Seasonal and temporal variation in the placenta during melatonin supplementation in a bovine compromised pregnancy model

Abstract

Compromised pregnancies result in a poorly functioning placenta restricting the amount of oxygen and nutrient supply to the fetus resulting in intrauterine growth restriction (IUGR). Supplementing dietary melatonin during a compromised pregnancy increased uteroplacental blood flow and prevented IUGR in a seasonal-dependent manner. The objectives were to evaluate seasonal melatonin-mediated changes in temporal alterations of the bovine placental vascularity and transcript abundance of clock genes, angiogenic factors, and nutrient sensing genes in 54 underfed pregnant Brangus heifers (Fall, n = 29; Summer, n = 25). At day 160 of gestation, heifers were assigned to treatments consisting of adequately fed (ADQ-CON; 100% NRC; n = 13), nutrient restricted (RES-CON; 60% NRC; n = 13), and ADQ or RES supplemented with 20 mg/d of melatonin (ADQ-MEL, n = 13; RES-MEL, n = 15). The animals were fed daily at 0900 hours until day 240 where Cesarean sections were performed in the morning (0500 hours) or afternoon (1300 hours) for placentome collections. In both seasons, we observed a temporal alteration of the core clock genes in the cotyledonary tissue in a season-dependent manner. In the fall, ARNTL, CLOCK, NR1D1, and RORA transcript abundance were decreased (P ≤ 0.05) in the afternoon compared to the morning; whereas in the summer, ARNTL, PER2, and RORA expression were increased (P ≤ 0.05) in the afternoon. Interestingly, in both seasons, there was a concomitant temporal increase (P ≤ 0.05) of cotyledonary blood vessel perfusion and caruncular melatonin receptor 1A transcript abundance. Melatonin supplementation did not alter the melatonin receptor 1A transcript abundance (P > 0.05), however, in the summer, melatonin supplementation increased cotyledonary VEGFA, CRY1, and RORA (P ≤ 0.05) transcript abundance. In addition, during the summer the placentomes from underfed dams had increased average capillary size and HIF1α transcript abundance compared to those adequately fed (P ≤ 0.05). In conclusion, these data indicate increased cotyledonary blood vessel size and blood distribution after feeding to better facilitate nutrient transport. Interestingly, the maternal nutritional plane appears to play a crucial role in regulating the bovine placental circadian clock. Based on these findings, the regulation of angiogenic factors and clock genes in the bovine placenta appears to be an underlying mechanism of the therapeutic effect of dietary melatonin supplementation in the summer.

Changes in placental vascularity were observed in a seasonally dependent manner, whereas dietary melatonin ameliorated vascular anomalies during the summer months. In addition, the maternal nutritional plane appears to play a crucial role in regulating bovine placental circadian clock genes.

Introduction

Placental insufficiency negatively impacts approximately 8% of all pregnancies and is responsible for 60% of the intrauterine growth restriction (IUGR) observed in humans (Ghidini, 1996; Limesand and Rozance et al., 2017). A poorly functioning placenta restricts oxygen and nutrient supply to the fetus resulting in fetal nutrient deficiency and IUGR. This IUGR drastically increases neonate mortality by nearly 3-fold (Bernstein et al., 2000) and surviving offspring are predisposed to metabolic diseases (Barker, 2004). Therefore, the proper functioning of the placenta plays a pivotal role in the developmental origins of health and diseases. Placental functional capacity is dependent on size, nutrient transporter abundance, and blood supply (Fowden et al., 2006) which are negatively impacted in compromised pregnancies. Therefore, evaluating therapeutics that support a healthy placenta and prevent IUGR in livestock is essential to reduce offspring morbidity and mortality and prevent economical losses.

