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The question of whether behavioral traits are heritable is under debate. An obstacle in demonstrating transgenerational inheritance in mammals originates from the maternal environment's effect on offspring phenotype. Here, we used in ovo embryonic heat conditioning (EHC) of first-generation chicks, demonstrating heredity of both heat and immunological resilience, confirmed by a reduced fibril response in their untreated offspring to either heat or LPS challenge. Concordantly, transcriptome analysis confirmed that EHC induces changes in gene expression in the anterior preoptic hypothalamus (APH) that contribute to these phenotypes in the offspring. To study the association between epigenetic mechanisms and trait heritability, DNA-methylation patterns in the APH of offspring of control versus EHC fathers were evaluated. Genome-wide analysis revealed thousands of differentially methylated sites (DMSs), which were highly enriched in enhancers and CCCTC-binding factor (CTCF) sites. Overlap analysis revealed 110 differentially expressed genes that were associated with altered methylation, predominantly on enhancers. Gene-ontology analysis shows pathways associated with immune response, chaperone-mediated protein folding, and stress response. For the proof of concept, we focused on HSP25 and SOCS3, modulators of heat and immune responses, respectively. Chromosome conformational capture (3C) assay identified interactions between their promoters and methylated enhancers, with the strongest frequency on CTCF binding sites. Furthermore, gene expression corresponded with the differential methylation patterns, and presented increased CTCF binding in both hyper- and hypomethylated DMSs. Collectively, we demonstrate that EHC induces transgenerational thermal and immunological resilience traits. We propose that one of the mechanisms underlying inheritance depends on three-dimensional (3D) chromatin reorganization.

A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks’ IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

Stressful environmental events in early life have long-lasting consequences on later stress responses. We previously showed that heat conditioning of 3-day-old chicks during the critical period of heat-response development leads to heat vulnerability later in life. Here we assessed the role of early-life heat stress on the inflammatory response in the chick anterior hypothalamus (AH), focusing on hypothalamic microglia. We identified the microglial cell population in the chick AH using anti-KUL01 and anti-CD45 antibodies. Specific microglial features were also confirmed by expression of their signature genes. Under normal environmental conditions, hypothalamic microglia isolated from lipopolysaccharide (LPS)-injected chicks displayed a classical activated proinflammatory profile accompanied by a decreased homeostatic signature, demonstrating similarity of immune response with mammalian microglial cells. In accordance with our previous observations, conditioning of 3-day-old chicks under high ambient temperature decreased the number of newborn cells in the AH, among them microglial precursors. Although heat exposure did not affect microglial cell viability, it had a long-term impact on LPS-induced inflammatory response. Exposure to harsh heat led to heat vulnerability, and attenuated recruitment of peripheral monocytes and T cells into the AH following LPS challenge. Moreover, heat conditioning altered microglial reactivity, manifested as suppressed microglial activation in response to LPS. Innate immune memory developed by heat conditioning might underlie suppression of the microglial response to LPS challenge. We describe alterations in genome-wide CpG methylation profile of hypothalamic microglia, demonstrating probable epigenetic involvement in the reprogramming of microglial function, leading to heat-induced inflammatory cross-tolerance.

Resilience to stress can be obtained by adjusting the stress-response set point during postnatal sensory development. Recent studies have implemented epigenetic mechanisms to play leading roles in improving resilience. We previously found that better resilience to heat stress in chicks can be achieved by conditioning them to moderate heat stress during their critical developmental period of thermal control establishment, 3 days posthatch. Furthermore, the expression level of corticotropin-releasing hormone (CRH) was found to play a direct role in determining future resilience or vulnerability to heat stress by alterations in its DNA-methylation and demethylation pattern. Here we demonstrate how intraperitoneal injection of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) influences the DNA methylation pattern, thereby affecting the long-term heat-stress response. Single PARPi administration, induced a reduction in both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), without affecting body temperature. The accumulated effect of three PARPi doses brought about a long-term decrease in 5mC% and 5hmC%. These changes coincided with a reduction in body temperature in non-conditioned chicks, similar to that occurring in moderately conditioned heat-stress-resilient chicks. The observed changes in DNA methylation can be explained by decreased activity of the enzyme DNA methyltransferase as a result of the PARPi injection. Furthermore, evaluation of the DNA-methylation pattern along the CRH intron showed a reduction in 5mC% as a result of PARPi treatment, alongside a reduction in CRH mRNA expression. Thus, PARPi treatment can affect DNA methylation, which can alter hypothalamic–pituitary–adrenal (HPA) axis anchors such as CRH, thereby potentially enhancing long-term resilience to heat stress.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

