EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE DEVELOPMENTAL BIOLOGY Copyright © 2021 The Authors, some Disruption of paternal circadian rhythm rights reserved; exclusive licensee affects metabolic health in male offspring via American Association for the Advancement nongerm cell factors of Science. No claim to original U.S. Government Works. Distributed Maximilian Lassi1,2, Archana Tomar1,2, Gemma Comas-Armangué1,2, Rebekka Vogtmann3, under a Creative Dorieke J. Dijkstra4, David Corujo5, Raffaele Gerlini1,2,6, Jonatan Darr1,2, Fabienne Scheid1,2, Commons Attribution Jan Rozman1,2,7, Antonio Aguilar-Pimentel1,2,6, Omry Koren8, Marcus Buschbeck5,9, License 4.0 (CC BY). Helmut Fuchs1,2,6, Susan Marschall1,2,6, Valerie Gailus-Durner1,2,6, Martin Hrabe de Angelis1,2,6,10, Torsten Plösch4†, Alexandra Gellhaus3†, Raffaele Teperino1,2* Circadian rhythm synchronizes each body function with the environment and regulates physiology. Disruption of normal circadian rhythm alters organismal physiology and increases disease risk. Recent epidemiological data and studies in model organisms have shown that maternal circadian disruption is important for offspring health and adult phenotypes. Less is known about the role of paternal circadian rhythm for offspring health. Here, we disrupted circadian rhythm in male mice by night-restricted feeding and showed that paternal circadian disrup- tion at conception is important for offspring feeding behavior, metabolic health, and oscillatory transcription. Mechanistically, our data suggest that the effect of paternal circadian disruption is not transferred to the offspring Downloaded from https://www.science.org on May 23, 2023 via the germ cells but initiated by corticosterone-based parental communication at conception and programmed during in utero development through a state of fetal growth restriction. These findings indicate paternal circadian health at conception as a newly identified determinant of offspring phenotypes. INTRODUCTION ZT that allows the SCN to synchronize the PCCs with the environ- Our lives are organized around 24-hour cycles, which include light and ment, but other environmental cues, such as feeding and stress, also dark phases. Organisms have developed an innate temporal program influence circadian rhythm by modulating the interaction between known as circadian rhythm that couples sleep-wake and fasting-feeding the SCN and the PCCs (3). Desynchronization between the PCCs and cycles with the light-dark cycle and allows the body to anticipate daily the environment is known as circadian disruption (6). environmental changes and coordinate physiological activities (1, 2). We live in a “24-hour society” with light and food available 24/7 In mammals, circadian rhythm is achieved through an internal and increasingly tight and stressful schedules, which interfere with clock. The hypothalamic suprachiasmatic nucleus (SCN) functions our normal rhythms and induce a certain degree of circadian dis- as a central oscillator, which is synchronized by the light-dark cycle ruption. Epidemiological studies and more controlled studies in (3). The SCN then sends humoral and neuronal signals to the periph- model organisms have associated circadian disruption with in- eral circadian clocks (PCCs), present in almost all tissues and organs creased risk to complex conditions such as metabolic, psychiatric, of the body (3, 4). At the molecular level, circadian rhythms are con- and oncological disorders (7–11). One open question remains on trolled by cell autonomous transcription/translation feedback loops whether the consequences of circadian disruption are limited to the comprising transcriptional activators (CLOCK/BMAL1), which tran- exposed generation or persist across generations. While a growing scribe their own repressors (PERs/CRYs/REV-ERBs) in 24-hour body of evidence suggests that maternal circadian disruption, either cycles (5). While these cycles persist also in the absence of external before or during gestation, modifies offspring phenotypes (12–16), cues, circadian rhythm responds to environmental stimuli, known nothing has been published (to the best of our knowledge) on the role as Zeitgebers (ZTs) (or time giver) (3). Light is the most important of paternal circadian rhythm for offspring physiology and health. Glucocorticoids (GCs) are a potent internal ZT (17). Their endog- 1 enous secretion in mice and humans is characterized by a prominent Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health Neuherberg, Germany. 2German Center for Dia- and robust circadian oscillation with peak anticipatory of the active betes Research (DZD) Neuherberg, Germany. 3Department of Gynecology and phase (18, 19). Partial or complete loss of this rhythm is associated to Obstetrics–University Hospital Essen – Essen, Germany. 4University of Groningen, profound circadian disruption with alterations in multiple physio- University Medical Center Groningen, Department of Obstetrics and Gynecology, logical functions including energy metabolism, stress response, immu- Groningen, Netherlands. 5Cancer and Leukemia Epigenetics and Biology Pro- gram, Josep Carreras Institute for Leukemia Research (IJC) Badalona, Spain. 6German nity, and cognition (20–22). GCs are also essential for fetal development Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, (23, 24) and likely involved in fetal entrainment to the environment German Research Center for Environmental Health Neuherberg, Germany. 7Czech through placental-fetal communication (12). Exposure to high levels of Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, 252 50, Vestec, Czech Republic. 8Azrieli Faculty GCs during pregnancy predisposes offspring to metabolic and neuro- of Medicine, Bar Ilan University, Safed, Israel. 9Program for Predictive and Personal- logical diseases later in life, most likely through impaired placental ized Medicine of Cancer, Germans Trias i Pujol Research Institute (PMPPC-IGTP), function and induction of fetal growth restriction (FGR) (15, 25–31). 08916 Badalona, Spain. 10Chair of Experimental Genetics, School of Life Science With the aim of understanding the effects of paternal circadian Weihenstephan, Technische Universität München Freising, Germany. *Corresponding author. Email: raffaele.teperino@helmholtz-muenchen.de disruption on offspring health, we have used an established en- †These authors contributed equally to this work. vironmental model of circadian disruption in male mice—i.e., Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 1 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE night-restricted feeding (32, 33)—and analyzed offspring metabo- C57BL6/J control mating and wild-type offspring of ClockD19 fa- lism. Our data show that paternal circadian rhythm is important for thers (CLOCKWT) phenotyped against isogenic wild-type offspring offspring feeding behavior, glycemia, and oscillatory transcription of the control mating (CTRL). in liver and hypothalamus. Mechanistically, our data suggest that the effects of paternal circadian disruption are transmitted to the Metabolic phenotyping offspring via the seminal plasma. They further highlight a potential Monitoring of weight and feeding trajectories role for corticosterone in seminal plasma in the pathogenesis of FGR F1 mice were weaned at 3 weeks of age, caged in groups of four per and developmental programming of offspring metabolic health cage with F1 mice from the same experimental group, and kept on through transcriptional alterations in placenta and fetal tissues. ad libitum access to food and water. Their growth trajectories were monitored by biweekly measurement of body weight on a precision scale with two decimal places. METHODS Food intake was measured manually every 2 months for 2 weeks Animal housing by calculating the amount of food that was put in the cage minus the C57BL6/J mice (males and females) were purchased from Charles