Hasegawa Chinatsu, Yokoyama Toshifumi, Umemura Yuria, Kawanishi Kohei, Miura Yuuka, Takada Nanako, Ohno Shuji, Onaru Kanoko, Omotehara Takuya, Hirano Tetsushi, Mantani Yohei, Miki Takanori, Hoshi Nobuhiko
Department of Animal Science, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Department of Anatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku, Tokyo 160-8402, Japan.
J Vet Med Sci. 2020 Apr 9;82(4):414-421. doi: 10.1292/jvms.20-0036. Epub 2020 Feb 21.
Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.
器官培养系统对于阐明哺乳动物未分化的遗传雄性性腺向睾丸分化的过程很有用，因为它们允许进行各种实验，包括涉及基因表达控制的实验。然而，如果不添加与睾丸分化相关的因子，很难在早于对应于交配后11.0天（dpc）的12个尾节（ts）的时间点之前从未成熟性腺诱导形成睾丸索。在本研究中，我们试图建立一种器官培养系统，该系统能在Sry（Y染色体性别决定区域）表达之前从未成熟性腺（约8个ts：10.5 dpc）诱导形成睾丸。将约8个ts的一对性腺-中肾复合体置于琼脂糖凝胶块的凹槽中，并放置半圆柱形琼脂糖凝胶片以维持性腺形态。性腺在气相中培养96小时。结果，许多遗传雄性性腺中出现了睾丸索样结构。观察到表达支持细胞标志物Sox9（SRY盒9）和Amh（抗苗勒管激素）的细胞，而未检测到颗粒细胞标志物Foxl2（叉头盒L2）。此外，在许多个体的整个性腺中都观察到了表达Sox9和Amh的细胞。Amh mRNA表达也上调。令人惊讶的是，从未成熟程度更高的性腺（6个ts）中观察到了部分睾丸结构的形成。这些结果表明，我们的性腺器官培养系统有助于阐明未分化的双潜能性腺中Sry表达的调控机制。
