Park Eun Young, Park Jin Hee, Mai Nhu Thi Quynh, Moon Byoung-San, Choi Jung Kyu
Department of Biotechnology, College of Life and Applied Sciences, Yeungnam University, Gyeongsan, 38541, South Korea.
Department of Biotechnology, Chonnam National University, Yeosu, 59626, Republic of Korea.
Mater Today Bio. 2023 Oct 7;23:100824. doi: 10.1016/j.mtbio.2023.100824. eCollection 2023 Dec.
This study aimed to derive mature oocytes from murine preantral follicles cultured in a biomimetic ovary with a porcine scaffold using decellularization technology. We evaluated the DNA content and the presence of cell and extracellular matrix (ECM) components, including collagen, elastin, and glycosaminoglycans (GAGs), in decellularized (decell) porcine ovaries. The DNA content inthe decell ovarian tissues was approximately 94 % less than that in native tissues (66 ± 9.8 ng/mg vs. 1139 ± 269 ng/mg). Furthermore, the ECM component integrity was maintained in the decell ovarian tissue. The soluble collagen concentration of native ovarian tissue (native) was 195.34 ± 15.13 μg/mg (dry wt.), which was less than 878.6 ± 8.24 μg/mg for the decell ovarian tissue due to the loss of cellular mass. Hydrogels derived from decell porcine ovaries were prepared to develop an in vitro biomimetic ovary with appropriate ECM concentration (2-6 mg/mL). Scanning electron microscope (SEM) imagining revealed that the complex fiber network and porous structure were maintained in all groups treated with varying ECM concentration (2-6 mg/mL). Furthermore, rheometer analysis indicated that mechanical strength increased with ECM concentration in a dose-dependently. The preantral follicles cultured with 4 mg/mL ECM showed high rates of antral follicle (66 %) and mature oocyte (metaphase II) development (47 %). The preantral follicles cultured in a biomimetic ovary with a decell porcine scaffold showed a higher rate of antral follicle and mature oocytes than those cultured in other biomaterials such as collagen and Matrigel. In mature oocytes derived from antral follicles, meiotic spindles and nuclei were stained using a tubulin antibody and Hoechst, respectively. Two-cell embryos were developed from MII oocytes following parthenogenetic activation. Preantral follicles were cultured in a biomimetic ovary derived from the ECM of a decell porcine ovary, and embryos were generated from MII oocytes. This biomimetic ovary could contribute to restoring fertility in infertile women with reduced ovarian function, benefit mating efforts for endangered species, and maintain animals with valuable genetic traits.
本研究旨在利用去细胞技术，从在具有猪支架的仿生卵巢中培养的小鼠腔前卵泡中获得成熟卵母细胞。我们评估了去细胞猪卵巢中DNA含量以及细胞和细胞外基质（ECM）成分（包括胶原蛋白、弹性蛋白和糖胺聚糖（GAGs））的存在情况。去细胞卵巢组织中的DNA含量比天然组织减少了约94%（66±9.8 ng/mg对1139±269 ng/mg）。此外，去细胞卵巢组织中ECM成分的完整性得以维持。天然卵巢组织（天然组）的可溶性胶原蛋白浓度为195.34±15.13μg/mg（干重），由于细胞质量的损失，去细胞卵巢组织的该浓度为878.6±8.24μg/mg，低于天然组。制备了源自去细胞猪卵巢的水凝胶，以开发具有适当ECM浓度（2 - 6 mg/mL）的体外仿生卵巢。扫描电子显微镜（SEM）成像显示，在所有用不同ECM浓度（2 - 6 mg/mL）处理的组中，复杂的纤维网络和多孔结构得以维持。此外，流变仪分析表明，机械强度随ECM浓度呈剂量依赖性增加。用4 mg/mL ECM培养的腔前卵泡显示出较高的窦状卵泡发育率（66%）和成熟卵母细胞（中期II）发育率（47%）。在具有去细胞猪支架的仿生卵巢中培养的腔前卵泡，其窦状卵泡和成熟卵母细胞的发育率高于在其他生物材料（如胶原蛋白和基质胶）中培养的卵泡。在源自窦状卵泡的成熟卵母细胞中，分别使用微管蛋白抗体和Hoechst对减数分裂纺锤体和细胞核进行染色。孤雌激活后，从MII期卵母细胞发育出了二细胞胚胎。将腔前卵泡培养在源自去细胞猪卵巢ECM的仿生卵巢中，并从MII期卵母细胞生成胚胎。这种仿生卵巢有助于恢复卵巢功能减退的不孕女性的生育能力，有利于濒危物种的交配繁殖，并维持具有宝贵遗传特征的动物。
