Nottola Stefania A, Heyn Rosemarie, Camboni Alessandra, Correr Silvia, Macchiarelli Guido
Laboratory for Electron Microscopy Pietro M. Motta, Department of Anatomy, University of Rome La Sapienza, Rome, Italy.
Microsc Res Tech. 2006 Jun;69(6):508-16. doi: 10.1002/jemt.20309.
The use of somatic cells for cocultures during in vitro fertilization (IVF) is currently finalized to obtain a higher number of healthy and viable embryos with a high potential of implantation. Among the different cell lines that can be used as feeder cells for cocultures, granulosa cells (GCs) are autologous cells, safe and easy to recover. The aim of the present study was to analyze the fine structure of human GCs used in a coculture system to evaluate, from a morphodynamic point of view, their role in supporting embryo development. GCs were collected during oocyte pick-up, 36 h after human chorionic gonadotropin administration, from patients undergoing IVF procedures, who had given their informed consent to be included in this protocol. After coculture, GCs were fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM). By LM, GCs appeared as clusters of loosely packed cells, irregularly rounded or polyhedral in shape, varying in diameter from 18 to 25 microm. Mitotic cells, as well as regressing elements (with pyknotic nuclei or dense cytoplasm) and cell fragments were occasionally observed. By TEM, the plasma membrane was irregular due to the presence of cytoplasmic evaginations. Linear and annular gap junctions between neighboring GCs were found. GC nuclei, rounded and eccentrically located, contained finely dispersed chromatin, one (often two) prominent nucleoli and, infrequently, peripheral patches of heterochromatin. Numerous organelles populated the GC cytoplasm, among them, mitochondria were rod-shaped or elongated, usually provided with tubular-vesicular cristae but occasionally showing atypical, longitudinally oriented cristae. Membranes of smooth endoplasmic reticulum, Golgi stacks and vesicles, secretory-like granules, cisternae of rough endoplasmic reticulum (RER), free ribosomes and polysomes, lysosomal-like bodies, microfilaments, and lipid droplets were also seen in the GC cytoplasm. In most cells, RER was scarcely represented and numerous lipid droplets filled the perinuclear space. On the contrary, some GCs contained an abundant RER and rare lipid droplets scattered in the cytoplasm. In conclusion, our data demonstrated the presence, in a coculture system, of GCs provided with ultrastructural characteristics typical of healthy, metabolically active, mostly steroidogenic cells. Protein-synthetic cells have also been detected. These data, evaluated at the light of biochemical and clinical studies, sustain the capability of human GCs cocultures to positively affect early embryo development in vitro by the secretion of steroids and proteins, putative "embryotrophic" factors.
在体外受精（IVF）过程中使用体细胞进行共培养，目前已确定是为了获得更多具有高着床潜力的健康且有活力的胚胎。在可用于共培养的不同细胞系中，颗粒细胞（GCs）是自体细胞，安全且易于获取。本研究的目的是分析在共培养系统中使用的人颗粒细胞的精细结构，从形态动力学角度评估它们在支持胚胎发育中的作用。在给予人绒毛膜促性腺激素后36小时，从接受IVF程序且已签署知情同意书纳入本方案的患者中，在取卵时收集颗粒细胞。共培养后，将颗粒细胞固定并进行光学显微镜（LM）和透射电子显微镜（TEM）处理。通过光学显微镜观察，颗粒细胞呈现为松散堆积的细胞簇，形状不规则圆形或多面体，直径在18至25微米之间变化。偶尔观察到有丝分裂细胞以及退化成分（核固缩或细胞质致密）和细胞碎片。通过透射电子显微镜观察，由于细胞质的内陷，质膜不规则。在相邻颗粒细胞之间发现了线性和环形缝隙连接。颗粒细胞核呈圆形且偏心定位，含有精细分散的染色质、一个（通常两个）突出的核仁，偶尔还有周边异染色质斑块。颗粒细胞的细胞质中有许多细胞器，其中线粒体呈杆状或细长形，通常具有管状泡状嵴，但偶尔也显示出非典型的纵向嵴。在颗粒细胞的细胞质中还可见到滑面内质网、高尔基体堆叠和小泡、分泌样颗粒、粗面内质网（RER）的池、游离核糖体和多聚核糖体、溶酶体样小体、微丝和脂滴。在大多数细胞中，粗面内质网很少，大量脂滴充满核周空间。相反，一些颗粒细胞含有丰富的粗面内质网，细胞质中散在着少量脂滴。总之，我们的数据表明，在共培养系统中存在具有健康、代谢活跃、主要为类固醇生成细胞典型超微结构特征的颗粒细胞。也检测到了蛋白质合成细胞。根据生化和临床研究评估这些数据，支持人颗粒细胞共培养通过分泌类固醇和蛋白质（假定的“胚胎营养”因子）对体外早期胚胎发育产生积极影响的能力。