weight of the food pellets that were left in the cage at the end of the River Laboratories Germany (Sandhofer Weg 7, 97633 Sulzfeld) day. Daily food intake was indirectly calculated by dividing the net and housed in ventilated cages at constant temperature (22° ± 1°C) amount of food by the number of mice per cage per day. with moderate humidity (50 ± 5%). The mice had unlimited access Indirect calorimetry to water and, unless stated otherwise in the experiments, were fed Ten-month-old mice randomly picked from each experimental ad libitum on a 12-hour light-dark cycle from 6:00 a.m. to 6:00 p.m. group (10 to 12 per group) were singly housed in a home cage indi- All housing protocols and animal experiments were performed rect calorimetry system (TSE Systems). They were monitored over according to the Institutional Animal Care and Use Committee a 4-day period for 21 hours per day and fed an ad libitum chow diet. Downloaded from https://www.science.org on May 23, 2023 guidelines and the European Union directive 2010/63/EU and were Data from the first day were discarded to reduce variation intro- approved by the responsible authorities of the government of Upper duced by acclimatization. Data from consecutive days were treated Bavaria (Tierversuchsantrag no.: ROB-55.2-2532.Vet_02-17-33). as technical replicates, acquired, averaged, and graphed at 20-min All efforts were made to reduce the number of animals and mini- intervals for single mouse. Food consumption was measured direct- mize suffering by considerate housing and husbandry. All pheno- ly as cumulative data. To visually inspect animals’ alignment to the typing procedures were examined for potential refinements. Animal light-dark cycle, activity (number of beam breaks in the X and Y welfare was assessed routinely for all mice involved. dimension of an ActiMot cage frame) and energy expenditure (H3, For the generation of the founder-RF (restricted feeding) fathers kcal/hour) data was graphed as a heatmap for single mouse and as a (four per experimental cohort in three independent cohorts), 6-week- function of time. old C57BL6/J males were accustomed to the mouse facility for 1 week Blood collection and then put on a 30-day RF schedule with access to food limited to Blood was collected from the tail vein of 10-month-old mice around 12 hours from 6:00 a.m. to 6:00 p.m. Control F0 males (CTR) were the clock at 6-hour intervals (max. 50 l of blood per time point), fed ad libitum. After 30 days, F0 males were mated to age-matched and EDTA serum was used for determination of corticosterone and unexposed females on ad libitum feeding schedule. To reduce [enzyme-linked immunosorbent assay (ELISA), Abcam #108821, the male-female interaction at conception and limit the paternal according to the manufacturer’s instructions] and insulin (ELISA, influence on the mother, we timed the mating to the day-night tran- MSD #K152BZC, according to the manufacturer’s instructions). sition from ZT10 to ZT12 (or in the first hours of the day—from One drop of blood was directly used to measure blood glucose (us- ZT1 to ZT3—when requested) and removed the fathers after positive ing the Accu-Check Aviva glucometer and test stripes, Roche). vaginal plug inspection. To minimize variations in intrauterine and Intraperitoneal glucose tolerance test early postnatal environment, pregnant females from the same experi- Overnight-fasted 10-month-old mice were intraperitoneally inject- mental group were cohoused until weaning, and litters were restricted ed with glucose (1.5 g/kg of body mass), and blood glucose excur- to six pups per dam to avoid over/undernutrition during lactation. sions were monitored at 15- to 30-min intervals for 120 min (using Glucocorticoid receptor heterozygous (GRhet) mice were pro- the Accu-Check Aviva glucometer and test strips, Roche). vided by the group of S. Herzig at the Helmholtz Research Center in Terminal bleeding and tissue collection Munich and genotyped using custom primers (GRflox1: 5′-GGC Ten-month-old mice were anesthetized with a ketamine-xylazine mix ATG CAC ATT ACG GCC TTC T-3′/GRflox4: 5′-GTG TAG CAG and terminally bled by heart puncture at 6-hour intervals around CCA GCT TAC AGG A-3′) and an optimized polymerase chain the clock and at groups of three to four animals per time point. reaction (PCR) amplification protocol [95 to 30 s ➔ 35× (55 to 30 s Blood was collected in EDTA monovettes (Sarstedt), immedi- ➔ 72 to 60 s) ➔ 72 to 10 min]. For the generation of the GRhet ex- ately centrifuged in a 1.5-ml Eppendorf tube to separate the plasma perimental cohort, 10-week-old GRhet females (n = 6) were mated (10,000g, 4°C, 10 min), and either directly used or stored at −80°C to age-matched C57BL6/J males in parallel with a C57BL6/J control for downstream measurements. Liver, adrenal glands, and hypo- mating and wild-type offspring of GRhet mothers (GRWT) pheno- thalamus were collected in RNAlater (Thermo Fisher Scientific) typed against isogenic wild-type offspring of the control mat- and either directly used for RNA extraction (TRIzol reagent, Ther- ing (CTRL). mo Fisher Scientific) or stored for further analyses. Clock mutant mice (ClockD19) (34) were provided by the group of H. Oster at the University of Lübeck. For the generation of the Sperm and epididymal fluid collection Clock experimental cohort, 10-week-old ClockD19 males (n = 6) Sperm and epididymal fluid were collected from 10-week-old F0 were mated to age-matched C57BL6/J females in parallel with a mice. Briefly, the cauda epididymis was dissected on both sides, Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 2 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE placed in a 2-ml Eppendorf tube containing sperm motility medi- useshortkmers=t; mink=5; qtrim=r; trimq=10; minlength=20; um, and cut into small pieces using precision scissors. Sperm was ref=Illumina TruSeq adapter sequences, A18 sequence. isolated using a double swim-up procedure. Briefly, after 20 min of Heatmap and principal components analyses (PCAs) were per- incubation at 37°C, the sperm cloud was separated from the diluted formed with the web application ClustVis using default parameters epididymal fluid by centrifuging the mixture at room temperature (39). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for 1 min at 1000g and then by reincubating it at 37°C for 10 min. was performed with DAVID (Database for Annotation, Visualization This gave a floating layer of sperm on top of the epididymal fluid. and Integrated and Discovery) (40, 41) using default parameters. The sperm and epididymal fluid were separated and placed into Detection and analysis of oscillating transcripts were performed using new Eppendorf tubes. The diluted epididymal fluid was subse- JTK_CYCLE according to the developer’s instructions (42). Briefly, quently used for ELISA. Epididymal fluid is referred in the text as TMM-normalized read counts were obtained for each sample. To seminal plasma. filter the lowly expressed genes, we only included genes with an average TMM > 0.3 among replicates at all time points (ZT0 to ZT18). In vitro fertilization and embryo transfer The following settings were used for JTK_CYCLE analysis: jtkdist In vitro fertilization (IVF) and oocyte isolation were conducted (4,2), periods (4:4), jtk.init (periods, 6). A gene was observed as according to the standardized procedures of the INFRAFRONTIER cycling if the adjusted P value was below 0.05. The genes with same consortium (35, 36). Briefly, unexposed females were superovulated lag phase were grouped together, displayed as radar plot, and anno- with 7.5 U of pregnant mare serum gonadotropin and 7.5 U of tated using the DAVID pathway analysis tool. human chorionic gonadotropin before being sacrificed for oocyte collection. The oocytes were transferred into human tubal fluid (HTF) cDNA synthesis and real-time PCR analysis at 37°C and 5% CO2. Sperm was isolated from cauda epididymis