Abstract
Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus–oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT–qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8–16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5–50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.
Highlights
Juglone is a phenolic compound identified in the roots, barks, and leaves of many Juglans species comprising J. nigra, J. regia, and J. cinerea, as well as Proteaceae, Caesalpiniaceae, Fabaceae, and the hickory tree [1]
Oocyte maturation comprises the transfer of naturally arrested oocytes from the germinal vesicle (GV) stage to the metaphase II (MII) stage [10]
Checking cumulus–oocyte complexes (COCs) under the microscope at days 0 and 1 from the onset of culturing revealed that cumulus cells surrounding the majority of oocytes of the untreated group were fully expanded, whereas a significant reduction in the properly expanded cumulus cells was observed in the three juglone-treated groups in a dose-dependent manner (64.0 ± 5.09%, 36.0 ± 6.78%, and 6.0 ± 2.45% for 12.5, 25.0, and 50.0 μM juglone vs. 90.0 ± 3.54% for control; Figure 1A,B; p < 0.05)
Summary
Juglone is a phenolic compound identified in the roots, barks, and leaves of many Juglans (walnut trees) species comprising J. nigra, J. regia, and J. cinerea, as well as Proteaceae, Caesalpiniaceae, Fabaceae, and the hickory tree [1]. Juglone suppressed lipopolysaccharides-induced inflammatory responses and NLRP3 inflammasome activation in tumor-derived J774.1 cells [9]. For these reasons, it has long been used in traditional medicine against various health conditions, including gastrointestinal disorders, cancers, allergies, and different microbial infections [6]. Oocyte maturation comprises the transfer of naturally arrested oocytes from the germinal vesicle (GV) stage to the metaphase II (MII) stage [10] During this process, the oocyte acquires the ability to generate an activation response, triggered by sperm during fertilization, and initiate subsequent embryonic development [11,12]. Mitochondria play an essential role in maintaining the functionality of oocytes by providing the main supply of ATP essential for maturation, fertilization, and embryo development [15,16]
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