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 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-tagged SERBP1 detected when the pull-down was done in control cells expressing either MYC-SPIN1 or HA-SERBP1, indicating the binding was specific. We further confirmed the interaction by detecting HAtagged SPIN1 in MYC-tagged SERBP1 pull-downs prepared from cells co-expressing MYC-SERBP1 and HA-SPIN1. Similar observations were made when immunoprecipitation assays were performed in cells co-expressing MYC-SPIN1 and HAHABP4. In summary, we identified and validated the physical interactions of SPIN1 with hyaluronan/mRNA-binding protein family members: SERBP1 and HABP4. SPIN1 and SERBP1 are Co-expressed in Growing and Fully Grown Oocytes Our findings that SPIN1 interacts physically with SERBP1 and HABP4 suggest these proteins are co-expressed in the same tissues or cells. To this end, we examined the expression patterns of Spin1, Serbp1, and Habp4 in Relebactam web ovulated oocytes, pre-implantation embryos, and ovaries. Serbp1 mRNA, similar to Spin1 mRNAs, was detected in oocytes, zygotes, 2- and 8- cell embryos, blastocysts, and ovaries. Habp4 mRNA, however, was co-detected with Spin1 mRNAs only in blastocyst-stage embryos and ovarian tissues. Expression of Spin1 and Serbp1 in ovulated oocytes and early embryos suggests that these genes are maternally expressed, and deposited during oocyte growth. When expression of SPIN1 and SERBP1 17110449 was examined in ovarian follicles, by immunostaining ovarian tissue sections with antibodies recognizing SPIN1 and SERBP1, these two proteins were detected in follicles of pre-antral and antral stages. In these follicles, SERBP1 was detected in 20719936 granulosa cells in addition to oocytes, whereas SPIN1 was predominantly expressed in oocytes. SPIN1 is detectable in the nucleolus of the oocyte in the antral follicle but not in the denuded fully grown oocyte. This could be the result of the differences in sample treatment, since the oocytes in the antral follicle were examined after fixation and cryo-sectioning whereas the fully grown oocytes were fixed and permeabilized for whole-mount staining. Our expression analyses of Spin1, Serbp1 and Habp4 suggested that SPIN1 and SERBP1, but not HABP4, function as a protein complex in oocytes. Thus, in subsequent studies we focused on the SPIN1/ SERBP1 inter