卵巢内瘦素受体缺失对卵巢生殖功能和全身糖脂代谢的影响

Ovary Defective Leptin Signal Does not Induce Ovarian Failure in the db/db Mouse

为探索卵巢内瘦素信号传导缺失对卵巢自身功能和全身糖脂代谢的影响,阐明瘦素对外周生殖系统的直接作用,进行了以下研究:①对瘦素长受体缺失(LR-)的雌性B6.Cg-m+/+Leprdb小鼠和其同窝出生的有完整LR的雌性野生型C57BL/6J小鼠(LR+),在性成熟期后的12周,进行卵巢相互移植,构建实验小鼠的LR基因型组合分别为A(躯体+,卵巢-)、B(躯体+,卵巢-)、C(躯体+,卵巢+)、D(躯体-,卵巢+)、E(躯体-,卵巢-)共5组(n=5),移植后观测2个动情周期取材;②检测各自的全身糖脂代谢指标、卵巢周期和生殖激素;③卵泡刺激素(0.75IU/g)刺激试验后,用免疫组化和蛋白质印迹(WB)方法,检测卵巢内脂代谢信号蛋白包括Janus激酶(JAK2)、磷酸化的丝裂原激活蛋白激酶(p-ERK)和低密度脂蛋白受体(LDLR)的表达。结果显示:①D、E组与A、B、C3组比较,体重相差约1倍,性腺周围脂肪垫重量相差约10倍(P<0.01);血葡萄糖、低密度脂蛋白胆固醇(LDL-C)和胰岛素水平相差2～3倍(P<0.01);A、B、C3组的全身血糖脂指标相似,D和E2组的高糖脂血症升高程度也相似。②A、B、C3组小鼠均恢复正常的动情周期,而D、E2组则始终处于动情间期;小鼠16周龄时D、E组与A、B、C3组比较:卵巢重量及E2、P、FSH、LH水平显著下降(P<0.05)。③瘦素刺激后JAK2在A、B、D和E组的卵巢中表达均呈弱阳性,而C组卵巢内呈强阳性表达;FSH刺激后p-ERK和LDLR在A、B、C3组卵巢内均呈强阳性表达,而D和E组则表达下降。进而得出结论:①单纯卵巢内瘦素信号缺失并不影响卵巢自身的生殖功能、脂质代谢和全身的糖脂代谢。②瘦素受体全身缺失小鼠的卵巢功能障碍,与卵巢外的全身因素即高糖脂血症和低促性腺激素状态有关。③高糖脂血症降调节卵巢内的瘦素信号蛋白JAK2的表达,诱导小鼠卵巢脂质化和功能障碍;FSH刺激可诱导ERK磷酸化后LDLR的表达增强,缓解小鼠卵巢的脂质化和功能障碍。

To study the role of impaired leptin signaling played in ovary reproductive failure and abnormal lipid profiles in the db/db mouse with mutant leptin receptor （LR-）, adult, female C57BL/6J mice were used in our experiments, littermate wide types （LR＋）, as well as diabetic （db/db） mutant genotypes （LR-）. By ovary transplantation on the day of 12 weeks, five genotypic groups were constructed with body LR＋ and ovary LR- in Group A, body LR＋ and ovary LR- in Group B, body LR＋ and ovary LR＋ in Group C, body LR- and ovary LR＋ in Group D, and body LR-and ovary LR-in Group E . The vaginal smear was performed to evaluate the recovery and cyclicity of transplanted ovaries with mutant or intact LR. Before execution, all mice were injected with recombination FSH （4 h before）, and then the ovaries were collected for the detection of the expression of JAK2, ERK, p-ERK and the LDLR, and the serum were reserved for the assay of the glucose, insulin, LDL-c, E2, P, FSH and LH. The weights of mice, their ovary and their fat pads were also recorded. The results show that mice in D and E groups have similar profiles of the glucose and fat metabolism, that is, hyperglycemia, hyperinsulinemia and hyperlipidemia, in significantly higher levels than those of A, B and C groups, although still in comparably normal levels. Moreover, mice with mutant body LR in D and E groups are acyclic and anovulatory, which means diminished steroidogenic capacity and gonadotropin levels, although ovaries in two groups are genetically different in LR. Mice with intact body LR in A, B, and C groups show normal cycles with similar gonadotropin and steroid levels and each group bear ovary with mutant or intact LR. The protein analysis shows a significantly higher FSH induced expression of p- ERK and LDLR in ovaries of A, B and C groups as compared with D and E groups, and significant higher leptin activated JAK2 expression in LR＋ ovaries in C group as compared with other groups. Thus it is concluded that ovary failure in the db/db mice is due to the hyperlipidemia state and the low gonadotropin levels, not due to defective leptin signal within ovaries. Ovarian hypercytolipidemia could be induced by overall hyperlipidemia through defective ovarian JAK2 expression or rescued by FSH induced phosphrylation of ERK and LDLR expression.