Initiation in vitro of growth of bovine primordial follicles
Initiation in vitro of growth of bovine primordial follicles
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DOI：
10.1095/biolreprod55.5.942
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发表时间：
1996-11-01
影响因子：
3.6
通讯作者：
Fortune, JE
中科院分区：
文献类型：
--
作者：
Wandji, SA;Srsen, V;Fortune, JE
控制原始卵泡生长的因素尚不清楚。我们已经验证了胎牛原始卵泡可以在无血清条件下存活并开始体外生长的假设。从6-8月龄的牛胎儿中分离主要含有原始卵泡的卵巢皮质的表面碎片，并在添加有胰岛素、转铁蛋白、硒、亚油酸和BSA（ITS+）的Waymouth MB 752/1培养基中培养0、2、4或7天。培养2、4和7天后的皮质片的组织学检查显示，健康原始卵泡的数量分别减少了88%、90%和94（p < 0.01），而健康初级卵泡数分别增加到260%、209%和197%，在第0天存在的数量的百分比（p < 0.05）。显示闭锁迹象的卵泡的百分比不随培养时间而变化，原始卵泡和初级卵泡分别约为28%和50%。在培养7天后，少数剩余的健康原始卵泡的平均直径是第0天存在的原始卵泡的平均直径的1.2倍（p < 0.01）。相比之下，在培养2、4和7天后，初级卵泡的直径分别是第0天的1.2、1.3和1.4倍（p < 0.01）。原始卵泡的卵母细胞直径在培养过程中变化不大，而初级卵泡的卵母细胞直径在培养4天和7天后明显增加（分别为1.1和1.2倍，p < 0.01）。卵泡生长在体外启动进一步证实了免疫定位的增殖细胞核抗原（PCNA），细胞生长和增殖的标志物，在培养和新鲜分离的卵巢皮质片。在新鲜分离的组织中，PCNA染色不存在于静止原始卵泡的前颗粒细胞和卵母细胞中，但在少数生长的初级卵泡的颗粒细胞和卵母细胞中染色强烈。培养2、4和7天后，PCNA在卵母细胞和许多新激活的初级卵泡的颗粒细胞中强烈表达，表明牛原始卵泡在体外可以进入生长期，颗粒细胞和卵母细胞的PCNA表达与原始卵泡的开始生长密切相关。事实上，高比例的原始卵泡启动生长在体外表明，卵巢基质施加抑制控制原始卵泡生长的启动在体内。我们所描述的文化系统可能提供了检验这一假设和其他假设的手段。
Factors that control the onset of primordial follicle growth are unknown. We have tested the hypothesis that primordial follicles from fetal calves can survive and initiate growth in vitro in serum-free conditions. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were isolated from bovine fetuses 6-8 mo old and cultured for 0, 2, 4, or 7 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and BSA (ITS+). Histological examination of cortical pieces after 2, 4, and 7 days in culture showed that the number of healthy primordial follicles had decreased by 88%, 90%, and 94%, respectively (p < 0.01), whereas the number of healthy primary follicles had increased to 260%, 209%, and 197%, respectively, of the number present on Day 0 (p < 0.05). The percentage of follicles that showed signs of atresia did not change with time in culture and was about 28% and 50% for primordial and primary follicles, respectively. After 7 days in culture, the mean diameter of the few remaining healthy primordial follicles was 1.2 times the average diameter of primordial follicles present on Day 0 (p < 0.01). In contrast, after 2, 4, and 7 days in culture, primary follicles were 1.2, 1.3, and 1.4 times larger in diameter, respectively, relative to Day 0 (p < 0.01). There was little change in the diameter of oocytes in primordial follicles during culture, whereas in primary follicles an increase in oocyte diameter became apparent after 4 and 7 days (1.1 and 1.2 times, respectively, p < 0.01). That follicle growth was initiated in vitro was further confirmed by immunolocalization of proliferating cell nuclear antigen (PCNA), a marker for cell growth and proliferation, in cultured and freshly isolated pieces of ovarian cortex. In freshly isolated tissue, PCNA staining was absent from pre-granulosa cells and oocytes of the quiescent primordial follicles but was intense in granulosa cells and oocytes of the few growing primary follicles. After 2, 4, and 7 days in culture, PCNA was expressed intensely in the oocyte and many granulosa cells of newly activated primary follicles.These results demonstrate that bovine primordial follicles can enter the growth phase in vitro and that PCNA expression by granulosa cells and oocytes is closely associated with the onset of primordial follicle growth. The fact that a high percentage of primordial follicles initiated growth in vitro suggests that the ovarian stroma exerts inhibitory control over the initiation of primordial follicle growth in vivo. The culture system we describe may provide the means to test this hypothesis and others.