Departamento de Bioquímica Clínica, Centro de Investigaciones en Bioquímica Clínica e Inmunología, Facultad de Ciencias Químicas-Universidad Nacional de Córdoba, Córdoba, CP 5000, Argentina.
Insect Biochem Mol Biol. 2011 Oct;41(10):832-41. doi: 10.1016/j.ibmb.2011.06.005. Epub 2011 Jul 7.
In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.
在这项工作中，我们以吸血昆虫载体双翅目最大作为模型，探索了表征从卵黄生成到滤泡闭锁过渡的生化变化。通过在卵黄生成后剥夺雌性血液餐来建立标准化的昆虫饲养条件，以诱导逐渐的滤泡退化阶段。在研究中，在卵黄生成前、卵黄生成和早期和晚期滤泡闭锁的代表性日子采集血淋巴和卵巢。当通过扫描电子显微镜检查时，处于闭锁初始阶段的卵巢小管很小，但仍显示出一定程度的不同步性，这种特征在高级退化状态下丧失。在晚期滤泡闭锁中，荧光标记的卵黄蛋白（Vg-FITC）的体内摄取测定显示卵母细胞摄取卵黄蛋白的竞争力丧失。在闭锁中循环卵黄蛋白水平明显高于卵黄生成前的水平，如果有第二餐血液，很可能为另一个生殖周期维持适当的条件。滤泡闭锁还表现为卵黄磷蛋白的部分蛋白水解，这在卵巢匀浆中通过 Western blot 得到证实。当测试卵巢肽酶对血红蛋白（非特异性底物）的活性时，在早期和晚期闭锁中检测到较高的活性，而在卵黄生成中检测到最低的活性。血红蛋白的活性被胃蛋白酶抑制剂 A（一种天冬氨酸肽酶抑制剂）显著抑制，并且在任何测试条件下都不受 E64（一种半胱氨酸肽酶抑制剂）的影响。使用特定的荧光底物表明，早期滤泡闭锁的卵巢匀浆显示出高组织蛋白酶 D 样活性，而没有检测到组织蛋白酶 B 或 L 的活性。底物 Abz-AIAFFSRQ-EDDnp 的消化产物的质谱分析进一步证实了卵巢组织中存在组织蛋白酶 D 样肽酶。根据我们的发现，组织蛋白酶 D 样肽酶的早期激活可能与促进卵黄蛋白再循环和/或增强滤泡去除有关。
