scholarly journals Regulation of ovulatory genes in bovine granulosa cells: lessons from siRNA silencing of PTGS2

Reproduction ◽  
2015 ◽  
Vol 149 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Ketan Shrestha ◽  
Karolina Lukasik ◽  
Anja Baufeld ◽  
Jens Vanselow ◽  
Uzi Moallem ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Reproductive Tract ◽  
Mrna Levels ◽  
Necrosis Factor Alpha ◽  
Lh Surge ◽  
Endometrial Cells ◽  
Promising Tool ◽  
Prostaglandin Endoperoxide Synthase

Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.

201. Effects of relaxin deficiency on matrix metalloproteinase expression in the cervix and vagina of pregnant mice

2005 ◽  
Vol 17 (9) ◽  
pp. 76
Author(s):  
J. T. McGuane ◽  
H. M. Gehring ◽  
L. J. Parry
Keyword(s):  
Reproductive Tract ◽  
Late Gestation ◽  
Mrna Levels ◽  
Biochemical Processes ◽  
Fluorogenic Probes ◽  
Pregnant Mice ◽  
Ecm Degradation ◽  
Stromal Collagen

The major functions of relaxin are associated with female reproductive physiology, especially the regulation of biochemical processes involved in the remodelling of the reproductive tract at term. Studies in relaxin deficient mice (Rlx–/–) demonstrate that although females give birth to live young without apparent dystocia, they have abnormal cervices and vaginae. This phenotype is attributed to an increase in stromal collagen, but the mechanism(s) by which relaxin regulates extracellular matrix (ECM) production in reproductive tissues is poorly understood. In this study, we assessed the expression of matrix metalloproteinases (MMPs) in the cervix and vagina of pregnant wild-type (Rlx+/+) and Rlx–/– mice. Tissues were obtained from adult C57/Blk6J Rlx+/+ mice on days 7.5, 14.5, 17.5, 18.5 pc and Rlx–/– littermates on days 7.5, 14.5 and 18.5 pc. Real-time PCR using dual-labelled fluorogenic probes was performed in an Opticon 2 cycler (MJ Research) to quantify MMP-2, -3, -7, -9 and -13 gene expression. In the cervix and vagina of Rlx+/+ mice, only MMP-2 mRNA levels were significantly higher at term compared with earlier stages of gestation. There were significant decreases in MMP-7 and -13 expression at term, but no change in MMP-3 and -9. In contrast, MMP-3, -7, -9 and -13 mRNA levels were significantly higher in the cervix and vagina of late pregnant Rlx–/– mice. The expression of MMP-2 did not differ between Rlx+/+ and Rlx–/– mice at term. Despite the higher expression of the majority of MMPs we examined in Rlx–/– mice, there was no histological evidence of increased ECM degradation in the cervix and vagina in late gestation. Although previous in vitro studies suggest that relaxin positively regulates MMP activity, our data demonstrate that relaxin deficiency does not result in decreased MMP expression in the mouse cervix and vagina in vivo.

scholarly journals Ovarian Expression of Insulin-Like Peptide 3 (INSL3) and Its Receptor (RXFP2) During Development of Bovine Antral Follicles and Corpora Lutea and Measurement of Circulating INSL3 Levels During Synchronized Estrous Cycles

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1897-1906 ◽  
Author(s):  
Leanne Satchell ◽  
Claire Glister ◽  
Emma C. Bleach ◽  
Richard G. Glencross ◽  
Andrew B. Bicknell ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Major Product ◽  
Corpora Lutea ◽  
Potential Contribution ◽  
Mrna Levels ◽  
Estrous Cycles ◽  
Lh Surge ◽  
Antral Follicles

Abstract Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

scholarly journals HDAC3 maintains oocyte meiosis arrest by repressing amphiregulin expression before the LH surge

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Huarong Wang ◽  
Han Cai ◽  
Xiao Wang ◽  
Meiling Zhang ◽  
Bingying Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Molecular Mechanisms ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Lh Surge ◽  
Oocyte Meiosis ◽  
H3k14 Acetylation

AbstractIt is known that granulosa cells (GCs) mediate gonadotropin-induced oocyte meiosis resumption by releasing EGF-like factors in mammals, however, the detailed molecular mechanisms remain unclear. Here, we demonstrate that luteinizing hormone (LH) surge-induced histone deacetylase 3 (HDAC3) downregulation in GCs is essential for oocyte maturation. Before the LH surge, HDAC3 is highly expressed in GCs. Transcription factors, such as FOXO1, mediate recruitment of HDAC3 to the amphiregulin (Areg) promoter, which suppresses AREG expression. With the LH surge, decreased HDAC3 in GCs enables histone H3K14 acetylation and binding of the SP1 transcription factor to the Areg promoter to initiate AREG transcription and oocyte maturation. Conditional knockout of Hdac3 in granulosa cells in vivo or inhibition of HDAC3 activity in vitro promotes the maturation of oocytes independent of LH. Taking together, HDAC3 in GCs within ovarian follicles acts as a negative regulator of EGF-like growth factor expression before the LH surge.

