Depletion of GPSM1 enhances ovarian granulosa cell apoptosis via cAMP-PKA-CREB pathway in vitro.
ABSTRACT: BACKGROUND:Genetic causes of premature ovarian insufficiency (POI) account for approximately 20?~?25% of patients. So far, only a few genes have been identified. RESULTS:Here, we first identified the c.1840C?>?A on G-protein signaling modulator 1 (GPSM1) as a susceptibility locus for POI in 10 sporadic POI patients by whole-exome sequencing. The frequency of GPSM1 c.1840C?>?A was then verified as 3/20 in a POI sample of 20 patients (including the above 10 patients) by Sanger sequencing. RT-PCR and western blot analysis showed the expression of GPSM1 in rat ovaries was increased in the large antral follicle stage compared to the primordial follicle stage (P??A of POI for the first time. Gpsm1 was related to rat follicle development, and silencing of Gpsm1 increased apoptosis and decreased proliferation in rat GCs, possibly through inhibition of the cAMP-PKA-CREB pathway.
Project description:Retinoic acid (RA) is a metabolite of vitamin A and has important roles in development, differentiation, and reproduction. Activin has been shown to regulate the RA pathway and affect granulosa cell (GC) proliferation, suggesting that RA is important for early follicle development. However, little is known about the effects of RA on GC functions, particularly steroidogenesis, during the early follicle stage. The aim of this study was to investigate the effects of all-trans-RA (atRA) on progesterone production in immature rat GCs cultured without gonadotropin. Our results demonstrated that atRA enhanced progesterone production by upregulating the levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450scc (Cyp11a1) mRNAs, but not 3?-hydroxysteroid dehydrogenase mRNA in immature rat GCs. Additionally, analysis of the mechanisms through which atRA upregulated StAR and Cyp11a1 mRNAs revealed that atRA enhanced intracellular cAMP accumulation and phosphorylation of cAMP response-element binding protein (CREB). In addition, H-89, an inhibitor of protein kinase A (PKA), abolished the stimulatory effects of atRA, indicating that atRA enhanced progesterone synthesis through cAMP/PKA signaling. In conclusion, our data demonstrated that atRA has a crucial role in progesterone synthesis in rat GCs during the early follicle stage.
Project description:Chemotherapy treatment in women can frequently cause damage to the ovaries, which may lead to primary ovarian insufficiency (POI). In this study, we assessed the preventative effects of hyaluronic acid (HA) in immunosuppressive drug-induced POI-like rat models and investigated the possible mechanisms. We found that HA, which was reduced in primary and immunosuppressant-induced POI patients, could protect the immunosuppressant-induced damage to granulosa cells (GCs) in vitro. Then we found that HA blocked the tripterygium glycosides (TG) induced POI-like presentations in rats, including delayed or irregular estrous cycles, reduced 17 beta-estradiol(E2) concentration, decreased number of follicles, destruction of follicle structure, and damage of reproductive ability. Furthermore, we investigated the mechanisms of HA prevention effects on POI, which was associated with promotion of GC proliferation and PGRMC1 expression. In conclusion, HA prevents chemotherapy-induced ovarian damage by promoting PGRMC1 in GCs. This study may provide a new strategy for prevention and treatment of POI.
Project description:Dysfunction of granulosa cells (GCs) leading to follicle atresia has been extensively studied as a major cause of premature ovarian insufficiency (POI), but the regulatory role of long non-coding RNAs (lncRNAs) in this process is still poorly understood. Here, we show that the lncRNA <i>LINC02690</i> or <i>GCAT1</i> (granulosa cell-associated transcript 1) is downregulated in GCs from patients with biochemical POI (bPOI), and we show a significant correlation between downregulated <i>GCAT1</i> and serum levels of follicle-stimulating hormone and anti-Müllerian hormone. Downregulation of <i>GCAT1</i> inhibited G1/S cell cycle progression and thus inhibited the proliferation of GCs. Mechanistically, we show that <i>GCAT1</i> competes with cyclin-dependent kinase inhibitor 1B (<i>CDKN1B</i>) mRNA for polypyrimidine tract-binding protein 1 (PTBP1) binding, and thus decreased <i>GCAT1</i> might promote PTBP1 binding to <i>CDKN1B</i> mRNA and thereby initiate CDKN1B protein (p27) translation. Together, our results suggest that downregulation of <i>GCAT1</i> under conditions of bPOI inhibits the proliferation of GCs through PTBP1-dependent p27 regulation, thus suggesting a novel form of lncRNA-mediated epigenetic regulation of GC function that contributes to the pathogenesis of POI.
