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Proceedings of the Royal Society B: Biological Sciences logoLink to Proceedings of the Royal Society B: Biological Sciences
. 2020 Oct 28;287(1937):20201991. doi: 10.1098/rspb.2020.1991

Early life variations in temperature exposure affect the epigenetic regulation of the paraventricular nucleus in female rat pups

Samantha C Lauby 1,2, Patrick O McGowan 1,2,3,4,✉
PMCID: PMC7661289  PMID: 33109014

Abstract

Early life maternal care received has a profound effect on later-life behaviour in adult offspring, and previous studies have suggested epigenetic mechanisms are involved. Changes in thyroid hormone receptor signalling may be related to differences in maternal care received and DNA methylation modifications. We investigated the effects of variations in temperature exposure (a proxy of maternal contact) and licking-like tactile stimulation on these processes in week-old female rat pups. We assessed thyroid hormone receptor signalling by measuring circulating triiodothyronine and transcript abundance of thyroid hormone receptors and the thyroid hormone-responsive genes DNA methyltransferase 3a and oxytocin in the paraventricular nucleus of the hypothalamus. DNA methylation of the oxytocin promoter was assessed in relation to changes in thyroid hormone receptor binding. Repeated room temperature exposure was associated with a decrease in thyroid hormone receptor signalling measures relative to nest temperature exposure, while acute room temperature exposure was associated with an increase. Repeated room temperature exposure also increased thyroid hormone receptor binding and DNA methylation at the oxytocin promoter. These findings suggest that repeated room temperature exposure may affect DNA methylation levels as a consequence of alterations in thyroid hormone receptor signalling.

Keywords: maternal care, temperature, thyroid hormone, DNA methylation, oxytocin

1. Background

Maternal care in early life, typically assessed by licking/grooming in rodents, has a profound influence on neurodevelopmental trajectories in offspring and their later-life behaviour [1]. Early life maternal separation, which disrupts maternal–pup contact, or natural variations in maternal care persistently alter transcript abundance of corticotropin releasing factor (Crf) [2], arginine vasopressin (Avp) [3] and the glucocorticoid receptor [4,5] in the brain. These changes in transcript abundance have been linked to persistent epigenetic modifications of DNA and histones that alter the binding of transcription factors to DNA. Other maternal factors may also play a role in later-life offspring phenotypes either by themselves or via interactions with licking/grooming [6]. The exposure of pups to lower ambient (room) temperatures as a result of brief disruptions in mother–pup contact has been proposed to be an important component involved in the reduction in stress response following early life handling [6,7]. Pups have an inefficient thermoregulatory system [8] and are dependent on non-shivering thermogenesis, huddling with siblings and proximal contact with the rat mother [8–10] to maintain body temperature. Licking-like tactile stimulation has also been shown to reduce the body temperature of rat pups [11].

Variations in both ambient temperature and licking/grooming alter thyroid hormone physiology. Room temperature exposure can induce release of thyroid hormones to evoke non-shivering thermogenesis in brown adipose tissue. In addition, acute licking-like tactile stimulation can induce conversion of the thyroid hormone thyroxine to triiodothyronine (T3) in the periphery and lead to DNA demethylation of the glucocorticoid receptor promoter in the hippocampus [12].

Thyroid hormones, especially T3, play important roles in neurodevelopment by regulating gene transcription through thyroid hormone receptor [13]. In the developing mouse brain, transcription of DNA methyltransferase 3a (Dnmt3a), which catalyses de novo DNA methylation modifications, can occur through the binding of liganded thyroid hormone receptor to thyroid hormone response elements (TREs) in intragenic regions of Dnmt3a [14]. Mouse pups that receive higher levels of maternal care also show increased Dnmt3a in the hippocampus at postnatal day (PND) 7 [15]. Thyroid hormone is also a regulator of oxytocin (Oxt) [16], a neuropeptide produced in the paraventricular nucleus (PVN) of the hypothalamus and involved with stress attenuation, among other phenotypes. Thyroid hormone and other receptors can bind to the composite hormone response element (CHRE) in the promoter region of Oxt to activate transcription [16,17]. Genetic deletion of this region abolishes transcription, indicating the CHRE is required for proper regulatory control of Oxt [18]. A CpG dinucleotide flanks the 5′ end of the CHRE but its potential role in the epigenetic regulation of Oxt in the PVN has not been examined. Previous work in humans has investigated DNA methylation of the Oxt promoter, including the CHRE [19,20] and the enhancer in the Oxt/Avp intergenic region [21]. However, because peripheral tissues were examined in these studies, it is not known whether DNA methylation at this locus is involved in the regulation of Oxt transcript abundance in the brain.

