Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh A J
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305.
Endocrinology. 1995 Jan;136(1):5-12. doi: 10.1210/endo.136.1.7828558.
Recent studies have demonstrated apoptotic DNA fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis apoptotic DNA fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two injections at 2-day intervals). Pretreatment with the GnRH antagonist increased apoptotic DNA fragmentation in rats between 16-32 days of age but not in younger and adult animals demonstrating an age-related change in gonadotropin dependence. To identify the exact testis cell type undergoing apoptosis, in situ analysis of DNA fragmentation was performed. In rats at 16-24 days of age, spermatocytes in selected tubules were found to have increased DNA fragmentation. In contrast, neither Leydig cells nor Sertoli cells were affected. In 32-day-old and adult animals, increased DNA fragmentation was seen in early primary spermatocytes of some tubules. Treatment with GnRH antagonist increased the number of cells with DNA fragmentation as well as percentage of tubules affected. In animals between 16-32 days of age, meiotic spermatocytes were labeled, whereas early spermatids were also labeled in 24- and 32-day-old animals. In adult animals, the level of apoptotic DNA fragmentation was not affected by GnRH antagonist treatment. However, DNA isolated from specific stages of the seminiferous tubules of adult animals showed stage-specific changes of apoptotic DNA fragmentation with 2-fold higher levels found in stages I and XII-XIV compared with stage VIII. In situ analysis of adult testis demonstrated that spermatocytes were the major cell type affected. In conclusion, the present study demonstrated that at least three factors determine the onset of apoptosis of the male germ cells: 1) the developmental stage of the animal; 2) serum levels of gonadotropins, especially FSH; and 3) specific stage of the seminiferous epithelial cycle. The present approach provides the basis for future analysis of the role of gonadotropins and other factors in the regulation of testis cell degeneration in normal and pathological states.
近期研究表明，在缺乏促性腺激素的未成熟大鼠睾丸中存在凋亡性DNA片段化。然而，在睾丸发育过程中经历凋亡的确切细胞类型以及睾丸细胞凋亡对促性腺激素依赖的年龄差异尚不清楚。本研究采用凝胶分级分离法和原位法来定量睾丸细胞DNA片段化的发育变化，并确定在给予和未给予GnRH拮抗剂处理的发育中大鼠体内受影响的特定细胞类型。在8至70日龄的大鼠中测量了整个睾丸的凋亡性DNA片段化。与8日龄动物相比，16至28日龄的大鼠睾丸凋亡性DNA片段化逐渐增加（1.8至2.0倍），随后成年动物中该水平下降。为了研究睾丸凋亡对促性腺激素的依赖性，用长效GnRH拮抗剂（阿扎林 - B，250微克/千克体重，每隔2天注射两次）处理来抑制血清FSH以及在较小程度上抑制LH。用GnRH拮抗剂预处理增加了16至32日龄大鼠的凋亡性DNA片段化，但在年幼和成年动物中未增加，这表明睾丸凋亡对促性腺激素的依赖存在年龄相关变化。为了确定经历凋亡的确切睾丸细胞类型，进行了DNA片段化的原位分析。在16至24日龄的大鼠中，发现选定曲细精管中的精母细胞DNA片段化增加。相比之下，睾丸间质细胞和支持细胞均未受影响。在32日龄和成年动物中，一些曲细精管的早期初级精母细胞中可见DNA片段化增加。用GnRH拮抗剂处理增加了DNA片段化细胞的数量以及受影响曲细精管的百分比。在16至32日龄的动物中，减数分裂精母细胞被标记，而在24日龄和32日龄的动物中早期精子细胞也被标记。在成年动物中，凋亡性DNA片段化水平不受GnRH拮抗剂处理的影响。然而，从成年动物曲细精管特定阶段分离的DNA显示出凋亡性DNA片段化的阶段特异性变化，与VIII期相比，I期和XII - XIV期的水平高2倍。成年睾丸的原位分析表明精母细胞是受影响的主要细胞类型。总之，本研究表明至少有三个因素决定雄性生殖细胞凋亡的起始：1）动物的发育阶段；2）促性腺激素的血清水平，尤其是FSH；3）曲细精管上皮周期的特定阶段。本方法为未来分析促性腺激素和其他因素在正常和病理状态下睾丸细胞退化调节中的作用提供了基础。