Melatonin, an amino acid-derived hormone synthesized by the pineal gland crosses the placenta protecting cells from oxidative stress (Tamura et al., 2008). Besides being an endogenous circadian rhythm modulator, melatonin acts as an antioxidant, scavenging for reactive oxygen species, leading to an increase in nitric oxide bioavailability (Bonnefont-Rousselot et al., 2011). Moreover, melatonin differentially alters blood flow by binding to MTNR1A or MTNR1B (melatonin receptors) on vascular smooth muscle cells to cause vasoconstriction or vasodilation, respectively (Doolen et al., 1998; Cook et al., 2011). Previous research has shown that melatonin supplementation improved placental efficiency and restored birth weights in the undernourished pregnant rats by increasing expression of placental antioxidant enzymes (Richter et al., 2009). In addition, melatonin exerts different functions of neovascularization and anti-angiogenesis depending on physiological and pathological conditions (Ma et al., 2020). Recently, our group reported a seasonal effect of dietary melatonin supplementation in cattle during late gestational maternal nutrient restriction (Contreras-Correa et al., 2021). During the fall, underfed dams exhibited decreased uterine blood flow and fetuses with reduced body weight, while melatonin supplementation did not seem beneficial. Nevertheless, dietary melatonin supplementation during the summer to underfed dams increased total uterine blood flow and prevented IUGR in cattle (Contreras-Correa et al., 2021). Undoubtedly, melatonin’s antioxidant properties along with the ability to regulate vasoconstriction and vasodilation of vascular smooth muscle and endothelial cells might be of great interest to explore for potentially improving placental function and controlling placental circadian rhythms.

Circadian rhythms are influenced by environmental factors such as light, feeding time, stress, and temperature allowing the organisms to anticipate metabolic and physiological requirements facilitating their adaptation to daily needs (Gamble et al., 2014). For years, circadian rhythms were thought to be controlled only by the suprachiasmatic nuclei of the hypothalamus (SCN); but more recently researchers found the existence of semiautonomous clocks located in peripheral tissues throughout the organism and these oscillatory clock genes have shown to function in a tissue-specific manner (Lowrey and Takahashi, 2004). The circadian clock regulates homeostasis, and disruption could cause metabolic disorders (Wharfe et al., 2011). Moreover, during pregnancy, the fetal pineal gland does not synthesize melatonin, thus the fetal SCN circadian rhythm is entrained by maternal melatonin (Yellon and Longo 1988; Torres-Farfan et al., 2006). Thereby, compromised pregnancies such as maternal undernutrition could potentially impact not only the placental function but also the fetal circadian system. Most circadian rhythm studies have been performed in rodents which are nocturnal animals making these findings unreliable for extrapolation to diurnal livestock species. Thus, the current study aimed to determine melatonin-mediated temporal changes in the bovine placental vascularity and gene expression of clock genes, angiogenic factors, and nutrient sensing genes during late gestational maternal nutrient restriction in a fall and summer trial.

Materials and Methods

Animals and sample collection

All experimental procedures were performed under appropriate protocols minimizing the amount of pain and discomfort experienced by animals. Furthermore, all procedures were approved by the Mississippi State University Institutional Animal Care and Use Committee (#17-709) in compliance with institutional and national guidelines. Animal breeding, handling, diets, and treatments were previously published (Contreras-Correa et al., 2021). Briefly, 54 pregnant heifers were enrolled in the study for dietary treatments and supplementation from September to December 2019 (fall; n = 29) and June to September 2020 (summer; n = 25). At day 160 of gestation, animals were assigned to one of four treatment groups consisting of adequately fed (ADQ-CON; 100%; NRC, 2000) global nutrient restricted (RES-CON; 60%), adequately fed supplemented with 20 mg/day of melatonin (ADQ-MEL), and nutrient restricted supplemented with 20 mg/d of melatonin (RES-MEL). Melatonin (#14427; Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in absolute ethanol at a concentration of 10 mg/mL and 2 mL were top dressed in the vitamin mix grain, whereas the control groups received 2 mL of absolute ethanol as a placebo. The animal number distribution per treatment for the fall trial was ADQ-CON (n = 7), RES-CON (n = 7), ADQ-MEL (n = 7), and RES-MEL (n = 8). For the summer trial, the animal number distribution per treatment was ADQ-CON (n = 6), RES-CON (n = 6), ADQ-MEL (n = 6), and RES-MEL (n = 7). Treatments were administered once daily at 0900 hours and after total consumption (around 15 min) a TMR diet was offered. Maternal and fetal concentrations of melatonin in the blood were previously reported and dietary melatonin supplementation increased circulating concentrations of melatonin above physiological levels (Contreras-Correa et al., 2021).