Here, we investigated early life room temperature exposure and licking-like tactile stimulation on thyroid hormone receptor signalling in female rat pups. We focused on females because there are possible sex differences in pup thermoregulation [22] and maternal care received [23]. In addition, the increase in oestrogen during sexual differentiation in the male pup brain can interact with thyroid hormone for gene transcription [24]. We measured levels of circulating T3 and transcript abundance in the PVN of thyroid hormone receptors and the thyroid hormone-responsive genes Dnmt3a and Oxt. We hypothesized that early life room temperature exposure and tactile stimulation would synergistically increase T3 levels as well as increase Dnmt3a and Oxt transcript abundance via changes in thyroid hormone receptor signalling. We predicted that the increase in Oxt transcript abundance would correspond to altered DNA methylation at the CHRE locus and differential thyroid hormone receptor binding. We also predicted that transcript abundance of other genes not directly regulated by thyroid hormone binding, including other DNA methyltransferases, Crf and Avp, would not show a similar pattern, and that Crf and Avp transcript abundance would be reduced with additional tactile stimulation.

2. Methods

(a). Rat breeding

Seven-week-old female (n = 28) and male (n = 16) Long-Evans rats were obtained from Charles River Laboratories (Kingston, NY, USA). They were housed in same-sex pairs on a 12 : 12 h light–dark cycle (lights on at 7.00) with ad libitum access to standard chow diet and water. For breeding, one male was housed with two females for one week. Females were then housed separately and weighed weekly throughout pregnancy. All animal procedures were approved by the Local Animal Care Committee at the University of Toronto Scarborough and conformed to the guidelines of the Canadian Council on Animal Care.

Females were checked for parturition starting three weeks after breeding, at 9.00 and 17.00. PND 0 was determined if the birth occurred between 9.00 and 17.00 or if pups were found at 9.00 but had not nursed yet. Pups found at 9.00 with a milk band were considered PND 1. At PND 1, litters were culled to four to six female pups and individually weighed. All litters in this study were derived from primiparous rat mothers. A total of 154 female rat pups were used for this study. One group of female rat pups was assessed for transcript abundance and DNA methylation and a separate group of female rat pups was assessed for T3 levels and chromatin immunoprecipitation (ChIP) enrichment.

(b). Postnatal manipulations

Figure 1a shows the timeline of the experimental design. From PND 2 to PND 7, 19 whole litters were separated from their mother for approximately 25 min per day during the light phase (9.00–13.00) and placed in a small cage lined with corn cob bedding. Nine litters were placed in a cage warmed with a heating pad (33–35°C; ‘nest temperature’ condition) and 10 litters were exposed to room temperature without extra heat (19–22°C; ‘repeated room temperature’ condition). A total of 51 pups were in the nest temperature condition and 54 pups were in the repeated room temperature condition.

Figure 1.

Figure 1.

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Repeated room temperature exposure decreased thyroid hormone activity with minimal effects of additional tactile stimulation. (a) Schematic diagram of the experimental design between litters and within litters of female rat pups. (b–g) Repeated room temperature exposure led to a significant decrease in (b) circulating total T3 levels, (c) thyroid hormone receptor α1 (Thra1) transcript abundance, (d) thyroid hormone receptor beta (Thrb) transcript abundance, (e) Hairless (Hr) transcript abundance, and (f) DNA methyltransferase 3a (Dnmt3a) transcript abundance, and (g) a marginal effect on oxytocin (Oxt) transcript abundance. Acute exposure to room temperature led to significant increases in circulating total T3 levels and thyroid-related transcript abundance compared with pups with repeated room temperature exposure. (g) Pups provided additional tactile stimulation had decreased Oxt transcript abundance in the nest temperature condition. The serum T3 barplot is displayed with mean ± s.e.m. qPCR barplots are displayed as fold changes relative to the pups handled once and nonstimulated ± s.e.m. (recalculated as 2fold change±s.e.m. to fit the logarithmic scale). *,p < 0.05, #,p < 0.10 main effect of temperature condition; ▪ , p < 0.05 main effect of tactile stimulation condition. (Online version in colour.)

During the daily 25 min separation, two or three female rat pups within a litter received additional tactile stimulation with a camel hair paintbrush (Craftsmart) for 15 min (‘stimulated’ condition; n = 78) while the remaining pups were left undisturbed (‘nonstimulated’ condition; n = 76). Within the nest temperature condition, 26 pups were in the stimulated condition and 25 pups were in the nonstimulated condition. Within the repeated room temperature condition, 27 pups were in the stimulated condition and 27 pups were in the nonstimulated condition. Pups received the additional tactile stimulation on the dorsal region of their body at a rate of approximately two strokes per second. The same pups received additional tactile stimulation each day. All pups were individually weighed daily and interscapular temperature was measured with an infrared thermometer (VWR) before and after the tactile stimulation period from PND 3 to PND 7. From PND 2 to PND 6, female rat pups were individually marked using odourless and tasteless food colouring (Club House, London, Ontario, Canada) to distinguish between siblings, as described in previous work by our laboratory [25].