Early life encounters with stress can lead to long-lasting beneficial alterations in the response to various stressors, known as cross-tolerance. Embryonic heat conditioning (EHC) of chicks was previously shown to mediate resilience to heat stress later in life. Here we demonstrate that EHC can induce cross-tolerance with the immune system, attenuating hypothalamic inflammation. Inflammation in EHC chicks was manifested, following lipopolysaccharide (LPS) challenge on day 10 post-hatch, by reduced febrile response and reduced expression of LITAF and NFκB compared to controls, as well as nuclear localization and activation of NFκB in the hypothalamus. Since the cross-tolerance effect was long-lasting, we assumed that epigenetic mechanisms are involved. We focused on the role of ten-eleven translocation (TET) family enzymes, which are the mediators of active CpG demethylation. Here, TET transcription during early life stress was found to be necessary for stress resilience later in life. The expression of the TET family enzymes in the midbrain during conditioning increased in parallel to an elevation in concentration of their cofactor α-ketoglutarate. In-ovo inhibition of TET activity during EHC, by the α-ketoglutarate inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), resulted in reduced total and locus specific CpG demethylation in 10-day-old chicks and reversed both thermal and inflammatory resilience. In addition, EHC attenuated the elevation in expression of the stress markers HSP70, CRHR1, and CRHR2, during heat challenge on day 10 post-hatch. This reduction in expression was reversed by BPTES. Similarly, the EHC-dependent reduction of inflammatory gene expression during LPS challenge was eliminated in BPTES-treated chicks. Thus, TET family enzymes and CpG demethylation are essential for the embryonic induction of stress cross-tolerance in the hypothalamus.

Visual Abstract, Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 (EZH2) and brain-derived neurotrophic factor (BDNF), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2. Antisense “knock-down” of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions −77 and − 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX’s ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.

Stressful events in early life might lead to stress resilience or vulnerability, depending on an adjustable stress-response set-point, which can be altered during postnatal sensory development and involves epigenetic regulation of corticotropin-releasing hormone (CRH). During the critical developmental period of thermal-control establishment in 3-day-old chicks, heat stress was found to affect both body temperature and expression of CRH in the hypothalamic paraventricular nucleus. Both increased during heat challenge in vulnerable chicks, whereas they decreased in resilient chicks. Our aim was to elucidate the epigenetic mechanism underlying the regulation of stress resilience or vulnerability. Accordingly, DNA CpG methylation (5mC) and hydroxymethylation (5hmC) at the CRH intron, which we found to serve as a repressor element, displayed low 5mc% alongside high 5hmc% in resilient chicks, and high 5mc% with low 5hmc% in vulnerable ones. RE1-silencing transcription factor (REST), which has a binding site on this intron, bound abundantly during acute heat stress and was nearly absent during moderate stress, restricting repression by the repressor element, and thus activating CRH gene transcription. Furthermore, REST assembled into a protein complex with TET3, which bound directly to the CRH gene. Finally, the adjacent histone recruited the histone acetylation enzyme GCN5 to this complex, which increased H3K27ac during harsh, but not moderate heat conditioning. We conclude that an epigenetic mechanism involving both post-translational histone modification and DNA methylation in a regulatory segment of CRH is involved in determining a resilient or vulnerable response to stress later in life.