as Total RNA was extracted using TRIzol reagent (Thermo Fisher described before (37). The sperm and the oocytes were cocultured Scientific), and 1 g was reverse-transcribed into cDNA using com- Downloaded from https://www.science.org on May 23, 2023 for 4 to 6 hours. Subsequently, the oocytes were transferred and in- mercially available kits (Applied Biosystems). Quantitative PCRs cubated overnight in high-calcium HTF culture medium at 37°C were performed on a QuantStudio 6 Real-Time PCR instrument and 5% CO2. Proper embryonic development was microscopically (Thermo Fisher Scientific). Postamplification melting curve analy- checked before embryo transfer to foster mothers. The two-cell em- sis was performed to check for unspecific products, and primer-­ bryos obtained were used for surgical bilateral oviduct transfer into only controls were included to ensure the absence of contamination. foster mothers as previously described (37). For normalization, threshold cycles (Ct values) were normalized to four different housekeeping genes (namely, Actin, Gapdh, 36b4, and RNA sequencing and data analysis Rplp0). The 2-Ct (Ct treated–Ct control) method was used to cal- Total RNA was prepared from liver (F0-ZT0 and F1-ZT0/6/12/18), culate fold enrichments. Primers were designed using qPrimerDepot. hypothalamus (F1-ZT0/6/12/18), adrenal glands (F1-ZT0/6/12/18), and placenta and fetal liver using the TRIzol reagent (Thermo Fisher Placenta histological analysis Scientific) according to the manufacturer’s instructions. RNA con- Quantitative image analysis of mouse placenta centration and integrity were controlled on a Bioanalyzer system For all placental analyses, formalin-fixed and paraffin-embedded (Agilent), and only RNA samples with RIN (RNA integrity num- samples were sectioned parallel to the mesometrial-fetal axis at 5 m ber) values > 7 were used for downstream applications. Sequencing and mounted on Superfrost Plus Slides (R. Langenbrinck, Em- libraries were prepared by using either the Nextera Library Prep Kit mendingen, Germany). Stained slides were scanned with the Aperio (Illumina) for the liver F0 samples or the QuantSeq 3′ mRNA-Seq CS2 ScanScope slide scanner (Leica, Wetzlar, Germany) at ×20 mRNA Library Prep Kit FWD for Illumina (Lexogen) with i7 in- magnification, and images were converted to TIFFs via Image Scope dexes (Lexogen) according to the manufacturer’s instructions. Li- (version 12.3.2.8013; Leica). Scanned slides were opened (plugin braries were sequenced on an Illumina HiSeq 2500 at 75 bp (F0) or “bioformats_package.jar.”) and analyzed using Fiji/ImageJ (43). Histo- 50 bp (F1/placenta/fetal liver) single-end, with a minimum output logical characterization of placenta, placental compartments (labyrinth of 40 to 50 million reads per sample. Read mapping and differential and spongiotrophoblast layer), and placental glycogen cells was per- expression analysis was performed using the A.I.R. (Artificial Intel- formed on three serial sections at three different parts (100-m interval) ligence RNA-Seq) software from Sequentia Biotech with the following in the proximity of the umbilical cord from each experimental group, pipeline: BBDuk (reads trimming: http://jgi.doe.gov/data-and- respectively. Total placental area was calculated by combining mea- tools/bbtools/bb-tools-user-guide/bbduk-guide/), STAR [reads map- surements of labyrinth and spongiotrophoblast area; differences in ping to the mouse genome GRCm38 (ENSEMBL): https://github. placental compartment composition were measured by the ratio of com/alexdobin/STAR], featureCounts (gene expression quantifica- labyrinth to spongiotrophoblast area as previously described (44, 45). tion: http://bioinf.wehi.edu.au/featureCounts/), and NOISeq [statisti- For morphometric analysis and general morphological evaluation, cal analysis of differentially expressed genes (DEGs): http://bioinfo. sections were stained with hematoxylin and eosin (H&E) and Masson-­ cipf.es/noiseq/doku.php]. Compared to other methods to calculate Goldner trichrome (MGT) staining. Placental glycogen stores were differential expression, NOISeq is a data adaptive nonparametric visualized and quantified using periodic acid–Schiff (PAS) reaction. method specifically designed to account for high variability across MGT staining replicates and genes with low expression levels (38). Before submit- For morphometric analysis, sections were stained with an MGT ting to A.I.R., raw reads obtained from Lexogen libraries were staining kit (#3459; Carl Roth GmbH, Karlsruhe, Germany) accord- trimmed for 10 bp from the left end using seqtk, and further trim- ing to the manufacturer’s protocol. Briefly, sections were deparaf- ming and quality control of reads were done using the bbduk.sh finized, rehydrated, incubated for 3 min with iron hematoxylin script from the BBMAP software using parameters k=13; ktrim=r; solution, and blued for 15 min in flowing tap water, followed by Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 3 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE 5-min Goldner’s stain I, 20-min Goldner’s stain II, and 10-min 30 days of restricted feeding (RF; food access during the inactive Goldner’s stain III with rinsing with 1% acetic acid solution in be- phase from 6:00 a.m. to 6:00 p.m.) (Fig. 1A). After an acclimation tween and followed by a standard dehydration procedure and phase of 2 to 3 days, RF mice consumed in 12 hours the same mounting in Xylene Mountant. MGT staining was used for mor- amount of food as CTR mice, which had ad libitum food access phometric characterization of muscle and collagen fibers. (fig. S1A). Also, the cumulative food intake over the 30-day period PAS reaction staining was not different between the groups (Fig. 1B) nor was their body Placental glycogen stores were detected with the PAS reaction. Sec- weight (Fig. 1C). In keeping with a day-only feeding regimen, RF tions were deparaffinized, rehydrated, incubated for 10 min with mice were hyperinsulinemic during the day (Fig. 1D) and overly 1% periodic acid (#HP00.1; Carl Roth), washed in tap water, incu- circadian disrupted as shown by the 24-hour corticosterone levels, bated for 20 min with Schiff’s reagent (#X900.1; Carl Roth), and which lose its classical peak at the beginning of the night phase treated 3 × 2 min with sulfite water (18 ml of 10% sodium-disulfite (Fig. 1, E and F), anticipatory of feeding and activity in nocturnal solution + 300 ml of distilled water + 15 ml of 1 M HCl) to reduce individuals (such as mice). Liver gene expression analysis at ZT0 unspecific PAS reaction. Quantification of PAS-positive area (%) (6:00 a.m.) revealed a profound transcriptional reprogramming within the spongiotrophoblast, as well as characterization of PAS-­ with more than 8000 DEGs (more than 2000 with an absolute positive cell within the labyrinth compartment, was performed fold change more than 0.5; fig. S1B) annotated to key pathways for without counterstain of the nuclei. metabolic control [such as the insulin, the adenosine monophosphate– activated protein kinase (AMPK), the mammalian target of rapamycin Statistical analysis (mTOR), and the peroxisome proliferator–activated receptor (PPAR) All figures and statistical analyses were generated using GraphPad signaling pathways among others] (fig. S1C and table S1). Also, anal- Prism 8 (San Diego, CA). Statistical significance was tested by ysis of a set of 50 core clock genes (as reported by the REACTOME Student’s t test or analysis of variance (ANOVA) when appropriate database) revealed complete transcriptional switch of the liver Downloaded from https://www.science.org on May 23, 2023 (see figure legends for individual cases). All data were expressed as circadian machinery (Fig. 1, G and H), confirmed by quantitative means ± SEM unless otherwise specified, and a two-tailed P value < 0.05 reverse transcription–PCR expression analysis of selected candidates