To investigate acute effects of room temperature exposure and tactile stimulation, nine litters were separated once at PND 7 (‘acute room temperature’ condition) and two or three female rat pups within a litter received additional tactile stimulation. A total of 49 pups were in the acute room temperature condition; 25 pups were in the stimulated condition and 24 pups were in the nonstimulated condition, with weights and temperatures measured as above.

The interscapular temperature change (before the tactile stimulation period minus after the tactile stimulation period) was calculated for all groups at PND 7 to verify the room temperature exposure conditions had reduced pup temperature while the nest temperature exposure condition kept a stable pup temperature during the separation period.

At PND 7, all female rat pups were sacrificed following the tactile stimulation period, and blood and brain were collected. One stimulated and one nonstimulated sibling were decapitated at a time. To examine if the time elapsed since the tactile stimulation period would affect the physiology of the pups, we noted the order pups were sacrificed for use as a control variable. Brains were flash frozen in isopentane and kept on dry ice. Blood was kept on ice at least 30 min before being centrifuged at 4000g at 4°C for 30 min. Serum and brain samples were stored at −80°C. One or two pups per stimulation group per litter were used for all downstream molecular analyses.

(c). Maternal care observations

From PND 2 to PND 6, each litter was video recorded for 1 h two times during the light phase (13.00–14.00, 17.00–18.00) and three times during the dark phase (21.00–22.22, 1.00–2.00, 5.00–6.00). These videos were coded with Observer XT 10.5 (Noldus) for maternal behaviour by five coders with high inter-rater reliability (greater than or equal to 90%). Nursing, licking/grooming, nest-building and other self-directed behaviours were scored every 3 min using an ethogram based on previous literature [26]. A total of 100 observations per day per mother were coded and each behaviour was represented as a percentage of the frequency of behaviour coded over total observations multiplied by 100. Total nursing was calculated as the sum of low crouch, high crouch and supine nursing observations. Total licking was calculated as the sum of anogenital and body licking observations.

Two litters from the nest temperature condition had missing maternal care recordings at PND 2 and one litter from the repeated room temperature condition had missing maternal care recordings from PND 2 to PND 3 owing to technical errors.

(d). Serum total triiodothyronine

Total T3 was measured in the pup serum (n = 5–7 per group) using enzyme-linked immunosorbent assay (ELISA; MP Biomedicals Inc., USA) following the manufacturer's instructions. For each pup sample, technical duplicates were measured when possible and 50 µl of serum per well was used. To keep all samples within the linear phase of the standard curve, the serum was diluted 1 : 1 with the ‘0’ standard. Each plate was normalized with a control sample of known concentration of T3 (Control Set I; MP Biomedicals Inc., USA). Concentration of T3 was determined using a four-point logistic curve using an online software (https://elisaanalysis.com/app) and multiplied by 2 to account for the dilution factor.

(e). Transcript abundance

PND 7 brains (n = 5 or 6 per group) were cryosectioned with 50 µm slices using a Leica CM3050S cryostat. The PVN (−1.40 to −2.00 mm Bregma) was microdissected using an atlas for the developing rat brain [27] and a supplementary atlas for the PND 7 rat brain [28]. RNA was extracted using a RNeasy Micro Kit (Qiagen) following the manufacturer's instructions. Concentration and purity of RNA were assessed using a spectrophotometer (Nanodrop ND-2000C, Thermo Scientific). Up to 1 µg of RNA was converted to cDNA (Applied Biosystems High Capacity cDNA Conversion Kit) and diluted to 5 ng µl−1 assuming 100% conversion efficiency.

Transcript abundances of candidate genes were assessed using StepOnePlus Real-Time PCR software with Fast SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) using technical triplicates. Specifically, we analysed arginine vasopressin (Avp), corticotropin releasing factor (Crf), DNA methyltransferase 1 (Dnmt1), Dnmt3a, DNA methyltransferase 3b (Dnmt3b), Hairless (Hr [29]), Oxt, thyroid hormone receptor α1 (Thra1) and thyroid hormone receptor β (Thrb). Each plate was corrected with one randomly assigned cDNA sample that was measured on all plates. All transcripts were normalized to the GEOmean of actin-β (Actb) and ubiquitin C (Ubc) transcript, with quantification calculated by the ΔCT method. Electronic supplementary material, table S1 displays the primer sets created from Primer-BLAST software (National Center for Biotechnology Information) and previous literature [30,31].

