Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Campus UAB, Cerdanyola del Vallès 08193, Spain.
Hum Reprod. 2013 Feb;28(2):414-22. doi: 10.1093/humrep/des363. Epub 2012 Oct 9.
What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes?
TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes.
TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown.
STUDY DESIGN, SIZE, DURATION: For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes.
We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT).
LIMITATIONS, REASONS FOR CAUTION: We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited.
This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.
端粒重复含 RNA（TERRA）和端粒酶在人类胎儿卵母细胞中的分布如何？
TERRA 在人类胎儿卵母细胞的端粒处形成离散焦点，并且与端粒保护因子 2（TRF2）的庇护成分以及端粒酶的催化亚基在减数分裂染色体的端粒处共定位。
TERRA 是已在许多不同真核生物体细胞中描述的端粒染色质的结构元件。端粒酶酶在成年体细胞中不活跃，但在生殖细胞、干细胞和大多数肿瘤中活跃；然而，其在卵母细胞中的分布仍不清楚。
研究设计、大小和持续时间：在这项研究中，使用了四个 22 孕周的正常胎儿的卵巢样本。这些样本是在父母和 Vall d'Hebron 医院伦理委员会的同意下获得的。
参与者/材料、设置、方法：我们分析了来自人类胎儿卵巢的细胞中端粒酶和 TERRA 的分布。通过优化免疫荧光（IF）和 RNA-荧光原位杂交（RNA-FISH）技术的组合，进行了 TERRA、端粒酶和端粒的共定位。通过 IF 检测联会复合体蛋白 3（SYCP3）、TRF2 和端粒酶的蛋白质成分[端粒酶逆转录酶（TERT）]，而通过 RNA-FISH 使用（CCCTAA）（3）寡核苷酸揭示 TERRA。SYCP3 信号允许我们识别已进入减数分裂的卵母细胞，并将其分类为前期 I 的不同阶段，而 TRF2 则表示染色体的端粒区。
我们首次显示了人类胎儿卵巢细胞中端粒酶和 TERRA 的存在及其细胞内分布。TERRA 以离散焦点的形式存在于 75%的卵巢组织细胞中，并且大多数 TERRA 分子（≈83%）位于端粒处（与 TRF2 共定位）。TERRA 水平在卵母细胞中高于在卵巢组织细胞中（P=0.00），并且在前期 I 阶段的进展过程中没有变化（P=0.37）。TERRA 存在于人类胎儿卵巢衍生的所有细胞类型中约 23%的端粒上。此外，约 22%的 TERRA 焦点与端粒酶的蛋白质成分（TERT）共定位。
局限性、谨慎的原因：我们对人类胎儿卵巢组织中的 TERRA 进行了描述性/定性研究。由于胎儿样本的获取和操作困难，本研究中使用的胎儿卵巢数量有限。
这是第一个关于人类胎儿卵母细胞中端粒重复 RNA 表达的报告。在从细线期到粗线期的卵母细胞端粒处存在 TERRA 及其与端粒酶蛋白质成分的共定位表明，这种 RNA 可能参与端粒结构的维持，至少通过减数分裂前期 I 期间发生的过程。由于卵母细胞中的端粒主要是关于花束结构进行研究的，我们的结果为减数分裂期间端粒结构引入了一个新的观点。
