| The follicular development and oogenesis play an irreplaceable role in the reproductive success,and are the determinant of litter size in the pig breeding industry.Folliculogenesis and oocyte maturation are affected by a number of intra-and extra-ovarian factors.Alternative splicing is one of the most post-transcriptional regulations to generate a large number of mRNA and protein isoforms from the single gene,thus increasing complexity of gene expression and promoting protein diversity.Alternative splicing plays an important role in animal physiological processes,such as follicular development and oogenesis.Our previous study showed GGT1 gene was differentially expressed between the Chinese Meishan pigs（MS）and Large White（LW）ovaries,and was enriched in follicular development pathway.The alternative splicing event analysis of the sequencing data and two splice variants of GGT1 gene were screened out:full-length variant（GGT1-01）and exon 11 skipped variant（GGT1-02）.The functions of GGT1 two splice variants in porcine ovarian granulosa cells were explored,and we used CRISPR/cas9 gene editing technology to construct a GGT1（GGT1-02variant in pigs）-deficient mice.The main results are as follows:1.The study of GGT1 gene alternative splicing on porcine ovarian granulosa cell growth and prostaglandin synthesis（1）Differentially expressed GGT1 gene splice variants were detected by MISO software（|ΔPSI|≥10%）.The different splice variants were verified.GGT1-01 splice variant was highly expressed in MS ovaries.EdU,MTT,flow cytometry and Western blotting analysis showed that GGT1-01 splice variant promoted ovarian granulosa cell proliferation and inhibited ovarian granulosa cell apoptosis,but GGT1-01 has no role in cell growth.（2）GGT1-02 interacted with TMCO1 to upregulate Ca2+levels,activate the activity of cPLA2,and control PGE2 synthesis via the AA-PTGS2-PGE2 pathway;but GGT1-01has no role in PGE2 synthesis.（3）qRT-PCR and Western blot results demonstrated that the splicing factor SRSF1regulated GGT1 alternative splicing via its second RRM domain and RS domain,thus up-regulating the expression of GGT1-02 splice variant.By constructing the GGT1-minigene eukaryotic expression vectors with different point mutation and co-transfecting with pc-SRSF1,it was found that hn RNPH1 was essential for SRSF1 to regulate GGT1 alternative splicing.（4）MTT,flow cytometry and Western blotting analysis showed that SRSF1promoted porcine ovarian granulosa cell proliferation and inhibited porcine ovarian granulosa cell apoptosis.ELISA experiments showed that SRSF1 could promote PGE2synthesis in porcine ovarian granulosa cell.Plasmid’s co-transfection experiments showed that SRSF1 mediated the alternative splicing of GGT1 gene,thereby regulating porcine ovarian granulosa cell proliferation,apoptosis and promoting PGE2 synthesis in porcine ovarian granulosa cells.2.The function of Ggt1 gene on follicular development and ovulation was studied using gene knockout mice（1）In this study,we generated a Ggt1-deleted mouse model by CRISPR/Cas9 system showed a range of phenotypes similar to PCOS in women including anovulation,elevated testosterone and LH/FSH levels,and sterility.This model could be used as a mammalian model to study the molecular regulation underlying the anovulation and sterility in PCOS patients.The 3-week-old Ggt1-/-mice ovaries contained similar numbers of primary,secondary and antral follicles but more atretic follicles compared with WT mice.However,the ovaries of 5-week-old Ggt1-/-mice were deficient in follicles beyond the secondary stage and contained fewer antral follicles than the control ovaries.Furthermore,there were essentially no antral follicles and more atretic follicles in Ggt1-/-mice at 8 weeks of age.（2）EdU,TUNEL and Western blotting results showed that there were no significant changes in the apoptosis and cell proliferation in 3-week-old Ggt1-/-and WT ovaries.Compared with the WT ovaries,the increased number of TUNEL-positive GCs,the increased level of BAX protein,and the decreased level of BCL-2 protein were noticed in8-week-old Ggt1-/-ovaries（P<0.01）.Besides,fewer EdU-positive GCs and lower PCNA protein levels present in Ggt1-/-mice indicated a reduced GC proliferation（P<0.05）.（3）Transmission electron microscopy was performed on ovaries from 3-week-old mice and 8-week-old mice.Most mitochondria in WT mice ovaries contained visible inner membrane,outer membrane,and well-aligned cristae.The ovarian mitochondria of3-week-old Ggt1-/-mice contained similar numbers and structures as those of WT mice.In striking contrast,ultra-structural aberrations especially including the cristae loss and vacuole formation,were frequently observed in mitochondria of 8-week-old Ggt1-/-ovaries（P<0.05）.TheΔψin oocytes was remarkably reduced when Ggt1 was deleted,and fluorescent dye JC-1 shifted from green to red with increasingψ,indicating the compromised mitochondrial activity（P<0.01）.In addition,the ROS levels were increased in Ggt1-/-oocytes（P<0.01）.Consistent with this,the ATP content in Ggt1-/-oocytes was lowered compared with control oocytes（P<0.01）.We found that the GV oocytes derived from Ggt1-/-mice were unable to resume meiosis,and that the GVBD rate was dramatically decreased in Ggt1-/-mice（P<0.01）.The first polar body（Pb1）extrusion in WT oocytes took place at 8-10 h after GVBD,and Pb1 extrusion in Ggt1-/-oocytes occurred at 11 h after GVBD.And the Pb1 extrusion rate was significantly lower in Ggt1-/-oocytes than WT mice（P<0.01）.（4）We supplemented the cysteine intake of Ggt1-/-female mice by dissolving NAC in the drinking water（10 mg/mL）from 3 to 8 weeks old.The growth of the Ggt1-/-female mice supplemented with NAC partially recovered.In addition,we found no significant difference in ROS and ATP levels between Ggt1-/-female mice and wild type when NAC were supplemented.As expected,the recovery of the mitochondrial function through NAC supplementation was sufficient to protect the granulosa cells from apoptosis and to promote granulosa cell proliferation.Moreover,the Ggt1-/-female mice fed with NAC and wild-type mice fed with NAC had a minor difference in the GVBD rate and in the first pole body exclusion rate.In summary,Ggt1 is required for ovarian follicular development,ovulation,and female fertility.Ggt1-deletion generates mitochondrial dysfunction and ROS accumulation,leading to PCOS-like phenotypes in female mice;NAC can alleviate mitochondrial dysfunction and oocyte developmental defect caused by Ggt1 deficiency in female mice;GGT1 interacts with TMCO1 to activate the cPLA2 and accelerate PGE2synthesis through the AA-PTGS2-PGE2 pathway in granulosa cells,and promote follicular development.Our data demonstrate that NAC restores the PCOS-like phenotypes in Ggt1-/-female mice,and extend the understanding of the potential therapeutic schedule associated with PCOS in human patients. |
