Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome.

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.

Inflammatory Markers in Uterine Lavage Fluids of Pregnant, Non-Pregnant, and Intrauterine Device Implanted Mares on Days 10 and 15 Post Ovulation.

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) (only EXP 1), prostaglandin F2α (PGF2α), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF2α was lower (p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15.

Leptin Signaling in the Ovary of Diet-Induced Obese Mice Regulates Activation of NOD-Like Receptor Protein 3 Inflammasome.

Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficient ob/ob (+/? and -/-) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1ß were upregulated in 16 L and downregulated in ob/ob. Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation in ob/ob-/-. We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.

Luteolysis and the Auto-, Paracrine Role of Cytokines From Tumor Necrosis Factor α and Transforming Growth Factor ß Superfamilies.

Successful pregnancy establishment demands optimal luteal function in mammals. Nonetheless, regression of the corpus luteum (CL) is absolutely necessary for normal female cyclicity. This dichotomy relies on intricate molecular signals and rapidly activated biological responses, such as angiogenesis, extracellular matrix (ECM) remodeling, or programmed cell death. The CL establishment and growth after ovulation depend not only on the luteinizing hormone-mediated endocrine signal but also on a number of auto-, paracrine interactions promoted by cytokines and growth factors like fibroblast growth factor 2, vascular endothelial growth factor A, and tumor necrosis factor α (TNF), which coordinate vascularigenesis and ECM reorganization as well as steroidogenesis. With the organ fully developed, the release of the uterine prostaglandin F2α activates luteolysis, an intricate process supported by intraluteal interactions that ensure the loss of steroidogenic function (functional luteolysis) and the involution of the organ (structural luteolysis). This chapter provides an overview of the local action of cytokines during luteal function, with particular emphasis on the role of TNF and transforming growth factor ß superfamilies during luteolysis.

Understanding the Inguinal Sinus in Sheep (Ovis aries)-Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors.

Post-parturient behavior of mammalian females is essential for early parent-offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Interleukins affect equine endometrial cell function: modulatory action of ovarian steroids.

The aim of the present study was to investigate the interaction between ovarian steroids, interleukins and prostaglandins (PG) in equine epithelial and stromal cells in vitro. In Experiment 1, cells were exposed to IL-1α (10 ng/mL), IL-1ß (10 ng/mL) or IL-6 (10 ng/mL) for 24 h and cell proliferation was determined using MTT. In Experiment 2, cells were exposed to progesterone (P4; 10(-7) M); 17-ß estradiol (E2; 10(-9) M) or P4+E2 for 24 h and later medium was replaced with a fresh one treated with IL-1α, IL-1ß or IL-6 (10 ng/mL, each) for 24 h. The oxytocin (OT; 10(-7) M) was used as a positive control. In Experiment 3, cells were exposed to P4 (10(-7) M), E2 (10(-9) M) or P4+E2 for 24 h and the IL receptor mRNAs transcription was determined using Real-time PCR. Prostaglandins concentration was determined using the direct enzyme immunoassay (EIA) method. Our findings reveal a functional linking between ovarian steroids and IL-stimulated PG secretion by equine endometrial cells. This interaction could be one of the mechanisms responsible for endometrial local orchestrating events during the estrous cycle and early pregnancy.

Ovarian steroid-dependent tumor necrosis factor-α production and its action on the equine endometrium in vitro.

Tumor necrosis factor-α (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2-5 of the estrous cycle; n=20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 10(-7)M), 17-ß estradiol (E2; 10(-9)M) or P4+E2 (10(-7)/10(-9)M) for 24h. Then, TNF mRNA transcription was determined using Real-time PCR. Additionally, TNF protein production was investigated in response to ovarian steroids for 24h using Enzyme-Linked Immunosorbent Spot (EliSpot). In Experiment 2, epithelial and stromal cells and endometrial explants (mid-luteal phase of the estrous cycle; n=5) were exposed in vitro to TNF (10 ng/ml) and to oxytocin (OT; positive control; 10(-7)M) for 24h. The concentrations of PGE2 and PGF2α were determined using a direct enzyme immunoassay (EIA) method. The transcription of prostaglandin-endoperoxide synthase-2 (PTGS-2), prostaglandin E2 synthase (PGES) and PGF2α synthase (PGFS) mRNAs in the endometrial explants was determined using Real-time PCR. Results showed that TNF is produced by two types of equine endometrial cells and its production is up-regulated by ovarian steroids (P<0.05) in stromal cells and by P4 (P<0.05) and E2 (P<0.01) in epithelial cells. Epithelial and stromal cells can also produce PG in response to TNF. In endometrial explants, TNF stimulated PGE2 production to a large extent and PGF2α secretion to a lesser extent. These actions are mediated by up-regulation of PG synthases mRNA transcription. The study indicates that TNF production is closely related to ovarian steroid actions and that the interaction between TNF and PG regulates physiologic processes in the equine endometrium.

