In vitro development of mechanically and enzymatically isolated cat ovarian follicles.
In vitro development of mechanically and enzymatically isolated cat ovarian follicles.
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DOI：
10.1530/raf-20-0067
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发表时间：
2021
影响因子：
2.1
通讯作者：
Songsasen N
中科院分区：
文献类型：
--
作者：
Nagashima JB;Hill AM;Songsasen N
在大型哺乳动物的培养系统中，卵泡的分离是促进卵泡在腔前阶段继续生长的关键步骤，以保持生育能力。尽管如此，机械分离方法依赖于使用者的技能且耗时，而酶方法则增加了破坏膜细胞层和基底膜的风险。在此，我们试图确定一种从卵巢组织中回收家猫(Felis Catus)早期卵泡的最佳方法，并评价分离策略对海藻酸水凝胶体外培养14天的卵泡发育、存活和基因表达的影响。用0.7L和1.4L的Liberase blendzyme分别用0.7和1.4Wünsch单位的混合酶消化90min和机械分离。与1.4L(P<；0.05)相比，机械分离可改善卵泡的生长和存活，并能更好地维持窦腔和卵泡膜细胞，但孵育后的细胞凋亡率高于酶分离的卵泡。然而，在体外培养7天以上，卵泡生长和存活的差异不明显。在不同的隔离策略中，细胞色素P19A1、GDF9、LHR或VEGFA的表达相似。与机械或0.7L新鲜分离的卵泡相比，来自所有分离方法的培养卵泡的STAR表达均降低，提示长期培养会导致膜细胞的存在和/或功能的丧失。综上所述，体外早期有腔和有腔卵泡的发育受到隔离策略的显著影响，但在没有扩大培养的情况下不一定能观察到。这些结果表明，在卵泡分离和优化基因组拯救和生育保护工作中必须格外小心。卵巢包含数百个卵子，在动物一生中，只有少数卵子从未成熟阶段发育到排卵。从这个池子里抢救卵子，并在培养中将它们培养到成熟阶段的能力，对于人类和濒危物种的生育能力保存工作都将是令人难以置信的宝贵价值。目前，分离卵泡(卵子及其周围的辅助细胞)是大型哺乳动物物种培养系统中促进持续生长的关键步骤。然而，隔离方法可能会影响卵泡未来的发育能力。我们评估了两种分离方法，机械显微剥离法(针/刀刃)和酶消化法(使用Liberase blendzyme)对通过常规程序获得的家猫卵巢进行了分离。与高浓度(1.4Wünsch单位/毫升)的酶分离卵泡相比，机械分离的卵泡在14天的培养中显示出更好的生长、存活和发育能力。然而，在这些指标上，机械分离与低酶(0.7Wünsch单位/毫升)并无不同，也没有显示毛囊细胞功能的关键基因的表达。此外，在培养7天以上时，卵泡生长/存活的差异并不明显。因此，卵泡分离策略会影响培养中的发育潜力，因此需要延长培养时间以确定最佳的育性保存方法。
Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts. The ovary contains hundreds of eggs with only a select few developing from an immature stage through to ovulation over the course of an animal's lifetime. Rescue of eggs from this pool, and the ability to grow them in culture to a mature stage, would be incredibly valuable for fertility preservation efforts in both humans and endangered species. Currently, the isolation of ovarian follicles (eggs with their surrounding helper cells) is a key step in culture systems for large mammalian species, to promote continued growth. Yet, isolation methods may affect the follicle’s future developmental capacity. We evaluated two isolation strategies, mechanical micro-dissection (needle/scalpel blade) and enzymatic digestion (using Liberase blendzyme) on ovaries of domestic cats obtained via routine spay procedures. Mechanically isolated follicles displayed improved growth, survival, and indications of developmental competence in 14-day culture, compared with high concentration (1.4 Wünsch units/mL) enzyme-isolated follicles. However, mechanical isolation was not different from low (0.7 Wünsch units/mL) enzyme for these metrics, or for expression of key genes indicative of follicular cell functions. Further, differences in follicle growth/survival were not apparent until 7+ days in culture. Thus, ovarian follicle isolation strategies influence developmental potential in culture, and extended culture will be required to identify optimal methods for fertility preservation efforts.
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DOI：
10.1530/jrf.0.1000333
发表时间：
1994-03-01
期刊：
JOURNAL OF REPRODUCTION AND FERTILITY
影响因子：
--
作者：
HIRAO, Y;NAGAI, T;KATO, S
通讯作者：
KATO, S
影响因子：
6.1
作者：
Demeestere, I;Delbaere, A;Englert, Y
通讯作者：
Englert, Y
影响因子：
2.8
作者：
Chansaenroj, A.;Songsasen, N.;Chatdarong, K.
通讯作者：
Chatdarong, K.
影响因子：
--
作者：
CLARK, BJ;SOO, SC;STOCCO, DM
通讯作者：
STOCCO, DM
影响因子：
6.1
作者：
Kristensen, Stine Gry;Rasmussen, Annette;Andersen, Claus Yding
通讯作者：
Andersen, Claus Yding