(f). DNA methylation analysis

DNA from six PVN from each additional tactile stimulation group in the repeated room temperature condition and nest temperature condition (total n = 24) was extracted using the Masterpure Complete DNA and RNA Extraction Kit (Epicentre) and 300 ng of DNA was used for bisulfite conversion using the EpiTect Fast Bisulfite Conversion Kit (Qiagen) following the manufacturer's instructions. Semi-nested PCR was performed with primers created from the Pyromark Q-CpG 1.0.9 software (electronic supplementary material, table S1) and targeted one CpG site flanking the oxytocin CHRE in the promoter region (chr3:123106520; rn6). The biotinylated amplicons were verified with gel electrophoresis and extracted with the MinElute Gel Extraction Kit (Qiagen). Pyrosequencing was done using a Pyromark Q106 ID pyrosequencer with technical triplicates. CpG methylation levels were calculated using Pyromark Q-CpG 1.0.9 software.

(g). Chromatin immunoprecipitation

Two PVN from the same litter and additional tactile stimulation group were pooled (total n = 3 for repeated room temperature and n = 4 for nest temperature) for ChIP based on the protocol from Stefanelli et al. [32]. Two batches were done with 1–2 pooled PVN samples per temperature condition per batch.

Pooled PVN tissue samples were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 min at 26°C. The samples were quenched with 1.25 M glycine and left at room temperature for 5 min, centrifuged at 21 100g for 30 s and washed five times with ice-cold PBS and a protease inhibitor cocktail (Roche) dissolved in PBS. The PVN tissue was homogenized with SDS lysis buffer (0.25 M sucrose, 60 mM KCl, 15 mM NaCl, 10 mM MES (pH 6.5), 5 mM MgCl2, 0.5% Triton X-100) and centrifuged at 4700g to pellet the cell nuclei. The SDS lysis buffer was removed and a salt buffer (50 mM NaCl, 10 mM PIPES (pH 6.8), 5 mM MgCl2, 1 mM CaCl2) was added prior to sonication (3–4 W output; three times for 10 s on, 30 s off; Fisher Scientific Sonic Dismembrator Model 100). The samples were incubated with 150 units of micrococcal nuclease (Cell Signaling) at 37°C for 10 min before quenching with 5 µl 0.5 M EDTA and placed on ice. SDS (10%) was added to each sample before being centrifuged at 17 000g for 5 min, aliquoted and diluted 4× with a ChIP dilution buffer. Each ChIP aliquot contained 20 µl of Millipore Protein G magnetic beads and 10 µg of thyroid hormone receptor α/β (Thermo-Fisher Scientific, cat. no. MA1–215) or 2 µg of H3K27ac (Abcam, cat. no. ab177178) antibody and was incubated at 4°C overnight. The beads were then pelleted using a magnetic separator and washed with ice-cold low-salt, high-salt, LiCl (Millipore) and Tris-EDTA buffers. Cross-links were reversed for ChIP aliquots and input chromatin samples using 10 µg of proteinase K in Tris-EDTA buffer (with 1% SDS) at 65°C for at least 2 h before purification using a PCR clean-up kit (BioBasic).

Primers for the Oxt CHRE were created with Primer-BLAST software (electronic supplementary material, table S1). Primers for the Dnmt3a 30.3 and 49.3 kbp TRE were created with Primer-BLAST software using the rat homologous sequences from previous literature using mouse ([14]; electronic supplementary material, table S1). Enrichment was measured for each gene locus using qPCR for the ChIP DNA and input chromatin samples with technical triplicates. The enrichments for thyroid hormone receptors and H3K27ac were normalized and calculated as relative percentages of input chromatin.

(h). Statistical analysis

The datasets analysed in this study can be found in the electronic supplementary material. All statistical analyses were performed using SPSS (IBM Corporation). To examine the effects of temperature condition on maternal care received, a repeated-measures 3 (nest temperature, repeated room temperature and acute room temperature) × 5 (PND 2–6) linear mixed model was used to correct for missing datapoints and random factors. To examine the effects of room temperature exposure and tactile stimulation on interscapular temperature, serum T3 concentration and transcript abundance, a 3 (nest temperature, repeated room temperature and acute room temperature) × 2 (stimulated and nonstimulated) general linear model was used. As there was a main effect of sacrifice order on Thra1, Hr and Dnmt1 transcript abundance, a linear mixed model was used with order of sacrifice as a random factor. Significant effects of temperature condition were followed with a post hoc test using Fisher's least significant differences. Significant effects of tactile stimulation or a significant temperature condition × tactile stimulation interaction were followed with a post hoc test within each temperature condition. To examine the effects of room temperature exposure and tactile stimulation on DNA methylation of the Oxt CHRE, a 2 (nest temperature and repeated room temperature) × 2 (stimulated and nonstimulated) general linear model was used. To examine the effects of room temperature exposure on thyroid hormone receptor and H3K27ac enrichment on Dnmt3a and Oxt, a one-way (nest temperature and repeated room temperature) linear mixed model was used with batch as a random factor. All effects were considered statistically significant at p ≤ 0.05 and marginally significant at p ≤ 0.10.