Project description:Patients with primary ovarian insufficiency (POI) often have a high prevalence of autoimmune disorders. To identify antigenic molecules associated with ovarian autoimmunity, we performed immunoprecipitation (IP) screening using serum from patients with POI and the established human granulosa cell line (HGrC1). POTE ankyrin domain family member E (POTEE) and POTE ankyrin domain family member F (POTEF), proteins specific to primates, were identified as candidate antigens. Using immunohistochemistry (IHC) with human ovarian tissue, POTEE or POTEF was weakly seen in the granulosa cells (GCs) of primordial follicles and primary follicles, and strongly in large antral follicles and luteal cells. Interestingly, no signals were detected in growing GCs in secondary, preantral, and small antral follicles. Thus, to explore the function of POTEE and POTEF in human folliculogenesis, we established HGrC1 cell lines with drug-inducible expression of POTEF. Expression of POTEF significantly suppressed cell proliferation in HGrC1 cells. Furthermore, chaperonin containing TCP-1 complex (CCT) components, which affect folding proteins required for cell proliferation, was bound to the actin domain of POTEF protein. Although CCT is normally localized only around the Golgi apparatus, TCP-1α, a component of CCT, co-migrated closer to the cell membrane when POTEF expression was induced. These data suggest that the interaction between POTEF and CCT components impairs the usual function of CCT during cell growth. In addition, over-accumulation of POTEF in HGrC1 cells leads to autophagic failure. It was recently reported that knockout of an autophagic gene in mice leads to a phenotype similar to human POI. These results suggested that a proper amount of POTEF is required for the maintenance of GCs in follicle pools, whereas POTEF overaccumulation might be involved in follicle atresia and the development of POI. We also showed the possibility that POTEF could be an antigen involved in ovarian autoimmunity.
Project description:<h4>Background</h4>Premature ovarian insufficiency (POI) is one of the major causes of infertility. We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) effectively improved ovarian function in a murine model of POI. Recent studies indicated that mesenchymal stem cell-derived exosomes were important components in tissue repair. In this study, we investigated the therapeutic effects of MenSCs-derived exosomes (MenSCs-Exos) in a rat model of POI and its mechanism in restoring ovulation.<h4>Methods</h4>Ovaries of 4.5-day-old Sprague Dawley rats (SD rats) were cultured in vitro to evaluate the effects of MenSCs-Exos exposure on early follicle development. Furthermore, POI in rats was induced by intraperitoneal administration of 4-vinylcyclohexene diepoxide (VCD). Forty-eight POI rats were randomly assigned to four groups, each receiving a different treatment: PBS, MenSCs, MenSCs-Exos, and Exo-free culture supernatant of MenSCs. Estrous cyclicity, ovarian morphology, follicle dynamics, serum hormones, pregnancy outcomes, and molecular changes were investigated.<h4>Results</h4>Exposure to MenSCs-Exos promoted the proliferation of granulosa cells in primordial and primary follicles in vitro and increased the expression of early follicle markers Deleted In Azoospermia Like (DAZL) and Forkhead Box L2 (FOXL2) while inhibiting follicle apoptosis. In vivo, MenSCs-Exos transplantation effectively promoted follicle development in the rat model of POI and restored the estrous cyclicity and serum sex hormone levels, followed by improving the live birth outcome. In addition, transplantation of MenSCs-Exos regulated the composition of the ovarian extracellular matrix and accelerated the recruitment of dormant follicles in the ovarian cortex and increased proliferation of granulosa cells in these follicles.<h4>Conclusion</h4>MenSCs-Exos markedly promoted follicle development in vitro and in vivo and restored fertility in POI rats, suggesting a restorative effect on ovarian functions. The therapeutic effect of MenSCs-Exos transplantation was sustainable, consistent with that of MenSCs transplantation. Our results suggested that MenSCs-Exos transplantation may be a promising cell-free bioresource in the treatment of POI.
Project description:<h4>Background</h4>Emerging drug-free in vitro activation (IVA) technique enables patients with premature ovarian insufficiency (POI) to restore ovarian function and conceive their own genetic offspring. However, various issues have greatly restricted its clinical application. Transplantation of adipose-derived stem cells (ADSCs) has promising roles in restoring ovarian function of rats with POI, but insufficient retention has greatly hampered their efficiency. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and ADSCs, which may prolong the retention of ADSCs and construct an early vascular microenvironment, thus compensating for the disadvantages of drug-free IVA to some extent and ameliorating impaired ovarian function in the POI rats.<h4>Methods</h4>After intraperitoneal injection of cyclophosphamide, the POI model rats were randomized into 5 groups: (1) POI group; (2) ovarian fragments group; (3) 3D scaffold combined with ovarian fragments group; (4) ovarian fragments combined with ADSCs group; (5) 3D scaffold with ADSCs combined with ovarian fragments as 3D-bioprinted engineering ovary group. Normal rats were identified as the control group. The localization of CM-Dil-labeled ADSCs and co-localization with CD31 were observed to examine the distribution and underlying mechanism of differentiation. Histomorphological and immunohistochemical analyses were performed to calculate follicle number and assess proliferation and apoptosis of granulosa cells (GCs). Immunofluorescence staining was used to evaluate angiogenesis. Hormone levels were measured to evaluate the restoration of endocrine axis. Western blot analysis and RT-PCR were conducted to explore the potential mechanism.<h4>Results</h4>CM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Several regions of the co-staining for CM-Dil and CD31 were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation, neoangiogenesis, and hormone levels were significantly improved in the 3D-bioprinted engineering ovary group, as compared with other groups. Furthermore, the ovarian function was ameliorated and angiogenesis was promoted through regulating the PI3K/AKT pathway.<h4>Conclusion</h4>Our results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of rats with POI, which could compensate for the disadvantages of drug-free IVA to some extent.
