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.
Highlights
In mammalian, more than 99% of the ovarian follicles undergo atresia during development [1,2]
Using an established in vitro hypoxic model [30,31], we verified the hypoxia state by detecting the protein level of hypoxia-inducible factor 1α (HIF-1α), which showed a marked upregulation in granulosa cells (GCs) cultured with 1% O2 (Figure 1A)
We further affirmed that MT at 10−7 M attenuated the apoptosis of porcine GCs triggered by hypoxia (Figure 1D)
Summary
More than 99% of the ovarian follicles undergo atresia during development [1,2]. Apoptosis of ovarian granulosa cells (GCs) occurs much earlier than the morphological changes of follicular atresia, which could be observed only when. GCs apoptosis is considered to be the initiating factor leading to follicular atresia [3,4]. The GCs showed typical features of apoptotic death, such as nuclear pyknosis and DNA fragmentation [5]. The identification of internucleosomal DNA fragmentation during atresia in porcine ovaries suggests that apoptosis is a basic mechanism associated with ovarian follicular atresia in mammalian as well as avian species [5]. The phenomenon of GCs apoptosis has been detected in atretic follicles of mice, pigs, cattle, sheep, and other animals [6,7,8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