3. Results

(a). Postnatal day 7 pup characteristics and maternal care received

There was a main effect of temperature exposure on interscapular temperature change at PND 7 (F2,139 = 91.777, p < 0.001). Female rat pups in the nest temperature condition maintained a relatively stable interscapular temperature. By contrast, the acute and repeated room temperature conditions induced a significant reduction in interscapular temperature during the separation period. This reduction in temperature was in the repeated compared with the acute room temperature condition (electronic supplementary material, figure S1A).

There was no main effect of temperature exposure on total licking received (F2,25.383 = 1.498, p = 0.243; electronic supplementary material, figure S1B) and total nursing received (F2,25.340 = 1.276, p = 0.296; electronic supplementary material, figure S1C). However, total nursing significantly declined over the first postnatal week (F2,24.461 = 9.160, p < 0.001) and there was a significant temperature exposure × PND interaction (F2,24.438 = 2.480, p = 0.040; electronic supplementary material, figure S1C). Rat mothers with litters in the repeated room temperature condition and nest temperature condition provided significantly more nursing than mothers with litters in the acute room temperature condition at PND 4.

(b). Circulating total triiodothyronine levels

There was a main effect of temperature exposure on total T3 levels in the PND 7 rat pup serum (F2,42 = 35.141, p < 0.001; figure 1a). The repeated room temperature condition led to significantly lower levels of total T3 than the nest and acute room temperature conditions (post hoc p-values < 0.001). The acute room temperature condition led to higher levels of total T3 than the nest temperature condition (post hoc p < 0.001). There was no main effect of additional tactile stimulation and no interaction (p-values > 0.1) on serum T3 levels.

(c). Transcript abundance

Repeated room temperature exposure led to reduced thyroid hormone receptor signalling-related transcript abundance and additional tactile stimulation led to reduced Oxt transcript abundance. There were main effects of temperature exposure on Thra1 (F2,28.755 = 113.774, p < 0.001; figure 1c), ThrB (F2,29 = 13.233, p < 0.001; figure 1d), Hr (F2,28.555 = 18.850, p < 0.001; figure 1e), Dnmt3a (F2,29 = 8.481, p = 0.001; figure 1f) and Oxt (F2,29 = 3.794, p = 0.034; figure 1g) transcript abundance. Pups in the repeated room temperature condition had a significant reduction of Thra1 (post hoc p < 0.001), Thrb (post hoc p < 0.001), Hr (post hoc p < 0.001), Dnmt3a (post hoc p = 0.001) and Oxt (post hoc p = 0.008) compared with pups in the acute room temperature condition. Pups in the repeated room temperature condition also had a significant reduction of Thra1 (post hoc p < 0.001), Thrb (post hoc p = 0.011), Hr (post hoc p < 0.001) and Dnmt3a (post hoc p = 0.003) and a marginal reduction in Oxt (post hoc p = 0.092) compared with pups in the nest temperature condition. In addition, pups in the acute room temperature condition had a significant reduction of Thra1 (post hoc p < 0.001) and a significant increase of Thrb (post hoc p = 0.018) compared with the nest temperature condition.

There was a marginal effect of additional tactile stimulation on Oxt transcript abundance (F1,29 = 3.890, p = 0.058). Pups with additional tactile stimulation had a decrease in Oxt if they were in the nest temperature condition (F1,10 = 7.011, p = 0.024; figure 1g). There were no main effects of temperature or tactile stimulation on Avp (electronic supplementary material, figure S2A) and Crf (electronic supplementary material, figure S2B) and no interactions in any of the genes measured (p-values > 0.1).

There was also a main effect of temperature exposure on Dnmt1 (F2,28.757 = 9.233, p = 0.001; electronic supplementary material, figure S2C). Repeated room temperature exposure induced a significant reduction in Dnmt1 relative to acute room temperature (post hoc p = 0.015) and nest temperature exposure (post hoc p < 0.001). There was a marginal reduction in Dnmt1 among pups with acute room temperature compared with nest temperature exposure (post hoc p = 0.087). There were no main effects of temperature or tactile stimulation on Dnmt3b (electronic supplementary material, figure S2D).

(d). Oxytocin DNA methylation at the composite hormone response element

To investigate the long-term changes of room temperature exposure and additional tactile stimulation, we examined DNA methylation levels with repeated exposures. Repeated room temperature exposure led to increased DNA methylation levels flanking the Oxt CHRE (see figure 2a for schematic diagram) compared with the nest temperature condition (F1,20 = 5.256, p = 0.033; figure 2b). There was no main effect of additional tactile stimulation and no interaction (p > 0.1) on DNA methylation levels.