// Moshe Reichenstein 1 , Gat Rauner 1, 2 , Shenhav Kfir 1, 2 , Tatiana Kisliouk 1 , Itamar Barash 1 1 Institute of Animal Science, ARO, The Volcani Center, Bet-Dagan, Israel 2 The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Correspondence to: Itamar Barash, email: itamar.barash@mail.huji.ac.il Keywords: STAT5, H2AX, mammary gland, methylation, paracrine Received: March 15, 2016 Accepted: May 20, 2016 Published: May 30, 2016 ABSTRACT Deregulated STAT5 activity in the mammary gland causes parity-dependent tumorigenesis. Epithelial cell cultures transfected with constitutively active STAT5 express higher levels of the histone H2AX than their non-transfected counterparts. Higher H2AX expression may be involved in tumorigenesis. Here, we aimed to link high STAT5 activity to H2AX–GFP expression by looking for distinct types of mammary cells that express these proteins. In vitro and in transgenic mice, only 0.2 and 0.02%, respectively, of the cells expressed the H2AX–GFP hybrid gene. Its expression correlated with that of the endogenous H2AX gene, suggesting that detectable H2AX–GFP expression marks high levels of H2AX transcript. Methylation of the H2AX promoter characterized non-GFP-expressing H2AX–GFP cells and was inversely correlated with promoter activity. Administration of 5-azacytidine increased H2AX promoter activity in an activated STAT5-dependent manner. In transgenic mice, H2AX–GFP expression peaked at pregnancy. The number of H2AX–GFP-expressing cells and GFP expression decreased in a Stat5a-null background and increased in mice expressing the hyperactivated STAT5. Importantly, H2AX–GFP activity was allocated to basal mammary cells lacking stem-cell properties, whereas STAT5 hyperactivity was detected in the adjacent luminal cells. Knockdown of RANKL by siRNA suggested its involvement in signaling between the two layers. These results suggest paracrine activation of H2AX via promoter demethylation in specific populations of basal mammary cells that is induced by a signal from neighboring luminal cells with hyper STAT5 activity. This pathway provides an alternative route for the luminally confined STAT5 to affect basal mammary cell activity.

Stressful events in early life might lead to stress resilience or vulnerability, depending on an adjustable stress-response set-point, which can be altered during postnatal sensory development and involves epigenetic regulation of corticotropin-releasing hormone (CRH). During the critical developmental period of thermal-control establishment in 3-day-old chicks, heat stress was found to affect both body temperature and expression of CRH in the hypothalamic paraventricular nucleus. Both increased during heat challenge in vulnerable chicks, whereas they decreased in resilient chicks. Our aim was to elucidate the epigenetic mechanism underlying the regulation of stress resilience or vulnerability. Accordingly, DNA CpG methylation (5mC) and hydroxymethylation (5hmC) at the CRH intron, which we found to serve as a repressor element, displayed low 5mc% alongside high 5hmc% in resilient chicks, and high 5mc% with low 5hmc% in vulnerable ones. RE1-silencing transcription factor (REST), which has a binding site on this intron, bound abundantly during acute heat stress and was nearly absent during moderate stress, restricting repression by the repressor element, and thus activating CRH gene transcription. Furthermore, REST assembled into a protein complex with TET3, which bound directly to the CRH gene. Finally, the adjacent histone recruited the histone acetylation enzyme GCN5 to this complex, which increased H3K27ac during harsh, but not moderate heat conditioning. We conclude that an epigenetic mechanism involving both post-translational histone modification and DNA methylation in a regulatory segment of CRH is involved in determining a resilient or vulnerable response to stress later in life.

Determining whether a stressful event will lead to stress-resilience or vulnerability depends probably on an adjustable stress response set point, which is most likely effective during postnatal sensory development and involves the regulation of corticotrophin-releasing hormone (CRH) expression. During the critical period of thermal-control establishment in 3-day-old chicks, heat stress was found to render resilient or sensitized response, depending on the ambient temperature. These two different responses were correlated with the amount of activation of the hypothalamic-pituitary-adrenal (HPA) axis. The expression of CRH mRNA in the hypothalamic paraventricular nucleus was augmented during heat challenge a week after heat conditioning in chicks which were trained to be vulnerable to heat, while it declined in chicks that were trained to be resilient. To study the role of CRH in HPA-axis plasticity, CRH or Crh-antisense were intracranially injected into the third ventricle. CRH caused an elevation of both body temperature and plasma corticosterone level, while Crh-antisense caused an opposite response. Moreover, these effects had long term implications by reversing a week later, heat resilience into vulnerability and vice versa. Chicks that had been injected with CRH followed by exposure to mild heat stress, normally inducing resilience, demonstrated, a week later, an elevation in body temperature, and Crh mRNA level similar to heat vulnerability, while Crh-antisense injected chicks, which were exposed to harsh temperature, responded in heat resilience. These results demonstrate a potential role for CRH in determining the stress resilience/vulnerability balance.

