Role of neurotropins in rat embryonic testis morphogenesis (cord formation)
Role of neurotropins in rat embryonic testis morphogenesis (cord formation)
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DOI：
10.1095/biolreprod62.1.132
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发表时间：
2000-01-01
影响因子：
3.6
通讯作者：
Skinner, MK
中科院分区：
文献类型：
--
作者：
Levine, E;Cupp, AS;Skinner, MK
生精索形成过程是区分睾丸和卵巢并指示男性性别决定的第一个形态学事件。大鼠胚胎第 14 天（第 0 天插入日期；E14）发生脐带形成。进行了一系列实验以确定神经营养因子及其受体对于大鼠胚胎索形成过程是否重要。使用 p75/LNGFR 抗体，通过免疫组织化学法测定从 E13 到出生（第 0 天，PO）睾丸和卵巢切片上的低亲和力促神经素受体 (p75/LNGFR) 的表达。 p75/LNGFR 的染色存在于 E13 性腺的中肾中，并以性别特异性方式出现在胚胎睾丸中 E14 发育中的索索周围。出生时，p75/LNGFR 的染色局限于生精索周围的单层细胞（即管周细胞）。发现神经营养素 3 (NT3) 和相应的高亲和力神经营养素 trkC 受体的基因以及其他神经营养素和受体在 E14 大鼠睾丸中表达。 E14 大鼠睾丸的免疫细胞化学分析表明，NT3 定位于支持细胞，trkC 存在于 E16 的间质单个细胞中和 E18 的选定管周细胞中。此前，与脊髓相邻的管周细胞被证明源自脊髓形成时迁移的中肾细胞。为了确定神经营养素是否参与脊髓形成，神经营养素的作用被抑制。高亲和力促神经素受体 (trk) 特异性激酶抑制剂 K252a 用于在脊髓形成前处理 E13 大鼠睾丸的器官培养物。用 K252a 处理 E13 睾丸器官培养物完全抑制了索的形成。经过 K252a 处理的含有索的 E14 睾丸器官培养物不会改变索的形态。第二个抑制神经营养素作用的实验使用了特定的拮抗剂 trk-IgG 嵌合融合蛋白和 E13 睾丸器官培养物。 trk-IgG 分子与内源性 trk 受体形成二聚体，并抑制受体信号传导和配体功能的激活。 40% 的 E13 睾丸器官培养物经 trkC-IgG 处理后，索带形成显着减少。 TrkA-IgG 对脐带的起始没有影响；然而，在百分之五十的接受治疗的器官中，观察到了索结构的“肿胀”外观。在 E13 器官培养物上使用 trkB-IgG 嵌合蛋白进行的实验对脐带形成或脐带形态没有影响。检查 trkC 和 NT3 敲除小鼠的睾丸以确定睾丸是否存在任何形态差异。 NT3 敲除在 E15 和 E17 睾丸中似乎具有正常的索状形态。 TrkC 基因敲除小鼠的 E14 和 PO 睾丸也具有正常的索状形态。 NT3 和 trkC 敲除小鼠的睾丸间质面积均小于野生型对照小鼠。此外，与对照组或 NT3 敲除小鼠相比，trkC 敲除小鼠的脊髓内表达 p75LNGFR 的细胞数量增加。综合观察表明，不同的神经营养蛋白配体、受体和/或可能不同的生长因子之间对这一关键的生物过程进行补偿。总之，结果表明神经营养素在胚胎大鼠睾丸发育期间的索形成过程中具有新的非神经元作用。所提出的假设是神经营养素参与雄性性别分化的进程，并且对于诱导胚胎睾丸索形成至关重要。
The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, PO) with an antibody to p75/LNGFR, The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and PO testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.