Tischkau S A, Bahr J M
Department of Animal Sciences, University of Illinois at Urbana-Champaign, 61801, USA.
Biol Reprod. 1996 Apr;54(4):865-70. doi: 10.1095/biolreprod54.4.865.
Microscopic analysis of ovarian follicles in the domestic hen has revealed differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germinal disc, which contains the female gamete. These differences appear as a morphological gradient, which implies variations in granulosa cell function. This observation prompted us to hypothesize that the germinal disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions: 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of a preovulatory follicle? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis? Incorporation of 3H-thymidine was used as a measure of proliferation, and production of progesterone was used as a measure of steroidogenesis. In the first experiment, 8-mm-diameter sections were obtained from three morphologically distinct regions of the granulosa monolayer: 1) the GDR, 2) granulosa cells distal to the GDR (distal granulosa) and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa cells). The GDR incorporated the most 3H-thymidine and produced the least progesterone. Distal granulosa cells incorporated the least 3H-thymidine and produced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of 3H-thymidine and produced an intermediate amount of progesterone. To answer the second question, conditioned medium was prepared from GDRs and distal granulosa cells (control) obtained from the F1 (largest preovulatory follicle) and F3 (the third-largest preovulatory follicle) follicles. Sections (8-mm in diameter) of the distal granulosa layer (F3 for 3H-thymidine incorporation, F1 for progesterone production) were incubated in GDR-conditioned medium or granulosa cell-conditioned medium to determine whether factors secreted into the medium by the GDR and distal granulosa cells affect granulosa cell proliferation and/or steroidogenesis. Certain samples of GDR-conditioned medium and granulosa cell-conditioned medium were boiled, protease-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and protease-sensitive factors that promoted proliferation and inhibited progesterone production by granulosa cells. These data indicate that diametrically opposed gradients of proliferation and steroidogenesis are present within the granulosa layer of an individual preovulatory follicle. Furthermore, the GDR produces proliferation-stimulating and steroidogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cells.
对家鸡卵巢卵泡的显微镜分析显示，颗粒细胞的细胞结构存在差异，这种差异取决于颗粒细胞相对于包含雌配子的胚盘的位置。这些差异表现为一种形态学梯度，这意味着颗粒细胞功能存在变化。这一观察结果促使我们提出假设，即禽类排卵前卵泡的胚盘区域（GDR）通过产生以旁分泌方式作用的因子来参与卵泡生长过程，这些因子可刺激颗粒层细胞增殖并抑制其类固醇生成，从而在颗粒层的形态和生理方面建立一个梯度。为了验证我们的假设，我们提出了两个问题：1）排卵前卵泡的颗粒层内是否存在增殖和类固醇生成的生理梯度？2）GDR是否分泌影响颗粒细胞增殖和/或类固醇生成的因子？采用3H-胸腺嘧啶核苷掺入法作为增殖的指标，孕酮生成量作为类固醇生成的指标。在第一个实验中，从颗粒单层的三个形态学上不同的区域获取直径为8毫米的切片：1）GDR，2）GDR远端的颗粒细胞（远端颗粒细胞），3）GDR与远端颗粒细胞之间中间位置的颗粒细胞（近端颗粒细胞）。GDR掺入的3H-胸腺嘧啶核苷最多，产生的孕酮最少。远端颗粒细胞掺入的3H-胸腺嘧啶核苷最少，产生的孕酮最多。近端颗粒细胞掺入的3H-胸腺嘧啶核苷量居中，产生的孕酮量也居中。为了回答第二个问题，从F1（最大的排卵前卵泡）和F3（第三大的排卵前卵泡）卵泡获取的GDR和远端颗粒细胞（对照）制备条件培养基。将远端颗粒层的切片（直径8毫米，F3用于3H-胸腺嘧啶核苷掺入实验，F1用于孕酮生成实验）在GDR条件培养基或颗粒细胞条件培养基中孵育，以确定GDR和远端颗粒细胞分泌到培养基中的因子是否影响颗粒细胞增殖和/或类固醇生成。对某些GDR条件培养基和颗粒细胞条件培养基样品进行煮沸、蛋白酶处理或活性炭处理。F3和F1的GDR产生对热和蛋白酶敏感的因子，这些因子可促进颗粒细胞增殖并抑制其孕酮生成。这些数据表明，在单个排卵前卵泡的颗粒层内存在截然相反的增殖和类固醇生成梯度。此外，GDR产生刺激增殖和抑制类固醇生成的因子，这些因子可能以自分泌或旁分泌方式影响颗粒细胞的增殖和类固醇生成。
