Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival.
Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival.
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DOI：
10.3390/ani12111434
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发表时间：
2022-06-02
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许多鸟类被列为濒危物种，配子保存技术的发展是必要的，以保护其遗传多样性。由于卵巢组织的冷冻保存和移植是目前唯一的方法，雌性生育力保存鸟类出生后，运输卵巢到实验室在良好的条件是成功的雌性生育力保存的关键。在这里，我们报告使用鸡作为野生鸟类的模型，在低温（4 °C）下储存，而不是室温，可以保持卵巢，确保卵母细胞存活，形态和生殖细胞标志物的蛋白质表达至少3天。此外，玻璃化冷冻研究证实，卵巢低温保存后的卵母细胞可以通过卵巢组织冷冻保存。另一方面，卵巢在室温下储存降低卵母细胞的存活率，同时增加细胞凋亡和氧化应激标志物的基因表达。我们的研究不仅证实了母鸡卵巢在低温下储存可以保护卵母细胞的事实，可以通过玻璃化冷冻保存，但它也有助于了解卵巢储存对母鸡卵巢内未成熟卵泡的完整性和耐冷性的影响。为了保护濒危鸟类，发展配子保存技术是必不可少的。然而，在卵母细胞中的研究尚未广泛进行。在这项研究中，假设卵巢被运送到一个研究设施死亡后，我们调查了卵巢储存卵母细胞冷冻保存鸟类雌性配子的目的，通过使用鸡作为濒危鸟类物种的模型的影响。切除后，将卵巢在低温（4 °C）或室温下储存1-3天。通过中性红染色、组织学、基因和蛋白表达分析，对不同条件下保存的每个时期的卵巢卵泡进行检查。此外，测量保存卵巢后储存介质的pH。然后，将卵巢组织玻璃化冷冻以测定冷冻能力。将卵巢组织储存在4 °C下可使卵泡存活且形态正常3天，并缓慢下降。相比之下，虽然不同的储存温度并没有影响卵泡活力和形态后，只有1天的存储，在室温下存储的卵巢组织迅速下降，在结构正常的卵泡，并很少看到有活力的卵泡后，存储3天。基因和蛋白质表达分析表明，细胞凋亡已经开始在第一天，如在室温条件下的CASP 9的较高表达所示。此外，SOD 1的高表达和室温储存条件下储存介质中pH值的快速下降表明，在这种条件下与低pH值相关的氧化应激对母鸡卵巢组织中卵泡存活率的影响。我们的冷冻保存研究还表明，在4 °C下储存的卵巢组织在冷冻保存后甚至在储存3天后也可以恢复。所描述的保存条件和冻存方法，使鸡卵泡得以存活，为进一步研究野生雌性鸟类卵巢组织的保存，以保存其生育力奠定了基础。
Many avian species are classified as endangered, and the development of the gamete preservation technique is essential to preserve their genetic diversity. Since cryopreservation and transplantation of ovarian tissues are currently the only methods of female fertility preservation in birds after birth, transporting the ovary to the laboratory in good condition is key for successful female fertility preservation. Here, we report using chickens as a model of wild birds for which storage at low temperature (4 °C), but not room temperature, can maintain the ovary, ensuring oocyte survival, morphology and protein expression of germ cell markers for at least 3 days. Furthermore, a vitrification study confirmed that oocytes after ovary storage at a low temperature can be preserved by ovarian tissue cryopreservation. On the other hand, ovaries stored at room temperature decreased oocyte survival, while increasing gene expression of apoptosis and oxidation stress markers. Our study not only verifies the fact that the storage of hen ovaries at low temperatures protects oocytes, which can be preserved by vitrification, but it also contributes to understanding the effect of ovary storage on the integrity and cryotolerance of immature follicles within the hen ovary. For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.
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