Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss)

Lhx8 is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. In this study, we report the characterization of rainbow trout lhx8 genes and identification of a novel germ cell-specific nuclear factor that interacts with Lhx8. Two lhx8 genes, lhx8a and lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Collectively, results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout.

Lhx8是一类重要的转录因子（transcription factor），特异性高表达于生殖细胞。Lhx8基因敲除小鼠因缺乏卵母细胞、且原始卵泡向初级卵泡的转化过程受损而不育。Lhx8缺陷还会影响诸多重要的卵母细胞特异性基因的表达。
本研究对虹鳟的lhx8基因进行了鉴定与功能表征，并成功筛选得到一种可与Lhx8互作的新型生殖细胞特异性核因子。本研究共鉴定出两个lhx8基因，即lhx8a与lhx8b，二者分别编码含344个和361个氨基酸的蛋白质。两种蛋白的序列一致性达83%，且两者的转录本均特异性表达于卵巢组织中。
实时定量PCR（quantitative real time PCR）分析显示，两个基因在卵黄发生前卵巢及早期胚胎中均呈现高表达水平。通过酵母双杂交筛选系统（yeast two-hybrid screening system），研究者鉴定出一种可与Lhx8互作的新型蛋白——Borealin-2。Lhx8a或Lhx8b与Borealin-2之间的互作，进一步通过双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验得到了验证。
Borealin-2由255个氨基酸组成，包含一个Nbl1结构域，其mRNA的表达仅限于卵巢与精巢组织。GFP报告基因实验证实，Borealin-2是一种核蛋白。
综上，本研究结果表明，Lhx8a与Lhx8b均可通过与Borealin-2互作发挥功能，这一过程可能在虹鳟的卵子发生及早期胚胎发育过程中发挥重要作用。