n efficiently, in 2883-98-9 web contrast to Spin1 mutant oocytes. After 6 hours incubation in IBMX-free medium, 50% of Spin1 mutant oocytes remained as GV oocytes, whereas less than 10% of control oocytes retained a GV. Overnight incubation in IBMX-free medium showed that a significant number of Spin1 mutant oocytes failed to resume meiosis as compared to control oocytes. Our findings suggest SPIN1 plays a role in meiotic resumption of mouse oocytes. The fact that half of the Spin1 mutant oocytes retained the ability to resume meiosis suggested that Spin1 functions in oocytes are partly complemented by other mechanisms involved in meiotic resumption. Yeast Two-hybrid Screening The CytoTrap Yeast Two-hybrid system was used to screen for interacting proteins of SPIN1. The Spin1 cDNA was first cloned into an pSOS vector, and co-transformed with a mouse ovarian cDNA library. Yeast transformation was performed using Yeastmaker Yeast Transformation System 2. Bioinformatics and Statistical Analyses The three-dimensional structure of human SPIN1 was visualized and edited using Molmol. The protein structure was downloaded from Protein Data Bank. Statistical significance was determined using Student’s t-test where appropriate. Calculations of average, standard error of the mean, and statistical significance, were done using Prism 5.03. Results 26507655 Ovarian Folliculogenesis and Oocyte Growth Appear Normal in Spin1 Mutant Ovary To understand the physiological roles of SPIN1, we characterized a mouse genetrap line in which a cassette containing a splice acceptor site was inserted in the intron between exon 3 and 4 of the Spin1 genomic locus. Heterozygous mice containing a Spin1 allele inserted by the genetrap cassette were viable and fertile. When the Spin1 genetrap heterozygotes were intercrossed, only wild type and heterozygous offsprings, but no homozygous mice, were obtained at weaning. Further analysis showed that homozygous genetrap pups are present at E18.5 but exhibit early post-natal death, with homozygous pups dying within 2 days of birth. Characterization of Spin1 genetrap homozygous fetal gonads at E18.5 shows that Spin1 mRNA and proteins are barely detectable in these tissues, indicating that the Spin1 genetrap homozygote is a null allele for Spin1 function . SPIN1 Interacts with Hyaluronan/mRNA-binding Protein Family Members: SERBP1 and HABP4 To understand the molecular functions of SPIN1 in regulating meiotic resumption, we aimed to identify binding partners of SPIN1. We used the CytoTrap yeast two-hybrid system to screen the mouse ovarian cDNA library for proteins that interact with SPIN1. Out of 151 yeast colonies that grew on the selective medium at restrictive temperature after yeast transformation, 26 colonies displayed reproducible growth after two rounds of selection. Sequencing analysis of the recovered cDNA clones revealed 23 clones encoded Serpine1 RNA binding protein, and 3 clones encoded Hyaluronan binding protein 4 . Further bioinformatic analysis of Serbp1 and Habp4 suggested that Serbp1 is expressed in mouse oocytes, and that both genes belong to the hyaluronan/ mRNA-binding protein family. Next, we validated the physical interactions of SPIN1 with SERBP1 and HABP4 by 20065018 performing co-immunoprecipitation Roles of SPIN1 in Mouse Oocytes assay in a heterologous expression system. When we pulled down MYC-tagged SPIN1 in cells co-expressing MYC-SPIN1 and HASERBP1, HA-tagged SERBP1 could be detected by Western blotting. Moreover, there was no HA-