Analysis of mechanism of changes in function and size of golden hamster testis with special reference to TGF-β signal transduction

首先，在精子发生前睾丸明显增大时，用BrdU法和电镜检查了性腺细胞的增殖和定位。卵泡细胞从17.5dpc开始移动，从18.5dpc开始迁移到基底膜上。在1.5dpp时首次检测到brdu标记的淋细胞。它们的数量从2.5dpp开始突然增加，然后逐渐增加。其次，研究了两种不同体型蝙蝠（爪哇果蝠，平均体重：567g；日本小马蹄蝠，平均体重：7g）睾丸的组织学和凝集素结合模式。这两个物种的睾丸形态和凝集素结合模式几乎相似。第三，利用RACE技术从小鼠脑中克隆出包含开放阅读框的小鼠Smad3 cDNA，并通过northern blot分析其表达模式。预测的小鼠Smad3氨基酸序列与人类Smad3（99.3%）和小鼠Smad2（85.4%）具有较高的同源性。Northern blot分析显示Smad3在脑和卵巢中高表达。原位杂交结果显示，海马锥体细胞、大脑皮层颗粒细胞和卵巢颗粒细胞中均检测到Smad3。接下来，在长和/或短光周期的影响下，我们检测了Smad2和Smad3 mRNA的表达。原位杂交在两个光周期中均检测到Smad2和Smad3 mRNA。Northern blots显示，Smad2 mRNA在两个光周期的所有阶段都被检测到，而Smad3 mRNA在短光周期中表达。光周期条件会改变Smad2和Smad3转录本之间的平衡。此外，在两个光周期中检测了Smad2和Smad3蛋白的定位。Smad2和Smad3蛋白在长光周期内定位于精母细胞细胞质中。它们在短的光周期内积聚在精母细胞核内。

First, during prespermatogenesis when the testis prominently increases in size, the reproliferation and relocation of gonocytes were examined by BrdU method and electron microscopy. Gonocytes began to move from 17.5dpc and relocated on the basement membrane from 18.5dpc. BrdU-labeled gonocytes were first detected on 1.5dpp. They increased in number abruptly from 2.5dpp and then gradually increased. Secondly, histology and lectin-binding patterns in the testes of two different-body sized bats (Java fruit bat, average b.w. : 567g ; Japanese lesser horseshoe bat, average b.w. : 7g) were investigated. Both testicular morphology and lectin-bindig patterns were almost similar between these two species. Thirdly, the mouse Smad3 cDNA including the open reading frame was cloned from the mouse brain using RACE technique, and its expression pattern was analyzed by northern blot. The predicted amino acid sequences of mouse Smad3 showed a high homology with human Smad3 (99.3%) and mouse Smad2 (85.4%). Northern blot analysis revealed that Smad3 was highly expressed in brain and ovary. In situ hybridization revealed that Smad3 was detected in pyramid cells of hippocampus, granular cells of cerebral cortex, and granulosa cells of ovary. Next, the expression of Smad2 and Smad3 mRNA was examined under the influence of long and/or short photoperiod in hamsters. In situ hybridization detected both Smad2 and Smad3 mRNA in spermatogonia and spermatocytes in both photoperiods. Northern blots showed that Smad2 mRNA was detected at all stages in both photoperiods, whereas Smad3 mRNA was expressed in a short photoperiod. The photoperiodic condition would change the balance between Smad2 and Smad3 transcripts. Moreover, the localization of Smad2 and Smad3 protein was examined in both photoperiods. The Smad2 and Smad3 proteins were localized in spermatocyte cytoplasm in a long photoperiod. They accumulated in spermatocyte nucleus in a short photoperiod.