Zeebaree Bayar K, Kwong Wing Y, Mann George E, Gutierrez Carlos G, Sinclair Kevin D
University of Nottingham, School of Biosciences, Sutton Bonington, Leicestershire, LE12 5RD, UK.
University of Nottingham, School of Biosciences, Sutton Bonington, Leicestershire, LE12 5RD, UK; Universidad Nacional Autonoma de Mexico, Facultad de Medicina Veterinaria, Mexico City 04510, Mexico.
Theriogenology. 2018 Jan 1;105:107-114. doi: 10.1016/j.theriogenology.2017.09.014. Epub 2017 Sep 15.
Our understanding of the effects of temperature on granulosa cell (GC) physiology is primarily limited to in vitro studies conducted under atmospheric (∼20% O) conditions. In the current series of factorial experiments we identify important effects of O level (i.e. 5% vs 20% O) on GC viability and steroidogenesis, and go onto report effects of standard (37.5 °C) vs high (40.0 °C) temperatures under more physiologically representative (i.e. 5%) O levels in the presence of different levels of melatonin (0, 20, 200 and 2000 pg/ml); a potent free-radical scavenger and abundant molecule within the ovarian follicle. Cells aspirated from antral (4-6 mm) follicles were cultured in fibronectin-coated wells using serum-free M199 for up to 144 h. At 37.5 °C viable cell number was enhanced and luteinization reduced under 5 vs 20% O. Oxygen level interacted (P < 0.001) with time in culture to affect aromatase activity and cell estradiol (E) production (pg/mL/10 cells). These decreased between 48 and 96 h for both O levels but increased again by 144 h for cells cultured under 5% but not 20% O. Progesterone (P) concentration (ng/mL/10 cells) was greater (P < 0.001) under 20 vs 5% O at 96 and 144 h. Cell number increased (P < 0.01) with time in culture under 5% O irrespective of temperature. However, higher doses of melatonin increased viable cell number at 40.0 °C but reduced viable cell number at 37.5 °C (P = 0.004). Melatonin also reduced (P < 0.001) ROS generation at both O levels across all concentrations. E increased with time in culture at both temperatures under 5% O, however P declined between 96 and 144 h at 40.0 but not 37.5 °C. Furthermore, melatonin interacted (P < 0.001) with temperature in a dose dependent manner to increase P at 37.5 °C but to reduce P at 40.0 °C. Transcript expression for HSD3B1 paralleled temporal changes in P production, and those for HBA were greater at 5% than 20% O, suggesting that hemoglobin synthesis is responsive to changes in O level. In conclusion, 5% O enhances GC proliferation and reduces luteinization. Elevated temperatures under 5% O reduce GC proliferation and P production. Melatonin reduces ROS generation irrespective of O level and temperature, but interacts with temperature in a dose dependent manner to influence GC proliferation and luteinization.
我们对温度对颗粒细胞（GC）生理影响的理解主要局限于在大气（约20%氧气）条件下进行的体外研究。在当前一系列析因实验中，我们确定了氧气水平（即5%与20%氧气）对GC活力和类固醇生成的重要影响，并继续报告在更具生理代表性的（即5%）氧气水平下，在存在不同水平褪黑素（0、20、200和2000 pg/ml）的情况下，标准温度（37.5°C）与高温（40.0°C）的影响；褪黑素是一种强大的自由基清除剂，在卵巢卵泡中含量丰富。从窦状（4 - 6毫米）卵泡中吸出的细胞在纤连蛋白包被的孔中使用无血清M199培养长达144小时。在37.5°C时，与20%氧气相比，5%氧气下活细胞数量增加，黄体化减少。氧气水平与培养时间相互作用（P < 0.001）以影响芳香化酶活性和细胞雌二醇（E）产生（pg/mL/10个细胞）。两种氧气水平下，这些在48至96小时之间均下降，但在5%氧气而非20%氧气下培养的细胞在144小时时再次增加。在96和144小时时，20%氧气下的孕酮（P）浓度（ng/mL/10个细胞）高于5%氧气下（P < 0.001）。在5%氧气下，无论温度如何，细胞数量随培养时间增加（P < 0.01）。然而，较高剂量的褪黑素在40.0°C时增加活细胞数量，但在37.5°C时减少活细胞数量（P = 0.004）。在所有浓度下，两种氧气水平下褪黑素均减少（P < 0.001）活性氧生成。在5%氧气下，两种温度下培养时E均随时间增加，然而在40.0°C而非37.5°C时，P在96至144小时之间下降。此外，褪黑素与温度以剂量依赖方式相互作用（P < 0.001），在37.5°C时增加P，但在40.0°C时减少P。HSD3B1的转录本表达与P产生的时间变化平行，且在5%氧气下HBA的转录本表达高于20%氧气下，表明血红蛋白合成对氧气水平变化有反应。总之，5%氧气增强GC增殖并减少黄体化。5%氧气下升高的温度降低GC增殖和P产生。褪黑素无论氧气水平和温度如何均减少活性氧生成，但与温度以剂量依赖方式相互作用以影响GC增殖和黄体化。
