自噬对小鼠围着床期子宫内膜容受性的影响

Effects of autophagy on endometrial receptivity during the peri-implantation period in mice

摘要:

目的 探究自噬对小鼠围着床期子宫内膜容受性的影响。方法 将36只ICR妊娠雌鼠随机分为空白组与自噬抑制剂组，每组18只。自噬抑制剂组自妊娠第1天（PD1）起，每日腹腔注射自噬抑制剂3-MA直至处死，空白组每日腹腔注射等量磷酸盐缓冲液（PBS）作为对照，各组分别于PD4、PD5、PD6以颈椎脱臼法处死6只小鼠。观察各组小鼠子宫形态，计数胚胎着床位点数，分别采用实时荧光定量聚合酶链反应、Western blotting检测子宫内膜组织中自噬相关因子Beclin-1、微管相关蛋白轻链3-Ⅰ（LC3-Ⅰ）、LC3-Ⅱ及子宫内膜容受性相关标志物雌激素受体α（ERα）、孕激素受体（PR）、同源框基因A10（HOXA10）mRNA与蛋白表达量，采用扫描电镜观察各组小鼠子宫内膜胞饮突形态，采用透射电镜观察子宫内膜细胞自噬体结构。结果 空白组PD4子宫无明显串珠状改变，PD5、PD6着床部位宫体膨大，形成串珠状，均匀分布，色泽红润；自噬抑制剂组PD4子宫色淡白、较细，无明显串珠状改变；PD5、PD6胚胎着床部位宫体轻度膨大，着床数目较少，分布不均匀，色淡红。自噬抑制剂组PD5、PD6胚胎着床位点数较空白组减少（P ＜0.05）。与空白组PD4、PD5、PD6比较，自噬抑制剂组PD4、PD5、PD6 LC3-Ⅱ mRNA相对表达量均降低（P <0.05）。与空白组PD4、PD5、PD6比较，自噬抑制剂组LC3-Ⅱ蛋白相对表达量、LC3-Ⅱ/LC3-Ⅰ比值均降低（P <0.05），LC3-Ⅰ蛋白相对表达量升高（P <0.05）。与空白组PD4、PD5、PD6比较，自噬抑制剂组PR mRNA相对表达量降低（P <0.05）；与空白组PD4、PD5比较，自噬抑制剂组HOXA10 mRNA相对表达量降低（P <0.05）。与空白组PD4、PD5、PD6比较，自噬抑制剂组ERα蛋白相对表达量升高（P <0.05）；与空白组PD5、PD6比较，自噬抑制剂组PD5、PD6 PR、HOXA10蛋白相对表达量均降低（P <0.05）。扫描电镜下，空白组PD4子宫内膜表面可见少量胞饮突，PD5、PD6胞饮突数目多，大小均一；自噬抑制剂组PD4子宫内膜表面胞饮突结构不明显且皱缩，PD5、PD6胞饮突数目少，大小不均。透射电镜下，空白组PD4子宫内膜细胞中自噬溶酶体数量较多，PD5、PD6数量减少；自噬抑制剂组PD4可见少量自噬溶酶体，PD5、PD6未见明显自噬溶酶体，较空白组明显减少。结论 自噬可能影响围着床期子宫内膜容受性的建立与调节，参与胚胎着床过程。

Abstract:

Objective To explore the effects of autophagy on endometrial receptivity during the peri-implantation period in mice.Methods Thirty-six pregnant ICR female mice were randomly divided into a blank group and an autophagy inhibitor group, with eighteen mice in each group. The autophagy inhibitor group received daily intraperitoneal injections of the autophagy inhibitor 3-MA starting from the 1st day of pregnancy (PD1) until sacrifice. The blank group received daily intraperitoneal injections of an equal volume of PBS as a control. Six mice in each group were sacrificed by cervical dislocation on PD4, PD5, and PD6. The uterine morphology of the mice in each group was observed, and the number of embryo implantation sites was counted. The mRNA and protein expression levels of the autophagy-related factors Beclin-1, LC3-I, and LC3-II, as well as the endometrial receptivity markers ERα, PR, and HOXA10 in the endometrial tissues were detected by qRT-PCR and Western blotting. The morphology of pinopodes on the endometrial surface was observed by scanning electron microscopy, and the autophagic structures in the endometrial cells were observed by transmission electron microscopy.Results In the blank group, the uterus on PD4 showed no obvious bead-like changes, while the implantation sites were enlarged on PD5 and PD6, exhibiting a bead-like appearance with uniform distribution and a pinkish coloration. In the autophagy inhibitor group, the uterus on PD4 appeared pale and thin, with no obvious bead-like changes. The implantation sites in the uterine body were slightly enlarged, with fewer embryos, uneven distribution, and a pale red coloration. Compared to the blank group, the implantation site numbers in the autophagy-inhibitor groups PD5 and PD6 were reduced (P < 0.05). Compared with the blank group, the expression of LC3-II mRNA was decreased in the autophagy inhibitor group on PD4, PD5, and PD6 (P < 0.05). The expression of LC3-II protein and the LC3-II/LC3-I ratio were decreased (P < 0.05), while the expression of LC3-I protein was increased on PD4, PD5, and PD6 in the autophagy inhibitor group (P < 0.05). Compared with the blank group, the expression of PR mRNA was decreased on PD4, PD5, and PD6 (P < 0.05), and the expression of HOXA10 mRNA was decreased on PD4 and PD5 in the autophagy inhibitor group (P < 0.05). The expression of ERα protein was increased on PD4, PD5, and PD6 (P < 0.05), while the expressions of PR and HOXA10 protein were decreased on PD5 and PD6 in the autophagy inhibitory group (P < 0.05). Scanning electron microscopy showed a small number of pinopodes on the endometrial surface on PD4 in the blank group, while numerous pinopodes homogeneous in size presented on PD5 and PD6. In the autophagy inhibitor group, pinopodes on the endometrial surface were unobvious and wrinkled on PD4, and a few of pinopodes heterogenous in size were observed on PD5 and PD6. Transmission electron microscopy demonstrated abundant autolysosomes in endometrial cells of the blank group on PD4, with a decline on PD5 and PD6. In the autophagy inhibitor group, only a small number of autolysosomes were observed on PD4, and none were evident on PD5 and PD6, indicating a significant reduction compared with the control group.Conclusions Autophagy may participate in the establishment and regulation of endometrial receptivity during the peri-implantation period and is involved in the process of embryo implantation.