背景:体外胚胎生产是马的一种高度需求的生殖技术,这需要卵母细胞的恢复(体内或死后)和体外成熟(IVM)。与体内对应物相比,接受IVM的卵母细胞表现出较差的发育能力,这与商业成熟培养基的次优组成有关。这项工作的目的是研究在IVM期间从马排卵前卵泡液(FF)获得的不同浓度的分泌组对卵丘-卵母细胞复合物(COCs)的影响。在存在或不存在分泌组的情况下,对通过卵拾取(OPU)或宰后从屠宰场(SLA)体内回收的COCs进行IVM(对照:0µg/ml,S20:20µg/ml或S40:40µg/ml)。在IVM之后,用于卵母细胞成熟前(Pre-IVM)和IVM后(Post-IVM)的培养基的代谢组,COCsmRNA表达,并对卵母细胞减数分裂能力进行了分析。
结果:IVM导致从OPU和SLA获得的COC产生乳酸和乙酸消耗。然而,当添加S40时,来自OPU的COCs在IVM后的葡萄糖消耗较高(控制前IVM与S40IVM后:117.24±7.72vs.82.69±4.24;平均值µM±SEM;p<0.05),而在SLA的COC中未观察到这一点。同样,分泌组增强苏氨酸的摄取(对照前IVM与S20后IVM与S40IVM后:4.93±0.33vs.3.04±0.25vs.2.84±0.27;OPU回收的COC的平均值µM±SEM;p<0.05)。关于代谢相关候选基因的相对mRNA表达,在OPU衍生的COC中,当在IVM期间以20-40µg/ml添加分泌组时,乳酸脱氢酶A(LDHA)表达显着下调(对照与S20vs.S40:1.77±0.14vs.1±0.25vs.1.23±0.14;倍数变化±SEM;p<0.05),但不是在SLACOC中。
结论:体外成熟(IVM)过程中分泌组的添加影响LDHA的基因表达,葡萄糖代谢,和马卵丘-卵母细胞复合物(COCs)中的氨基酸周转,在使用卵子拾取(OPU)和屠宰场衍生的COC(SLA)检索的COC之间观察到不同的结果。
BACKGROUND: In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of
secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of
secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed.
RESULTS: IVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise,
secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when
secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs.
CONCLUSIONS: The addition of
secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA).