The Molecular Mechanism Of Isg15 Gene Affecting Mammalian Ovarian Follicle Development And Ovulation

The follicle is the basic structure and functional unit of the mammalian ovary.These follicles from various developmental stages are contemporaneously present within animal ovaries,while few follicles complete development and expel mature oocyte by gonadotropins.Ovarian follicular development and ovulation is a complex physiological process coordinated by several endocrine,paracrine factors and signaling pathways.Studies on the regulation mechanisms of folliculogenesis and ovulation are of important theoretical significance and practical value to better understand the complex reproductive processes in the pig.In previous studies,transcriptome sequencing of the pre-ovulatory ovarian follicles from Taihu（Meishan and Erhualian）and Large White sows revealed interferon-stimulated gene 15（ISG15）was significantly differently expressed.In this study,we constructed Isg15 knockout mice by CRISPR/Cas9 technology,and explored the roles of Isg15 in follicle development and ovulation.The main results are as follows:1.The role of Isg15 gene in follicle development and ovulation（1）Isg15 deficiency improves female fertility in mice.In this study,we generated a Isg15-deleted mouse model by CRISPR/Cas9 system.Cumulative litter size was significantly higher in Isg15^-/-female mice compared with Isg15^+/+mice.And the number of implanted embryos and naturally ovulated oocytes in Isg15^-/-female mice were more than Isg15^+/+female mice.Although Isg15^-/-female mice displayed hyperfertility,Isg15 deletion did not affect their estrous cycle and gonadotropin（FSH,LH）secretion.（2）Knockout of Isg15 promotes follicular development in mice.H&E staining were performed on ovaries tissues:Isg15^-/-ovaries were similar to wildtype ones in histology at 3 weeks of age.But,by 8 weeks of age,Isg15^-/-ovaries contained more corpora lutea（CL）than Isg15^+/+ovaries.（3）Isg15 deletion impacts ovulation in mice.To further confirm whether the hyperfertility in Isg15^-/-females was attributed to high ovulation rate,we observed ovulation in vivo using PMSG/h CG-primed immature mice.H&E staining revealed that the number of antral follicles was similar between Isg15^+/+and Isg15^-/-mice treated with PMSG.There were more pre-ovulatory follicles,ruptured follicles and corpora lutea in Isg15^-/-ovaries than in normal ones treated with PMSG and h CG.Isg15 depletion markedly increased the number of super-ovulated oocytes.The enzyme-linked immunosorbent analysis showed that the concentrations of serum estradiol and progesterone were elevated in Isg15^-/-mice after PMSG/h CG treatment.（4）Isg15 deficiency enhances cumulus expansion of cumulus-oocyte complexes and suppresses apoptosis in murine granulosa cells.The cumulus expansion of cumulus-oocyte complexes was then checked and showed Isg15 deletion promoted cumulus expansion.Moreover,TUNEL,fluorescence-activated cell sorting and Western blot showed that knockdown Isg15 could suppress apoptosis,which facilitated the formation of preovulatory follicles.2.The mechanisms of the Isg15 gene in murine ovarian granulosa cells（1）Isg15 and ISGylation system genes（Uba7,Ube2l6,and Herc6）affect the expressions of follicle development and ovulation-related genes and estradiol synthesis.RT-PCR,Western blot and ELISA results demonstrated that Isg15 and ISGylation system genes（Uba7,Ube2l6,and Herc6）could downregulate the expressions of follicle development and ovulation-related genes,and suppressed estradiol synthesis.While,the deubiquitinating enzyme Usp18 showed opposite results.Subsequently,ISGylation-inactivated mutant Isg15aa was constructed and further showed that Isg15aa overexpression hardly affected the expressions of ovulation-related genes and estradiol synthesis.（2）Immunoprecipitation-mass spectrum analysis identifies potential ISG15interacting proteins in murine granulosa cells.ADAMTS1 was found as a ISG15interacting protein in protein immunoprecipitation and mass spectrometry analysis.Co-immunoprecipitation assays manifested that HERC6 efficiently modified ISGylation of ADAMTS1 as the E3 ligase.In addition,USP18 was able to efficiently reverse the ISG15 modification from ADAMTS1.（3）Lys309,Lys593,Lys597,and Lys602 are the alternative ISGylation sites on ADAMTS1.Loss-of-protein domain assay was carried out to construction of Adamts1 deletion mutants.Protein domain deletion and site-directed mutagenesis showed that Lys309,Lys593,Lys597 and Lys602 are the alternative ISGylation sites on ADAMTS1.（4）ISG15 induces the proteasome-dependent degradation of ADAMTS1.Western blot revealed that Isg15 overexpression caused a sharp increase ISGylation of ADAMTS1but a marked decrease of ADAMTS1 protein.Exogenous MG132（proteasome inhibitor）addition could rescue the degradation of ADAMTS1 protein.Moreover,purified ISGylated ADAMTS1 was incubated with different proteasomes in vitro and the ISGylation of ADAMTS1 almost disappeared when incubated with 20S proteasome.In summary,Isg15 participates in ovarian development and ovulation.Isg15knockout enhances ovulation and causes hyperfertility in mice.Moreover,immunoprecipitation-mass spectrum analyses reveal that ISG15 could bind to ADAMTS1via ISGylation-conjugating system（UBA7,UBE2L6,and HERC6）and degrade ADAMTS1 protein via a 20S proteasome dependent pathway,thereby inhibiting the expressions of ovulation-related genes and estradiol synthesis.Taken together,ISG15 and ISGylation play an indispensable role in follicle development and ovulation,which provides a new strategy for improving female fertility in mammals.