At day 240 of gestation, 80 days post-treatment initiation, heifers underwent Cesarean sections for fetal removal and placentome collection from 0400 to 0600 hours (0500 hours; morning; AM) or 1200 to 1400 hours (1300 hours; afternoon; PM). Timepoints were selected to be in an average 4 h before and after feeding time. After fetal removal, two placentomes adjacent to the umbilical cord were collected. We have previously reported no differences in placentome blood perfusion or cotyledonary transcriptomics from small, medium, or large-sized placentomes within the same animal and approximated location (Reid et al., 2022). One placentome was submerged in cold 1 × phosphate buffered saline (PBS) and immediately transported to the laboratory for placentome blood vessel perfusion (described below); while the middle portion of the second placentome was selected and sectioned into a 1- by 1-cm, placed in an embedding mold containing Optimal Cutting Temperature media (OCT; Fisher Scientific, Pittsburgh, PA) and frozen by submersion in super-cooled bath of 2-methylbutane and stored at −80 °C for further immunohistochemistry analysis. The remainder of the middle portion of the placentome was separated into the maternal caruncle and fetal cotyledonary villi, placed in cryogenic tubes, snap frozen in liquid nitrogen, and stored at −80 °C for further mRNA expression analysis. Vaginal temperatures, uterine artery blood flow, fetal morphometrics, and maternal and fetal blood levels of melatonin from these animals are reported in Contreras-Correa et al. (2021).

Placentome perfusion and macroscopic blood vessel density

Macroscopic blood vessel density was determined according to Lemley et al. (2018) with minor modifications. Briefly, the cotyledonary artery from an intact placentome was catheterized with a 20-g by 2-in. catheter (Exel Safalet Cath, Exelint International, Los Angeles, CA). After catheterization of the cotyledonary artery, placentomes were perfused with 1 × PBS to remove blood. The placentomes were then perfused with approximately 35 mL of 100-µg/mL of Concanavalin A, Alexa Fluor 647 conjugate (ThermoFisher Scientific, Waltham, MA) until the fluorophore was drained through the cotyledonary vein. Subsequently, the cotyledonary artery and vein were ligated with a silk suture to ensure a closed system and were immediately photographed with an in vivo imaging system, Lumina XRMS Series III (IVIS, PerkinElmer, Waltham, MA). The radiance signal (photon/s/cm²/sr) was determined from each sample using the region of interest function from the IVIS. A negative control placentome was imaged following PBS perfusion alone.

