Abnormal expression of Prolyl oligopeptidase (POP) and its catalytic products Ac-SDKP contributes to the ovarian fibrosis change in Polycystic ovary syndrome (PCOS) mice

Abnormal expression of Prolyl oligopeptidase (POP) and its catalytic products Ac-SDKP contributes to the ovarian fibrosis change in Polycystic ovary syndrome (PCOS) mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Abnormal expression of Prolyl oligopeptidase (POP) and its catalytic products Ac-SDKP contributes to the ovarian fibrosis change in Polycystic ovary syndrome (PCOS) mice Suo Han , Shimeng Wang , Xiang Fan , Xiaojie Wang , Yingtong Huang , Hongdan Zhang , Yinyin Ma , Jing Wang , Chunping Zhang doi: https://doi.org/10.1101/2023.01.04.522712 Suo Han 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shimeng Wang 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiang Fan 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiaojie Wang 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yingtong Huang 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hongdan Zhang 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yinyin Ma 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jing Wang 2 Department of Microbiology, College of Medicine, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chunping Zhang 1 Department of Cell Biology, Nanchang University , Nanchang, Jiangxi, People’s Republic of China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: zhangcp81{at}163.com Abstract Full Text Info/History Metrics Preview PDF Abstract Polycystic ovary syndrome (PCOS) is an endocrine disorder and metabolic syndrome. Ovarian fibrosis pathological change in PCOS gradually attracted people’s attention. In this study, we constructed PCOS mice model through dehydroepiandrosterone. Sirius red staining showed that the ovarian tissues in PCOS mice had obvious fibrosis. Prolyl oligopeptidase(POP) is a serine protease and N-acetyl-Seryl-aspartyl-Lysyl-proline (Ac-SDKP) is its catalytic products. Studies show that abnormal expression and activity of POP and Ac-SDKP are closely related to tissue fibrosis. We found that the expression of POP and Ac-SDKP was decreased in ovaries of PCOS mice. Further studies showed that POP and Ac-SDKP promoted the expression of Matrix metalloproteinases 2 (MMP-2) expression and decreased the expression of transforming growth factor beta 1 (TGF-β1) in granulosa cells. Hyperandrogenemia is a typical symptom of PCOS. We found that testosterone induced the low expression of POP and MMP2, and high expression of TGF-β1 in granulosa cells. POP overexpression and Ac-SDKP treatment inhibited the effect of testosterone on TGF-β1 and MMP2 in vitro and ovarian fibrosis in PCOS mice model. In conclusion, PCOS ovarian tissue showed obvious fibrosis. Low expression of POP, Ac-SDKP and changes in fibrotic factors contribute to the ovarian pathological fibrosis induced by androgen. Introduction The ovary is the reproductive organ of female animals. Its main function is to secrete sex hormones and produce mature eggs. The follicle is the basic functional unit of the ovary. According to different stages of development, follicles can be divided into primordial follicles, primary follicles, secondary follicles, antral follicles and pre-ovulatory follicles. After puberty, ovarian follicle development shows periodic changes under the action of the hypothalamic-pituitary-ovarian axis ( 1 ). During the ovarian cycle, follicles grow in an external environment containing extracellular matrix (ECM) such as collagen, laminin and fibronectin( 2 – 5 ). ECM not only provides structural support for developing follicles, but also regulates the growth of granulosa cells and oocytes by combining growth factors and hormones ( 6 , 7 ). During ovulation, luteinizing hormone peak induces oocyte maturation, cumulus cell expansion, follicular rupture and luteal formation. During oocyte maturation, cumulus cells express hyaluronic acid synthase 2 and secrete hyaluronic acid. This mucinous elastic matrix accumulates between cumulus cells and causes the separation and diffusion of cumulus cells, which is a key step for the maturation and ovulation of oocytes. The dysfunction of synthesis of cumulus matrix components can lead to reduced fertility or infertility ( 8 ). So, ECM remodeling plays an important role in ovulation. Therefore, the normal development, maturation and ovulation of follicles depend on the periodic degradation and remodeling of ECM. Ovarian fibrosis, characterized by excessive proliferation of ovarian fibroblasts and ECM deposition, is one of the main causes of ovarian dysfunction ( 9 – 11 ). Studies have shown that ovarian fibrosis is closely related to the occurrence of polycystic ovary syndrome (PCOS) and premature ovarian failure (POF). In the androgen-induced PCOS model, the ovaries showed severe fibrosis ( 12 , 13 ). Studies have found that the expression of MMPs and TIMPs in PCOS is also unbalanced, which leads to abnormal deposition of ECM components such as collagen, resulting in ovulation disorders and polycystic changes in ovaries ( 14 – 16 ). In addition, CTGF and PPARγ, extracellular matrix regulators were abnormally expressed in PCOS patients ( 17 ). These studies suggest that ovarian fibrosis is related to the pathological changes of PCOS. Prolyl oligopeptidase (POP) is a unique enzyme in the serine protease superfamily that is widely expressed in mammalian tissues and organs, such as brain, ovary, liver, and testis ( 18 ). The enzymatic activity of POP is mainly involved in the metabolism of functional polypeptides, such as vasopressin, substance P and thyroid stimulating hormone releasing hormone, through hydrolysis of prolyl (Pro) -XAA peptide bond (X is any amino acid except Pro)( 19 , 20 ). Its function has been extensively studied in the central nervous system. POP is mainly associated with learning and cognition, and abnormal expression and activity of POP are closely associated with Alzheimer’s disease and Parkinson’s disease ( 21 , 22 ). POP also has high activity in the liver. It has been reported that circulating POP activity is significantly decreased in patients of multiple sclerosis (MS) and cirrhotic patients and rat model ( 23 – 25 ).Studies showed that POP attenuated the activation of HSCs through inhibition of TGF-β signaling and induction of PPAR-γ and have anti-fibrosis potential in liver ( 26 ). The decrease of POP also contributes to the development of fibrosis in progressive nephropathy ( 27 ). Studies also showed that POP produces N-acetyl-Seryl-aspartyl-Lysyl-proline (Ac-SDKP) by participating in the cleavage of thymosin β4. Thymosin β4-POP-Ac-SDKP axis has the anti-fibrotic properties in liver and kidney ( 28 , 29 ). These studies suggest that POP and its lysis product Ac-SDKP play an important role in inhibiting fibrosis, and their abnormal expression and activity are closely related to tissue fibrosis. In mammalian ovaries, POP has been found to be expressed in oocytes, granulosa cells, and theca cells ( 18 , 30 , 31 ). During estrus, POP activity has been found to be significantly increased in ovaries ( 32 ). In previous study, we reported that POP is highly expressed in murine luteal cells. POP also regulates progesterone synthesis in luteal cells through the extracellular signal-regulated kinase (ERK) signaling pathway ( 33 ). Considering the role of POP in tissue fibrosis, we detected the expression of POP in ovaries of PCOS mice and found that POP was lowly expressed. In this study, we will explore the possible mechanism of POP on PCOS ovarian fibrosis to provide 4 a new theoretical and experimental basis for understanding the mechanism of PCOS ovarian fibrosis. Materials and Methods Animals Female Kunming mice (21 days) were purchased from the Animal Facility of Nanchang University. The mice were randomly divided into the following two groups: Control group and PCOS group (n = 15).The Control group mice were injected subcutaneously daily with 0.2 ml olive oil. The PCOS group received subcutaneous injection of DHEA (60mg/kg body weight dissolved in 0.2 ml of olive oil) for 21 days. All mice were housed in a temperature and light controlled facility with free access to water and food. Throughout the whole treatment period, the animals were weighed each three day. Estrus cycle was monitored through vaginal smears. The control and PCOS group were sacrificed at the diestrus stage of the estrous cycle. Blood samples were centrifuged at 1,000 g for 10 min and the serum was collected. Concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were assessed by a commercial laboratory (Beijing Sinouk Institute of Biological Technology, Beijing, China). To observe the in vivo effect of POP overexpression on ovarian fibrosis in PCOS model, we injected subcapsularly concentrated retrovirus into the ovaries in PCOS mice. In detail, 20μl concentrated pMIG-POP retrovirus was injected subcapularly into the left ovary with a 10 μl-syringe and 20μl concentrated pMIG retrovirus was injected subcapularly into the right ovary of the same mice. From the day after surgery, the mice received subcutaneous injection of DHEA (60 mg/kg body weight dissolved in 0.2 ml of olive oil) for 21 days. To observe the in vivo effect of Ac-SDKP on ovarian fibrosis in PCOS model, the mice were randomly divided into the two groups: PCOS group and PCOS+ Ac-SDKP group. The mice in PCOS group received 60 mg/kg DHEA dissolved in 0.2 ml of olive oil and 100μl saline for 21 days. The mice in PCOS+ Ac-SDKP group received 60 mg/kg DHEA and 800μg/kg Ac-SDKP for 21 days. The experimental protocols were approved by the ethical committee of Nanchang University. Hematoxylin-eosin (HE) staining The ovaries were isolated and immediately fixed with 4% paraformaldehyde for 24 hours. The tissues were dehydrated and embedded in paraffin. Sections of 5 μm thickness were stained with hematoxylin for 2 minutes and eosin for 10 second. After dehydration and transparency, the sections were sealed with neutral gum. Immunohistochemistry After deparaffinization and rehydration through degraded ethanol, the slides received antigen retrieval in 10 mM sodium citrate buffer for 20 minutes. The sections were inactivated through 3% H2O2 for 10 minutes, incubated with 3%BSA to block nonspecific binding and were incubated with primary Prolyl oligopeptidase antibody (dilution 1: 300, BS60084, BIOWORLD) at 4°C overnight. After washing with phosphate-buffered saline (PBS), the secondary antibody was incubated for 30 min at room temperature, and the color was developed with 3,3_-diaminobenzidine for 2 min. The sections were counterstained with hematoxylin for 30s and sealed with neutral gum. Sirius Red Staining According to Sirius Red Staining Kit instruction, the 5μm sections were deparaffinized, rehydrated, and stained with sirius red staining solution for 1 hour. The sections were re-immersed in alcohol and xylene. The sections were sealed with neutral gum. Plasmids Construction and virus packaging To construct POP overexpression plasmid, the whole sequence of POP was amplified by POP forward primer 5’-CCGCTCGAG ATGCTGTCCTTCCAGTACCC-3’ and POP reverse primer 5’-CCGGAATTC TTACTGGATCCACTCGATGTT-3’, digested with Xhol and EcoRI and cloned into pMig plasmid vector. DNA sequencing was used to confirm successful insertion. To produce retrovirus, 293T cells were seeded in 10cm dishes and transfected with a mixture of DNA containing 10μg of pCL-Eco (IMGENEX), 10μg of pMIG vector, or pMIG-POP expression vectors by lipofectamine 3000 reagent. The media containing retroviruses were harvested 24 hours after transfection, and filtered through a 0.45 μm pore-size filter. To concentrate retrovirus, the filtered media mixed with virus precipitation solution at a volume ratio of 4:1. The mixture was gently inverted and left for 45 minutes at 4 ° C. After centrifuged at 7000xg for 45 minutes at 4°C, the supernatant was completely removed and the white precipitate was resuspended in 200μl PBS. Granulosa cell culture and transfection 21-day-old Kunming female mice were injected intraperitoneally with 5IU of Pregnant Mare Serum Gonadotropin (PMSG) to induce follicle development. After 48 hours, they were sacrificed by cervical dislocation, and the ovaries were removed aseptically and washed three times with PBS. The surrounding fat and connective tissue were removed, and the ovaries were punctured with a 25-gauge injection needle under a stereoscope to release the granulosa cells. Cells were collected by centrifugation at 1500 rpm/min for 5 minutes, resuspended in F12/DMEM medium, and seeded into different culture plates according to experimental needs. For transfection, 2μg plasmids were transfected into granulosa cells using FUGENE-6, when cell confluence reached 70%-80%. After 8 hours, fresh medium was changed. The cells were treated with different reagents for 48 hours and were lysed for RNA and protein extraction after. Quantitative Real-Time PCR Total RNA was extracted using TRIzol reagent following the instruction (Invitrogen, America), and cDNA was synthesized using a reverse transcription kit (Trans, Beijing, China). Quantitative real-time PCR was carried out in a 20μl reaction volume, including 10μl 2X Brilliant SYBR Green qPCR Master Mix, 1μl cDNA, 0.5μl primers and 8.5μl H 2 O. The relative expressions of each gene were determined and normalized to the expression of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated using the 2-ΔΔCT method. Primers were listed in Table 1. Western Blot Total proteins were extracted using RIPA lysis buffer. Western blot was used to detect the expression of POP, MMP2, β-actin and TGF-β1.The total protein was separated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to PVDF membrane. The blot was blocked with 5% skim milk solution for 2 h at room temperature and incubated with primary POP antibody (dilution 1: 1000, BS60084, BIOWORLD), TGF-β1(dilution 1:500, BA2120, BOSTER), MMP2(dilution 1:1000, BA2120, BOSTER) and β-actin (dilution 1:5000, 66009-1-Ig, proteintech) overnight at 4°C. After washed three times with TBST, the blot was incubated with anti-mouse IgG (dilution 1:20000, BS12478, BIOWORLD) or anti-rabbit IgG (dilution 1:20000, BS13278, BIOWORLD) for 1 hour at room temperature. Enhanced Easy See Western Blot Kit was employed to visualize the target bands, and the intensity of bands was quantified by Bio-Rad Image Laboratory software. β-actin was used as an internal reference for detecting relative expression levels. Ac-SDKP measurement Ac-SDKP levels in the ovary were detected with an Enzyme-Linked Immunosorbent Assay (ELISA) kit (MyBiosource, America). The mice were sacrificed by cervical dislocation, and the ovaries were removed aseptically and washed with PBS. The surrounding fat and connective tissue were removed. After weighing, the ovaries were homogenized in PBS (tissue weight (g): PBS (mL) volume=1:9).The homogenates were then centrifuged for 5 minutes at 5000×g to get the supernatant. The Ac-SDKP levels in the sample were determined following the manufacturer’s protocol. Statistical analysis All data were statistically analyzed using GraphPad Prism 7.00. The data were shown in the form of the mean and standard error of the mean (SEM). Statistical comparison between the two groups was performed by independent sample T test. One-way analysis of variance followed by the Student– Newman–Keuls test was used for statistical comparisons among multiple groups. p<0.05 was considered statistically significant. Results PCOS mouse model was successfully constructed We constructed the PCOS mouse model by subcutaneous injection of DHEA for 21 days. HE staining showed that the ovaries of mice in the control group contained various stages of growing follicles and corpus luteum. The follicular cysts in PCOS group were obvious, the granular cell layer of follicle was reduced, and the number of corpus luteum in ovary was reduced compared with the control group ( Fig1D ). The body weight of mice in PCOS group was increased significantly compared with control group ( Fig1A ). Hormone level detection showed that LH and FSH levels in PCOS group were lower than those in control group, while LH/FSH levels was increased( Fig1B ). Estrous cycle showed that mice in the control group had regular estrous cycles, while mice in the PCOS group were stopped in the diestrus phase ( Fig1C ). These results were consistent with the characteristics of PCOS, indicating that the PCOS mouse model was successfully constructed. Download figure Open in new tab Figure 1 Parameters and ovarian morphological change of PCOS model mice (n=15). A shows the body weight change. B shows the representative change of estrous cycle. C shows the serum LH and FSH concentration and LH/FSH value. D show the representative HE staining of the ovary of control and PCOS mice. * indicates p<0.05. ** indicates p<0.01. Scale bar=200 μm Ovarian fibrosis was increased in PCOS group Collagen is the main component of ECM, which can combine with the highly acidic dye Sirius red, so Sirius red staining is often used to detect tissue fibrosis changes( 34 ). The ovarian tissues were stained with Sirius red, and the results showed that the ECM of the PCOS group was significantly thicker than that of the control group ( Fig2A ). TGF-β1 is an important cytokine that promotes organ fibrosis ( 35 ). Matrix metalloproteinases (MMPs)/Tissue inhibitors of metalloproteinases (TIMPs) also play important roles in the process of tissue fibrosis ( 36 ). We detected the expression changes of these fibrosis related factors by real-time quantitative PCR and western blot, and found that the mRNA expression level of MMP-2 in PCOS group were decreased, while the mRNA expression levels of TGF-β1 were increased. The mRNA expression levels of TIMP1 and MMP-9 were not significantly changed compared with the control group ( Fig2B ). Western blotting also confirmed that TGF-β1 protein expression was increased in PCOS group, while MMP-2 protein expression was decreased ( Fig2C ). The results of morphology and expression of fibrosis factors showed that ovarian fibrosis was significantly increased in PCOS mice. Download figure Open in new tab Figure 2 Changes of ovarian fibrosis in PCOS model mice. The changes of tissue fibrosis were detected by Sirius red staining. A shows the representative Sirius red staining of ovary in control and PCOS model mice. Scale bar=200 μm. B shows the mRNA expression of fibrosis related factors, including MMP-2, MMP-9, TIMP1, and TGF-β1. C shows the protein expression of MMP-2 and TGF-β1. The experiment was independently repeated for three times. * indicates p<0.05. POP and Ac-SDKP were decreased in ovaries of PCOS mice It has been reported that POP attenuates the activation of hepatic stellate cells by inhibiting TGF-β1 signal transduction and inducing Peroxisome proliferator-activated rector-gamma (PPAR-γ)( 26 ). Chronic infusion of POP inhibitors increases renal medullary fibrosis ( 37 ).We also reported the expression of POP in mouse ovaries ( 33 ). We detected the expression of POP by real-time PCR and western blot, and found that the expression of POP in the PCOS group was significantly lower than that in the control group ( Fig3A and 3B ). Immunohistochemical staining showed that POP was expressed in ovarian granulosa cells and corpus luteum, and the staining of POP in PCOS group was significantly weaker than that in control group ( Fig3C ). As metabolite of POP, Ac-SDKP inhibit collagen synthesis, participate in the regulation of extracellular matrix deposition and fibrosis in tissues and organs, and has a significant inhibitory effect on the injury and fibrosis of heart, liver, lung, kidney and other organs ( 38 ). We also examined the production of Ac-SDKP in ovaries and found that the concentration of Ac-SDKP in PCOS group was decreased compared with control group, which is consistent with the change of POP expression ( Fig 3D ). Download figure Open in new tab Figure 3 Changes in POP and Ac-SDKP expression in PCOS model mice. A shows the relative mRNA expression of POP in the ovary of control and PCOS mice. B shows the relative protein level of POP in the ovary of control and PCOS mice. C shows the representative immunohistochemical staining of POP in control and PCOS model mice Scale Bar is indicated in the Figure. D shows the concentration of Ac-SDKP in control and PCOS model mice.* indicates p<0.05. POP and Ac-SDKP promoted the expression of MMP-2 expression and decreased the expression of TGF-β1 in granulosa cells To further investigate whether the decreased expression of POP and Ac-SDKP in PCOS model mediates the changes of ovarian fibrosis, we treated primary granulosa cells with S-17092, an inhibitor of POP, and found that S-17092 inhibited the expression of MMP-2 and promoted the expression of TGF-β1. There was no significantly change of MMP9 and TIMP1 ( Fig4A-B ). After overexpression of POP, MMP2 expression was increased, while TGF-β1 expression was decreased. These results suggest that POP regulates the expression of TGF-β1 and MMP2 in ovarian granulosa cells ( Fig4C-D ). After treatment with Ac-SDKP, MMP2 expression was also increased, while TGF-β1 expression was decreased.These results suggest that Ac-SDKP also regulates the expression of TGF-β1 and MMP2 in ovarian granulosa cells ( Fig4E-F ). Download figure Open in new tab Figure 4 The effect of POP and Ac-SDKP on fibrosis related factors in granulosa cells. The granulosa cells were treated with 100μM S-17902(POP inhibitor) or were transfected with POP overexpression plasmid for 48 hours. A shows relative mRNA expression of fibrosis related factors, including MMP-2, MMP-9, TIMP1, and TGF-β1 after treatment with S-17902. B shows the protein expression of MMP-2 and TGF-β1 after treatment with S-17902. C and D show relative mRNA and protein expression of MMP-2 and TGF-β1 after overexpression of POP. The granulosa cells were treated with 100nM Ac-SDKP for 48 hours. E and F show relative mRNA and protein expression of MMP-2 and TGF-β1 after treatment with Ac-SDKP * indicates p<0.05. POP and Ac-SDKP mediated the effect of testosterone on the expression of fibrosis factors in granulosa cells Hyperandrogenemia is a typical symptom of PCOS ( 39 ), and androgen has also been widely used to induce PCOS animal models ( 40 ). We treated primary granulosa cells with testosterone to observe the effect of testosterone on the expression of fibrosis factors and POP in granulosa cells. The results showed that testosterone promoted the expression of TGF-β1 and TIMP1, and inhibited the expression of MMP-2. There was no significantly influence on MMP 9. Protein levels also confirmed that testosterone inhibited MMP-2 expression and promoted TGF-β1 expression. Meanwhile, real-time PCR and western blot assay showed that testosterone inhibited the expression of POP ( Fig5A-B ). We also observe the effect of overexpression of POP and Ac-SDKP on the expression of MMP2 and TGF-β1 induced by testosterone. We found that POP and Ac-SDKP inhibited the effect of testosterone on MMP2 and TGF-β1 ( Fig5C-D ). Download figure Open in new tab Figure 5 The effect of testosterone on the expression of POP and fibrosis related factors in granulosa cells and the role of POP and Ac-SDKP in testosterone induced fibrosis factor change. The granulosa cells were treated with 10μM testosterone for 48 hours. A shows the mRNA expression of MMP-2, MMP-9, TIMP1, TGF-β1 and POP after treatment with testosterone. B shows the protein expression of MMP-2, TGF-β1 and POP. C shows the effect of POP overexpression on the expression of MMP-2 and TGF-β1 induced by testosterone. D shows the effect of Ac-SDKP on the expression of MMP-2 and TGF-β1 induced by testosterone. * indicates p<0.05. Groups with different superscript letters are significantly different (p<0.05). POP overexpression and Ac-SDKP decreased the ovarian fibrosis in PCOS mice To further observe the effect of POP and Ac-SDKP in ovarian fibrosis in PCOS model, we injected POP overexpression retrovirus into the ovaries and found that POP overexpression reversed the pathological changes of ovarian fibrosis ( Fig6A ). Ac-SDKP also rescued the fibrosis phenotype of PCOS ( Fig6B ). Download figure Open in new tab Figure 6 The effect of POP overexpression and Ac-SDKP treatment on ovarian fibrosis in PCOS model. The changes of tissue fibrosis were detected by Sirius red staining. A shows the representative Sirius red staining of ovary after injection of pMIG retrovirus and pMIG-POP retrovirus. B shows the representative Sirius red staining of ovary after treatment with Ac-SDKP in PCOS mice model. Scale bar=200 μm. Discussion PCOS is an endocrine disorder and metabolic syndrome caused by both heredity and environment ( 41 , 42 ). In addition to typical polycystic change, fibrosis, as a pathological change, has gradually attracted people’s attention ( 9 , 12 , 43 ). We constructed the PCOS model through DHEA induction, and Sirius red staining showed that PCOS mice had obvious fibrosis in the ovarian tissues compared with control mice. TGF-β1 is a multidirectional regulatory cytokine, which play important roles in regulating cell growth, differentiation and immune function ( 44 ). TGF-β1 is also an important promoting organ fibrosis cytokine, which can promote the expression of ECM, and inhibit the degradation of ECM. TGF-β1 inhibits the expression and activation of MMPs, up-regulates the expression of protease inhibitors such as TIMPs, and promotes the synthesis of ECM components such as type I,II and IV collagen and fibronectin through autocrine and paracrine pathways( 45 – 47 ). TGF-β1 antibody alleviates injury response and fibrosis by inhibiting TGF-β1 signaling pathway, suggesting that blocking TGF-β1 signaling may be an effective way to prevent and treat fibrosis ( 48 ). In the ovary, TGF-β1 is expressed in granulosa cells, theca cells and oocytes, and play important roles in follicle development through autocrine and paracrine pathways, including regulation of steroid hormone synthesis, ECM remodeling, and COC expansion ( 49 ).Serum TGF-β1 levels were significantly higher in PCOS patients than in control group ( 50 ). TGF-β1 also plays an important role in the deposition of extracellular matrix in chocolate cysts ( 51 , 52 ). In the testosterone induced PCOS model, TGF-β mediates ovarian fibrosis by regulating the expression of fibrosis factors ( 13 , 53 ).We also found that there was significantly increased ovarian TGF-β1 expression in PCOS model. MMPs are a class of highly conserved zinc ion-dependent proteolytic enzymes that play important roles in various protein degradation processes and function in tissue ECM remodeling. It is expressed in a variety of tissues and cells, but the expression level and activity are low. When the tissues are stimulated or in pathological state, the expression of MMPs will be increased and the activity is activated. Activated MMPs can degrade a variety of ECM components including collagen, laminin, and fibronectin. TIMPs are specific inhibitors of MMPs. The dynamic balance of MMPs/TIMPs plays an important role in tissue remodeling and injury repair. Changes in TIMPs/MMPs concentration can also affect the pathogenesis of various diseases, such as PCOS, pregnancy disorders, obesity and metabolic syndrome ( 36 ). Studies have shown that disruption of MMPS/TIMPs balance in PCOS can lead to abnormal degradation of ECM components such as collagen in follicles, resulting in ovulation disorders and polycystic changes in ovary. However, the expression and activity of these enzymes vary greatly in different studies. Gomes et al reported that there were no significant differences in serum level of MMP-2, MMP-8, MMP-9 and TIMP-1 between PCOS patients and healthy volunteers, but the levels of TIMP-2 in PCOS patients was decreased ( 14 ). Lewandowski et al reported that serum MMP-2, MMP-9 and TIMP-1 were increased in PCOS patients, whiie there was no difference in TIMP-2 ( 15 ). Lahav-baratz et al found that MMPs (mainly MMP-1, MMP-2 and MMP-9) activity in follicular fluid of PCOS patients was similar to that of the control group, but the expression of TIMP-1 protein was greatly reduced ( 16 ). Henmi T et al induced PCOS rat model through DHEA and found that MMP-2 expression and activity in the PCOS model group were significantly lower than those in the control group ( 54 ). We constructed DHEA-induced PCOS mouse model, and found that the expression of MMP-2 was decreased significantly, while the expression of MMP-9 and TIMP-1 did not change significantly. Combined with the abnormal expression of TGF-β1 in PCOS model, we inferred that the abnormal expression of TGF-β1 and MMP-2 may be involved in the ovarian fibrosis of PCOS. POP is a serine protease expressed in multiple organs, which promoted the release of the antifibrotic peptide Ac-SDKP from thymosin-β4 (Tβ4). POP and Ac-SDKP