Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Theriogenology. 2011 Dec;76(9):1706-15. doi: 10.1016/j.theriogenology.2011.07.002.
During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocyte maturation on their developmental capacity after fertilization (Exp. 1), incidence of apoptosis in cumulus oocyte complexes (COCs) (Exp. 2) or on relative mRNA abundances of genes in cumulus cells and oocytes (Exp. 3). COCs were matured in serum-free medium containing 1 mg/mL polyvinyl alcohol and 0, 10, 100, or 1000 ng/mL leptin (L0, L10, L100, and L1000, respectively), or in medium supplemented with 10% fetal calf serum (FCS) as a positive control. Addition of leptin during oocyte maturation had no effect on cleavage rates after fertilization (FCS, 68.6%; L0, 62.9%; L10, 66.9%; L100, 63.4%; L1000, 60.9%). Similarly, no significant differences in blastocyst rates were observed when oocytes were matured in the presence of L0 (8.4%), L10 (9.3%), L100 (6.7%), L1000 (8.2%), compared to control FCS (9.4%). In Experiment 2, maturation in the presence of 1000 ng/mL of leptin increased the proportion of TUNEL-positive cumulus cell (6.9%) with respect to those matured in the presence of FCS (4.96%), but not at the lower leptin doses. When relative mRNA abundances were examined for seven genes by qRT-PCR, five (TP53, BAX, DNMT3A, PGTS2 and LEPR) showed differences among groups. LEPR expression was significantly higher in the oocytes matured with FCS compared with the other groups and in those matured with PVA (L0) without leptin compared with the three groups of oocytes matured in the presence of leptin. In conclusion, the addition of leptin to the in vitro maturation medium used for prepubertal bovine oocytes does not increase the development potential of the oocytes or reduce the percentage of apoptosis in cumulus cells. Leptin blocks transcription of the leptin receptor (LEPR) probably reflecting selective, differential degradation by doses of leptin.
在成年牛卵母细胞的体外成熟过程中，瘦素对囊胚发育、细胞凋亡和发育相关基因的转录水平具有有益作用。本研究分析了瘦素对未成年牛卵母细胞和卵丘细胞的差异影响。实验 1 中，研究了在卵母细胞成熟过程中添加瘦素对受精后发育能力的影响；实验 2 中，研究了添加瘦素对卵丘-卵母细胞复合物（COCs）中细胞凋亡的影响；实验 3 中，研究了添加瘦素对卵丘细胞和卵母细胞中基因相对 mRNA 丰度的影响。COCs 在含有 1 mg/mL 聚乙烯醇和 0、10、100 或 1000ng/mL 瘦素（L0、L10、L100 和 L1000）的无血清培养基中成熟，或在补充有 10%胎牛血清（FCS）的培养基中成熟作为阳性对照。在卵母细胞成熟过程中添加瘦素对受精后的卵裂率没有影响（FCS：68.6%；L0：62.9%；L10：66.9%；L100：63.4%；L1000：60.9%）。同样，当卵母细胞在 L0（8.4%）、L10（9.3%）、L100（6.7%）、L1000（8.2%）存在的情况下成熟时，囊胚率与对照 FCS（9.4%）相比没有显著差异。在实验 2 中，与成熟于 FCS 相比，在 1000ng/mL 瘦素存在下，TUNEL 阳性的卵丘细胞比例增加（6.9%），但在较低剂量的瘦素下没有增加。通过 qRT-PCR 检查 7 个基因的相对 mRNA 丰度时，其中 5 个（TP53、BAX、DNMT3A、PGTS2 和 LEPR）在各组之间存在差异。与其他组相比，用 FCS 成熟的卵母细胞 LEPR 表达显著升高，与未添加瘦素的 PVA（L0）相比，在添加瘦素的三组卵母细胞中表达升高。总之，在未成年牛卵母细胞的体外成熟培养基中添加瘦素不会增加卵母细胞的发育潜力，也不会降低卵丘细胞的凋亡率。瘦素可能通过剂量选择性地降解瘦素受体（LEPR）来阻止其转录。
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