Jorssen E P A, Langbeen A, Marei W F A, Fransen E, De porte H F M, Leroy J L M R, Bols P E J
Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
Theriogenology. 2015 Jul 15;84(2):301-11. doi: 10.1016/j.theriogenology.2015.03.020. Epub 2015 Mar 28.
To provide new insights in the molecular mechanism controlling preantral follicular development and to unravel the needs to support in vitro follicular development of early-stage preantral follicles (PAFs), there is a need for alternative in vitro bovine follicle culture methods. In this study, we aimed to characterize follicular dynamics using an IVC system of isolated and individually cultured bovine early PAFs during 10 days to generate individual follicle follow-up data. Preantral follicles (<50 μm) were isolated from slaughterhouse ovaries and cultured individually for 10 days. Individual follicle morphology, growth, survival, quality, and cell proliferation were evaluated in time by combining noninvasive and invasive assessment methods. The PAFs were light microscopically evaluated during culture to assess follicular dynamics, stained with neutral red to determine follicle viability, stained with 4',6-diamidino-2-phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling to evaluate cell proliferation and follicle quality, and processed for histologic evaluation to assess follicle morphology. On the basis of their morphology, follicles were subdivided in three categories, with category 1 follicles showing the best morphologic features. On Day 0, only category 1 follicles were selected, but follicle categories were reassigned on evaluation Days 1, 2, 4, 7, or 10. Although 67% of the follicles survived 10 days of IVC, the number of follicles exhibiting a normal morphology decreased significantly from Day 7 onward and the apoptotic index increased significantly from Day 10. Both category 1 and 2 follicles showed a significant increase in follicular diameter (Day 10: 21.80 ± 0.86 and 11.82 ± 0.80, respectively). This increase in follicular diameter showed to be correlated with an increase in the total cell number. In conclusion, this culture system showed to support follicular development until Day 10, although the proportion of follicles showing normal morphologic features and the follicular quality decreased after 10 days of IVC. Follicles maintaining their category 1 morphologic features over time seem to be of a better quality and show a higher developmental competence as compared to category 2 and 3 follicles.
为了深入了解控制窦前卵泡发育的分子机制，并阐明支持早期窦前卵泡（PAF）体外卵泡发育的需求，需要采用替代的体外牛卵泡培养方法。在本研究中，我们旨在使用分离并单独培养的牛早期PAF的体外培养（IVC）系统，在10天内对卵泡动态进行表征，以生成单个卵泡的跟踪数据。从屠宰场的卵巢中分离出窦前卵泡（<50μm）并单独培养10天。通过结合非侵入性和侵入性评估方法，及时评估单个卵泡的形态、生长、存活、质量和细胞增殖情况。在培养过程中，对PAF进行光学显微镜评估以评估卵泡动态，用中性红染色以确定卵泡活力，用4',6-二脒基-2-苯基吲哚和末端脱氧核苷酸转移酶dUTP缺口末端标记染色以评估细胞增殖和卵泡质量，并进行组织学评估以评估卵泡形态。根据形态，卵泡被分为三类，其中1类卵泡显示出最佳的形态特征。在第0天，仅选择1类卵泡，但在评估的第1、2、4、7或10天重新分配卵泡类别。尽管67%的卵泡在IVC 10天后存活，但从第7天起，形态正常的卵泡数量显著减少，凋亡指数从第10天起显著增加。1类和2类卵泡的卵泡直径均显著增加（第10天：分别为21.80±0.86和11.82±0.80）。卵泡直径的增加与总细胞数的增加相关。总之，该培养系统显示能够支持卵泡发育至第10天，尽管在IVC 10天后，形态正常的卵泡比例和卵泡质量有所下降。与2类和3类卵泡相比，随时间保持1类形态特征的卵泡似乎质量更好，发育能力更高。
