The Mechanism of Cryptorchidism in LH Receptor Knockout Animals

描述(申请人提供)：到目前为止，隐睾症是人类最常见的性别分化缺陷。睾丸下降在解剖学上可分为两个阶段，即经腹阶段和腹股沟阴囊阶段。已知胰岛素样肽3(INSL3)和雄激素信号通路在控制经腹期睾丸下降中起关键作用。然而，尽管腹股沟阴囊畸形的发生率在临床上更为常见，但调节腹股沟阴囊时相的机制并不完全清楚。靶向阻断小鼠促黄体激素受体(LHRKO)会损害成人型间质细胞的发育，从而导致INSL3和睾酮水平的急剧下降。令人惊讶的是，血清雌二醇水平显著上升。LHRKO男性由于腹股沟阴囊睾丸下降缺陷而表现为双侧隐睾型。组织学、形态计量学、细胞增殖和分化分析表明，该缺陷是由于在睾丸下降的第二阶段，间充质细胞分裂和分化为分泌肌细胞的减少所致。Hoxa10、Hoxa11、Wt1、Dll1和Desrt等几个已知参与引带发育的基因在突变小鼠中的表达没有改变，而Lgr8、Notch1和Numb的mRNA水平与年龄匹配的野生型小鼠相比显著下降。相反，ESR1(ER1)和Esr2(ER2)的表达显著升高。出生后睾酮替代治疗(TRT)完成了睾丸下降到阴囊的过程，并伴随着循环中雌二醇浓度和引带中ESR1、Esr2、Lgr8、Notch1和Numb mRNA水平的正常化。TRT对这种缺陷的修复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，而需要引带中雄激素受体的活性。在体外的引带器官培养中，如果没有通过INSL3或松弛素激活富含亮氨酸重复序列的G蛋白偶联受体8(LGR8)信号通路，单用雄激素并不能显著刺激引带间充质细胞的增殖。雌激素与雄激素联合治疗可阻止雄激素诱导的Lgr8表达。INSL3和雄激素共同作用可增加Notch1的mRNA水平，但不能增加Numb的mRNA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系统以刺激引带细胞的增殖、分化和肌肉发生是控制引带发育促进腹股沟阴囊睾丸下降的基本分子途径。除了雄激素缺乏外，雄激素/雌激素信号失衡也是导致LHR缺失动物隐睾体表型的原因之一。在这项赠款申请中，我们提出了四个具体的目标来验证我们的假设：(1)确定雄激素调节引带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缺失雄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信号在LHR阴性雄性腹股沟阴囊睾丸下降过程中的重要性；以及(4)研究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。隐睾症是迄今为止最常见的男性先天缺陷。虽然已知腹股沟阴囊睾丸下降是雄激素依赖性的，但其潜在的机制仍不清楚。目前的研究重点是在腹股沟阴囊睾丸下降过程中确定雄激素下游靶基因和后续的信号网络，这可能为睾丸下降的原因提供新的见解。

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.