oxidation-related gene expression in cumulus cells. Simultaneously, melatonin significantly increased the amount of LDs, FAs, ATP, and enhanced the lipid metabolism-related gene expression in oocytes. Finally, the oocyte quality was improved by increasing GDF9, BMP15 and GSH and decreasing ROS levels.
Conclusion: These findings revealed that the MT2-mediated cAMP/PKA signaling pathway promotes intracellular lipolysis and FA production in cumulus cells, which provided an essential energy source for COCs development.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.jpg
Figure S1. Expression of mRNA and proteins in cumulus cells by Real-time PCR and Western blot analysis, respectively. (A) Relative protein expression and (B) mRNA expression. Melatonin/MTn, 10-9 mol/L melatonin; Luzindole/Lu, 10-9 mol/L Luzindole; 4P-PDOT/4P, 10-9 mol/L 4P-PDOT. Different letters denote significant difference (P < 0.05).
- SupplementaryFigure2.jpg
Figure S2. Expression of oocyte developmental indicators and lipid metabolism-related genes in oocytes. (A and D) GDF9, (B and E) BMP15, (C, F and G) GSH and ROS, and (H) mRNA expression related to lipid metabolism. Melatonin/MTn, 10-9 mol/L melatonin; Luzindole/Lu, 10-9 mol/L Luzindole; 4P-PDOT/4P, 10-9 mol/L 4P-PDOT. Scale bar = 25 μm. Different letters denote significant difference (P < 0.05).
- SupplementaryTable1.docx
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