Wandji S A, Srsen V, Voss A K, Eppig J J, Fortune J E
Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Biol Reprod. 1996 Nov;55(5):942-8. doi: 10.1095/biolreprod55.5.942.
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.
控制原始卵泡生长起始的因素尚不清楚。我们检验了这样一个假说，即来自胎牛的原始卵泡能够在无血清条件下于体外存活并开始生长。从6 - 8月龄的牛胎儿中分离出主要含有原始卵泡的卵巢皮质浅表组织块，在添加了胰岛素、转铁蛋白、硒、亚油酸和牛血清白蛋白（ITS+）的Waymouth MB 752/1培养基中培养0、2、4或7天。培养2、4和7天后对皮质组织块进行组织学检查发现，健康原始卵泡的数量分别减少了88%、90%和94%（p < 0.01），而健康初级卵泡的数量分别增加到第0天的260%、209%和197%（p < 0.05）。显示闭锁迹象的卵泡百分比在培养过程中未随时间变化，原始卵泡和初级卵泡的该百分比分别约为28%和50%。培养7天后，少数剩余健康原始卵泡的平均直径是第0天原始卵泡平均直径的1.2倍（p < 0.01）。相比之下，培养2、4和7天后，初级卵泡的直径相对于第0天分别增大了1.2、1.3和1.4倍（p < 0.01）。培养过程中原始卵泡的卵母细胞直径变化不大，而在初级卵泡中，4天和7天后卵母细胞直径明显增大（分别为1.1倍和1.2倍，p < 0.01）。通过对培养的和新分离的卵巢皮质组织块中增殖细胞核抗原（PCNA，一种细胞生长和增殖的标志物）进行免疫定位，进一步证实了卵泡在体外开始生长。在新分离的组织中，静止原始卵泡的颗粒前体细胞和卵母细胞中没有PCNA染色，但少数生长中的初级卵泡的颗粒细胞和卵母细胞中PCNA染色强烈。培养2、4和7天后，PCNA在新激活的初级卵泡的卵母细胞和许多颗粒细胞中强烈表达。这些结果表明，牛原始卵泡能够在体外进入生长阶段，并且颗粒细胞和卵母细胞中PCNA的表达与原始卵泡生长的起始密切相关。高比例的原始卵泡在体外开始生长这一事实表明，卵巢基质在体内对原始卵泡生长的起始施加抑制性控制。我们描述的培养系统可能提供检验这一假说及其他假说的方法。
