Liyuan Fu

Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Jianbo Yao

Committee Co-Chair

Vagner A Benedito

Committee Member

Kenneth P Blemings

Committee Member

Hao Ma

Committee Member

Shuo Wei

Abstract

LIM homeobox 8 (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. To date, no attempts have been made to investigate the existence of any cellular factors that might interact with Lhx8 protein in oocytes and early embryos. In this study, we report the characterization of rainbow trout and bovine Lhx8 genes and identification of important germ cell-specific nuclear factors that interact with Lhx8 protein in both species. In rainbow trout, 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. 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. The open reading frame (ORF) of bovine Lhx8 gene was amplified from cDNA of a bovine fetal ovary using primers designed based on a predicted bovine Lhx8 cDNA sequence and a partial 5 'end transcript. The ORF of bovine Lhx8 cDNA is 1,134 bp in length encoding a protein of 377 amino acids. A splicing variant of Lhx8 (Lhx8_v1) was identified, which results from alternative splicing of exon 2 and 3, and encodes a protein of 293 amino acids. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in the splicing variant, Lhx8_v1, is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 mRNA are specifically expressed in fetal ovaries and testis but not detectable in the somatic tissues as well as in granulosa and theca cells. Lhx8 mRNA is highly abundant in GV and MII stage oocytes as well as in early stage embryos but not detectable in morula and blastocyst stage embryos. Lhx8_v1 mRNA expression is detectable in oocytes and early embryo but not in morula and blastocyst stage embryos. A GFP reporter assay revealed that Lhx8 is a nuclear protein and the predicted monopartite NLS is required for its transport into the nucleus. Direct yeast two-hybrid analysis revealed that bovine Lhx8 protein interacts with Figla, a basic helix-loop-helix transcription factor. The interaction between Lhx8 and Figla was confirmed by a co-immunoprecipitation assay. This is the first time that a direct protein-protein interaction between two germ cell-specific transcription factors essential for oocyte and follicular development is demonstrated. The study provides new information for studying the mechanisms of the regulatory roles of Lhx8 in oocyte/follicular development and early embryogenesis.

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