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Human granulosa-luteal cells initiate an innate immune response to pathogen-associated molecules. Ibrahim LA et al. (2016) The microenvironment of the ovarian follicle is key to the developmental success of the oocyte. Minor changes within the follicular microenvironment can significantly disrupt oocyte development, compromising the formation of competent embryos and reducing fertility. Previously described as a sterile environment, the ovarian follicle of women has been shown to contain colonizing bacterial strains, whereas in domestic species, pathogen-associated molecules are concentrated in the follicular fluid of animals with uterine infection. The aim of this study is to determine whether human granulosa-luteal cells mount an innate immune response to pathogen-associated molecules, potentially disrupting the microenvironment of the ovarian follicle. Human granulosa-luteal cells were collected from patients undergoing assisted reproduction. Cells were cultured in the presence of pathogen-associated molecules (LPS, FSL-1 and Pam3CSK4) for 24h. Supernatants and total RNA were collected for assessment by PCR and ELISA. Granulosa-luteal cells were shown to express the molecular machinery required to respond to a range of pathogen-associated molecules. Expression of TLR4 varied up to 15-fold between individual patients. Granulosa-luteal cells increased the expression of the inflammatory mediators IL1B, IL6 and CXCL8 in the presence of the TLR4 agonist E. coli LPS. Similarly, the TLR2/6 ligand, FSL-1, increased the expression of IL6 and CXCL8. Although no detectable changes in CYP19A1 or STAR expression were observed in granulosa-luteal cells following challenge, a significant reduction in progesterone secretion was measured after treatment with FSL-1. These findings demonstrate the ability of human granulosa-luteal cells to respond to pathogen-associated molecules and generate an innate immune response.//////////////////
Conjugated Linoleic Acids Attenuate LPS-Induced Pro-Inflammatory Gene Expression by Inhibiting the NF-?B Translocation Through PPAR? in Buffalo Granulosa Cells. Yenuganti VR 2014 et al.
PROBLEM
In granulosa cells, TLR4-mediated LPS-induced immune response interferes with ovarian granulosa cell function.
METHOD OF STUDY
LPS-induced pro-inflammatory gene expression was monitored by real-time PCR and NF-?B was determined by the immuno-blotting and immuno-staining in granulosa cells in vitro.
RESULTS
The LPS (1g/mL) increased pro-inflammatory gene expression which was reverted back by treatment with TLR4 signaling inhibitor. Cotreatment of CLA (10m) with LPS did not show any effect on LPS-induced pro-inflammatory gene expression but granulosa cells pre-treated with CLA for 24hr, attenuated LPS-induced pro-inflammatory gene expression and nuclear NF-?B. GW9662, a PPAR?-inhibitor, further increased the expression of pro-inflammatory genes.
CONCLUSION
The present findings reiterated that pre-treatment with CLA can prevent LPS-induced granulosa cells dysfunction.
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Pathogen-associated molecular patterns initiate inflammation and perturb the endocrine function of bovine granulosa cells from ovarian dominant follicles via TLR2 and TLR4 pathways. Price JC 2013 et al.
Bacterial infections of the uterus or mammary gland commonly cause disease and infertility by perturbing growth and steroidogenesis of the dominant follicle in the ovary of cattle. Cells of the innate immune system use Toll-like receptors TLR2, TLR4 and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) expressed by bacteria, leading to activation of MAPK and NF?B pathways, and production of inflammatory cytokines such as IL-1?and IL-6, and the chemokine IL-8. The present study tested whether granulosa cells from dominant follicles have functional TLR2, TLR4 and TLR5 pathways. Supernatants of primary bovine granulosa cells accumulated IL-1? IL-6 and IL-8 when treated for 24 h with PAM that binds TLR2 or LPS that binds TLR4, but not flagellin that binds TLR5. Granulosa cell responses to PAM or LPS were rapid, with increased phosphorylation of p38 and ERK1/2 within 30 min and increased abundance of IL6, IL1B, IL10, TNF, IL8 and CCL5 mRNA after 3 h treatment. Accumulation of IL-6 in response to PAM and LPS was attenuated using siRNA targeting TLR2 and TLR4, respectively. Furthermore, treating granulosa cells with inhibitors targeting MAPK or NF?B reduced the accumulation of IL-6 in response to LPS or PAM. Treatment with LPS or PAM reduced the accumulation of estradiol and progesterone, and the PAMPs reduced granulosa cell expression of CYP19A1 mRNA and protein. In conclusion, bacterial PAMPs initiate inflammation and perturb the endocrine function of bovine granulosa cells from dominant follicles via TLR2 and TLR4 pathways.Pr?s: A rapid inflammatory response to pathogen-associated molecular patterns mediated by TLR2 and TLR4 pathways in granulosa cells provides a molecular explanation of how bacterial infections distant to the ovary can perturb dominant follicle function.
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Lipopolysaccharide Reduces the Primordial Follicle Pool in the Bovine Ovarian Cortex Ex Vivo and in the Murine Ovary In Vivo. Bromfield JJ et al. Infections of the uterus or mammary gland with Gram-negative bacteria cause infertility in cattle, not only during disease but also for some time afterward. Even though these infections are in organs distant from the ovary, metritis and mastitis perturb antral follicle development and function in vivo. Although granulosa cells from antral follicles express Toll-like receptor 4 (TLR4), and detect and mount an inflammatory response to lipopolysaccharide (LPS) from Gram-negative bacteria, it is not known whether LPS impacts preantral follicle development. The present study tested the hypothesis that LPS perturbs the development of primordial ovarian follicles. Exposure of bovine ovarian cortex ex vivo to LPS reduced the primordial follicle pool, associated with increased primordial follicle activation. Ovarian cortex culture supernatants accumulated the inflammatory mediators IL-1beta, IL-6 and IL-8 in a LPS concentration-dependent manner. In addition, LPS exposure modulated key intracellular regulators of follicle activation, with loss of primordial follicle PTEN and cytoplasmic translocation of FOXO3. Acute exposure of mice in vivo to LPS also reduced the primordial follicle pool, associated with increased follicle atresia. The increased follicle atresia was TLR4 dependent as Tlr4-deficient mice were insensitive to LPS-mediated follicle atresia. However, LPS did not affect the diameter of individually cultured bovine secondary follicles or their enclosed oocytes. In conclusion, LPS reduced the primordial ovarian follicle pool in the bovine ovarian cortex ex vivo and in the murine ovary in vivo. These observations provide an insight into how bacterial infections distant from the ovary have long term effects on fertility.
Induced expression of pattern recognition receptors (PRRs) in cumulus oocyte complexes (COCs): Novel evidence for innate immune-like functions during ovulation. Shimada M et al. Ovulation is the complex, inflammatory-like process by which the cumulus oocyte complex (COC) is released from a mature, preovulatory (PO) follicle through a rupture site at the ovarian surface and requires expression of genes that generate and stabilize the expanded extracellular COC matrix. Gene profiling analyses of COCs at selected time intervals during ovulation revealed that many genes associated with immune related surveillance functions were also induced in cumulus cells. Specifically, cell surface signaling molecules known as pattern recognition receptors (PRRs) that act as sensors of the external environment important for the innate immune system to detect 'self' from 'non-self' or 'altered self' are induced and/or expressed in cumulus cells as well as granulosa cells. These include the complement factor C1q, CD14 and the Toll-like receptors (TLRs) 4, 8 and 9 as well as mediators of TLR activation, MYD88 and IRF3. COCs exposed to bacterial LPS exhibit enhanced phosphorylation of p38MAPK, ERK1/2 and NF-kB and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and SCARBI and exhibited phagocytic uptake of fluorescently-tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process.
Ovarian follicular cells have innate immune capabilities that modulate their endocrine function. Herath S et al. Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of androstenedione from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-4 (TLR4), CD14, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR4 complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell androstenedione production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR4, CD14 and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.
Granulosa cell subtypes respond by autophagy or cell death to oxLDL-dependent activation of the oxidized lipoprotein receptor 1 and toll-like 4 receptor. Serke H et al. Autophagic cell death has been observed in granulosa cell cultures via the oxLDL-dependent activation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1). This activation might differ for cytokeratin-positive (CK(+)) and CK(-) granulosa cells. In particular, LOX-1 and toll-like receptor 4 (TLR4), one of the pattern recognition receptors of innate immunity, might be diversely regulated. Granulosa cell subtype cultures were established from the follicle harvests of patients undergoing in vitro fertilization (IVF) therapy. In response to oxLDL treatment, the fibroblast-like CK(-) cells upregulated LOX-1 and exhibited reparative autophagy, which could be blocked with anti-LOX-1 antibody. The epithelioid-like CK(+) cells did not regulate LOX-1 expression upon oxLDL application, but the expression of TLR4 and CD14 increased between 0 and 36 h of oxLDL/nDL treatment. This upregulation was associated with non-apoptotic cell death based on the absence of cleaved caspase-3. Reactive oxygen species (ROS) increased with 12 h oxLDL application and steroidogenic acute regulatory (StAR) protein expression was negligible. In CK(-) cells, the inhibition of TLR4 downregulated LOX-1 and induced apoptosis. We concluded that CK(-) granulosa cells are protected against oxLDL-dependent apoptosis by TLR4, whereas, in CK(+) cells, oxLDL-induced TLR4 activation triggers nonapoptotic cell death. The CK(+) cells might represent immune-like granulosa cells involved in ovarian remodeling processes.