with multiple comparison correction was used to indicate statistical (such as Cry1, Arntl/Bmal1, and Per2) over a 24-hour period (fig.S1, significance (* < 0.05, ** < 0.01, and *** < 0.001). D to F). Thus, 30 days of RF are sufficient to disrupt the circadian rhythm in male mice. RESULTS Paternal circadian disruption reprograms feeding behavior, Thirty days of night-restricted feeding disrupt circadian metabolic health, and oscillatory transcription in liver rhythm in male mice and hypothalamus of male offspring In keeping with published data on the effect of time-RF on circadian Ten-week-old, RF male mice (n = 4 in three independent experi- rhythm (32, 33), 6-week-old male C57BL6/J mice were subjected to ments) have been mated to isogenic, age-matched, and unexposed Fig. 1. Thirty days of night-restricted feeding disrupt circadian rhythm in male mice. (A) Experimental design of the circadian disruption by night-RF (RF) paradigm. (B and C) Cumulative food intake (B) and body weight trajectory (C) during the 30 days of night-RF (n = 26). (D) Circulating insulin levels after 30 days of night-RF (n = 4 to 6). (E and F) Circulating corticosterone levels (E) and rhythmicity (F) of corticosterone secretion after 30 days of night-RF (n = 4 to 8). (G) PCA representing the variance in the expression of core clock genes in liver at Zeitgeber 0 (ZT0). (H) Heatmap representation of the expression of core clock genes in CTRL and RF livers at ZT0. [***P < 0.001 (two-way ANOVA mixed effect model, time × experimental group.] Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 4 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE females to generate the F1 cohort. F1 males were born and devel- with the hyperphagia, F1-RF males also show higher metabolic rate oped normally, and weight monitoring from weaning to ~10 months [or carbohydrate oxidation rate as shown by the respiratory ex- of age showed normal growth curves and no significant difference change ratio (RER)] across the 24 hours (Fig. 2D), a mild but signifi- in body weight between the F1 groups (Fig. 2A). Unexpectedly, cant increase in energy expenditure selectively around ZT0 (Fig. 2E), monitoring of day and night food intake per cage in the same time- and remain hypoactive during the dark phase (Fig. 2F). Both energy frame revealed age-dependent disruption of feeding rhythm, with expenditure and locomotor activity are well in phase with the day- significant increase in day food intake in male offspring of RF fa- night transition (Fig. 2G), suggesting intact light-sensing mecha- thers (F1-RF) (fig. S2A). Indirect calorimetry-based analysis of en- nism and no overt alteration of circadian rhythm. F1-RF males are ergy metabolism in single animals (randomly selected from each also hyperglycemic (Fig. 2H) while remaining glucose tolerant as independent experiment as 1 per litter, i.e., n = 10 to 12) confirmed shown by normal and normally cycling circulating insulin levels overall hyperphagia in F1-RF males (Fig. 2, B and C). In keeping (fig. S2B) and intraperitoneal glucose tolerance testing (fig. S2C). Downloaded from https://www.science.org on May 23, 2023 Fig. 2. Paternal circadian disruption reprograms offspring feeding behavior, metabolic health, and oscillatory transcription in liver and hypothalamus. (A) Body weight trajectories. (B and C) Single animal cumulative food intake (B) and quantification of daily food intake (C). (D to G) Single animal respiratory exchange ratio (RER) (D), en- ergy expenditure (E), locomotor activity (F), and their heatmap representation (G). (H and I) Daily oscillations in circulating glucose (H) and corticosterone (I) levels. (J) Heatmap of the expression of genes involved in adrenal corticosterone biosynthesis. (K) Liver RNA-seq–based Nr3c1 expression. (L) Liver RNA-seq analysis of GR target genes from public- ly available GR Chip-seq datasets. (All expr., all expressed genes in the dataset.) (M to P) JTK_CYCLE analysis of liver (M) and (N) and hypothalamus (O) and (P) RNA-seq data (M) and (O). Radar plot presenting the circadian gene expression in liver (M) and hypothalamus (O). (N) KEGG pathway analysis of oscillating genes in liver from F1-C (gray bars), F1-RF (red bar), or both (blue bars) F1 groups. (P) KEGG pathway analysis of genes differentially expressed in the hypothalamus of F1 male mice at ZT0. (Q) Heatmap visualization of RNA-seq–based expression of neuropeptides in F1-C and F1-RF mice. (R to T) RNA-seq–based expression of selected differentially expressed neuropeptides in the hypothalamus at ZT0 (R) and ZT12 (T) (n = 3 biological replicates). (U) Quantification of average daily food intake around ZT0 (S) and during the night phase (U). Data from F1 male mice (n = 10 to 12 or n = 3 biological replicates/ZT for RNA-seq experiments). *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA mixed effect model, time × experimental group or two-tailed t test. ns, not significant; GABA, -aminobutyric acid; DCPA, Dopamine or Dihidroxphenylalanine; ccMP-PKG, cyclic-GMP (Guanine Mono Phosphate)-Protein Kinase G. Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 5 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE While not overly circadian disrupted, F1-RF males are hypercor- interestingly includes key components of orexigenic and anorexi- ticosteronemic (Fig. 2I) with significantly dampened rhythm (fig. genic signaling cascades (Fig. 2, P and Q). In particular, we found S2D) and out-of-phase expression of genes encoding for critical strong and robust up-regulation of key orexigenic neuropeptides, components of the corticosterone (GC) biosynthetic machinery in such as Npy and Hcrt (precursor of the two major orexins 1 and 2) the adrenal glands (e.g., StAR, Cyp11a1, and Cyp11b1) (Fig. 2J and and down-regulation of anorexigenic neuropeptides, such as Cartpt, fig. S2E). In keeping with these results, liver expression of the main Bdnf, and Pomc (Fig. 2Q and table S3). These differences occur ex- GR Nr3c1 in F1-RF males shows a shifted surge from ZT12 to ZT18 clusively at ZT0 (Fig. 2, R and T)—when nocturnal animals stop and a significant over expression at ZT0 (Fig. 2K) in line with the eating—suggesting prolonged and disrupted feeding cycles in F1- expression of GR target genes—extracted from the analysis of pub- RF males. Quantification of food intake in the different phases of licly available GR-Chip-seq datasets (46)—which are globally and sig- the day revealed that—albeit overall hyperphagic (Fig. 2C)—F1-RF nificantly up-regulated at ZT0 and down-regulated at ZT12 (Fig. 2L). males consume significantly more food at the night-day transition The effects of paternal circadian disruption are only partially (around ZT0) and in the first hours of the day (Fig. 2, S and U). detectable in female offspring. Despite no difference in feeding be- In keeping with the altered overall oscillatory transcription, fur- havior (fig. S2F), metabolic rate (fig. S2G), and energy expenditure ther analysis of a set of 50 core-clock genes (as reported by the (fig. S2H), female offspring of circadian disrupted fathers are hypo- REACTOME database) in liver and hypothalamus confirmed tran- active (fig. S2I) and interestingly lighter (fig. S2J) compared to offspring scriptional alterations of the clock machinery in both tissues (fig. of control fathers. Light-entrained circadian rhythm is maintained S3, H to J). In particular, unsupervised clustering of the core-clock (fig. S2K), although F1-RF females show significant reduction in geneset identified three distinct clusters in both liver and hypothal- corticosterone levels and dampened 24-hour rhythm (fig. S2, L and amus. In liver, cluster 1 includes day-expressed genes (e.g., Nr1d1; M). Furthermore, despite being hyperglycemic and hyperinsulinemic fig. S3, H and I) with a delayed transcriptional peak in F1-RF males; during the active phase (fig. S2, N and O), they remain glucose tol- clusters 2 and 