The Mechanism of Cryptorchidism in LH Receptor Knockout Animals

描述（由申请人提供）：隐睾症是迄今为止人类性别分化中最常见的缺陷。睾丸下降在解剖学上可分为两个阶段，即经腹期和腹股沟阴囊期。众所周知，胰岛素样肽3 （INSL3）和雄激素信号通路在睾丸下降经腹期的控制中起关键作用。然而，调节腹股沟-阴囊期的机制尚不完全清楚，尽管这种缺陷的发生率在临床上更为常见。有针对性地破坏小鼠黄体生成素受体（LHRKO）会损害成体间质细胞的发育，导致INSL3和睾酮水平的显著降低。令人惊讶的是，血清雌二醇水平显著升高。LHRKO男性表现出由腹股沟阴囊睾丸下降缺陷引起的双侧隐睾表型。组织学、形态学、细胞增殖和分化分析表明，这种缺陷是由于在睾丸下降的第二阶段间充质细胞分裂和向肌细胞分化的减少。在突变小鼠中，掌骨中几个已知参与掌骨发育的基因，如Hoxa10、Hoxa11、Wt1、Dll1和沙漠的表达没有改变，而与年龄匹配的野生型兄弟姐妹相比，Lgr8、Notch1和Numb mRNA水平急剧下降。相比之下，Esr1 （ER1）和Esr2 （ER2）的表达明显升高。产后睾酮替代疗法（TRT）完成了睾丸降至阴囊，并同时使循环中的雌二醇浓度和网膜中Esr1、Esr2、Lgr8、Notch1和Numb mRNA水平正常化。TRT治疗的缺陷既不依赖于成人型间质细胞的恢复，也不依赖于生殖股神经的恢复，而是需要在管骨中雄激素受体的活性。体外器官培养发现，如果不通过添加INSL3或松弛素激活富含亮氨酸的含重复g蛋白偶联受体8 （LGR8）信号通路，单雄激素不能显著刺激脐间充质细胞的增殖。雌激素与雄激素联合治疗可阻止雄激素诱导的Lgr8表达。INSL3和雄激素的共同处理增加了Notch1，但没有增加Numb mRNA的水平。综上所述，我们推测雄激素诱导Lgr8基因表达和激活Lgr8信号冲击Notch系统，刺激输卵管细胞增殖、分化和肌发生是控制输卵管发育促进腹股沟阴囊睾丸下降的重要分子途径。除了雄激素缺乏外，雄激素/雌激素信号不平衡也在LHR缺失动物的隐睾表型中起作用。在这项拨款申请中，我们提出了四个具体目标来验证我们的假设：(1)确定雄激素调节管骨中Lgr8基因表达的潜在机制；(2)评估LGR8信号通路在雄激素诱导的LHR阴性男性腹股沟阴囊睾丸下降中的作用；(3)确定Notch信号通路在调节雄激素和LGR8信号调控腹股沟阴囊睾丸下降过程中输卵管发育中的重要作用；(4)探讨雌激素对雄激素诱导的LHR阴性雄性腹股沟阴囊睾丸下降的影响。隐睾是迄今为止男性最常见的先天缺陷。虽然已知腹股沟阴囊睾丸下降是雄激素依赖性的，但其潜在机制尚不清楚。目前的研究重点是确定腹股沟阴囊睾丸下降过程中雄激素下游靶基因及其信号网络，这可能为睾丸下降的原因提供新的认识。

DESCRIPTION (provided by applicant): Cryptorchidism is by far the most common defect of sexual differentiation in humans. Testicular descent can be anatomically divided into two stages, i.e., transabdominal and inguinoscrotal phases. It is known that both insulin-like peptide 3 (INSL3) and androgen signaling pathways play critical roles in the control of the transabdominal phase of testis descent. However, the mechanism that regulates the inguinoscrotal phase is not completely understood, even though the incidence of this defect is much more common clinically. Targeted disruption of luteinizing hormone receptor (LHRKO) in mouse impaired development of adult-type Leydig cells which resulted in a dramatic reduction of INSL3 and testosterone levels. Surprisingly, serum estradiol levels were significantly increased. LHRKO males exhibited a bilateral cryptorchid phenotype resulting from a defect in inguinoscrotal testis descent. Histology, morphometry, cell proliferation and differentiation analyses demonstrated the defect was due to a reduction in mesenchymal cell division and differentiation into cremaster muscle cells during the second stage of testis descent. The expression of several genes in the gubernaculum that are known to be involved in gubernacular development, such as Hoxa10, Hoxa11, Wt1, Dll1 and Desrt, were not altered in mutant mice, while Lgr8, Notch1 and Numb mRNA levels were drastically deceased as compared with age-matched wild type siblings. In contrast, the expressions of Esr1 (ER1) and Esr2 (ER2) were significantly elevated. Postnatal testosterone replacement therapy (TRT) completed testicular descent into the scrotum and concomitantly normalized estradiol concentrations in the circulation and Esr1, Esr2, Lgr8, Notch1 and Numb mRNA levels in the gubernaculum. Remedy of the defect by TRT was neither dependent on recovery of adult-type Leydig cells nor the genitofemoral nerve but required androgen receptor activity in the gubernaculum. Using organ culture of the gubernaculum in vitro, androgen alone did not significantly stimulate proliferation of gubernacular mesenchymal cells without activation of the leucine-rich repeat-containing G-protein coupled receptor 8 (LGR8) signaling pathway by addition of INSL3 or relaxin. Co-treatment of estrogen with androgen prevented androgen-induced Lgr8 expression. Cotreatment of INSL3 and androgen increased Notch1 but not Numb mRNA levels. Taking these results together, we hypothesize that induction of the Lgr8 gene expression by androgen and activation of LGR8 signal impinging on the Notch system to stimulate gubernacular cell proliferation, differentiation and myogenesis are the essential molecular pathways in the control of gubernacular development to facilitate inguinoscrotal testis descent. In addition to androgen deficiency, unbalanced androgen/estrogen signals also play a role in contributing to the cryptorchid phenotype in LHR null animals. In this grant application, we propose four specific aims to verify our hypotheses: (1) to define the underlying mechanism by which androgen modulates the Lgr8 gene expression in the gubernaculum; (2) to assess the role of the LGR8 signaling pathway in androgen-induced inguinoscrotal testis descent in LHR null males; (3) to determine the importance of the Notch signaling pathway in mediating androgen and LGR8 signals to regulate gubernaculum development during inguinoscrotal testis descent and (4) to investigate the effect of estrogen on androgen-induced inguinoscrotal testis descent in LHR null males. Cryptorchidism is by far the most common birth defect in males. Although inguinoscrotal testis descent is known to be androgen dependent, the underlying mechanism remains unclear. The current proposal is focusing on determining androgen downstream target genes and subsequent signaling network during the process of inguinoscrotal testis descent, which may provide new insight into the causes of testis maldescent.