Microarrays reveal oocyte lipid storage droplet gene as key factor in reproductive dysfunction of captive-reared P. monodon. Penaeus monodon

Background: Aquaculture of the black tiger prawn Penaeus monodon remains severely constrained by an almost total dependence on wild-caught broodstock. Reliance on wild-caught broodstock stems, for the most part, from reduced reproductive potential of captive-reared females. Reproductive performance of captive-reared females is usually characterised by longer latency period, lower egg production, egg hatch rates and post-larval survivorship compared with their wild-caught counterparts. Improved understanding of the cellular and associated molecular events occurring during peneaid ovarian maturation could therefore be fundamental to improving reproductive success of captive-reared animals. Methodology/Principle Findings: In support of other studies, our histological analyses of developing oocytes revealed differences between wild-caught and captive-reared P. monodon, including reduced lipid accumulation in oocytes of captive-reared animals. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Custom oligonucleotide microarrays were constructed and screened with transcripts derived from the ovary, cephalothorax and eyestalk from animals of all ovarian maturation stages. Ovarian maturation-related differential expression patterns were observed for 111 transcripts, with 53 transcripts displaying differential expression between wild-caught and captive-reared animals. Notably transcripts encoding vitellogenin - the major egg yolk protein precursor, and a lipid storage droplet protein (which we named pmLSD) which is involved in lipid accumulation, were found to be more highly expressed in wild-caught animals. pmLSD transcripts localise to pre-vitellogenic oocytes of wild-caught animals and the pmLSD protein is exclusively localised to the surface of lipid droplets of oocytes at vitellogenic and cortical rod stages. Overall design: We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Target preparation and microarray hybridisation. Ovarian RNA samples from nine wild-caught animals representing six ovarian maturation stages (P, 2, 24, V, R, E) were used in microarray hybridisations. Similarly, RNA samples from three captive-reared animals representing four maturation stages (P, 24, V, E) were used in microarray hybridisations. For wild-caught animals, samples from each ovarian maturation stage were pooled into groups of four and five, enabling two hybridisations. For captive-reared animals, samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage. Importantly, as the four stages for captive-reared animals were (1) pre-ablation pre-vitellogenic, (2) post-ablation pre-vitellogenic, (3) post-ablation vitellogenic, (4) post-ablation vitellogenic with cortical rods, this arrangement allowed for 2 samples of captive-reared pre-vitellogenic and 2 samples of captive-reared vitellogenic, thereby enabling t-tests between samples, while also allowing analysis across the whole 4 stages via cluster analysis. All hybridisations were single channel hybridisations conducted using equal amounts of RNA pooled from each individual.

**背景**：斑节对虾（Penaeus monodon）的养殖仍严重受制于几乎完全依赖野生亲虾的现状。这种对野生亲虾的依赖，主要源于人工繁育雌亲虾的生殖潜能下降。与野生亲虾相比，人工繁育雌亲虾的生殖性能通常表现为生殖滞后期更长、产卵量更低、卵孵化率及幼体存活率更低。因此，深入解析对虾卵巢成熟过程中的细胞及相关分子事件，是提升人工繁育亲虾生殖成功率的核心基础。

**方法与主要发现**：与其他研究结果一致，我们对发育中卵母细胞的组织学分析显示，野生与人工繁育的斑节对虾存在诸多差异，其中包括人工繁育个体的卵母细胞脂质积累量显著降低。我们采用寡核苷酸微阵列（oligonucleotide microarray）技术，对比了野生与人工繁育斑节对虾卵巢成熟相关基因的表达谱。我们定制构建了寡核苷酸微阵列，并使用覆盖所有卵巢成熟阶段的个体的卵巢、头胸部及眼柄的转录本进行探针筛选。本研究共检测到111条与卵巢成熟相关的差异表达转录本，其中53条在野生与人工繁育个体间存在表达差异。值得注意的是，编码主要卵黄蛋白前体卵黄蛋白原（vitellogenin），以及参与脂质积累的脂滴储存蛋白（我们将其命名为pmLSD）的转录本，在野生个体中表达量更高。pmLSD转录本定位于野生个体的卵黄发生前期卵母细胞，而pmLSD蛋白则特异性定位于卵黄发生期及皮层杆期卵母细胞的脂滴表面。

**实验整体设计**：我们采用寡核苷酸微阵列技术，对比了野生与人工繁育斑节对虾卵巢成熟相关基因的表达谱。
**靶标制备与微阵列杂交**：选取9只处于6个卵巢成熟阶段（P、2、24、V、R、E）的野生个体的卵巢RNA样本用于微阵列杂交；同理，选取3只处于4个成熟阶段（P、24、V、E）的人工繁育个体的卵巢RNA样本用于杂交。对于野生个体，将每个卵巢成熟阶段的样本分别合并为4个和5个样本组，完成两次杂交；对于人工繁育个体，将3只个体各阶段的样本合并，每个阶段完成一次杂交。值得说明的是，人工繁育个体的4个成熟阶段分别为：(1) 眼柄切除前卵黄发生前期、(2) 眼柄切除后卵黄发生前期、(3) 眼柄切除后卵黄发生期、(4) 眼柄切除后伴皮层杆的卵黄发生期。该实验设计可获得2个人工繁育卵黄发生前期样本与2个人工繁育卵黄发生期样本，支持样本间的t检验分析，同时可通过聚类分析完成全部4个阶段的整体分析。所有杂交均为单通道杂交，使用来自每个个体的等量混合RNA完成。