This study aimed to determine whether epigenetic malprogramming induced by high-fat diet (HFD) has an obesogenic effect on nonmated and mated female rats and their offspring. Further, it aimed to reprogram offspring's epigenetic malprogramming and phenotype by providing normal diet after weaning. Body weight (BW) was measured, and plasma and hypothalamic arcuate nuclei were collected for analysis of hormones, mRNA, and DNA CpG methylation of the promoter of Pomc, a key factor in control of food intake. In nonmated females, HFD decreased Pomc/leptin ratio by ∼38%. This finding was associated with Pomc promoter hypermethylation. While heavier during pregnancy, during lactation HFD dams showed sharper BW decrease (2.5-fold) and loss of Pomc promoter hypermethylation. Moreover, their weight loss was correlated with demethylation (r=−0.707) and with gadd45b mRNA expression levels (r=0.905). Even though offspring of HFD dams ate standard chow from weaning, they displayed increased BW, Pomc promoter hypermethylation, and vulnerability to HFD challenge (3-fold kilocalorie intake increase). These findings demonstrate a long-term effect of maternal HFD on CpG methylation of the Pomc promoter in the offspring, which was not reprogrammed by standard chow from weaning. Further, the results suggest a possible mechanism of demethylation of the Pomc promoter following pregnancy and lactation.—Marco, A., Kisliouk, T., Tabachnik, T., Meiri, N., Weller, A. Overweight and CpG methylation of the Pomc promoter in offspring of high-fat-diet-fed dams are not “reprogrammed” by regular chow diet in rats.

Impaired response of the brain to the leptin signal leads to a persisting dysregulation of food intake and energy balance. High plasma leptin or insulin should activate proopiomelanocortin (POMC), the precursor of the anorexigenic neuropeptide α-melanocyte-stimulating hormone (α-MSH) in the hypothalamic arcuate nucleus (ARC). Nevertheless, in obesity, this signal transduction pathway might be impaired. In this study we investigated whether chronic high fat (HF) diet consumption from post-weaning to adulthood increases CpG methylation of the Pomc promoter. The hypothesis that this would disrupt the essential binding of the transcription factor Sp1 to the Pomc promoter was tested. Male rats were raised from postnatal day 21 till 90 on either HF or standard diet. As a result HF fed rats were significantly heavier, with high leptin and insulin levels in their plasma but almost no changes in ARC mRNA expression levels of Pomc. The Pomc promoter area in the HF-treated rats was found to be hypermethylated. Furthermore, there was a direct correlation in individual rats between CpG methylation at specific sites that affect Sp1 binding and plasma leptin levels and/or body weight. Although, as expected the HF diet resulted in up-regulation of Sp1, the binding of Sp1 to the hypermethylated Pomc promoter was significantly reduced. Therefore, we suggest that hypermethylation on the promoter region of the Pomc gene can emerge at post-lactation periods and interfere with transcription factor binding, thus blocking the effects of high leptin levels, leading to obesity.

