Whiteley Sarah L, Holleley Clare E, Georges Arthur
Institute for Applied Ecology, University of Canberra, Canberra, Australia.
Australian National Wildlife Collection, CSIRO, Canberra, Australia.
PLoS One. 2025 Jul 29;20(7):e0327930. doi: 10.1371/journal.pone.0327930. eCollection 2025.
Developing in vitro protocols for non-model species poses challenges, and yet are essential for advancing molecular biology studies. This is particularly true for sex determination research that relies on being able to functionally demonstrate the role of genes in determining sex and guiding the process of sex differentiation. Reptile species are attractive models for sex determination research as many species display thermolabile sex systems, allowing for exploration of gene-environment interactions. The first in vitro gonad culture technique for a turtle was published almost 35 years ago in 1990, but these techniques have seen limited use. This is likely because of challenges inherent to the system, where cultures need to be maintained for prolonged periods for gonadal differentiation to occur. All published techniques for long-term cultures involve removing the gonad from the surrounding mesonephros, which causes issues for proper testes development. Here we present the first protocol developed in the reptile model, Pogona vitticeps, that allows the long-term culture of the whole urogenital system supporting differentiation from biopotential gonads to ovaries or testes. Gross morphology is well maintained, and the gonad can be dissected from the mesonephros even after a culture period of up to 19 days. The cultured gonads display sex specific gene expression and morphology. This protocol will facilitate research in sex determination by providing an effective and low-cost alternative to existing protocols and expand capacity for functional manipulation studies in non-model species.
为非模式物种开发体外实验方案具有挑战性，但对于推进分子生物学研究至关重要。对于性别决定研究而言尤其如此，因为该研究依赖于能够从功能上证明基因在决定性别的过程中所起的作用，并指导性别分化过程。爬行动物是性别决定研究的理想模型，因为许多物种表现出温度依赖型性别决定系统，这使得对基因与环境相互作用的探索成为可能。近35年前，即1990年，第一篇关于乌龟的体外性腺培养技术的论文发表，但这些技术的应用有限。这可能是由于该系统固有的挑战，即需要长时间维持培养才能发生性腺分化。所有已发表的长期培养技术都涉及将性腺从周围的中肾中分离出来，这对睾丸的正常发育会产生问题。在此，我们展示了在鬃狮蜥这一爬行动物模型中开发的首个实验方案，该方案能够对整个泌尿生殖系统进行长期培养，支持从双潜能性腺向卵巢或睾丸的分化。大体形态得到良好维持，即使在长达19天的培养期后，性腺也能从中肾中分离出来。培养的性腺表现出性别特异性基因表达和形态。该方案将通过为现有方案提供一种有效且低成本的替代方案，促进性别决定研究，并扩大非模式物种功能操作研究的能力。