Placentome immunohistochemistry and microscopic blood vessel density

The immunofluorescence imaging of blood vessels was performed according to Lemley et al. (2018) with minor modifications. Briefly, placentomes were embedded in OCT molds and sectioned into 7 μm using a CRYOSTAR NX50 (Thermo Scientific, Waltham, MA), and placed on positively charged microscope slides. Two slides per cow were used and slides were blocked for nonspecific antigen binding using 10% goat serum in PBS containing 0.2% of Tween-20 for 30 min. Subsequently, a 1:50 dilution of rabbit polyclonal to Von Willebrand Factor (ab6994; Abcam, Cambridge, MA) was used as the primary antibody and incubated for1 h; followed by a 1:250 dilution of Goat Anti-Rabbit IgG H&L AlexaFluor 594 (ab150080; Abcam, Cambridge, MA) as the secondary antibody with an incubation time of 30 min. To contrast the caruncular crypts and cotyledonary villi, slides were incubated for 5 min with a 1:500 dilution of Fluorescein labeled Griffonia Simplicifolia Lectin I (FL-1101; Vector Laboratories, Burlingame, CA). The dilutions were made in 3% goat serum in PBS and slides were kept in humidified boxes protected from light sources. Lastly, slides were treated with Fluoroshield mounting medium with DAPI (ab104139; Abcam, Cambridge, MA) for nuclear staining. Six images per slide were captured using 10 × magnification with an EVOS microscope (AMAFD1000; Life Technologies, Carlsbad, CA). Ten to twelve representative images per animal were then analyzed by two technicians blinded to treatment groups using ImageJ (https://imagej.nih.gov/ij/download.html). Total capillary number per tissue area (vessel number/mm²), percent capillary area (%/mm²), average capillary size (μm²), and average capillary perimeter per tissue area (mm/mm² or mm⁻¹) were determined.

Placentome RT-PCR

This procedure was performed according to Contreras-Correa et al. (2020). Briefly, RNA was extracted from the caruncular and cotyledonary tissue by homogenizing approximately 25 mg of tissue in RLT Lysis buffer (QIAGEN, Hilden, Germany) and RNA was purified using RNeasy Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s recommendations. The quality of the total RNA extraction was assessed and quantified using a NanoDrop One spectrophotometer (Thermo Scientific, Waltham, MA). The High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Vilnius, Lithuania) was used to reverse transcribe 1 µg of total RNA into cDNA and stored at −20 °C. The quantifications of gene expression were obtained using TaqMan probe-based assays (Applied Biosystems, Pleasanton, CA) and a QuantStudio 3 real-time qPCR system (Applied Biosystems, Foster City, CA). A brief description of the target genes and housekeeping genes is provided in Table 1. Samples were run in duplicate and CT values were averaged and used for relative quantification using the 2 ^−ΔΔCT method. The geometric mean of the housekeeping genes was calculated and used to normalize the transcript abundance of target genes in the corresponding tissue. The housekeeping genes were previously shown to have stability in the bovine placenta (Contreras-Correa et al., 2020).

Table 1.

Assays, GenBank accession number, and amplicon length used for TaqMan probe-based real-time qPCR of bovine placentomes

	Genes	Assay ID	Accession	Amplicon	Significance
Circadian regulation	ARNTL	Bt04302511	NM_001191170.1	71	Molecular circadian transcription factor
	CLOCK	Bt04945311_m1	NM_001289769.1	66	Molecular circadian transcription factor
	CRY1	Bt03275639	NM_01105415.1	108	Inhibitor of ARNTL-CLOCK
	PER2	Bt04311404	NM_001192317.1	88	Glucocorticoid regulation
	RORA	Bt04298453_m1	NM_001192861.1	72	Transcriptional activator
	NR1D1	Bt03251200_m1	NM_001078100.2	72	Transcriptional repressor
	PPARα	Bt03220821_m1	NM_001034036.1	61	Nuclear receptor, lipid metabolism
	PPARɣ	Bt03217547_m1	NM_181024.2	85	Nuclear receptor, lipid metabolism
Angiogenic factors	HIF1α	Bt03259345	NM_174339.3	80	Transcriptional regulator of hypoxia
	ARNT	Bt01121917_m1	NM_173993.1	96	Co-factor for transcriptional regulation of HIF1α
	CITED2	Bt03247527_m1	NM_001075819.1	74	Inhibits transactivation of HIF1α-induced genes
	VEGFA	Bt02674021_m1	NM_174216.1	83	Proliferation and migration of vascular endothelial cells
	PGF	Bt03222872_m1	NM_173950.2	84	Placental growth factor
	NOS3	Bt03217682_g1	NM_181037.3	85	Endothelial nitric oxide synthase
	PTGS2	Bt03214492_m1	NM_174445.2	87	COX2 formation of prostanoids
	PF4	Bt03263741_g1	NM_001101062.1	104	Platelet factor 4
Nutrient sensing pathways	NAMPT	Bt04305356	NM_001244141.1	94	Rate limiting step in NAD + biosynthesis
	AKT1	Bt03212220_m1	NM_173986.2	57	Serine-threonine kinase 1, Amino acid sensing
	RPS6KB1	Bt03224957_m1	NM_205816.1	99	Regulates protein synthesis, cell growth and proliferation
	NR3C1	Bt04299834	NM_001206634.1	86	Glucocorticoid receptor
Melatonin receptors	MTNR1A	Bt07107181_s1	EU716172.1	88	Melatonin receptor 1A
	MTNR1B	Bt04304394_m1	NM_001206907.2	53	Melatonin receptor 1B
Reference genes	RPL19	Bt03229687	NM_001040516.1	82	Ribosomal protein L19
	SF3A1	Bt03254301	NM_001081510.1	60	Splicing factor 3a, subunit