3, instead, include genes whose transcription mostly Downloaded from https://www.science.org on May 23, 2023 erant as shown by normal glucose tolerance testing (fig. S2P). While changes at the day-night transition (e.g., Dbp, Clock, and Per2; fig. interesting, these results are in line with recently reported findings S3, H and I), showing either a dampened transcriptional rhythm showing profound sexual dimorphism in phenotypic traits (47), (e.g., Dbp and Clock) or a delayed peak (e.g., Per2) in F1-RF males. susceptibility to parental effects (48, 49), and circadian rhythm (50). In the hypothalamus, instead, the two major clusters are defined by To reduce the overall number of animals used in the study, we de- genes up- or down-regulated mainly at ZT0 (cluster 1, up-regulated, cided to mechanistically follow up the feeding behavior phenotype, including Nr1d1; cluster 3, down-regulated, including Nr3c1 and as the strongest and most sexual dimorphic; therefore, the rest of Per2) with a third cluster (cluster 2) of unaffected genes, which in- the results have been generated using exclusively male offspring. cludes Clock (fig. S3, J and K). These findings indicate that disruption of paternal circadian The intergenerational effects of paternal circadian disruption are rhythm reprograms feeding behavior and metabolic health in male phenocopied by paternal genetic alterations in the core-clock gene offspring. While not overly circadian disrupted, offspring of circadian Clock (fig. S4A). Wild-type offspring of heterozygous ClockD19 mutant disrupted fathers show altered GC rhythm and GR signaling with a sig- fathers (CLOCKWT) are hyperphagic despite a significantly reduced nificant shift toward day expression of GR target genes in the liver. body weight (fig. S4, B to D), show a higher metabolic rate (fig. S4E), To understand whether the altered GC rhythm in F1-RF males is and are hypoactive with reduced overall energy expenditure (fig. S4, F associated with a global alteration of oscillatory transcription, we and G). Also, these animals appear to have a normal light-entrained performed liver RNA sequencing (RNA-seq) around the clock and circadian rhythm (fig. S4H) while showing dampened GC rhythm (fig. used JTK_CYCLE to identify transcripts with robustly oscillating S4I) and reversed glucose oscillatory behavior (fig. S4J). Thus, envi- expression profiles (adj. P value < 0.05; table S2). We found a global ronmental or genetic disruption of paternal circadian rhythm repro- transcriptional shift of approximately 6 hours in F1-RF liver (F1-C grams feeding behavior, metabolic health, and corticosterone rhythm peak ~ZT18; F1-RF peak ~ZT0) (Fig. 2M), with more than 2000 in male offspring, with transcriptional alterations of oscillatory genes transcripts losing or gaining rhythmicity (fig. S3A), and 1390 tran- and core clock components in liver and hypothalamus. scripts maintaining rhythmicity with an overall 6-hour shift in peak expression (fig. S3, A and B). Functional annotation of differentially The intergenerational effects of paternal circadian oscillating transcripts (Fig. 2N) and of DEGs at ZT0 (fig. S3, C and disruption are associated with placental and developmental D) shows enrichment for genes involved in glucose, lipid and signatures of FGR amino acid metabolism, and mitochondrial oxidative capacity, Our results so far point to an important function of paternal cir- suggesting altered liver adaptation to daily fast/feeding cycles in cadian health at conception for offspring metabolic and circadian F1-RF males. homeostasis. Although the consequences of paternal Clock muta- To further understand the hyperphagia and circadian pheno- tion (fig. S4A) would suggest a direct involvement of the core clock types characterizing F1-RF males, we profiled hypothalamic tran- system in the intergenerational transmission, the same system does scription around the clock by RNA-seq and used JTK_CYCLE to not seem to be relevant in the male germ cells as the expression identify oscillating transcripts. We observed a profound alteration of key core-clock components (namely Cry1, Arntl/Bmal1, and of rhythmic transcription in the hypothalami of F1-RF males. Oscil- Per2) does not oscillate in 24 hours and is not affected by the RF lating hypothalamic transcripts peak around ZT18 in F1-C and at (fig.S4, K to M). ZT0 in F1-RF males (Fig. 2O, fig. S3E, and table S3) with a low On the other hand, according to the Developmental Origins of number of genes maintaining rhythmicity (198; fig. S3F). Function- Health and Disease (51) hypothesis, most of the individual metabolic al analysis of DEGs at ZT0 (fig. S3G) shows significant enrichment phenotypes are determined in utero. In particular, exposure to a hostile for lipid metabolism and adipocytokine signaling pathways, which uterine environment has been associated—in humans and model Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 6 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE organisms—with adult hyperphagia and late onset metabolic disorders, indicated that DEGs are strongly associated to placental alterations due to impaired placenta development and function (52, 53). To found in mammalian models of FGR (Fig. 3C and table S4) (54, 55). understand whether the effects of paternal circadian disruption on Despite the pronounced molecular signature of FGR, F1-RF placenta offspring health could have been programmed in utero, we generated and fetuses did not show overt developmental and morphological an independent cohort of F0 males, mated them to age-matched and changes apart from a mild reduction in placental efficiency (~10%, unexposed females as previously described, and analyzed male n = 19 to 20), measured as the ratio between fetal and placental weight placenta and fetal liver transcriptomes at Embryonic Day 18.5 (E18.5). (Fig. 3, D and E, and fig. S5, A to F) (45). The FGR-associated KEGG In placenta, RNA-seq revealed more than 6500 DEGs (more than and MGI terms are also significantly enriched among the circa 8000 1500 with an absolute fold change of more than 0.5) between F1-RF DEGs (3000 with an absolute fold change of more than 0.5) between and F1-C male placentas (Fig. 3A and table S4), which cluster to F1-RF and F1-C fetal livers (Fig. 3, F to H; fig. S5G; and table S5), pathways of cellular senescence (p53, insulin, and PI3K-Akt), hypoxia indicating that, albeit uncoupled from morphological phenotypes, and oxidative stress [Forkhead Box O pathway (FoxO), hypoxia-­ the placental FGR signature is sensed by the developing fetus. inducible factor (HIF), and vascular endothelial growth factor (VEGF)], Intriguingly, we also found significant and specific enrichment and energy deprivation (AMPK) and include genes critical for endo- of the circadian rhythm KEGG pathway in fetal liver (including cytosis and endoplasmic reticulum (ER) stress (Fig. 3B, fig. S5G, core clock genes as Per1-3, Cry1, Clock, Bmal1, Nr1d1, and Rora) and table S4). Further interrogation of the MGI (Mouse Genome (fig. S5H), suggesting in utero programming of adult circadian Informatics) database for mammalian gene/phenotypes associations rhythm as well. While few of the clock genes are also differentially Downloaded from https://www.science.org on May 23, 2023 Fig. 3. Paternal circadian disruption reprograms placenta and fetal liver transcriptomes. (A to C) Cake plot visualization of DEGs in placenta from F1-C and F1-RF male mice (A) (n = 4 biological replicates per group isolated between ZT2 and ZT4) and their functional annotation using KEGG pathway analysis (B) or the MGI phenotype database (C). D, Decidua; S, Spongiotrophoblast; L, Labyrinth. (D) Quantification of F1 placenta and fetal weight and placental efficiency at E18.5. (E) Representative placenta H&E staining (representative images of n = 4 placenta per group). (F to H) Cake plot visualization of DEGs in fetal livers from F1-C and F1-RF male mice (F) (n = 4 biological replicates per group) and their functional annotation using KEGG pathway analysis (G) or the MGI phenotype database (H). (I and J) Co-DEGs in fetal and adult liver (ZT0) (I) (red dots indicate genes with absolute log2FC > 0.5) and their functional annotation using KEGG pathway analysis (J). FC, fold change. (K to M) Venn diagram visualization of the fraction of reported GR target genes differentially expressed in the placenta and/or the fetal liver of F1-RF versus F1-C male mice (K) and their func- tional annotation using KEGG pathway analysis (L) or the MGI phenotype database (M). (N) Schematic summary of our findings reporting paternal circadian disruption associated to a signature of FGR. Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 7 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE expressed in the placenta (fig.S5H), the pathway is not significantly to test the hypothesis that reduced corticosterone in the seminal enriched, and the same genes are only mildly differentially ex- plasma is important for the intergenerational consequences of pressed. Globally, placenta and fetal liver share 38% of the DEGs paternal circadian disruption, we generated a new cohort by breed- (fig. S5I), which increases to a maximum of 60% for DEGs belong- ing parental mice during the day (between ZT1 to ZT3) when corti- ing to the significantly enriched KEGG pathways (fig. S5I). Further- costerone levels in the seminal plasma were not significantly more, comparison of fetal and adult liver (ZT0) transcriptional different between CTR and RF males (Fig. 4, A and B). In keeping responses to paternal circadian disruption shows more than 2000 with our hypothesis, day breeding normalized the hyperphagia shared DEGs (768 with an absolute fold change of more than 0.5) (Fig. 4C), the hyperglycemia (Fig. 4D), and the corticosterone (Fig. 3I and table S5), which are significantly coregulated (Fig. 3I; (Fig. 4, E and F) phenotypes observed in F1-RF animals [replotted Spearman r = 0.28, P < 10−4) and cluster to glucose (Gck, G6pc, and in Fig. 4 (C to F) as control reference]. Idh1), lipid (Hmgcr, Acss2, Ces1d, Acacb, Ckmt1, Lpin3, and Ndufa4), To further support these findings, we used two additional ap- and amino acid metabolic pathways (Ido2 and Mthfr) (Fig. 3J, proaches (fig. S6): (i) We analyzed a new cohort of F1 males gener- fig. S5J, and table S5). These findings provide evidence of an FGR- ated via IVF, thereby excluding paternal factors other than germ like intrauterine environment as a consequence of paternal precon- cells (61) and therefore also seminal plasma and the corticosterone ceptional circadian disruption and suggest it as a potential mechanistic from the conception (fig. S6, box 1); (ii) We adopted a mouse genet- link to the observed offspring phenotypes. ics approach to reduce the GR expression at conception. We there- GCs and parental stress play an important role in the pathogen- fore used females heterozygous for the glucocorticoid receptor esis of FGR and programming of adult metabolic phenotypes (GRhet) and mated them to age-matched and unexposed males with (23, 25, 26). Also—relevant to our experimental question and model—­ normal and normally cycling corticosterone in the seminal plasma GCs are important in entraining circadian rhythms (17, 56, 57) (fig. S6, box 2). Should our hypothesis be true, heterozygous loss of and can potentially transduce the effects of circadian disruption maternal GR would mimic the intrauterine environment derived Downloaded from https://www.science.org on May 23, 2023 (19, 21). GR target genes (46) are globally down-regulated in the from paternal circadian disruption and phenocopy its effects—at placenta but not in the fetal liver of F1-RF males (fig. S5K), with least to a certain extent and independently from genetic inheri- more than 60% of them being differentially expressed either in the tance. As a consequence, wild-type offspring of heterozygous moth- placenta or in the fetal liver (20% with an absolute fold change of ers (GRWT) should be phenotypically distinct from isogenic control more than 0.5) and a 42% (1286 of 3039 genes) overlap between the animals (CTRL) and similar to F1-RF males. tissues (Fig. 3K). KEGG and genotype/phenotype association anal- IVF-generated animals (iF1) were born and developed normally, ysis of differentially expressed GR target genes revealed significant and weight monitoring from weaning to ~10 months of age showed enrichment of pathways associated to cellular metabolism [PPAR normal growth curves with slight, although significant, reduction in and tricarboxylic acid (TCA) cycle; Fig. 3L and fig. S5L1–2] and se- body weight in iF1-RF males (Fig. 4G). Unexpectedly, analysis of nescence (FoxO and p53; Fig. 3L and fig. S5L3–4) and highlighted a single animal food intake revealed that iF1 animals are hyperphagic genetic signature of FGR (Fig. 3M). at levels similar to F1-RF males with no difference between the Thus, disruption of paternal circadian rhythm might lead to the groups (Fig. 4, H and I), indicating that the germ cells were not observed offspring phenotypes through FGR and altered GC/GR sufficient to mediate the observed paternal effects. Further quantifi- signaling in the female tract (Fig. 3N). Two questions still remain to be cation of food intake in the different phases of the day and compar- answered: (i) What is the source of GC to the female? (ii) Is the altered ison to the same animals generated via natural conception showed GC/GR signaling sufficient to the intergenerational phenotype? that iF1 males are hyperphagic at the night-day transition and in the (Fig. 3N). first hours of the day with no difference between the groups and at levels similar to F1-RF males (fig. S7A). Monitoring of energy me- Corticosterone signaling at conception is important tabolism through indirect calorimetry showed no further difference for the effects of paternal circadian disruption between the groups apart from a minor but significant increase in on offspring phenotype energy expenditure in iF1-C males in line with the increased body Seminal plasma contains trophic factors for mature spermatozoa weight (fig. S7, B to D). As for F1 males generated via natural concep- and a battery of cytokines, hormones, and metabolites important tion, iF1 males also have an intact light-sensing mechanism (Fig. 4J). for fertilization, implantation, placentation, and pregnancy outcome Notably, despite a slight difference in basal glucose (Fig. 4K) and no (58). Almost a decade ago, seminal plasma has been proposed as a difference in circulating corticosterone levels between the iF1 groups potential vehicle of parental acquired information to the offspring (Fig. 4M), iF1 males have blood glucose and circulating corticoster­ (59–62). To test the hypothesis that seminal plasma could be the one levels comparable to F1-RF males (Fig. 4, L and N) and a source of GC to the female at conception, we measured corticoste- dampened corticosterone diurnal rhythm (Fig. 4O). rone concentration in seminal plasma of CTR and RF F0 males at Thus, factors in the seminal plasma are important for the effects different ZT. Corticosterone is indeed present in seminal plasma of paternal circadian disruption on offspring phenotype. Intrigu- (Fig. 4A) at concentrations comparable with those in serum (see ingly, by phenocopying paternal circadian disruption, the IVF ex- Fig. 1E for comparison), and its appearance oscillates within 24 hours periment also suggests that seminal plasma plays a protective role. with a peak at the beginning of the night phase (Fig. 4, A and B). Both findings are in keeping with the reduction of corticosterone Thirty days of night-RF severely dampened corticosterone rhyth- levels in seminal plasma of circadian disrupted males and therefore micity in seminal plasma with a significantly less pronounced support our hypothesis that corticosterone