Kawada Tsuyoshi, Aoyama Masato, Yasuda Keiko, Satake Honoo
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto, Japan.
Department of Chemistry, Biology, and Environmental Science, Faculty of Science, Nara Women's University, Nara, Japan.
Front Endocrinol (Lausanne). 2025 Jun 5;16:1621348. doi: 10.3389/fendo.2025.1621348. eCollection 2025.
Cathepsin D is an aspartic protease responsible for the proteolytic processing of vitellogenin at the early stages of folliculogenesis in oviparous vertebrates. Previously, we identified a multifunctional neuropeptide, tachykinin (TK), as an inducer of cathepsin D that promotes vitellogenic follicle growth in the ascidian (), a sister group of vertebrates. However, no regulatory factor for cathepsin D in the ovary has been identified in vertebrates. Moreover, the involvement of cathepsin D in mammalian folliculogenesis has yet to be investigated.
Ovaries of 2-week-old ICR mice were used. gene expression in the ovaries was examined by real-time PCR. Localization of cathepsin D was shown by immunostaining of ovarian sections. Cathepsin D activity was measured using supernatants from the homogenized ovaries. Mouse follicle growth was evaluated using three-dimensional follicle culture system.
Immunohistochemical analysis revealed that cathepsin D is co-localized with TK receptors in granulosa cells of secondary follicles in the ovaries of two-week-old mice, which are sexually immature and in which the hypothalamus-pituitary-gonadal (HPG) axis is not yet functional. TK treatment of the ovaries significantly increased gene expression and its proteolytic activity. Moreover, inhibition of cathepsin D markedly suppressed the secondary follicle growth.
Collectively, these results indicate that cathepsin D plays essential roles in mouse secondary follicle growth. Recently, we also demonstrated that ovarian TKs promote secondary follicle growth in mice, primarily in a paracrine/autocrine manner. Combined with these findings, the present study leads us to propose an evolutionary scenario in which TK-cathepsin D signaling functions as a conserved mechanism for HPG axis-independent follicle growth across chordates, which may be more broadly conserved than the vertebrate-specific, HPG axis-dependent systems.
组织蛋白酶D是一种天冬氨酸蛋白酶，在卵生脊椎动物卵泡发生的早期阶段负责卵黄蛋白原的蛋白水解加工。此前，我们鉴定出一种多功能神经肽速激肽（TK），它是组织蛋白酶D的诱导剂，可促进海鞘（脊椎动物的姐妹类群）中卵黄生成卵泡的生长。然而，在脊椎动物中尚未鉴定出卵巢中组织蛋白酶D的调节因子。此外，组织蛋白酶D在哺乳动物卵泡发生中的作用尚未得到研究。
使用2周龄ICR小鼠的卵巢。通过实时PCR检测卵巢中的基因表达。通过卵巢切片的免疫染色显示组织蛋白酶D的定位。使用匀浆卵巢的上清液测量组织蛋白酶D的活性。使用三维卵泡培养系统评估小鼠卵泡的生长。
免疫组织化学分析显示，在2周龄性未成熟且下丘脑 - 垂体 - 性腺（HPG）轴尚未发挥功能的小鼠卵巢中，组织蛋白酶D与颗粒细胞中的TK受体共定位。用TK处理卵巢可显著增加基因表达及其蛋白水解活性。此外，抑制组织蛋白酶D可明显抑制次级卵泡的生长。
总体而言，这些结果表明组织蛋白酶D在小鼠次级卵泡生长中起重要作用。最近，我们还证明卵巢TK主要以旁分泌/自分泌方式促进小鼠次级卵泡的生长。结合这些发现，本研究使我们提出一种进化假说，即TK - 组织蛋白酶D信号传导作为一种跨脊索动物的HPG轴非依赖性卵泡生长的保守机制，可能比脊椎动物特有的、HPG轴依赖性系统具有更广泛的保守性。