play important roles in the process of anti-fibrosis ( 26 , 55 ). Studies showed that Ac-SDKP reversed the hypertension-induced cardiac fibrosis through downregulating TGF-β1( 56 ). We previously reported that POP was highly expressed in granulosa cells, theca cells and luteal cells. POP regulates progesterone synthesis in luteal cells ( 33 ). In this study, we detected the decreased expression of POP and Ac-SDKP in ovary of PCOS mouse model, suggesting that POP and Ac-SDKP may be involved in the ovarian fibrosis process of PCOS. To further confirm the relationship between POP-Ac-SDKP and fibrosis, we treated primary ovarian granulosa cells with POP inhibitor S-17092 and found that S-17092 promoted the expression of TGF-β1 and inhibited the expression of MMP-2. After overexpression of POP, TGF-β1 expression was decreased, while MMP2 expression was increased. Ac-SDKP treatment also decreased the expression of TGF-β1 and promoted the expression of MMP2.These results suggest that low level of POP and Ac-SDKP may mediate the progression of ovarian fibrosis by regulating the expression of TGF-β1 and MMP-2. Hyperandrogenemia is an important clinical feature in patients with PCOS ( 57 ). Hyperandrogen stimulates chronic ovarian inflammation, activates NLRP3 inflammasome, and further induces a series of pathological changes, including ovarian interstitial cell fibrosis ( 43 ). After we treated primary granulosa cells with testosterone, we found that the expression of TGF-β1 was up -regulated, MMP-2 and POP expression were down-regulated. Overexpression of POP and Ac-SDKP treatment inhibited the effect of testosterone on expression of TGF-β1 and MMP2. These results suggest that the decreased expression of POP-Ac-SDKP and disorder of fibrosis factors induced by high testosterone are involved in the pathological process of PCOS. Studies showed that Ac-SDKP has promising value in reducing fibrosis in heart, liver, vessels and kidneys. Our results also showed that POP overexpression and Ac-SDKP reversed the ovarian fibrosis change of PCOS model. In conclusion, we found that PCOS ovarian tissue showed significant fibrosis, and the expression level of POP and Ac-SDKP was reduced in PCOS ovarian tissue. POP and Ac-SDKP may mediate androgen-induced fibrosis by affecting the expression of TGF-β1 and MMP-2. 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Share Abnormal expression of Prolyl oligopeptidase (POP) and its catalytic products Ac-SDKP contributes to the ovarian fibrosis change in Polycystic ovary syndrome (PCOS) mice Suo Han , Shimeng Wang , Xiang Fan , Xiaojie Wang , Yingtong Huang , Hongdan Zhang , Yinyin Ma , Jing Wang , Chunping Zhang bioRxiv 2023.01.04.522712; doi: https://doi.org/10.1101/2023.01.04.522712 Share This Article: Copy Citation Tools Abnormal expression of Prolyl oligopeptidase (POP) and its catalytic products Ac-SDKP contributes to the ovarian fibrosis change in Polycystic ovary syndrome (PCOS) mice Suo Han , Shimeng Wang , Xiang Fan , Xiaojie Wang , Yingtong Huang , Hongdan Zhang , Yinyin Ma , Jing Wang , Chunping Zhang bioRxiv 2023.01.04.522712; doi: https://doi.org/10.1101/2023.01.04.522712 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (8003) Biochemistry (18709) Bioengineering (14841) Bioinformatics (44360) Biophysics (22560) Cancer Biology (19680) Cell Biology (26844) Clinical Trials (138) Developmental Biology (13942) Ecology (20969) Epidemiology (2067) Evolutionary Biology (25412) Genetics (16151) Genomics (23477) Immunology (18678) Microbiology (42413) Molecular Biology (18021) Neuroscience (93343) Paleontology (699) Pathology (2977) Pharmacology and Toxicology (5086) Physiology (8105) Plant Biology (15969) Scientific Communication and Education (2094) Synthetic Biology (4549) Systems Biology (10219) Zoology (2384) window.__CF$cv$params={r:'a3c6ac68d9f95de5',t:'MTc4OTYzMzAzNQ==',u:'01a0ae7105e57719a01a6bac2bae7b77',ut:'DUBD4TIi2BUFMJCwzM1E0yay28RmRskU426oTCrWWZk-1789633037-1.2.1.1-GDq_R7v.OgvelwEOH.ksIEofzzN_rrxaN9hHJG_T3Jb9f7tlf8oBRTmm9QK5iM9B5.i19Ih3ad3ZgpL37dNIPqUHFRmXfYHseQk3LB2HhAw',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();