Figure 2.

Figure 2.

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Repeated room temperature exposure increased DNA methylation and thyroid hormone receptor binding at the oxytocin CHRE in the paraventricular nucleus. (a) Schematic diagram of regulatory gene loci tested for Oxt and Dnmt3a. (b) DNA methylation at the CpG site (highlighted) flanking the composite hormone response element in the oxytocin promoter (underlined) increased in female rat pups with repeated room temperature exposure. (c) Female rat pups with repeated room temperature exposure had significant increases in thyroid hormone receptor enrichment at the Oxt CHRE but not at the Dnmt3a TRE. Barplots are displayed with mean ± s.e.m. *p < 0.05 main effect of temperature condition.

(e). Chromatin immunoprecipitation of thyroid hormone receptor and H3K27ac

The Oxt CHRE and two TREs within Dnmt3a (figure 2a) were analysed for thyroid hormone receptor binding and H3K27ac levels. Repeated room temperature induced a significant increase in thyroid hormone receptor enrichment at the Oxt CHRE relative to nest temperature (F1,4.015 = 17.811, p = 0.013; figure 2c). There were no effects of repeated temperature at the Dnmt3a +30.3 kbp TRE (F1,4.046 = 0.022, p = 0.889) or +49.3 kbp TRE (F1,4.031 = 0.234, p = 0.654). Likewise, there were no effects of temperature on H3K27ac enrichment in any gene loci tested (p-values > 0.1).

4. Discussion

In this study, we investigated how two factors rat pups commonly experience in the early life maternal environment, ambient (room) temperature exposure and licking-like tactile stimulation, would affect neurodevelopment by changes in thyroid hormone receptor signalling at the neonate stage. This is the first study to our knowledge to investigate the contribution of early life room temperature exposure on epigenetic modifications in the brains of rat pups. We found that female rat pups subjected to repeated room temperature exposure in early life showed a reduction in several measures of thyroid hormone receptor signalling relative to pups with early life nest temperature exposure, including circulating T3 and transcript abundance in the PVN of thyroid hormone receptors and the thyroid hormone-responsive genes Dnmt3a and Oxt. These effects were associated with increased DNA methylation and thyroid receptor binding at the CHRE in the Oxt promoter in room temperature-exposed pups. Female rat pups with acute room temperature exposure showed the highest levels of T3 and an increase in measures of thyroid hormone receptor signalling tested relative to rat pups with repeated room temperature exposure. There was no effect of additional tactile stimulation on most of the thyroid hormone receptor signalling measures and minor effects on transcript abundance of Oxt in the PVN. These findings indicate that early life room temperature exposure, a proxy for reduced maternal contact, may influence offspring phenotype via changes in thyroid hormone receptor signalling and downstream DNA methylation modifications. In addition, our results suggest that the changes in Dnmt3a transcript levels and DNA methylation at the Oxt CHRE may occur as a consequence of alterations in thyroid hormone receptor signalling.

(a). Effects on thyroid hormone receptor signalling

We predicted that room temperature exposure would increase circulating T3 levels in order to activate thermogenesis. However, we found decreased circulating T3 in response to repeated room temperature exposure, as well as decreased transcript abundance of thyroid hormone receptors and the thyroid hormone-responsive genes Dnmt3a and Oxt, compared with pups with nest temperature exposure. Though the decrease in Oxt is a nonsignificant trend, it followed a general pattern of repression of thyroid hormone receptor signalling with an increase in DNA methylation in the Oxt promoter region. Female rat pups exposed to acute room temperature showed the predicted increase in these measures, demonstrating that a suppression in thyroid hormone receptor signalling occurs with repeated exposures to room temperature. We also found similar effects on transcript abundance of the classic T3-responsive gene Hairless. We also observed minimal alterations in maternal care received between the different temperature exposure groups; therefore, the data suggest that these changes in the rat pups are more likely due to the temperature manipulations directly than to indirect changes in maternal care received.

Cold acclimatization in adult rats can decrease levels of thyroid hormone released in response to a cold stressor [33], possibly as thyroid hormone is no longer required to activate thermogenesis [34]. Interestingly, rat pups exposed to repeated room temperature also showed a more pronounced decrease in interscapular temperature, the main site for thermogenesis, than rat pups with an acute exposure to room temperature during the separation period at PND 7. It is unknown if these changes in thyroid hormone physiology persist into adulthood and if they would affect other phenotypes. However, studies in mice show a potential link between nest quality, with lower-quality nests associated with greater exposure of the pups to the ambient temperature, and increased metabolic rate, thermogenesis and thyroxine levels in adulthood [35,36]. In addition, the downstream alterations in early life Oxt transcript abundance could affect social interaction, response to stressors and other behaviours in adulthood [37]. Overall, these findings suggest that the effects of repeated room temperature exposure in early life may reflect a physiological adaptation to cold stressors in the rat pup. Future work is needed to elucidate mechanisms that underlie changes in peripheral thyroid hormone status, including measuring thyroid stimulating hormone in the pituitary gland and deiodinase activity in the liver.