might be the mediat- peak at the day-night transition (Fig. 4, A and B). Since our main ing factor. experimental cohorts have been generated by breeding parental GRhet females are healthy, fertile, and develop normally. They mice around the day-night transition (see Methods for details) and show reduced Nr3c1 expression in the uterus (fig. S7E) and unaltered Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 8 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from https://www.science.org on May 23, 2023 Fig. 4. Corticosterone signaling at conception is important for the effects of paternal circadian disruption on offspring phenotype. (A and B) Corticosterone levels (A) and rhythmicity (B) in F0 mice seminal plasma (n = 8). (C to F) Total food intake (C), blood glucose (D), plasma corticosterone (E), and corticosterone surge at ZT12 (F) in F1-C and F1-RF males sired by parents mated at the beginning of the day (ZT1 to ZT3) or at the day-night transition (ZT10 to ZT12) (G) Body weight trajectories. (H and I) Single animal cumulative food intake (H) and quantification of daily total food intake compared to F1 male mice generated via natural conception (I). (J) Heatmap representation of single animals’ daily energy expenditure and locomotor activity. (K and L) Daily oscillations in circulating glucose levels (K) and comparison to F1 male mice generated via natural conception (L). (M to O) Daily oscillations in circulating corticosterone levels (M and O) compared to F1 male mice generated via natural conception (N). (P to R) Cake plot of DEGs in placenta (P) and their functional annotation using KEGG (Q) or the MGI phenotype database (R). (S) Body weight trajectories. (T and U) Single animal cumu- lative food intake and quantification of daily total food intake and food intake at the night-day transition (ZT0 to ZT3) (U). (V) Heatmap representation of single animals’ daily energy expenditure and locomotor activity. Data from IVF-generated F1 male mice (G to O) or GRWT (offspring of GRhet mothers) and CTRL male mice (P to X) (n = 10 two-way ANOVA mixed effect model, time × experimental group or two-tailed t test; n = 3 biological replicates for RNA-seq experiments). (W and X) Daily oscillations in circulating corticosterone (W) and glucose (X) levels. (Y) PCA-based visualization of the phenotypic distance between F1 male mice from GRhet mothers (GRWT), RF fathers (F1-RF and iF1-RF generated via natural conception or IVF, respectively), and their respective controls (CTRL, F1-C, and iF1-C). AUC, area under the curve; NC, natural conception. circulating corticosterone levels (fig. S7F). We mated GRhet females (Fig. 4Q and table S6), and highlight a signature of FGR also con- to age-matched and unexposed males and analyzed males’ placenta firmed by interrogating the MGI database for gene/phenotypes as- and fetal liver transcriptomes at E18.5, as well as the development of sociations (Fig. 4R and table S6). Nr3c1 is not differentially expressed adult metabolic phenotypes in male wild-type offspring of GRhet between GRWT and CTRL placenta (fig. S7G). Despite the tran- mothers (GRWT) compared to a cohort of isogenic control animals scriptional signature of FGR in GRWT placenta, morphological and (CTRL). Differential expression and pathway analysis of placenta developmental analysis did not show overt alterations apart from a RNA-seq revealed almost 8000 DEGs (almost 2000 with an absolute significant reduction in placental efficiency (~25%, n = 6 to 12) (fig. fold change of more than 0.5) (Fig. 4P and table S6), which cluster to S7, H and I). The FGR-associated KEGG and MGI terms are also pathways of cellular senescence (insulin and PI3K-Akt), hypoxia and significantly enriched among the circa 6000 DEGs (circa 1100 with cellular stress (HIF, FoxO, VEGF, and ER stress), and metabolic control an absolute fold change of more than 0.5) between GRWT and CTRL (AMPK, fatty acid metabolism, neurotrophin, and adipocytokine) fetal livers (fig. S7, J to L, and table S7). These findings resemble Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 9 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE what we had previously observed in placenta and fetal livers from rhythm in male offspring on normal light-dark cycle and ad libitum F1-RF males (see Fig. 3), supporting our hypothesis that interfering fed. The hyperphagia is mostly contributed by food consumption at with corticosterone signaling in the maternal tract would phenocopy—­ the night-day transition (around ZT0), in keeping with hypothalamic at least to a certain extent—the effect of paternal circadian disrup- transcriptional profiles at the same time point showing deregulation tion on placenta and fetal liver transcriptional programming. In of orexigenic and anorexigenic circuits. These phenotypes remind keeping with this, placenta and fetal livers from the two offspring of a developmentally programmed hyperphagia and susceptibility cohorts share almost 60% of the DEGs (fig. S7M), which again sig- to metabolic diseases as a consequence of in utero adverse condi- nificantly cluster to key pathways for FGR (FoxO, p53, VEGF, HIF, tions and FGR (52, 53, 65–68). Analysis of placenta and fetal liver and PI3K-Akt), endocytosis, ER stress, and metabolic control (fatty reveals a transcriptional signature of FGR in offspring of circadian acid metabolism, insulin, TCA cycle, and oxidative phosphoryla- disrupted fathers, which is interestingly uncoupled from overt tion) (fig. S7N). morphological and developmental alterations (placenta morphology We then generated an independent cohort of GRWT (from GRhet and placental and fetal weights are normal) but associated to re- mothers; see fig. S6) and CTRL males and metabolically pheno- duced placental efficiency and in line with the adult phenotypes. typed them. GRWT and CTRL males were born and developed nor- FGR is one of the reported determinants of HPA (Hypothalamic mally, and weight monitoring from weaning to ~10 months of age Pituitary Axis) axis activity in adulthood (65, 67), and offspring of showed normal growth curves and no significant difference in body circadian disrupted fathers are constantly hypercorticosteronemic, weight between the groups (Fig. 4S). Indirect calorimetry-based with partially blunted diurnal corticosterone rhythm, altered ex- analysis of energy metabolism in single animals showed overall pression of adrenal components of the corticosterone biosynthetic hyperphagia in GRWT males (Fig. 4, T and U). In keeping with the machinery, desynchronized expression of GR target genes in the liver, data from F1-RF males, quantification of food intake in the differ- and a remarkable down-regulation of the GR in the hypothalamus ent phases of the day revealed that—albeit overall hyperphagic— at ZT0 [which is essential to sense circulating corticosterone and Downloaded from https://www.science.org on May 23, 2023 GRWT males consume significantly more food at the night-day control HPA axis activity (69)]. Thus, paternal circadian rhythm transition (around ZT0) and in the first hours of the day (Fig. 4U). controls offspring metabolism possibly by changing the developing Despite no difference in metabolic rate (fig. S7O), GRWT males fea- environment in utero. ture a mildly but significantly higher energy expenditure (fig. S7P) in line with the mildly increased physical activity (fig. S7Q). GRWT Seminal fluid factors signal the status of paternal males also have an intact light-sensing mechanism (Fig. 4V) while circadian rhythm showing dampened corticosterone rhythm (Fig. 4W) and hypergly- Genetic and acquired information flows from parent to offspring cemia (Fig. 4X). Last, and in keeping with our original hypothesis, mostly via the gametes at conception. Lifetime environmental chal- IVF-generated (iF1-RF and iF1-C) and GRWT males are phenotypi- lenges indeed reprogram the epigenome in germ cells, and the epi- cally very similar