Maternal obesity is a risk factor for offspring obesity. The melanocortin 4 receptor (Mc4r) is one of the mediators of food intake and energy balance. The present study examined the epigenetic mechanisms underlying altered Mc4r levels in the hypothalamic paraventricular nucleus in the offspring of high-fat diet (HFD)-induced obese dams and sought to elucidate the role of thyroid hormones in epigenetic regulation and tagging of their nucleosome at the Mc4r promoter. Female Wistar rats were fed an HFD or standard chow from weaning through gestation and lactation. Epigenetic alterations were analyzed in the offspring on postnatal day 21 at the Mc4r promoter using chromatin immunoprecipitation and bisulfite sequencing. To study the role of triiodothyronine (T3) in Mc4r downregulation, dams received methimazole (MMI), an inhibitor of thyroid hormone production. Offspring of HFD-fed dams had a greater body weight, elevated plasma T3 concentrations, and lower Mc4r messenger RNA levels than controls. At the Mc4r promoter, offspring of HFD-fed mothers demonstrated increased histone 3 lysine 27 acetylation (H3K27ac) with a greater association to thyroid hormone receptor-β (TRβ), an inhibitor of Mc4r transcription. Moreover, TRβ coimmunoprecipitated with H3K27ac, supporting their presence in the same complex. Maternal MMI administration prevented the HFD reduction in Mc4r levels, the increase in TRβ, and the increase in the TRβ-H3K27ac association, providing further support for the role of T3 in downregulating Mc4r levels. These findings demonstrate that a perinatal HFD environment affects Mc4r regulation through a T3 metabolic pathway involving histone acetylation of its promoter.

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

As with other sensory mechanisms, determination of the thermal-control set point is refined during a critical period of development by alterations in cellular properties in the frontal hypothalamus. These alterations in hypothalamic plasticity are achieved by renewal of the protein repertoire via activation or silencing of gene transcription, both of which are regulated by histone modifications. This study demonstrates induction of global histone H3 lysine 27 (H3K27) dimethylation, with no changes in its trimethylation levels, in the frontal hypothalamus, as well as at the promoter of the brain-derived neurotrophic factor (BDNF) gene during thermal-control establishment. Furthermore, antisense 'knockdown' of the H3K27-specific methyltransferase, enhancer of zeste 2, which was induced in correlation with the dimethylation of H3K27, inhibited Bdnf mRNA expression and disrupted the establishment of thermoregulation. This phenotypic effect was partially rescued by intracranial injection of BDNF. The presented findings highlight the specific epigenetic role of chromatin modifications in thermal-control establishment.

Thermal control establishment develops during a critical period by alterations in cellular properties in the frontal hypothalamus. These alterations may be modulated by the epigenetic code that determines the repertoire of transcribed proteins. Here we demonstrate transient changes in the expression of brain-derived neurotrophic factor (Bdnf) during both thermal conditioning and re-exposure of conditioned chicks to heat stress, relative to their age-matched naive counterparts. These changes coincide with changes in CpG methylation pattern in the avian Bdnf promoter region. Reduction in methylation during heat conditioning was observed at a cAMP response element-binding (CREB) site which coincided with both elevation in phospho-CREB levels and its binding to the Bdnf promoter. At the same time, an increase in methylation was observed at two other CpG sites, accompanied by elevation of the DNA methyltransferase 3a (DNMT3a) expression. DNMT3a was also found to bind to the two elevated methyl CpG sites, but not to the CREB binding site. These data suggest that complex and dynamic changes in DNA methylation are involved in the regulation of Bdnf expression during thermotolerance acquisition.

Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2α injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10–20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3–6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture.Granulosa-derived heparanase is a novel member of the luteinizing hormone-induced extracellular matrix-degrading enzymes contributing to follicular rupture and ovulation.

International audience; Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.

