Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
Member of the Oncofertility Consortium, Michigan State University, East Lansing, MI, USA.
Mol Hum Reprod. 2022 Sep 29;28(10). doi: 10.1093/molehr/gaac033.
Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.
卵泡是卵巢的功能单位，已经开发出几种方法来体外培养卵泡，这些方法可以重现卵子发生和卵泡发生的关键事件。酶消化方案通常用于从卵巢中增加卵泡的产量。然而，这些方案对最外层的卵泡膜和颗粒细胞的影响，以及对卵泡功能的影响，尚不清楚。为了研究酶消化对卵泡功能的影响，我们从 CD1 小鼠中收集原始卵泡，分别通过酶消化（Enzy-FL）或机械分离（Mech-FL），并比较了在体外卵泡生长系统中包裹的卵泡生长、类固醇生成和细胞分化，该系统维持卵母细胞及其周围体细胞的 3D 结构。卵泡用 0.5%藻酸盐包裹并培养 8 天。与 Enzy-FL 相比，Mech-FL 生长更快，产生的雄烯二酮、雌二醇和孕酮水平显著更高。卵泡膜间质细胞标记基因 Cyp17a1 的表达也更高，该基因编码 17-羟化酶/17、20-裂合酶，催化 pregnenolone 和 progesterone 羟化为 17-羟孕烯醇酮和 17-羟基孕酮，以及这些产物转化为脱氢表雄酮和雄烯二酮，以及 Star，该基因编码一种必需的转运蛋白胆固醇进入线粒体，在 Mech-FL 中的表达也高于 Enzy-FL。Mech-FL 在卵泡的外边缘保持完整的卵泡膜间质层，碱性磷酸酶染色证实这与体内模式相似，而 Enzy-FL 从培养开始时就没有卵泡膜间质细胞。因此，在培养开始时保留卵泡膜细胞层可以更好地支持卵泡生长和功能。有趣的是，Enzy-FL 中最外层的颗粒细胞在培养的第 4 天表达了 CYP17A1，同时保持了抑制素 α 亚单位的表达和立方核。因此，在没有卵泡膜间质细胞的情况下，颗粒细胞有可能分化为产生雄激素的细胞。这项工作可能对人类卵泡培养有影响，由于卵巢皮质的密度，需要进行酶分离。
