SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

中文翻译：

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基于形态、直径和细胞数量的分离牛腔前卵泡的表征


摘要腔前卵泡是辅助生殖技术中潜在的卵母细胞储存库。随着人们对开发腔前卵泡生长技术的兴趣日益浓厚体外随着牛成为了解人类卵泡发生的合适模型物种，需要建立发育阶段的准确分类。牛腔前卵泡的分类主要基于组织学分析和估计模型，这可能不能很好地转化为正确表征从卵巢分离的腔前卵泡。在本研究中，我们通过分离后的形态对牛窦前卵泡进行分类，通过直接计数确定颗粒细胞的直径和数量，并将我们的结果与以前报告牛窦前卵泡分类的研究进行比较。通过卵巢组织均质化分离卵泡，并根据颗粒细胞的形态和层数分为初级、早期次级和次级阶段。单独测量直径并使用 Hoechst 33342 作为核染色剂来计数颗粒细胞。我们发现按形态分为初级、早期次级和次级的卵泡具有不同的平均直径和细胞数量（磷< 0.01);细胞数量和直径呈正相关，每个发育阶段的细胞密度和细胞数量也呈正相关（磷< 0.01）。这里获得的结果大部分与之前基于组织学切片和孤立卵泡的分类一致，但存在一些差异。 目前的数据增加了牛腔前卵泡分类的准确性，这对于优化培养条件以产生具有发育能力的卵母细胞至关重要。