A maternal high-fat diet (HFD) alters the offspring's feeding regulation, leading to obesity. This phenomenon is partially mediated by aberrant expression of the hypothalamic anorexigenic neuropeptide proopiomelanocortin (POMC). Nevertheless, although some individual offspring suffer from morbid obesity, others escape the malprogramming. It is suggested that this difference is due to epigenetic programming. In this study, we report that in lean offspring of non-HFD–fed dams, essential promoter regions for Pomc expression were enriched with 5-hydroxymethylcytosine (5hmC) together with a reduction in the level of 5-methylcytosine (5mC). Moreover, 5hmC was negatively correlated whereas 5mC was positively correlated with body weight in offspring from both HFD- and control-fed dams. We further found that Pomc expression in obese offspring is determined by a two-step epigenetic inhibitory mechanism in which CpG methylation is linked with histone posttranslational modifications. An increase in CpG methylation at the Poxmc promoter enables binding of methyl-binding domain 1 (MBD1) to 5mC, but not to its derivative 5hmC. MBD1 then interacts with SET domain bifurcated 1 methyltransferase to promote bimethylation on the histone 3 lysine 9 residue, reducing Pomc mRNA expression. These results suggest an epigenetic regulatory mechanism that affects obesity-prone or resilient traits.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) or Prokineticin-1 (PK-1) is a novel cysteine-rich protein that belongs to the AVIT protein family. EG-VEGF/PK-1, described as selective angiogenic mitogen, is widely expressed in different tissues including steroidogenic endocrine glands. This review summarizes the expression and functions of EG-VEGF/PK-1 in corpus luteum (CL)-derived cells: endothelial and steroidogenic cell types. EG-VEGF/PK-1 mRNA is expressed by luteal steroidogenic cells of human, rat and bovine ovaries, but was absent from the luteal Endothelial cells CLEC. Luteal EC expressed high levels of both PK-receptors PK-R1 and PK-R2 - the two G protein-coupled PK-1 receptors. Interestingly, expression of EG-VEGF/PK-1 and VEGF were inversely regulated in human and bovine luteinized granulosa cells. EG-VEGF/PK-1 elevated [3H]-thymidine incorporation, MAPK activation and c-jun/fos mRNA expression and enhanced LEC proliferation. EG-VEGF/PK-1 also inhibited serum starvation-induced apoptosis in these cells. Stress conditions such as serum withdrawal, TNFalpha and chemical hypoxia markedly increase PK-R2 expression, whereas mRNA levels of PK-R1 remain unchanged, implying that the anti-apoptotic effect of PK-1 on LEC may be mediated via PK-R2. Besides its direct mitogenic and anti-apoptotic effects, EG-VEGF/PK-1 elevated VEGF mRNA expression in bovine luteal steroidogenic cells, which possesses only PK-R1. Together, these findings suggest an important role for PK-1 in luteal function by acting as a mitogen and survival factor in LEC. Nevertheless, the inverse regulation of EG-VEGF/PK1 and VEGF mRNA expression by ovarian cells and the distribution of its receptors may suggest that in addition to its angiogenic effects, EG-VEGF/PK-1 may also play other roles in ovary.

Endocrine gland vascular endothelial growth factor (EGVEGF) is a novel angiogenic mitogen selective for endothelial cells (EC) in endocrine glands. EG-VEGF is identical to a protein previously cloned and termed prokineticin (PK)-1. The present study examined the expression of EG-VEGF/PK-1 and its receptors in ovarian steroidogenic cells and EC and compared the regulation of EG-VEGF/PK-1 and VEGF expression in SV40 transformed luteinized human granulosa cell line (SVOG). Normal granulosa or SVOG cells expressed EG-VEGF/ PK-1 mRNA. Incubation of SVOG cells with forskolin augmented EG-VEGF/PK-1 expression in a dose-dependent manner. Chemical hypoxia induced by CoCl2 and desferrioxamine mesylate (100 M each) markedly reduced EG-VEGF/ PK-1. In contrast, hypoxia significantly elevated VEGF mRNA (VEGF165, 189) and protein secretion. Thrombin, like hypoxia, also induced an opposite effect on VEGF and EG-VEGF/ PK-1. Whereas EG-VEGF/PK-1 and VEGF were inversely regulated, steroidogenesis and EG-VEGF/PK-1 were positively correlated in SVOG cells. A distinct pattern of ovarian PK receptor (PK-R) expression was observed in which steroidogenic cells predominantly express PK-R1 receptors, whereas corpus luteum-derived EC express high levels of both PK-R1 and PK-R2. Therefore, acting via either PK-R2 or PK-R1, EGVEGF/PK-1 may have angiogenic as well as nonangiogenic functions in the ovary. (J Clin Endocrinol Metab 88: 3700 –3707, 2003)

