Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil.
Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, São Paulo, Brasil.
Theriogenology. 2014 Jan 15;81(2):326-31. doi: 10.1016/j.theriogenology.2013.10.002. Epub 2013 Oct 7.
In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.
体外培养的胚胎在细胞质脂滴 (LD) 中储存高脂质含量，减少或去除 LD 已被证明可以提高冷冻-解冻活力。Perilipin Adipophilin Tail-interacting Protein 47 kD（PAT）家族蛋白参与许多细胞类型中 LD 的形成和调节，但在牛卵母细胞或胚胎前体中尚未检测到它们的存在。因此，本研究旨在检测 PAT 家族转录物（Perilipin-2 [PLIN2] 和 Perilipin-3 [PLIN3]）在不成熟和体外成熟（IVM）卵母细胞以及 2-4 细胞、8-16 细胞、桑椹胚（MO）和囊胚（BL）阶段的体外胚胎中的表达。PLIN3 的表达在 IVM 时下调，PLIN2 在 IVM 卵母细胞中的表达比对 PLIN3 更高（P < 0.001）。在胚胎发育的早期阶段，PLIN2 的表达在 MO 阶段达到峰值（P < 0.001），然后在 BL 阶段再次下降。相比之下，PLIN3 在发育的最早阶段表达水平较低，在 MO 阶段略有上调（P < 0.05），在 BL 阶段大量上调（15 倍；P < 0.001）。在大多数分析阶段，PLIN3 的表达比对 PLIN2 更高（P < 0.05），除了在 8-16 细胞胚胎中。这些结果表明，PLIN2 可能参与维持 IVM 卵母细胞受精后胚胎发育所需的脂质库存。此外，我们假设 PLIN3 是主要的 PAT 蛋白，负责稳定胚胎发育过程中急性脂质负荷形成的 LD。我们使用免疫细胞化学在第 7 天的 BL 中证实了 PLIN2 和 PLIN3 蛋白的存在：这些 PAT 蛋白与 BODIPY 染色的 LD 共定位。PLIN3 在细胞质中的分布似乎比 PLIN2 更广泛，与脂肪细胞中的模式一致。这些发现表明，分别对 PLIN2 和 PLIN3 呈阳性的较大（旧）和新形成（较小）LD 共存于囊胚中。据我们所知，这是首次报道表明 PAT 家族的转录物存在于牛卵母细胞和胚胎中。