ARNTL, aryl hydrocarbon receptor nuclear; CLOCK, circadian locomotor output cycles kaput; CRY1, cryptochrome circadian regulator 1; PER2, period circadian regulator 2; RORA, RAR-related orphan receptor alpha, NR1D1, nuclear receptor subfamily 1, group D, member 1; PPARα, peroxisome proliferator activated receptor alpha; PPARɣ, peroxisome proliferator activated receptor gamma; HIF1α, hypoxia inducible factor 1 subunit alpha; ARNT, aryl hydrocarbon receptor nuclear translocator; CITED2, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2; VEGFA, vascular endothelial growth factor A; PGF, placental growth factor; NOS3, nitric oxide synthase 3, PTGS2, prostaglandin-endoperoxide synthase 2; PF4, platelet factor 4; NAMPT, nicotinamide phosphoribosyltransferase; AKT1, AKT serine/threonine kinase 1; RPS6KB1, ribosomal protein S6 kinase B1; NR3C1, nuclear receptor subfamily 3 group C; MTNR1A, melatonin receptor 1A; MTNR1B, melatonin receptor 1B; RPL19, ribosomal protein L19; SF3A1, splicing factor 3a subunit 1.

Statistical analysis

Normal distribution was determined by the Shapiro-Wilks statistics of the UNIVARIATE procedure in the SAS software version 9.4 (SAS Institute, Cary, NC). Utilizing the same animals our previous work (Contreras-Correa et al., 2021) showed significant differences in uterine artery hemodynamics, vaginal temperatures, and fetal morphometrics between seasons (P ≤ 0.05); therefore, data were individually analyzed for Fall and Summer trials. The placental macroscopic blood vessel density, microscopic capillary density, and gene expression data were analyzed using ANOVA of the MIXED procedure of SAS. In addition, since the microscopic capillary number is a quantitative discrete variable, the GENMOD procedure of SAS was used to perform a Poisson regression. The model included the fixed effect of the nutritional plane (ADQ and RES), treatment (CON and MEL), time (AM and PM), and corresponding interactions. Means were separated using the PDIFF option of the LSMEANS statement. Statistical differences were considered significant if P ≤ 0.05. Data are presented as means ± pooled SE. Fetal sex was included in the model as a covariate; mean separation was not performed for fetal sex.

