环磷酸鸟苷（cGMP）在牛卵母细胞脂解作用中的介导作用及其对胚胎发育和冷冻保存的影响。
The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.
作者信息
Schwarz Kátia R L, de Castro Fernanda C, Schefer Letícia, Botigelli Ramon C, Paschoal Daniela M, Fernandes Hugo, Leal Cláudia L V
机构信息
Universidade de São Paulo (USP), Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), Departamento de Medicina Veterinária, Pirassununga, São Paulo, Brasil.
Universidade Estadual Paulista (Unesp), Instituto de Biociências de Botucatu (IBB), Departamento de Farmacologia, Botucatu, São Paulo, Brasil.
出版信息
PLoS One. 2018 Jan 23;13(1):e0191023. doi: 10.1371/journal.pone.0191023. eCollection 2018.
This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP) and cGMP-dependent kinase (PKG) during in vitro maturation (IVM) on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs), and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII), cGMP levels, lipid content in oocytes (OO), transcript abundance for genes involved in lipolysis (ATGL) and lipid droplets (PLIN2) in cumulus cells (CC) and OO, and presence of phosphorylated (active) hormone sensitive lipase (HSLser563) in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme) with 10-5M sildenafil (SDF) during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05) and did not affect on maturation rate (84.3±6.4% MII). Fetal calf serum (FCS) in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA) + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05). FCS increased lipid content in OO (40.1 FI, p<0.05) compared to BSA (34.6 FI), while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05). PKG inhibitor (KT5823) reversed this effect (38.9 FI, p<0.05). ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI) compared to its use in either or none of the culture periods (34.2 FI, p<0.05). Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.
本研究旨在确定体外成熟（IVM）过程中环磷酸鸟苷（cGMP）和cGMP依赖性蛋白激酶（PKG）对牛卵丘-卵母细胞复合体（COCs）中脂解相关参数、胚胎发育及冷冻存活率的影响。用cGMP/PKG调节剂使COCs成熟，并评估其第二次减数分裂中期（MII）率、cGMP水平、卵母细胞（OO）中的脂质含量、卵丘细胞（CC）和OO中参与脂解的基因（脂肪甘油三酯脂酶，ATGL）及脂质滴（ perilipin 2，PLIN2）的转录丰度，以及OO中磷酸化（活性）激素敏感脂肪酶（HSLser563）的存在情况。还评估了胚胎发育、脂质含量及玻璃化冷冻存活率。在24小时IVM期间用10-5M西地那非（SDF）抑制磷酸二酯酶5（PDE5，一种cGMP水解酶）可增加COCs中的cGMP（未处理对照组为9.5 fMol/COC，处理组为56.9 fMol/COC，p<0.05），且不影响成熟率（MII为84.3±6.4%）。与牛血清白蛋白（BSA）+SDF相比，IVM培养基中的胎牛血清（FCS）降低了COCs中的cGMP（分别为19.6和66.5 fMol/COC，p<0.05）。与BSA（34.6 FI）相比，FCS增加了OO中的脂质含量（40.1 FI，p<0.05），而SDF降低了脂质含量（分别为29.8和29.6 FI，与BSA或FCS组合时，p<0.05）。PKG抑制剂（KT5823）逆转了这种作用（38.9 FI，p<0.05）。在CC和OO中检测到了ATGL和PLIN2转录本，但仅在CC中受cGMP和PKG影响。在有或无调节剂情况下成熟的OO中均检测到HSLser563。与在IVM和IVC任一阶段或均未使用SDF相比，仅在IVM和IVC期间添加SDF时观察到胚胎脂质含量降低（27.6 FI，p<0.05）。玻璃化冷冻存活率不受SDF影响。总之，cGMP和PKG参与了OO中的脂解过程，可能也参与了CC和胚胎中的脂解；血清对该途径有负面影响，导致脂质积累，cGMP调节可能会降低卵母细胞和胚胎中的脂质含量，但不会提高胚胎的冷冻耐受性。
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