scholarly journals WNK1 regulates uterine homeostasis and its ability to support pregnancy

2020 ◽  
Author(s):  
Ru-pin Alicia Chi ◽  
Tianyuan Wang ◽  
Chou-Long Huang ◽  
San-pin Wu ◽  
Steven Young ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Embryo Implantation ◽  
Ion Homeostasis ◽  
Female Reproductive Tract ◽  
Female Reproduction ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Akt Phosphorylation

AbstractWNK1 is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans which is associated with abnormal ion homeostasis. Our earlier findings demonstrated that WNK1 is critical for in vitro decidualization in human endometrial stromal cells – pointing towards an unrecognized role of WNK1 in female reproduction. Here, we employed a mouse model with conditional WNK1 ablation from the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyosis and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic and interactomic analyses revealed a novel regulatory pathway whereby WNK1 represses AKT phosphorylation through the phosphatase PP2A in endometrial cells from both humans and mice. We show that WNK1 interacts with PPP2R1A, an isoform of the PP2A scaffold subunit. This interaction stabilizes the PP2A complex, which then dephosphorylates AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the regulation of genes that are crucial for embryo implantation.

scholarly journals Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells

2021 ◽  
pp. 1-10
Author(s):  
Alireza Ghanavatinejad ◽  
Nesa Rashidi ◽  
Mahroo Mirahmadian ◽  
Simin Rezania ◽  
Mahdokht Mosalaei ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Inflammatory Responses ◽  
Female Reproductive Tract ◽  
In Vivo Studies ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Tnf Α ◽  
Tract Infections

<b><i>Objectives:</i></b> Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. <b><i>Design:</i></b> This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. <b><i>Participants/Materials, Setting, and Methods:</i></b> Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. <b><i>Results:</i></b> 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. <b><i>Limitations:</i></b> Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. <b><i>Conclusions:</i></b> 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

scholarly journals Involvement of Chemerin and CMKLR1 in the progesterone decrease by PCOS granulosa cells

Reproduction ◽  
2021 ◽  
Author(s):  
Anthony Estienne ◽  
Namya Mellouk ◽  
Alice Bongrani ◽  
Ingrid Plotton ◽  
Ingrid Langer ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Molecular Mechanisms ◽  
Normal Weight ◽  
Mrna Levels ◽  
Progesterone Secretion ◽  
And Control ◽  
Ovarian Syndrome ◽  
Human Receptor

Polycystic ovarian syndrome (PCOS) is the main cause of infertility in women. It is frequently associated with reduced progesterone production by human luteinised granulosa cells (hlGCs). However, the molecular mechanisms involved in these steroidogenesis alterations in PCOS patients are unclear. In a dihydrotestosterone-induced PCOS mouse model, steroid production is maintained in the setting of chemokine-like receptor 1 (CMKLR1) knockout. Thus, chemerin and chemerin receptors in terms of expression and progesterone regulation could be different in control and PCOS hlGCs. We firstly confirmed that progesterone levels in both plasma (p < 0.0001) and follicular fluid (FF) (p < 0.0001) were significantly reduced in PCOS normal weight women compared to control women. These data were associated with a lower STAR mRNA expression in both in vivo (p < 0.0001) and in vitro (p < 0.0001) hlGCs from PCOS women. Secondly, chemerin FF levels (p < 0.0001) and RARRES2 (p < 0.05) and CMKLR1 (p < 0.0001) mRNA levels in GCs were higher in PCOS normal weight patients. Thirdly, treatment of hlGCs with a specific nanobody (the VHH CA4910) targeting the human receptor for CMKLR1 leading to its inactivation, abolished chemerin-induced progesterone inhibition, suggesting the involvement of CMKLR1 in this process. Furthermore, the inhibition of progesterone secretion induced by chemerin was two-fold higher in PCOS hlGCs (p < 0.05). Moreover, the VHH CA4910 reinstated a normal progesterone secretion with lower concentrations in PCOS hlGCs, suggesting a different chemerin sensitivity between PCOS and control hlGCs. Thus, chemerin, through CMKLR1, could be involved in the steroidogenesis alterations in PCOS hlGCs.