Results

Placentome vascularity

In Fall, the time of placentome collection within a day significantly impacted the fluorescent signal radiance. Representative images of cotyledonary macroscopic blood vessel density for morning and afternoon are illustrated in Figure 1A. The total fluorescent signal radiance relative to placentome weight was increased (P = 0.0124) in placentomes collected in the afternoon vs. placentomes collected in the morning (Figure 1B). When analyzing the microscopic blood vessel density, a nutrition by treatment by time interaction was found for average capillary size and capillary area (P ≤ 0.05; Figure 1C and D). The average capillary size (µm²) of the placentomes from RES-CON dams decreased from morning to afternoon (P = 0.0325). Moreover, in the afternoon, the collected placentomes from RES-CON dams exhibited decreased average capillary size compared to RES-MEL (48.57 ± 10.34 vs. 81.44 ± 10.34 µm²; P = 0.0353), while no differences were observed between ADQ-CON and RES-MEL for this variable (P = 0.9017). Similarly, the percent capillary area of the RES-CON placentomes were decreased from morning to afternoon (P ≤ 0.05), whereas in the afternoon the placentomes from RES-CON dams (2.76 ± 1.07 mm²) displayed reduced (P = 0.0325) percent capillary area compared to ADQ-CON (6.48 ± 1.13 mm²) and RES-MEL (6.20 ± 1.07 mm²).

Figure 1.

Placentome vascularity for fall supplementation trial. (A) Visual representation of negative control perfused with PBS, and placentomes collected in the morning (AM) or afternoon (PM) perfused with Concanavalin A, Alexa Fluor 647 conjugate. Main effects of nutrition, treatment, and time are presented for the (B) the fluorescent signal radiance relative to placentome weight (g). A nutrition by treatment by time interaction for average capillary size and percent capillary area are presented on (C and D), respectively. The AM represents in average 0500 hours and PM represents on average 1300 hours. Least squares mean with different letters represent a difference (P ≤ 0.05).

For the summer trial, representative macroscopic blood vessel density images are illustrated for each treatment group in Figure 2A. A nutrition by treatment by time interaction was observed for macroscopic blood vessel density on the relative fluorescent signal radiance (P = 0.0166; Figure 2B). The relative fluorescent signal radiance increased (P ≤ 0.05) in placentomes of RES-CON animals from morning to afternoon. Furthermore, the placentomes collected in the afternoon from RES-CON dams displayed increased fluorescent signal radiance when compared to ADQ-CON and RES-MEL placentomes (P ≤ 0.05), while no differences were observed between ADQ-CON and RES-MEL (P > 0.05). The placentomes from RES dams had increased average capillary size compared to ADQ (60.0 ± 4.02 vs. 48.0 ± 3.99 µm2; P ≤ 0.05), while the capillary number, area, and perimeter were similar amongst treatments. Representative images of microscopic blood vessel density are illustrated in Figure 3A–C.

Figure 2.

Placentome vascularity for the summer supplementation trial. (A) Representation of negative control perfused with PBS, and placentomes from all treatment groups collected in the morning (AM) or afternoon (PM) perfused with Concanavalin A, Alexa Fluor 647 conjugate. (B) A nutrition by treatment by time interaction for the signal radiance relative to placentome weight (g). (C) A main effect of nutrition was observed for average capillary size. The AM represents on average 0500 hours and PM represents in average 1300 hours. Least squares mean with different letters represent a difference (P ≤ 0.05).

Figure 3.

Immunofluorescence images of heifer placentomes at day 240 of gestation. Capillaries stained with Anti-Von Willebrand Factor (red, Alexa Fluor 594), caruncular epithelium (green, FITC), and nuclei (blue, DAPI). (A) Negative control treated without Anti-Von Willebrand Factor. (B) Fully stained image with all three fluorescent channels overlaid. (C) Texas Red channel used for analysis using ImageJ. The white scale bar represents 400 μm.

Caruncular mRNA transcript abundance

In Fall, nutrition by time interaction was observed for VEGFA (P = 0.0173; Figure 4A) where the mRNA transcript abundance was increased in ADQ animals from morning to afternoon, and in the afternoon, it was greater than the RES counterparts (P = 0.014). Additionally, a treatment by time interaction was found for PF4 (P = 0.0142; Figure 4B) where the mRNA transcript abundance of CON animals was increased from morning to afternoon and in the morning the MEL heifers had increased mRNA transcript abundance compared to the CON (P = 0.0401). A main effect of time was observed for CRY1, MTNR1A, NAMPT, and NR3C1 where they were all increased in the afternoon compared to the morning (P < 0.05; Table 2). No significant differences were observed for the remainder of the target genes (Table 2).

