College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China.
College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China.
Peptides. 2018 Mar;101:106-111. doi: 10.1016/j.peptides.2018.01.006. Epub 2018 Jan 11.
RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), has been proposed as a key inhibitory regulator of mammal reproduction. Our previous studies have demonstrated that RFRP-3 inhibited the expression of proliferation-related proteins in porcine granulose cells (GCs), but the inhibitory mechanism causing this has not been discovered. Here, we aim to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on porcine GC proliferation. To this end, the viability of porcine GCs was initially estimated by cell counting kit-8 (CCK-8). We confirmed that different doses of RFRP-3 decreased the cellular viability, suggesting that RFRP-3 could inhibit the proliferation of GCs. Subsequently, we evaluated the direct effects of RFRP-3 on the expression of cell cycle regulators. Compared to the control treated cells, 10 and 10 M of RFRP-3 effectively reduced the transcription of Cyclin B1 and CDK1 mRNAs. However, treatment with RFRP-3 did not alter Cyclin A2, Cyclin D1, CDK2, or CDK4 mRNA levels. These results suggest that RFRP-3 might be inducing G2/M-phase arrest in porcine GCs. Finally, to further determine the molecular mechanism underlying RFRP-3-mediated G2/M cell cycle arrest, we observed the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. The results showed that RFRP-3 treatment significantly increased the expression of Myt1, p-Wee1 and p-Cdc2, whereas the level of Cyclin B1 significantly decreased in porcine GCs treated with 10 M of RFRP-3. Taken together, our data suggest that RFRP-3 regulates the phosphorylation or expression of G2/M cell cycle regulatory factors to induce G2/M-phase arrest via inhibition Cyclin B-CDK1 complex activation in porcine GCs, which might provide an unfavorable condition for porcine GC proliferation.
促性腺激素抑制激素相关肽-3（RFRP-3），作为哺乳动物促性腺激素抑制激素（GnIH）的同源物，被认为是哺乳动物生殖的关键抑制调节因子。我们之前的研究表明，RFRP-3 抑制了猪颗粒细胞（GCs）中增殖相关蛋白的表达，但尚未发现导致这种抑制的机制。在这里，我们旨在阐明其潜在机制，并确定 RFRP-3 对猪 GC 增殖的细胞周期调控作用位点。为此，我们首先通过细胞计数试剂盒-8（CCK-8）来评估猪 GCs 的活力。我们证实，不同剂量的 RFRP-3 降低了细胞活力，表明 RFRP-3 可以抑制 GCs 的增殖。随后，我们评估了 RFRP-3 对细胞周期调节蛋白表达的直接影响。与对照处理的细胞相比，10 和 10 M 的 RFRP-3 有效降低了 Cyclin B1 和 CDK1 mRNA 的转录。然而，RFRP-3 处理并未改变 Cyclin A2、Cyclin D1、CDK2 或 CDK4 mRNA 水平。这些结果表明，RFRP-3 可能诱导猪 GCs 发生 G2/M 期阻滞。最后，为了进一步确定 RFRP-3 介导的 G2/M 细胞周期阻滞的分子机制，我们观察了 RFRP-3 处理的猪 GCs 中 G2/M 细胞周期调节因子的水平。结果表明，RFRP-3 处理显著增加了 Myt1、p-Wee1 和 p-Cdc2 的表达，而用 10 M 的 RFRP-3 处理猪 GCs 时，Cyclin B1 的水平显著降低。综上所述，我们的数据表明，RFRP-3 通过抑制 Cyclin B-CDK1 复合物的激活来调节 G2/M 细胞周期调节因子的磷酸化或表达，从而诱导猪 GCs 中的 G2/M 期阻滞，这可能为猪 GC 增殖提供不利条件。
