t in the Spin1 mutant ovary, similar to ovarian follicles of wild type controls. We then punctured mutant and wild type ovaries to harvest fully grown oocytes, and immunostained the oocytes with antibodies recognizing SPIN1 and b-tubulin. SPIN1 was only detected in wild type control oocytes, but not in Spin1 mutant oocytes. The microtubules displayed interphase-like distribution and were indistinguishable between mutant and wild type control oocytes. Taken together, we show that Spin1 genetrap homozygous mice are deficient in Spin1 mRNA and proteins, but follicular development and oocyte growth are unaffected by the absence of SPIN1. Spin1 Mutant FGOs are Defective in Resuming Meiosis Fully grown oocytes arrested at prophase I, resume meiosis when released from ovarian follicles. To examine whether SPIN1 plays a role in meiotic resumption, we first harvested oocytes containing intact GV from Spin1 mutant and wild 19053768 type control ovarian follicles in medium consisting of IBMX, a phosphodiesterase inhibitor that prevents spontaneous GV breakdown. The denuded oocytes were then released into IBMX-free medium, and the ability of oocytes to resume meiosis was scored by GVBD. Upon release into IBMX-free medium, control oocytes underwent GV breakdown efficiently, in 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. Scopoletin biological activity Calculations of average, standard error of the mean, and statistical significance, were done using Prism 5.03. Results 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 15701837 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