Figure 4.

Caruncular mRNA transcript abundance of clock genes and angiogenic factors for fall and summer trials. In the Fall trial, (A) a nutrition by time interaction was observed for VEGFA and (B) a treatment by time interaction was observed for PF4. In the Summer trial, (C) a nutrition by treatment by time interaction was found for MTNR1A and (D) a nutrition by time interaction was found for CRY1, PER2, PPARα, and RORA. The caruncular mRNA transcript abundance was normalized using SF3A1 and RPL19 as housekeeping genes. Least squares mean with different letters represent a difference (P ≤ 0.05).

Table 2.

Fall trial mRNA transcript abundance of target genes in caruncular tissue

	Nutritional plane			Treatment			Time			P-value
	ADQ	RES	SE	CON	MEL	SE	AM	PM	SE	Nut	Trt	Time
AKT1	1.06	1.11	0.06	1.07	1.11	0.06	1.06	1.11	0.06	0.564	0.626	0.534
ARNT	1.03	0.98	0.07	1.01	1.00	0.07	0.95	1.05	0.07	0.581	0.949	0.317
ARNTL	0.76	0.98	0.20	1.02	0.72	0.20	0.72	1.02	0.20	0.416	0.282	0.284
CITED2	0.65	0.57	0.08	0.72	0.50	0.08	0.68	0.54	0.08	0.464	0.057	0.206
CLOCK	0.93	1.44	0.29	1.15	1.22	0.30	0.95	1.42	0.30	0.221	0.867	0.256
CRY1	0.97	1.30	0.21	1.14	1.12	0.21	0.82	1.44	0.21	0.272	0.944	0.047
HIF1α	1.10	1.05	0.12	1.13	1.02	0.12	0.94	1.20	0.12	0.781	0.533	0.128
MTNR1	1.28	0.67	0.37	0.86	1.08	0.38	0.32	1.63	0.36	0.252	0.678	0.024
NAMPT	1.05	0.86	0.23	0.95	0.96	0.23	0.57	1.34	0.23	0.541	0.964	0.025
NOS3	0.76	0.85	0.13	0.74	0.87	0.14	0.85	0.76	0.14	0.619	0.493	0.653
NR1D1	0.57	0.80	0.16	0.69	0.67	0.16	0.69	0.68	0.16	0.323	0.934	0.961
NR3C1	1.35	1.32	0.19	1.53	1.14	0.19	1.06	1.61	0.19	0.933	0.161	0.049
PER2	1.05	1.16	0.17	1.14	1.08	0.17	1.02	1.19	0.17	0.617	0.799	0.475
PF4	1.17	0.95	0.23	–	–	–	–	–-	–	0.497	–	–
PGF	0.60	0.55	0.08	0.56	0.59	0.08	0.54	0.61	0.08	0.659	0.760	0.544
PPARα	1.30	1.20	0.11	1.06	1.43	0.12	1.40	1.09	0.12	0.528	0.033	0.074
PPARɣ	1.02	0.93	0.17	1.12	0.82	0.18	0.97	0.98	0.18	0.727	0.231	0.976
PTGS2	0.34	0.23	0.08	0.37	0.20	0.08	0.24	0.34	0.08	0.317	0.132	0.419
RORA	0.66	0.84	0.18	0.74	0.76	0.18	0.73	0.77	0.19	0.457	0.932	0.907
RPS6KB1	1.02	1.16	0.13	1.15	1.03	0.12	0.98	1.20	0.12	0.422	0.494	0.222
VEGFA	–	–	–	0.87	0.79	0.08	–	–	–	–	0.445	–

ADQ (n = 14): 100% nutritional recommendations; RES (n = 15): 60 % nutritional recommendations; CON (n = 14): 2 mL ethanol as placebo; MEL (n = 15): 20 mg/d of melatonin diluted in ethanol; AM (n = 15): 0500 hours (Morning); PM (n = 14): 1300 hors (Afternoon). Significant differences declared at (P ≤ 0.05).