We also found that repeated room temperature exposure was associated with decreased transcript abundance of Dnmt1, which has not been previously shown to be responsive to thyroid hormone. However, one study has shown Dnmt3a and Dnmt1 can cooperatively add de novo methyl groups to both strands of DNA [38]. Incubation of a DNA fragment with Dnmt3a before Dnmt1 stimulates DNA methylation modifications while the inverse does not, suggesting that this relationship is mainly driven by changes in Dnmt3a [38], though follow-up studies have not been done to our knowledge. Therefore, it is possible that the changes in Dnmt1 transcript abundance in our study were a downstream effect of the changes in Dnmt3a transcript abundance.

We did not find effects of additional tactile stimulation on most of the thyroid hormone receptor signalling measures tested, which contrasts with the study results by Hellstrom et al. [12]; however, their study used male rat pups as subjects with one instance of additional tactile stimulation for 5 min. It is possible that the effects of tactile stimulation on T3 levels are transient and the deiodinase activity decreased over prolonged periods of tactile stimulation in our study. It is also possible that female rat pups respond differently to tactile stimulation compared with male pups, given that males and females can respond differently to natural variations in maternal care received [39].

We found that additional tactile stimulation was associated with a trend in decreased Oxt transcript abundance, though this did not correspond to changes in DNA methylation in the CHRE. We also found similar but nonsignificant decreases in Crf and Avp transcript abundance. There is some evidence that tactile stimulation can increase oxytocinergic neuron activity [40] and that variation in maternal care can be transmitted across generations through changes in the oxytocinergic system in female offspring [41,42]. However, other studies have shown that augmented maternal care and brief early life separations decrease oxytocin transcript abundance and oxytocin-positive neurons in the hypothalamus [43,44]. In addition, one study showed that the proximal thermotactile contact with the rat mother but not maternal licking increased oxytocin neuropeptide concentrations in the hypothalamus [45]. Therefore, the relationship between maternal care received and pup oxytocin appears to be complex, and variations in temperature exposure may be one confounding factor in these studies.

(b). Effects on DNA methylation and the role of thyroid hormone receptor

We hypothesized that changes in transcript abundance of Oxt and Dnmt3a would be mediated by differences in DNA methylation (in Oxt) and thyroid hormone receptor binding. We found that a decrease in Oxt transcript abundance in the repeated room temperature exposure condition corresponded to increased levels of DNA methylation and thyroid hormone receptor binding at the CHRE in the promoter region of oxytocin. There is some evidence that DNA methylation changes associated with variations in maternal care received occur as a consequence of differential transcription factor binding [5]. One possible explanation of our findings is that there is a repressive effect of unliganded thyroid hormone receptor on Oxt transcription. If unliganded by T3, thyroid hormone receptor can recruit repressors, including silencing mediator of thyroid and retinoic receptors (SMRT) and nuclear receptor corepressor 1 (NCOR1), and activate the histone deacetylase HDAC3 to silence gene transcription [46]. HDAC3 appears to mainly affect H3K9ac levels [47], which may explain the nonsignificant differences in H3K27ac observed in our study. However, no studies to our knowledge have found HDAC3 activity preceding DNA methyltransferase binding or DNA methylation modifications. These findings imply that higher DNA methylation at the oxytocin CHRE may occur as a consequence of unliganded thyroid hormone receptor binding, but future work is needed to elucidate this hypothesis more directly and to examine which thyroid hormone receptor isoforms are involved.

While repeated room temperature exposure decreased Dnmt3a transcript abundance, this was not associated with differences in thyroid hormone receptor binding in the regulatory TRE regions tested. We tested these two sites because of previous evidence of differential binding of thyroid hormone receptors in regulating Dnmt3a transcript in the developing mouse brain [14]. However, other TREs exist in the Dnmt3a gene and their responsiveness to thyroid hormone can be species-specific [14]. Future studies are needed to verify which Dnmt3a TREs would be most relevant for the transcription of Dnmt3a in the neonatal rat brain. In addition, it is possible that our sample size and therefore statistical power were not large enough to detect differences in thyroid hormone receptor binding at the Dnmt3a TREs.

