RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells

Abstract

Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs) and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3), decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL) and anti-oxidant genes (SOD1 and GPX4) without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A) and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

The Mechanism of Melatonin and Its Receptor MT2 Involved in the Development of Bovine Granulosa Cells

“…Unluteinized GCs play an important role in modulating the ovarian follicular development, such as folliculogenesis, follicle selection, oocyte maturation, follicular atresia, and also act as an important role in regulating the ovary function by local autocrine/paracrine systems. Our previous studies have demonstrated that melatonin was involved in modulating the GCs functions [ 25 , 27 , 33 ]. However, there is little information about the role of MT2 in modifying the reproduction related genes , hormone related genes, development related genes and hormone secretion, as well as whether MT2 affects the GCs response to melatonin.…”

“…Similarly, our recent studies have showed that MTNR1A silencing don’t affect bovine GCs respond to melatonin [ 27 ]. Furthermore, MTNR1B silencing didn’t disrupt the effects of melatonin on apoptosis and cell cycle in bovine GCs [ 33 ]. It has been reported that MT1 and MT2 could mediate the modulation of complex reproductive mechanisms [ 21 , 25 , 33 , 37 , 70 , 71 , 72 ].…”

“…Furthermore, MTNR1B silencing didn’t disrupt the effects of melatonin on apoptosis and cell cycle in bovine GCs [ 33 ]. It has been reported that MT1 and MT2 could mediate the modulation of complex reproductive mechanisms [ 21 , 25 , 33 , 37 , 70 , 71 , 72 ]. Therefore, MT1 and MT2 may act in a complementary way to mediate the melatonin actions on bovine GCs [ 27 , 32 ].…”

“…Our previous study found that MT2 was involved in regulating the GCs apoptosis and the apoptosis related genes expression. Moreover, MT2 knockdown didn’t affect the effects of melatonin on suppressing the GCs apoptosis or blocking the cell cycle [ 33 ]. In this study, we revealed the role of MT2 in modulating reproduction-related genes expression, hormones secretion and other regulators of folliculogenesis using MT2 silencing.…”

The effect of SNP rs400827589 in exon 2 of the MTNR1B gene on reproductive seasonality and litter size in sheep

“…To date, numerous studies have explored the relationship between polymorphism of MTNRs and litter size or reproductive seasonality traits in different mammals (Calvo et al., 2018; Chu, Cheng, Liu, Fang, & Ye, 2006, 2008; Chu et al., 2007; Gunwant et al., 2018; Hua, 2006; Liu, Wang, Zhou, Pang, & Wang, 2018; Notter, Cockett, & Hadfield, 2003; Pelletier et al., 2000; Wang, Liu, Ahmad, et al, 2012; Wang, Liu, Wu, et al, 2012). However, there were few studies about the polymorphism of MTNRIB .…”