Akingbemi B T, Ge R S, Klinefelter G R, Gunsalus G L, Hardy M P
Center for Biomedical Research, Population Council, New York, NY 10021, USA.
Biol Reprod. 2000 Mar;62(3):571-8. doi: 10.1095/biolreprod62.3.571.
Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1, 1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450(scc)) and cholesterol utilization. The reduced steady-state mRNA level for P450(scc) in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.
祖细胞、未成熟和成年睾丸间质细胞。分化过程伴随着睾丸间质细胞产生睾酮（T）能力的逐步增加。T在青春期前促进男性表型，并在成年期维持性功能；因此，睾丸间质细胞中T生物合成的破坏会对男性生育能力产生不利影响。本研究旨在评估一种外源性雌激素甲氧滴滴涕（滴滴涕[1,1,1-三氯-2,2-双（对氯苯基）乙烷]的甲氧基化异构体）改变睾丸间质细胞类固醇生成功能的能力。分别从用分级浓度的甲氧滴滴涕生物活性代谢物2,2-双（对羟基苯基）-1,1,1-三氯乙烷（HPTE）处理的21日龄、35日龄和90日龄的Sprague-Dawley大鼠中获得纯化的祖细胞、未成熟和成年睾丸间质细胞，并评估其T产生情况。HPTE对睾丸间质细胞基础和LH刺激的T产生具有剂量依赖性抑制作用。与对照值相比，祖细胞和未成熟睾丸间质细胞在培养中用HPTE处理10小时后，T产生明显减少；成年睾丸间质细胞的等效时间为18小时。通过将睾丸间质细胞孵育3、6、10、14或18小时并在允许恢复时间后测量T产生来评估HPTE诱导抑制的可逆性。用HPTE处理3小时后，未成熟和成年睾丸间质细胞在处理后18小时的T产生与对照值相似，但祖细胞睾丸间质细胞的T产生明显较低。HPTE作用的开始及其作用的可逆性表明，睾丸间质细胞在青春期分化期间比成年期对该化合物更敏感。当对照和HPTE处理的未成熟睾丸间质细胞与孕烯醇酮、孕酮和雄烯二酮一起孵育时，T产生相当，但当用22R-羟基胆固醇进行孵育时，HPTE处理的睾丸间质细胞产生的T量明显减少（P<0.01）。这一发现表明，HPTE诱导的T产生抑制与细胞色素P450胆固醇侧链裂解酶（P450(scc)）活性降低和胆固醇利用减少有关。通过逆转录-聚合酶链反应和光密度测定法证明了HPTE处理的睾丸间质细胞中P450(scc)的稳态mRNA水平降低。总之，本研究表明，HPTE在发育的所有阶段均直接抑制睾丸间质细胞的T生物合成。这种作用表明，T产生减少可能是与甲氧滴滴涕以及可能与其他滴滴涕相关化合物有关的男性不育的一个促成因素。
