Mishra A, Reddy I J, Gupta Psp, Mondal S
Animal Physiology Division, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, India.
Reprod Domest Anim. 2016 Dec;51(6):1020-1029. doi: 10.1111/rda.12789. Epub 2016 Sep 30.
The objective of this study was to find out the impact of L-carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post-fertilization medium separately. Subsequent objective was to observe the L-carnitine-mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L-carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L-carnitine during post-fertilization period. So it is suggested to use L-carnitine during maturation than post-fertilization period. Antiapoptotic and proliferative effects of L-carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L-carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but supplementation of L-carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control. L-carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L-carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16-cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L-carnitine supplementation. In conclusion, L-carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L-carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.
本研究的目的是分别在成熟培养基和受精后培养基中添加L-肉碱（10 mM），以探究其对体外培养的绵羊植入前胚胎发育调控的影响。后续目的是观察L-肉碱介导的绵羊卵母细胞和体外产生的发育胚胎中凋亡基因（Bcl2、Bax、Casp3和PCNA）表达的变化。与在受精后阶段用L-肉碱培养的假定合子相比，用L-肉碱成熟的卵母细胞的卵裂率（67.23%对43.12%）、桑葚胚率（47.65%对28.58%）和囊胚率（(32.12%对13.24%）显著更高（p <.05）。因此，建议在成熟阶段而非受精后阶段使用L-肉碱。分别用放线菌素D（凋亡剂）和TNFα（抗增殖剂）诱导培养基，并添加或不添加L-肉碱，证实了L-肉碱的抗凋亡和增殖作用。用放线菌素D和TNFα培养的卵母细胞和胚胎出现发育停滞，桑葚胚和囊胚率显著降低（p <.05），但在放线菌素D和TNFα诱导的培养基中添加L-肉碱显示出与对照相似的结果。在体外成熟过程中添加L-肉碱在大多数发育阶段显著上调了Bcl2和PCNA基因的表达。虽然L-肉碱在发育初期上调了Bax的表达，但在后期下调，而Casp3的表达在到16细胞阶段之前上调，但之后表达没有差异。L-肉碱的添加对GAPDH基因的表达没有影响。总之，L-肉碱在胚胎发育过程中起抗凋亡和增殖化合物的作用，在体外成熟过程中添加L-肉碱改变了胚胎发育阶段凋亡基因的表达。