Project description:Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both ?-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3?-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.
Project description:BACKGROUND:With the development of regenerative medicine and tissue engineering technology, almost all stem cell therapy is efficacious for the treatment of premature ovarian failure (POF) or premature ovarian insufficiency (POI) animal models, whereas little stem cell therapy has been practiced in clinical settings. The underlying molecular mechanism and safety of stem cell treatment in POI are not fully understood. In this study, we explored whether fetal mesenchymal stem cells (fMSCs) from the liver restore ovarian function and whether melatonin membrane receptor 1 (MT1) acts as a regulator for treating POI disease. METHODS:We designed an in vivo model (chemotherapy-induced ovary damage) and an in vitro model (human ovarian granulosa cells (hGCs)) to understand the efficacy and molecular cues of fMSC treatment of POI. Follicle development was observed by H&E staining. The concentration of sex hormones in serum (E2, AMH, and FSH) and the concentration of oxidative and antioxidative metabolites and the enzymes MDA, SOD, CAT, LDH, GR, and GPx were measured by ELISA. Flow cytometry (FACS) was employed to detect the percentages of ROS and proliferation rates. mRNA and protein expression of antiapoptotic genes (SURVIVIN and BCL2), apoptotic genes (CASPASE-3 and CASPASE-9), and MT1 and its downstream genes (JNK1, PCNA, AMPK) were tested by qPCR and western blotting. MT1 siRNA and related antagonists were used to assess the mechanism. RESULTS:fMSC treatment prevented cyclophosphamide (CTX)-induced follicle loss and recovered sex hormone levels. Additionally, fMSCs significantly decreased oxidative damage, increased oxidative protection, improved antiapoptotic effects, and inhibited apoptotic genes in vivo and in vitro. Furthermore, fMSCs also upregulated MT1, JNK1, PCNA, and AMPK at the mRNA and protein levels. With MT1 knockdown or antagonist treatment in normal hGCs, the protein expression of JNK1, PCNA, and AMPK and the percentage of proliferation were impaired. CONCLUSIONS:fMSCs might play a crucial role in mediating follicular development in the POI mouse model and stimulating the activity of POI hGCs by targeting MT1.
Project description:<h4>Background</h4>Granulosa cells (GCs) are multilayered somatic cells within the follicle that provide physical support and microenvironment for the developing oocyte. In recent years, the role of Neuregulin-1 (NRG1), a member of the EGF-like factor family, has received considerable attention due to its neurodevelopmental and cardiac function. However, the exact physiological role of NRG1 in GC is mainly unknown. In order to confirm that NRG1 plays a regulatory role in rat GC functions, endogenous NRG1-knockdown studies were carried out in GCs using RNA interference methodology.<h4>Results</h4>Knockdown of NRG1 in GCs resulted in the enhanced expression and secretion of the cytokines and chemokines. In addition, the phosphorylation of PI3K/Akt/ERK1/2 was significantly low in GCs under these experimental conditions. Moreover, in vitro experimental studies suggest that tumor necrosis factor-α (TNFα) treatment causes the physical destruction of GCs by activating caspase-3/7 activity. In contrast, exogenous NRG1 co-treatment of GCs delayed the onset of TNFα-induced apoptosis and inhibited the activation of caspase-3/7 activity. Furthermore, current experimental studies suggest that gonadotropins promote differential expression of NRG1 and ErbB3 receptors in GCs of the antral follicle. Interestingly, NRG1 and ErbB3 were intensely co-localized in the mural and cumulus GCs and cumulus-oocyte complex of pre-ovulatory follicles in the estrus stage.<h4>Conclusions</h4>The present studies suggest that gonadotropins-dependent NRG1-signaling in GCs may require the balance of the cytokines and chemokines expression and secretion, ultimately which may be supporting the follicular maturation and oocyte competence for ovulation and preventing follicular atresia.
Project description:Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development.