230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation

2005 ◽  
Vol 17 (9) ◽  
pp. 90
Author(s):  
K. R. Dunning ◽  
C. X. Yeo ◽  
D. L. Russell
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
In Vitro Maturation ◽  
Full Length ◽  
Matrix Composition ◽  
In Vitro Fertilisation ◽  
Lh Surge ◽  
Matrix Expansion

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.

Erythropoietin gene expression is suppressed after lipopolysaccharide or interleukin-1 beta injections in rats

1997 ◽  
Vol 273 (3) ◽  
pp. R1067-R1071 ◽  
Author(s):  
S. Frede ◽  
J. Fandrey ◽  
H. Pagel ◽  
T. Hellwig ◽  
W. Jelkmann
Keyword(s):  
Gene Expression ◽  
Inflammatory Diseases ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Mrna Levels ◽  
Necrosis Factor Alpha ◽  
Hypoxia Exposure ◽  
Factor Alpha

Proinflammatory cytokines play an important role in the pathogenesis of anemia in inflammatory diseases. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been reported to inhibit the synthesis of erythropoietin (EPO) in vitro. To evaluate the in vivo significance of this observation, we have investigated effects of the administration of bacterial lipopolysaccharide (LPS) and IL-1 beta on renal EPO production in rats. Measurements by competitive reverse-transcription polymerase chain reaction showed that EPO mRNA levels were significantly reduced in the kidneys of normoxic rats 6 h after the injection of LPS (0.1 or 1 mg/kg). In addition, LPS and IL-1 beta (1 microgram/kg) inhibited the increase in EPO mRNA and plasma EPO levels when administered to rats before hypoxia exposure (8% O2 in the inspiratory gas). Evidence for an inflammatory reaction in the kidneys of LPS-treated rats was provided by measurements of greatly elevated renal TNF-alpha mRNA levels. Furthermore, kidneys isolated from LPS-created rats produced less immunoreactive EPO when perfused hypoxically in vitro for 2 h. Thus mediators of the immune response inhibit renal EPO gene expression in vivo, which is relevant with respect to the impaired synthesis of EPO in inflammatory diseases in humans.

scholarly journals Mechanisms of Adiponectin Action in Fertility: An Overview from Gametogenesis to Gestation in Humans and Animal Models in Normal and Pathological Conditions

2019 ◽  
Vol 20 (7) ◽  
pp. 1526 ◽  
Author(s):  
Alix Barbe ◽  
Alice Bongrani ◽  
Namya Mellouk ◽  
Anthony Estienne ◽  
Patrycja Kurowska ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Polycystic Ovary ◽  
Embryo Implantation ◽  
Female Reproductive Tract ◽  
Trophoblast Invasion ◽  
Endometrial Cells ◽  
Foetal Growth ◽  
Foetal Growth Restriction

Adiponectin is the most abundant plasma adipokine. It mainly derives from white adipose tissue and plays a key role in the control of energy metabolism thanks to its insulin-sensitising, anti-inflammatory, and antiatherogenic properties. In vitro and in vivo evidence shows that adiponectin could also be one of the hormones controlling the interaction between energy balance and fertility in several species, including humans. Indeed, its two receptors—AdipoR1 and AdipoR2—are expressed in hypothalamic–pituitary–gonadal axis and their activation regulates Kiss, GnRH and gonadotropin expression and/or secretion. In male gonads, adiponectin modulates several functions of both somatic and germ cells, such as steroidogenesis, proliferation, apoptosis, and oxidative stress. In females, it controls steroidogenesis of ovarian granulosa and theca cells, oocyte maturation, and embryo development. Adiponectin receptors were also found in placental and endometrial cells, suggesting that this adipokine might play a crucial role in embryo implantation, trophoblast invasion and foetal growth. The aim of this review is to characterise adiponectin expression and its mechanism of action in male and female reproductive tract. Further, since features of metabolic syndrome are associated with some reproductive diseases, such as polycystic ovary syndrome, gestational diabetes mellitus, preeclampsia, endometriosis, foetal growth restriction and ovarian and endometrial cancers, evidence regarding the emerging role of adiponectin in these disorders is also discussed.

Nanocurcumin: A Double-Edged Sword for Microcancers

2020 ◽  
Vol 26 (45) ◽  
pp. 5783-5792
Author(s):  
Kholood Abid Janjua ◽  
Adeeb Shehzad ◽  
Raheem Shahzad ◽  
Salman Ul Islam ◽  
Mazhar Ul Islam
Keyword(s):  
Intracellular Signaling ◽  
Dosage Forms ◽  
The Body ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Anticancer Activities ◽  
Road Map ◽  
Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

Sign in / Sign up

Export Citation Format

Share Document