Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium
Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium
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DOI：
10.1111/rda.12022
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发表时间：
2012-12-01
影响因子：
1.7
通讯作者：
Songsasen, N.
中科院分区：
文献类型：
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作者：
Fujihara, M.;Comizzoli, P.;Songsasen, N.
目的探讨不同培养液、不同蛋白质补充量及不同微环境对猫、犬原始卵泡活力的影响。卵巢皮质切片在α-最低必需培养液(α-MEM)或添加10%胎牛血清(FBS)、10%敲除血清替代物(KSR)或0.1%聚乙烯醇(不含蛋白质)的MEM中培养3、9或15天。在另一项单独的研究中，猫和狗的卵巢组织分别在无蛋白的α-MEM和MEM中进行培养，分别在细胞培养板上、1.5%琼脂糖凝胶上或24孔细胞培养板上培养(对照)。用钙黄素AM/乙锭二聚体和苏木精/伊红染色组织学评价卵泡活力。猫卵泡在α-MEM中体外培养9天后无一存活，而在无蛋白条件下培养15天的猫卵泡存活率为37.5%(p<；0.05)。相反，在无蛋白条件下15天，α-MEM在维持犬卵泡存活率(32.7%比8.1%)方面优于MEM(p<；0.05)。血清对两种动物的卵泡存活都是不利的(p<；0.05)。剔除血清替代补充剂和无蛋白条件支持了猫卵泡的存活，而后者在维持犬卵泡存活方面优于前者(p<；0.05)。同样，在培养3天和9天后，与细胞培养组和对照组相比，琼脂糖凝胶提高了犬卵泡的存活率(p<；0.05)。对于猫来说，琼脂糖凝胶(p<；0.05)比对照组更好地支持卵泡存活，但与细胞培养插入物相当。因此，对于猫和狗来说，维持皮质内卵巢切片中原始卵泡的存活需要不同的体外微环境。在培养中提高这些早期卵泡存活率的一个关键因素似乎是琼脂糖凝胶的使用，它可能通过促进气体交换来提高卵泡的活力。
Contents Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in alpha-minimum essential medium (alpha-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free alpha-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in alpha-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, alpha-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.