N‐Acetyl‐l‐cysteine restores reproductive defects caused by Ggt1 deletion in mice

Dear Editor, Polycystic ovary syndrome (PCOS) is an autoimmune disease that is characterized by follicular growth arrest and chronic anovulation and linked with female infertility in 5%–20% of reproductive-aged women.1 γ-Glutamyltranspeptidase (GGT1), which is one of the membrane-associated enzymes in mammalian cells, may lead to an altered glucose metabolism and abnormal lipid profile that are main clinical phenotypes of PCOS.2 Here, we discovered that Ggt1-deficient female mice had similar phenotypes to those of women with PCOS. The reproductive defects in Ggt1-null mice were restored by N-acetyl-l-cysteine (NAC), which is an antioxidant commonly used in the treatment of PCOS patients.3 Ggt1−/− mice generated by CRISPR/Cas9 system (Figure 1A,B) showed smaller body size and ovaries (Figure 1C,D). Ggt1−/− females were anovulatory and infertile with elevated testosterone and LH/FSH levels and decreased prostaglandin (PGE2) levels, which are similar to PCOS in women (Figure 1E–M). Moreover, Ggt1−/− mice showed abnormal follicular development with no antral follicles and more atretic follicles, and insensitivity to exogenous gonadotropins stimulation (Figure 1N–W). Granulosa cells (GCs) provide the microenvironment required for developing oocytes and follicles, and ovarian granulosa cell apoptosis is the main cause of follicular atresia.4 There were no significant changes in apoptosis and cell proliferation between 3-week-old Ggt1+/+ and Ggt1−/− ovaries (Figure S1A,B). ButGCswithmore apoptosis and less proliferation were found in 8-week-oldGgt1−/− ovaries compared with the Ggt1+/+ mice (Figure 2A–E). We further checked the mitochondrial structure in Ggt1−/− ovaries by transmission electron microscopy. There were similar numbers and structures of the ovarian mitochondria in 3-week-old Ggt1−/− mice and Ggt1+/+ mice (Figure S1C–G). Interestingly, ultra-structural aberrations, especially vacuole formation and cristae loss were frequently observed inmitochondria of 8-week-oldGgt1−/−

ovaries ( Figure 2F-J). In addition, reactive oxygen species (ROS) level was increased, whereas mitochondrial membrane potential, ATP content, and mtDNA levels were remarkably reduced in Ggt1 −/− oocytes ( Figure 2K-P). Furthermore, Germinal vesicle (GV) oocytes derived from 3-week-old Ggt1 −/− mice were unable to resume meiosis ( Figure S1H). GV breakdown (GVBD) rate and the first polar-body extrusion rate were dramatically decreased in Ggt1 −/− oocytes compared with Ggt1 +/+ oocytes ( Next, we used pig as an animal model to further explore the role of GGT1 in folliculogenesis. Two independent cDNA libraries from pre-ovulatory ovarian follicles in Large White (LW) and Meishan (MS) sows were sequenced with the high-throughput Illumina Solexa system ( Figure  S2A,B). KEGG pathway analysis showed that the differentially expressed genes (DEGs) were enriched in multiple biological processes including metabolic activity, arachidonic acid metabolism, and steroid biosynthesis ( Figure  S2C,D). GGT1 was involved in multiple biological processes such as arachidonic acid (AA) metabolism (Figure S2C). Porcine GGT1 pre-mRNA was subjected to alternative splicing analysis, generating a full-length isoform (GGT1-01) and an exon11 skipped isoform (GGT1-02) (Figure 3A and Figure S2E,F). GGT1-02 which is equivalent to Ggt1 in mice was highly expressed in MS ovaries ( Figure  S2G) and conserved in mammals ( Figure S2H,I).
GGT1-02 knockdown significantly increased porcine ovarian granulosa cell (pGC) apoptosis and inhibited cell proliferation ( Figure 3B-G). In GGT1-02 repressed pGCs, ROS level was increased, whereas ATP content, PGE2 synthesis, and the expression of follicular developmentrelated genes were decreased ( Figure 3H-N  GGT consists of one large subunit and one small subunit. 5 Only when the large subunit and the small one form a complex, the GGT is highly active. 6 The two subunits of GGT1-02 were shown to directly interact with each other, but there was an unknown structure between the large and small subunits of GGT1-01 ( Figure S2E). ELISA results showed that GGT1-02 had a higher GGT activity than GGT1-01 ( Figure 4A and Figure S4A,B). We found that GGT1-02 regulated the Ca 2+ stores, and promoted AA synthesis through activating the cPLA2 activity in pGCs ( Figure 4B-K and Figure S4C-H). TMCO1 acts as a Ca 2+ load-activated calcium channel to release Ca 2+ when there is an overload of ER Ca 2+ , thereby maintaining calcium homeostasis. 7 The Co-IP assay results verified that GGT1-02 interacted with TMCO1, and the 949-1155 bp domain in GGT1-02 was indispensable for PGE2 synthesis ( Figure 4L-R). In addition, murine GGT1 interacted with TMCO1 to regulate PGE2 synthesis via the cPLA2-AA-PTGS2 pathway ( Figure S5A-F).
In this study, we also explored the mechanism underlying porcine GGT1 splicing. Our results demonstrated that both RNA recognition motif 2 (RRM2) and arginine/serine-rich (RS) domains of SRSF1 were necessary for GGT1 splicing ( Figure S6A-H). RNAbinding-protein-immunoprecipitation results indicated that SRSF1 is directly bound to the GGT1 pre-mRNA through the RRM domain ( Figure S6I). The RS domain of serine/arginine-rich (SR) protein is involved in protein-protein interaction. 8 Here, analysis on pc-SRSF1 co-transfected with GGT1-minigene carrying deletions of regulatory elements showed that GGT1-minigene-G4, which had a binding site of hnRNPH1, was a critical element for the SRSF1-mediated GGT1 splicing. SRSF1 interacted with hnRNPH1 and then promoted the production of the GGT1-02 transcript ( Figure S6J-S). Meanwhile, SRSF1 mediated GGT1 splicing to regulate porcine ovarian granulosa cell growth and PGE2 synthesis ( Figure S7A-L).
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 PGE2 synthesis through the AA-PTGS2-PGE2 pathway in granulosa cells and promote fol-licular development ( Figure S8). 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.

A C K N O W L E D G M E N T S
We thank the anonymous reviewers for critical reading and discussions of the manuscript. We greatly thank Prof. Chunyan Mou (Huazhong Agricultural University) for her efforts in revising this manuscript. This work was supported financially by Hubei Science and Technology Major Projects (2020ABA016), Key Research and Development Project of Hubei Province (2020BBB069), National Key R&D Program of China, Hubei Agricultural Science and Technology Innovation Action Project (2018skjcx05), and the Fundamental Research Funds for the Central Universities (2662019PY017).

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest.

E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Huazhong Agricultural University.