Determining whether a stressful event will lead to stress-resilience or vulnerability depends probably on an adjustable stress response set point, which is most likely effective during postnatal sensory development and involves the regulation of corticotrophin-releasing hormone (CRH) expression. During the critical period of thermal-control establishment in 3-day-old chicks, heat stress was found to render resilient or sensitized response, depending on the ambient temperature. These two different responses were correlated with the amount of activation of the hypothalamic-pituitary-adrenal (HPA) axis. The expression of CRH mRNA in the hypothalamic paraventricular nucleus was augmented during heat challenge a week after heat conditioning in chicks which were trained to be vulnerable to heat, while it declined in chicks that were trained to be resilient. To study the role of CRH in HPA-axis plasticity, CRH or Crh-antisense were intracranially injected into the third ventricle. CRH caused an elevation of both body temperature and plasma corticosterone level, while Crh-antisense caused an opposite response. Moreover, these effects had long term implications by reversing a week later, heat resilience into vulnerability and vice versa. Chicks that had been injected with CRH followed by exposure to mild heat stress, normally inducing resilience, demonstrated, a week later, an elevation in body temperature, and Crh mRNA level similar to heat vulnerability, while Crh-antisense injected chicks, which were exposed to harsh temperature, responded in heat resilience. These results demonstrate a potential role for CRH in determining the stress resilience/vulnerability balance.

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.

Thermal control set point is regulated by thermosensitive neurons of the preoptic anterior hypothalamus (PO/AH) and completes its development during postnatal critical sensory period. External stimuli, like increase in environmental temperature, influence the neuronal protein repertoire and, ultimately, cell properties via activation or silencing of gene transcription, both of which are regulated by the "histone code.''" Here, we demonstrated an increase in global histone H3 lysine 9 (H3K9) acetylation as well as H3K9 dimethylation in chick PO/AH during heat conditioning at the critical period of sensory development. In contrast to the global profile of H3K9 modifications, acetylation and dimethylation patterns of H3K9 at the promoter of the catalytic subunit of eukaryotic translation initiation factor 2B (Eif2b5) were opposite to each other. During heat conditioning, there was an increase in H3K9 acetylation at the Eif2b5 promoter, simultaneously with decrease in H3K9 dimethylation. These alterations coincided with Eif2b5 mRNA induction. Moreover, exposure to excessive heat during the critical period resulted in long-term effect on both H3K9 tagging at the Eif2b5 promoter and Eif2b5 mRNA expression. These data suggest a role for dynamic H3K9 post-translational modifications in global translation regulation during the thermal control establishment.

Thermal control establishment develops during a critical period by alterations in cellular properties in the frontal hypothalamus. These alterations may be modulated by the epigenetic code that determines the repertoire of transcribed proteins. Here we demonstrate transient changes in the expression of brain-derived neurotrophic factor (Bdnf) during both thermal conditioning and re-exposure of conditioned chicks to heat stress, relative to their age-matched naive counterparts. These changes coincide with changes in CpG methylation pattern in the avian Bdnf promoter region. Reduction in methylation during heat conditioning was observed at a cAMP response element-binding (CREB) site which coincided with both elevation in phospho-CREB levels and its binding to the Bdnf promoter. At the same time, an increase in methylation was observed at two other CpG sites, accompanied by elevation of the DNA methyltransferase 3a (DNMT3a) expression. DNMT3a was also found to bind to the two elevated methyl CpG sites, but not to the CREB binding site. These data suggest that complex and dynamic changes in DNA methylation are involved in the regulation of Bdnf expression during thermotolerance acquisition.

International audience; Prokineticins (PKs), multifunctional secreted proteins, activate two endogenous G protein-coupled receptors (R) termed PK-R1 and PK-R2. It was suggested that PK1 acts selectively on the endothelium of endocrine glands, yet PK-Rs were also found in endothelial cells (EC) derived from other tissues. Therefore we examined here the characteristics of PK - system in EC derived from different vascular beds. Corpus luteum (CL)-derived EC (LEC) expressed both PK-R1 and PK-R2. In contrast, EC from the aorta (BAEC) only expressed PK-R1. Interestingly, also EC from brain capillaries (BCEC) expressed only PK-R1. The distinct pattern of PK-R expression may define EC phenotypic heterogeneity. Regulation of receptor expression also differed in BAEC and LEC: TNFalpha markedly reduced PK-R1 only in BAEC, but serum removal decreased PK-R1 in both cell types. Therefore, if cells were initially serum-starved, the anti-apoptotic effect of PKs was retained only in LEC. Yet, addition of PKs concomitant with serum removal enhanced the proliferation and survival of both BAEC and LEC. Immunohistochemical staining showed that in CL and aorta PK1 was expressed in smooth muscle cells in vessel walls, suggesting a paracrine mode of action. PK1 enhanced the net paracellular transport (measured by electrical resistance and Mannitol transport) in LEC but not in BAEC or BCEC. Collectively, these findings indicate that PKs serve as mitogens and survival factors for microvascular (LEC) and macrovascular (BAEC) EC. However, the distinct expression and function of PK receptors suggest different physiological roles for these receptors in various EC types.