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Distinct expression patterns of TLR transcripts in human oocytes and granulosa cells from primordial and primary follicles. Ernst EH et al. (2020) Ovulation has long been regarded as a process resembling an inflammatory response. Previously, luteinizing hormone (LH) was shown to induce Toll-like receptor 2 (TLR2) and TLR4 in granulosa cells from preovulatory hormone-dependent follicles. However, whether this could already initiate before the hormone-dependent phase is currently unknown. The aim of this study was to investigate TLR genes in human oocytes and granulosa cells from primordial and primary ovarian follicles during the hormone-independent phase. A class-comparison study of existing oocyte and granulosa cell RNA sequencing transcriptomes from primordial (n = 539 follicles) and primary (n = 261) follicles collected from three patients was examined. This revealed a distinct expression pattern of TLR3, TLR4 and TLR5 transcripts. Interestingly, the TLR3 protein was differentially detected in both the oocyte and the granulosa cells in primordial and primary follicles, suggesting that TLR3 is maternally contributed both as mRNA and protein. Intracellularly, the compartmentalized TLR3 dot-like staining in the intersection between the oocyte and the surrounding primordial granulosa cells. The TLR4 protein was detected in both primordial and primary follicles, with a notable staining in the granulosa cells. We functionally challenged ovaries in vitro, by polyinosinic:polycytidylic acid (poly I:C) and LPS, known to activate TLR3 and TLR4, respectively, and found a tendency for increased IL-6 production, which was particular evident in the LPS-treated group. Based on the expression of TLRs, it is notably that human primordial and primary follicles express genes that would allow them to respond to innate immune proteins and cytokines during follicle activation.////////////////// Lipopolysaccharide Initiates Inflammation in Bovine Granulosa Cells via the TLR4 Pathway and Perturbs Oocyte Meiotic Progression in Vitro. Bromfield JJ et al. Infections of the reproductive tract or mammary gland with Gram-negative bacteria perturb ovarian function, follicular growth, and fecundity in cattle. We hypothesized that lipopolysaccharide (LPS) from Gram-negative bacteria stimulates an inflammatory response by ovarian granulosa cells that is mediated by Toll-like receptor (TLR) 4. The present study tested the capacity of bovine ovarian granulosa cells to initiate an inflammatory response to pathogen-associated molecular patterns and determined subsequent effects on the in vitro maturation of oocytes. Granulosa cells elicited an inflammatory response to pathogen-associated molecular patterns (LPS, lipoteichoic acid, peptidoglycan, or Pam3CSK4) with accumulation of the cytokine IL-6, and the chemokine IL-8, in a time- and dose-dependent manner. Granulosa cells responded acutely to LPS with rapid phosphorylation of TLR signaling components, p38 and ERK, and increased expression of IL6 and IL8 mRNA, although nuclear translocation of p65 was not evident. Targeting TLR4 with small interfering RNA attenuated granulosa cell accumulation of IL-6 in response to LPS. Endocrine function of granulosa cells is regulated by FSH, but here, FSH also enhanced responsiveness to LPS, increasing IL-6 and IL-8 accumulation. Furthermore, LPS stimulated IL-6 secretion and expansion by cumulus-oocyte complexes and increased rates of meiotic arrest and germinal vesicle breakdown failure. In conclusion, bovine granulosa cells initiate an innate immune response to LPS via the TLR4 pathway, leading to inflammation and to perturbation of meiotic competence.
Changes in the Expression of Toll-Like Receptor mRNAs During Follicular Growth and in Response to Lipopolysaccharide in the Ovarian Follicles of Laying Hens. Subedi K et al. The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1 beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increases with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.
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