Identification Of Target Protein Of DPF-1 Rat Oocytes Can Attain Maturation In Mass In Combination Culture System

The nonciliated epithelial cells of the oviduct actively synthesize and secrete a high-molecular-weight family of glycoproteins termed oviductins. Due to oviductins bind to the zona pellucida , cell- membranes of gametes and early embryos , it was suggested that oviductins has a potential biological role during the transport of gametes, fertilization and early embryonic development. A rabbit oviductin, here we termed Development Promoting Factor-1(DPF-1), has cloned from cDNA library derived from rabbit oviduct mucosal epithelial cells. DPF-1 appears to be highly conserved, and shares a high degree of identity and similarity in the N-terminal region of oviductins. The results obtained from "loss of function" experiments, indication that the binding site to recognize its target protein is localized in the c-terminal region of DPF-1. Since we decide to identify its target protein/proteins by using yeast two hybrid system. In the same time, we decided to establish a system that can provide mature oocytes in mass to fulfil the need for the further establishment of the target cDNA library.A "combination culture system" , containing primary differentiated granulosa cells, proestrus anterior pituitary lobe and Δ-4-androstenedione, can act in a synergistic manner to effectively induce the maturation, fertilization and development of oocytes from early tertiary. follicles of diethylstilbestrol (DES)-treated rats. This combination culture system provides an experimental model in which the molecular mechanisms of egg maturation, fertilization, and embryonic development can be studied. After DES stimulation, an average of 189 oocytes were obtained from early tertiary follicles of unilateral ovaries; 77.6% of these oocytes, when cultured in our combination culture system, were shown to extrude a first polar body, a criterion of oocyte maturation. Of the mature oocytes, the rate of normal fertilization and egg cleavage were 87.8% and 93%, respectively. After 96 h of culturing in vitro, 59% of the zygotes developed into morulae or blastocysts. Embryos implanted at the two-cell stage were able to develop into healthy individuals.To identify proteins that interact with DPF-1, 1.9kb DPF-1 cDNA was fused to pSos plasmid. And the target cDNA library, rat oocyte pMyr cDNA plasmid library, was constructed. 1 putative positive clone derived from the yeast two hybrid screening was isolated, and its nucleic acid sequence analysis showed that it shared 93% identity with activating signal cointegrator 1 complex subunit 2 (ASC-1 complex subunit p100). Since p100 was expressed in many tissues, especially in ovary and pre-implantation embryos, and ASC-1 complex, including the interactions of ASC-1 with p100, p200 and p50, which is essential for SRF(serum response factor), AP-1 (activating protein 1) and NF-B(nuclear factor B) transactivation. So identification of p100 as a novel target for DPF-1, will bring us some supposes to study the mechanism of DPF-1 in regulation of early embryo development.