Ovarian steroids affect prostaglandin production in equine endometrial cells in vitro.

This study aimed to evaluate the influence of ovarian steroids on equine endometrial epithelial and stromal cells, specifically i) prostaglandin (PG) production in a time-dependent manner, ii) specific PG synthases mRNA transcription and protein expression, and iii) cell proliferation. After passage I, cells were exposed to vehicle, oxytocin (OT, positive control, 10(-7) M), progesterone (P4, 10(-7) M), 17ß estradiol (E2, 10(-9) M), or P4+E2 for 12, 24, 48, or 72 h. Following treatment, PG concentration was determined using the direct enzyme immunoassay (EIA) method. Alterations in PG synthases mRNA transcriptions, PG synthases protein expression, and cell proliferation in response to the treatments were determined after 24 h using real-time PCR, western blot, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide respectively. After 24 h, E2 and P4+E2 increased PGE2 and PGF2α secretion as well as specific prostaglandin-endoperoxide synthase-2 (PTGS2), PGE2 synthases (PGES), and PGF2α synthases (PGFS) expression in the epithelial cells (P<0.05). Additionally, E2 and P4+E2 increased PTGS2 expression in stromal cells after 24 h (P<0.05). In stromal cells, P4+E2 increased PGE2 production as well as PGES expression after 24 h (P<0.05). Both E2 and P4+E2 increased PGF2α production by stromal cells after 24 h (P<0.05). Ovarian steroids affected proliferation of stromal and epithelial cells during the 24-h incubation period (P<0.05). We provide evidence that ovarian steroids affect PG production in equine endometrial cells, upregulating PTGS2, PGES, and PGFS expression. Ovarian steroid-stimulated PG production could be an important mechanism occurring in the equine endometrium that is involved in the regulation of the estrous cycle and early pregnancy.

Impairment of the interleukin system in equine endometrium during the course of endometrosis.

The aim of the study was to characterize endometrial mRNA transcription, immunolocalization, and protein expression of interleukin (IL) 1alpha, IL1beta, IL6, and IL1RI, IL1RII, and IL6Ralpha/beta in the course of endometrosis during the estrous cycle. Additionally, the influence of IL1alpha, IL1beta, and IL6 on prostaglandin (PG) secretion and PG synthase mRNA transcription in endometrial tissue during endometrosis was investigated. The endometrial samples were obtained at the early (n = 12), mid- (n = 12), and late (n = 12) luteal phases and at the follicular (n = 12) phase of the estrous cycle. Within each of these phases, there were four samples within each category I, II, and III of endometrium, according to the Kenney classification. In experiment 1, transcription of IL1alpha, IL1beta, IL6, and their receptor's (IL1RI, IL1RII, and IL6Ralpha/beta) mRNAs and their immunolocalization and protein expression were determined using real-time PCR and immunohistochemistry, respectively. In Experiment 2, endometrial samples (n = 5 samples within categories I, II, and III) were obtained for tissue culture in the midluteal phase of the estrous cycle. The endometrial tissues were stimulated with IL1alpha (10 ng/ml), IL1beta (10 ng/ml), IL6 (10 ng/ml), and oxytocin (positive control; 10⁻7 M) for 24 h. The PG concentration was determined using ELISA. In addition, transcription of PTGS-2, PGES, and PGFS mRNAs was determined using real-time PCR. ILs were found to regulate PG secretion via modulation of PG synthases in equine endometrium. The alterations in IL and the expression of their receptors, and in endometrial secretory functions, were observed during the course of endometrosis, and suggest serious changes in the endometrial microenvironment. The described disturbances may be closely related to impaired endometrial processes responsible for the subfertility or the infertility in endometrosis.

Cytokines and angiogenesis in the corpus luteum.

In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.

Is FAS/Fas ligand system involved in equine corpus luteum functional regression?

Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic and luteolytic factors, cell viability, and apoptosis. FASL and FAS protein expression and mRNA transcription were evaluated in different luteal stages of the equine CL by Western blotting and real-time PCR assays, respectively. Protein expression and FASL mRNA transcription increased in the late CL. Also, FAS and FASL proteins were present in large steroidogenic and endothelial CL cells throughout the luteal phase, as demonstrated by immunohistochemistry. Equine luteal cells isolated from midluteal phase CL were stimulated without (control) or with exogenous cytokines: FASL (10 ng/ml); TNF+IFNG (10 ng/ml each; positive control) or FASL+TNF+IFNG (10 ng/ml each). FASL clearly inhibited in vitro progesterone and prostaglandin E(2) (PGE(2)) production by equine luteal cells but increased prostaglandin F(2alpha) (PGF(2alpha)). Furthermore, FASL effect on equine luteal cell viability depended on the presence of cytokines TNF and IFNG. In conclusion, this study shows the presence of FASL and FAS in the equine CL and suggests their importance in functional luteolysis.