ACTIVATION OF SPERMATOGENIC RECOVERY AFTER TOXIC INSULT

男性暴露在某些环境和医疗毒物中会导致长期的无精子症。有时精子发生恢复，表明干细胞(A型)精原细胞存活，但其分化有限。照射或二溴氯丙烷(DBCP)处理的大鼠睾丸含有A精原细胞，这种精原细胞可保留一年以上，但无法分化，可能是人类情况的模型。在大鼠中，短暂的睾酮抑制会导致精子发生的进展和生育能力的恢复。这一假说提出，在中毒处理的大鼠中，可以通过操纵各种类固醇激素和FSH来实现更大程度的生育恢复，这些激素和FSH通过作用于睾丸的支持细胞和可能的另一个类固醇受体阳性的体细胞来调节特定基因的表达，从而调节精原细胞的发育。最初，将在所有目标中使用辐射，但将使用DBCP诱导的性腺损伤来检查主要发现对其他毒物的共性。为了确定是否可以增强和延长恢复，将在毒物暴露前后用GnRH拮抗剂、睾酮、雌激素或孕激素的不同组合对大鼠进行治疗。毒物剂量、激素时机和特定激素治疗对恢复持续时间的影响将进一步阐明抑制机制，并建议恢复精子发生的程序。为了评估不同的类固醇反应细胞(支持细胞、小管周围细胞、间质细胞、血管细胞)的作用，将使用三种方法。这些包括组织碎片、分离的小管和小管组件的体外培养；体内间质细胞的清除；以及确定毒物处理的睾丸中的水肿(可能是由于血管损伤)是否与抑制精原发育有关。将采取初步步骤来识别相关基因。从单纯照射和激素处理的照射大鼠的靶细胞的纯化群体中提取的RNA将进行消减杂交，以获得差异表达克隆的文库。这项研究应该确定激素或其他因素，这些激素或其他因素可能被用作干预措施，以促进男性在某些类型的毒物暴露、免疫抑制治疗癌症、衰老和导致生殖细胞发育受阻的特发性不育症后的生育力恢复。

Exposure of men to certain environmental, and medical toxicants results in prolonged azoospermia. Occasionally spermatogenesis recovers, indicating that stem (type A) spermatogonia survived but their differentiation was limited. Testes of irradiated or dibromocholoropropane (DBCP) treated rats contain A spermatogonia that remain for over one year but fail to differentiate, and may be a model for the human situation. In the rat, transient suppression of testosterone results in progression of spermatogenesis and restoration of fertility. The hypothesis is proposed that, in toxicant-treated rats, greater restoration of fertility can be achieved by manipulating various steroid hormones and FSH, which are regulating spermatogonial development by acting on Sertoli cells and possibly another steroid-receptor positive somatic cell of the testis to modulate the expression of specific genes. Initially, irradiation will be used in all the Aims, but the generality of major findings to other toxicants will be checked using DBCP-induced gonadal damage. To determine whether enhanced and prolonged recovery can be achieved, rats will be treated both before and after toxicant exposure with different combinations of a GnRH antagonist, testosterone, estrogens, or progestins. The effects of toxicant dose, timing of hormones, and specific hormone treatment on the duration of recovery will further elucidate inhibitory mechanisms and suggest procedures for restoration of spermatogenesis. To evaluate the roles of different steroid-responsive cells (Sertoli, peritubular, Leydig, vascular), three approaches will be used. These are in vitro culture of tissue fragments, isolated tubules, and tubule components; in vivo elimination of Leydig cells; and determining whether the edema (likely from vascular damage) in toxicant-treated testes correlates with the inhibition of spermatogonial development. Initial steps will be taken to identify genes involved. RNA from purified populations of the target cell from irradiated-only and hormone-treated irradiated rats will be subjected to subtractive hybridization to obtain libraries of differentially expressed clones. This study should define hormones or other factors that might be used as intervention to enhance recovery of fertility in men following certain types of toxicant exposure, cancer of immunosuppressive therapy, aging, and idiopathic infertility that result in blocks in germ cell development.