Sharma G Taru, Dubey Pawan K, Meur S K
Reproductive Physiology & ETT Laboratory, Division of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, UP 243122, India.
Anim Reprod Sci. 2009 Aug;114(1-3):115-24. doi: 10.1016/j.anireprosci.2008.09.009. Epub 2008 Sep 24.
The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro growth and survival of buffalo preantral follicles with or without growth factors. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal growth factor (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 microg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like growth factor (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic fibroblast growth factor (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 degrees C in 5% CO(2) and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at -30 degrees C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and growth by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly (P<0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved growth and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P(4)) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P(4) was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10-15 days before degenerating, but follicles cultured with growth factors were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with growth factors was found to be essential for antrum formation.
本研究的目的是开发一种三维（3D）胶原凝胶培养系统，用于有无生长因子情况下水牛腔前卵泡的体外生长和存活。从当地屠宰场收集水牛卵巢，通过显微解剖分离腔前卵泡。将分离的腔前卵泡置于胶原凝胶包被的培养皿中或包埋于胶原凝胶微滴中。培养基为添加了胎牛血清（10%）、胰岛素转铁蛋白硒溶液（ITS，1%）、表皮生长因子（EGF，20 ng/ml）和促卵泡激素（FSH，0.5 μg/ml）的TCM-199。卵泡分为三组，在上述培养基中培养（a组，对照组），添加胰岛素样生长因子（IGF-I，100 ng/ml，b组），或添加IGF-I和碱性成纤维细胞生长因子（bFGF，10 ng/ml，c组）。腔前卵泡在38.5℃、5% CO₂和最大湿度条件下孵育。每72小时更换一次培养基，废弃培养基储存于-30℃用于激素分析。我们发现，胶原凝胶的细胞外基质通过提供表面相互作用和增加卵泡附着来维持卵泡的活力和生长。与整个表面包被培养方法相比，包埋于胶原凝胶微滴中的腔前卵泡有更好的卵泡腔形成和发育。与对照组相比，在胶原凝胶基质上用IGF-I培养的卵泡在培养第10天显示出显著更高的存活率和更大的平均卵泡直径，生长和黏液形成得到改善。然而，用IGF-I与bFGF联合培养的卵泡存活率降低，平均卵泡直径小于IGF-I组（b组）。与对照组相比，在IGF-I培养的卵泡中，培养第9天孕酮（P₄）积累更多；而在IGF-I与bFGF联合培养中，P₄明显降低。对照组的卵泡在退化前可存活长达10 - 15天，但用生长因子培养的卵泡能够存活长达20天，并显示出早期卵泡腔形成的迹象。总之，我们表明胶原凝胶是用于水牛腔前卵泡延长培养的一种新型且有效的3D微环境。发现向培养基中添加生长因子对于卵泡腔形成至关重要。