A highly vascular endocrine gland, the corpus luteum (CL) is an excellent model for the study of angiogenic factors. Prokineticins (PK-1 and -2), also termed endocrine-gland-derived vascular endothelial growth factor (VEGF) and BV8 are newly identified proteins described as selective angiogenic mitogens. We previously identified PK binding sites, two closely homologous G protein-coupled receptors (PK-R1 and PK-R2) in human and bovine ovarian cells, but their function remained unknown. In this study we examined the presence and effects of PK in CL-derived endothelial and steroidogenic cell types (LEC and LSC, respectively). PK-1 mRNA was identified in CL and follicles by real-time PCR, using primers specific for the bovine PK-1 sequence (retrieved from Bos taurus whole genome shotgun database). PK were potent angiogenic mitogens for LEC; they enhanced cell proliferation, elevated [3H]thymidine incorporation, MAPK activation, and c-jun/fos mRNA expression. The effects of PK proteins on cell survival were examined by nuclear morphology (4′,6-diamidino-2-phenylindole dihydrochloride staining), measurement of DNA fragmentation (terminal dUTP nucleotide end labeling assay), and caspase-3 cleavage. Results obtained by these techniques demonstrated that PK protected LEC from serum starvation-induced apoptosis. Stress conditions such as serum withdrawal, TNF-α, and hypoxia markedly increased PK-R2 expression, whereas mRNA levels of PK-R1 remained unchanged. These suggest that the antiapoptotic effect of PK-1 on LEC may be mediated via PK-R2. PK-1 increased VEGF mRNA expression by LSC, implying that it could also indirectly, via VEGF, affect luteal angiogenesis. Together, these findings suggest an important role for PK-1 in luteal function by acting as a mitogen and survival factor in LEC.

Endothelium-derived endothelin-1 (ET-1) and nitric oxide (NO) are pivotal regulators of corpus luteum (CL) function. To have a better insight into their synthesis and action, members of the ET system (ET-1, ET converting enzyme (ECE-1) isoforms a–d, ETAand ETBreceptors) along with NO synthase (NOS) isoforms – endothelial (e)NOS and inducible (i)NOS – were quantified in CL-derived endothelial cells (CLEC). The expression of these genes in microvascular CLEC, obtained by lectin-coated magnetic beads, was compared with cells removed from the luteal microenvironment and maintained in culture for different durations, and with endothelial cells (EC) derived from a large blood vessel (i.e. bovine aortic endothelial cells, BAEC). The profile of gene expression in the different EC types was determined by quantitative real-time PCR. Freshly isolated EC from mid-cycle CL exhibited high ET-1 receptor expression (both ETAand ETB), low ET-1 synthesizing ability (both prepro (pp) ET-1 and ECE-1), but elevated iNOS – the high throughput NOS isoform. The distinct phenotype of CLEC was lost soon after an overnight culture. ETAand ETBreceptor levels declined, ppET-1 levels increased while iNOS was reduced. These changes were extenuated during long-term culture of CLEC. The general pattern of gene expression in BAEC and long-term cultured CLEC was similar yet some differences, reminiscent of freshly isolated CLEC, remained: ECE-1c, ETBreceptor and NOS isoforms were expressed differently in BAEC as compared with lines of CLEC.This study suggests that the luteal microenvironment is necessary to sustain the selective phenotype of its resident endothelial cells. The inverse relationship between ppET-1 and iNOS observed in freshly isolated CLEC and in cultured cells is physiologically significant and suggests that ET-1 and NO may modulate the production of each other.