to F1-RF males (Fig. 4Y). These findings provide genetic alterations are either transferred to the developing embryos evidence for a role of seminal plasma corticosterone in the intergen- or indirectly affect development and adult phenotypes (64). Almost erational consequences of paternal circadian disruption. Together, a decade ago, also seminal plasma—the acellular part of the semen—­ our results show that paternal circadian rhythm is important for has been proposed as a potential vehicle of parental acquired in- offspring feeding behavior and metabolic health, reinforce the role formation to the offspring (59–62). Here, we have performed IVF of seminal plasma in acquired inheritance, and propose corticoster­ experiments, which eliminate seminal plasma at conception and one as an important molecule for parental communication at con- therefore its influence on the maternal tract, to isolate the role of ception and offspring phenotype. germ cells in transmitting the effects of paternal circadian disrup- tion to the offspring. Unexpectedly, the results of this set of experi- ments indicated that IVF per se phenocopied paternal circadian DISCUSSION disruption with no difference between the experimental groups. Paternal circadian rhythm is important for offspring They further suggest that not only the germ cells are not sufficient metabolic health to mediate the effects of paternal circadian disruption but also that Circadian rhythm controls physiology, and disruption of normal some factor(s) in the seminal plasma possibly act as a phenotypic circadian rhythm alters whole body homeostasis and predisposes to on/off switch. several complex conditions, including metabolic, oncological, and Seminal plasma contains trophic factors for mature spermato- neurological disorders (8, 9, 11, 63). Parental health at conception zoa and a battery of cytokines, hormones, and metabolites impor­ or maternal health during pregnancy are important determinants of tant for fertilization, implantation, placentation, and pregnancy offspring development and adult health (64). Among a series of en- outcome (58). Components of the seminal plasma have a high turn- vironmental challenges affecting intergenerational health, recent over rate indicating a capacity to rapidly modulate fluid composi- reports have highlighted the relevance of maternal circadian rhythm tion in response to environmental challenges (70). Studies have for pregnancy success and offspring health in humans and model shown that seminal plasma has a primary immunomodulatory role organisms (13–16). So far, instead, nothing is known about the rel- on the female tract at conception, which facilitates female receptiv- evance of paternal circadian rhythm. Here, we have used a validated ity and embryo implantation, therefore improving pregnancy out- environmental model of circadian disruption through night-RF in come (58). Seminal plasma composition is altered in obese mice fathers and showed that paternal circadian rhythm is important and men (71–74), and the absence of seminal plasma at conception for offspring feeding behavior, glucose control, and oscillatory tran- modifies placenta structure and function and induces metabolic scription. As short as 30 days of night-RF in fathers is sufficient to phenotypes in the offspring (59). Our IVF data suggest that seminal induce hyperphagia, hyperglycemia, and disrupted corticosterone plasma is also important to control food intake, glycemia, and possibly Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 10 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE the activity of the HPA axis, as control male mice generated via IVF 4. S.-H. Yoo, S. Yamazaki, P. L. Lowrey, K. Shimomura, C. H. Ko, E. D. Buhr, S. M. Siepka, are hyperphagic, hyperglycemic, and hypercorticosteronemic. H.-K. Hong, W. J. Oh, O. J. Yoo, M. 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Pathol. 228, the assistance with the IVF experiments; and the group of S. Herzig at the Helmholtz Diabetes 554–564 (2012). Center and H. Oster at the University of Lübeck for providing the GRhet and the ClockD19 56. N. D. Horseman, C. F. Ehret, Glucocorticosteroid injection is a circadian zeitgeber animals. Funding: This work has been supported by grants of the German Diabetes Research in the laboratory rat. Am. J. Physiol. 243, R373–R378 (1982). Center (DZD NEXT grant 2019) and the Fritz Thyssen Stiftung (Az. 10.19.2.027MN) to R.T. and Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 12 of 13 EMBARGOED UNTIL 2:00 PM US ET WEDNESDAY, 26 MAY 2021 SCIENCE ADVANCES | RESEARCH ARTICLE by the ERC Recognition Award from the Helmholtz Research Center Munich to R.T. We thank Data and materials availability: Raw sequencing data are available at GEO under the the Helmholtz Association and the German Diabetes Research Center for funding the accession number: PRJNA71394. Analysis of RNA-seq experiments is reported in the positions of R.G., J.D., M.L., F.S., A.T., and R.T. M.B.’s and D.C.’s contribution to the project was supplementary tables. All other data needed to evaluate the conclusions in the paper are supported by the RTI2018-094005-B-I00 and ISCIII PIE16/00011 from FEDER/Ministerio de present in the paper and/or the Supplementary Materials. Additional data related to this paper Ciencia e Innovación–Agencia Estatal de Investigación (to M.B.). A.G.’s and R.V.’s contribution may be requested from the authors. to the project was supported by Mercator Research Center Ruhr (MERCUR) An-2015-0009, German Research Foundation (DFG) GE2223/2-1; Programm zur internen Submitted 19 January 2021 Forschungsförderung Essen (IFORES) project grant D/107-81240 (Medical Faculty, University of Accepted 2 April 2021 Duisburg-Essen) (to A.G.). Work in the German Mouse Clinic is supported by grants of the Published 26 May 2021 German Federal Ministry of Education and Research (Infrafrontier grant 01KX1012 to M.H.d.A.) 10.1126/sciadv.abg6424 and German Center for Diabetes Research (DZD) (to M.H.d.A.). Author contributions: R.T. conceptualized the project, supervised the experiments, analyzed and interpreted the results, Citation: M. Lassi, A. Tomar, G. Comas-Armangué, R. Vogtmann, D. J. Dijkstra, D. Corujo, wrote the manuscript, and acquired funds. M.L., G.C.-A., A.T., R.V., D.J.D., D.C., R.G., J.D., A.A.-P., R. Gerlini, J. Darr, F. Scheid, J. Rozman, A. Aguilar-Pimentel, O. Koren, M. Buschbeck, H. Fuchs, and F.S. performed the experiments and analyzed the results. J.R., O.K., M.B., H.F., S.M., V.G.-D., S. Marschall, V. Gailus-Durner, M. Hrabe de Angelis, T. Plösch, A. Gellhaus, R. Teperino, M.H.d.A., T.P., and A.G. supervised experiments, acquired funds, and reviewed and edited the Disruption of paternal circadian rhythm affects metabolic health in male offspring via nongerm manuscript. Competing interests: The authors declare that they have no competing interests. cell factors. Sci. Adv. 7, eabg6424 (2021). Downloaded from https://www.science.org on May 23, 2023 Lassi et al., Sci. Adv. 2021; 7 : eabg6424 26 May 2021 13 of 13 Disruption of paternal circadian rhythm affects metabolic health in male offspring via nongerm cell factors Maximilian Lassi, Archana Tomar, Gemma Comas-Armangu, Rebekka Vogtmann, Dorieke J. Dijkstra, David Corujo, Raffaele Gerlini, Jonatan Darr, Fabienne Scheid, Jan Rozman, Antonio Aguilar-Pimentel, Omry Koren, Marcus Buschbeck, Helmut Fuchs, Susan Marschall, Valerie Gailus-Durner, Martin Hrabe de Angelis, Torsten Plsch, Alexandra Gellhaus, and Raffaele Teperino Sci. Adv., 7 (22), eabg6424. DOI: 10.1126/sciadv.abg6424 Downloaded from https://www.science.org on May 23, 2023 View the article online https://www.science.org/doi/10.1126/sciadv.abg6424 Permissions https://www.science.org/help/reprints-and-permissions Use of this article is subject to the Terms of service Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science. 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).