(c). Limitations

We did not find differences in maternal care received throughout the first postnatal week between temperature groups, which does not appear to support previous literature on differences in maternal licking received immediately following reunion of the pups in neonatal handling studies [48]. While we did not observe long-term differences in maternal licking received or nursing between groups, maternal care received could still be a contributing factor to the thyroid hormone measures or transcript abundance of the neuropeptides tested in this study since our manipulations are proxies for maternal licking and maternal contact. In addition, we measured maternal care received by proportion of target behaviours observed over total observations, but it is unknown whether this may relate to the duration of these behaviours. It would be useful to observe maternal care received by live observations along with undisturbed video recordings in future work. It is also possible that the all-female litters used in this study may not reflect typical maternal care patterns in mixed litters as male pups typically receive more anogenital licking from their mother [23].

Another potential caveat is our use of nonstimulated pups exposed to nest temperature as our ‘unmanipulated’ control group. There could be other influential factors involved during the brief early life separations that we did not consider, such as exposure to a novel environment or the indirect effects of the separations on the rat mother's endocrine stress response [49]. Therefore, it would be important in future studies to compare the nonstimulated pups exposed to nest temperature with a group of pups that are sacrificed immediately at PND 7.

Finally, although we observed statistically significant differences between temperature exposure groups on DNA methylation at the oxytocin CHRE, the differences are modest (around 3.5%). However, a previous study in humans also reported modest differences in DNA methylation at the oxytocin promoter region [20]. Given that oxytocin is selectively expressed in neurons, this may reflect a change in the number of oxytocin-positive neurons in the PVN, as observed in other studies using neonatal handling [43,44,50], and therefore could be biologically relevant. Although the number of oxytocin-positive neurons cannot be inferred from qPCR data in the present study, this would be important to examine in future work.

5. Conclusion

Overall, our findings indicate that early life room temperature exposure may affect DNA methylation indirectly by changes in DNA methyltransferases and directly by modifications at specific gene loci (oxytocin CHRE) as a consequence of differences in thyroid hormone receptor signalling. There is accumulating evidence that the epigenome is dynamic and responsive to environmental exposures, but studies that have investigated the underlying mechanisms between environmental exposures and DNA methylation are limited. In addition, there is evidence that DNA methyltransferase recruitment is targeted based on transcriptional activity and transcription factor binding at specific gene loci [51]. Other studies have proposed that the effects of other steroid hormones are mediated by their effects on DNA methylation modifications, through either differential steroid receptor binding or DNA methyltransferase activity [52,53], typically in light of transcriptional activation. Our findings support the hypothesis that steroid hormones can affect the epigenome through both steroid receptor binding and DNA methyltransferase activity as well as induce transcriptional repression.

More broadly, these findings also indicate the importance of variations in temperature exposure in a variety of models of early life experience. Some studies separate rat pups at room temperature (e.g. [43,44]) or at thermoneutral conditions (e.g. [50]) but do not consider the effects of variations in temperature exposure on later-life phenotype. In addition, in human populations, preterm infants have challenges with thermoregulation similar to neonatal rat pups [54]. This population is also more likely to be diagnosed with congenital hypothyroidism than infants born at full term [55]. Studies on the effects of variations in temperature exposure on the human epigenome are currently limited [56] but would be important to consider in the future.

Supplementary Material

Supplementary Table and Figures
rspb20201991supp1.docx (380.9KB, docx)
Reviewer comments
rspb20201991_review_history.pdf (756.9KB, pdf)

Supplementary Material

Supporting Datasets
rspb20201991supp2.xlsx (25.4KB, xlsx)

Acknowledgements

We thank Iva Zovkic and Gilda Stefanelli for assistance with the chromatin immunoprecipitation, and the undergraduate research students for assistance with the behavioural experiments.

Ethics

All animal procedures were approved by the Local Animal Care Committee at the University of Toronto Scarborough and conformed to the guidelines of the Canadian Council on Animal Care.

Data accessibility

The datasets that support this research article can be accessed in the electronic supplementary material.

Authors' contributions

S.C.L. and P.O.M. designed the study. S.C.L. performed the rat pup manipulations and molecular work, and analysed the data. P.O.M. supervised the research. S.C.L. and P.O.M. wrote the manuscript.

Competing interests

We declare we have no competing interests

Funding

S.C.L. holds a Connaught Scholarship for International Doctoral Students at the University of Toronto. Funding for this study was provided by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to P.O.M.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table and Figures
rspb20201991supp1.docx (380.9KB, docx)
Reviewer comments
rspb20201991_review_history.pdf (756.9KB, pdf)
Supporting Datasets
rspb20201991supp2.xlsx (25.4KB, xlsx)

Data Availability Statement

The datasets that support this research article can be accessed in the electronic supplementary material.

Articles from Proceedings of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

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