Melatonin Alleviates Hypoxia-Induced Apoptosis of Granulosa Cells by Reducing ROS and Activating MTNR1B–PKA–Caspase8/9 Pathway

Abstract

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT–MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

“…Consistent with these data, previous studies reported that EP4 receptor blockade hindered proliferation and induced apoptosis in cervical cancer cells via the EP4-PKA-CREB pathway. Moreover, reduced PKA induced apoptosis through Bcl-2/Bax-induced apoptosis [23] and activation of the caspase-8/9/11 pathway and pyroptosis [24,25]. The PKA inhibitor blocked PGE2 through EP2-and EP4-induced apoptosis, indicating that the PKA pathway is mainly responsible for PGE2-mediated inhibition of apoptosis [26].…”

Sulforaphane Acts Through NFE2L2 to Prevent Hypoxia-Induced Apoptosis in Porcine Granulosa Cells via Activating Antioxidant Defenses and Mitophagy

“…ROS was mainly brought forth through the electron transfer chain and the ROS-detoxifying enzyme pathway. Our previous study demonstrated that hypoxia induced GC apoptosis through ROS. ,, In the present study, we show that SFN supplemented under hypoxic exposure can relieve GC apoptosis by decreasing the level of ROS. Further studies revealed that SFN could increase the expression of NFE2L2-guided antioxidant enzymes such as NQO1, HO-1, SOD1, and SOD2.…”

“…After 36 h, porcine GCs were washed with PBS two times and treated with different SFN concentrations (1, 5, and10 μM) or inhibitors. With GCs being cultured in a hypoxic environment, we cultured the cells under hypoxic conditions according to the previous method (1% O 2 , 5% CO 2 , and balanced N 2 ) …”

“…It has been demonstrated that the oxygen partial pressure (pO 2 ) within follicular fluid is decreased during follicular development, , leading to a hypoxic environment surround GCs. Since the basement membrane separates GCs from blood capillaries outside the follicular wall, the enlargement of follicular sizes exacerbates the hypoxic status in GCs. − One of the major stresses is excessive production of intracellular reactive oxygen species (ROS). Low oxygen tension increased ROS generation, which had been demonstrated to have a antisteroidogenic effect in GCs. , ROS accumulation leads to oxidative stress, which stimulates apoptotic and necrotic cell death. − …”

Turn-on silicon-based fluorescent probe for visualizing endogenous CO during hypoxia

“…The production of endogenous carbon monoxide (CO) in cells comes from the degradation of heme, which is caused by the up-regulation of heme oxygenase-1 (HO-1). HO-1 expression is induced by various stimuli, 1 including reactive oxygen species (ROS), 2 hypoxia, 3 inflammation, viral infection, and stress. Moreover, HO-1 typically catalyzes the degradation of heme into bilirubin, free iron and CO. 4,5 Bilirubin is considered to have an antioxidant effect; 6,7 free iron is usually stored in ferritin, thereby limiting its ability to participate in the Fenton reaction to produce cytotoxic free radicals; 8 CO restrains apoptosis by taking part in the activation of mitogen-activated protein kinase (MAPK) pathways.…”