In Summer, a nutrition by treatment by time interaction was observed for MTNR1A where the mRNA transcript abundance in RES-CON caruncle was increased from morning to afternoon (P < 0.0001; Figure 4C). Furthermore, in the afternoon, the MTNR1A transcript abundance in RES-CON was increased compared to the ADQ-CON, ADQ-MEL, and RES-MEL (P < 0.05). A nutrition by treatment interaction was observed for ARNT and PGF (P < 0.05; data not shown). The mRNA transcript abundance of ARNT was increased in ADQ-MEL compared to ADQ-CON (P = 0.046). Lastly, the mRNA transcript abundance of PGF was decreased in RES-MEL placentomes compared to ADQ-MEL. Interestingly, a nutrition by time interaction was found for the circadian rhythm modulators CRY1, PER2, PPARα, and RORA (P < 0.05; Figure 4D) where the mRNA transcript abundance of the aforementioned genes increased in the ADQ group from morning to afternoon whereas no temporal difference was observed in RES animals. After evaluating the main effects of nutrition and time, no significant differences were observed. Nevertheless, a main effect of time was observed for ARNT, HIF1α, NAMPT, and NR1D1 where they were all increased in the afternoon compared to the morning (P < 0.05; Table 3).

Table 3.

Summer trial mRNA transcript abundance of target genes in caruncular tissue

	Nutritional plane			Treatment			Time			P-value
	ADQ	RES	SE	CON	MEL	SE	AM	PM	SE	Nut	Trt	Time
AKT1	1.01	1.06	0.07	1.14	0.94	0.07	1.07	1.01	0.08	0.600	0.068	0.594
ARNT	–	–	–	–	–	–	1.17	1.46	0.10	–	–	0.049
ARNTL	1.06	1.05	0.22	1.04	1.07	0.22	0.84	1.27	0.23	0.973	0.929	0.180
CITED2	1.14	1.34	0.19	1.18	1.29	0.19	1.37	1.10	0.22	0.477	0.675	0.373
CLOCK	0.86	0.81	0.18	0.87	0.80	0.18	0.64	1.03	0.19	0.862	0.778	0.145
HIF1α	1.38	1.25	0.16	1.33	1.29	0.16	1.04	1.58	0.16	0.547	0.876	0.025
NAMPT	0.71	0.73	0.11	0.74	0.70	0.11	0.55	0.89	0.12	0.920	0.766	0.049
NOS3	1.10	1.21	0.20	1.10	1.21	0.21	1.30	1.00	0.20	0.692	0.705	0.301
NR1D1	0.49	0.30	0.13	0.49	0.29	0.14	0.17	0.61	0.14	0.324	0.297	0.049
NR3C1	1.11	0.97	0.13	1.03	1.05	0.13	0.86	1.22	0.14	0.434	0.924	0.080
PPARɣ	2.29	2.51	0.36	2.17	2.63	0.36	2.27	2.53	0.40	0.685	0.389	0.641
PTGS2	1.13	1.93	0.31	1.50	1.55	0.31	1.22	1.83	0.31	0.082	0.904	0.179
RPS6KB1	1.10	1.28	0.15	1.30	1.08	0.16	1.04	1.35	0.17	0.418	0.339	0.212
VEGFA	0.79	0.75	0.07	0.76	0.78	0.07	0.67	0.87	0.07	0.650	0.798	0.051

ADQ (n = 11): 100% nutritional recommendations; RES (n = 11): 60 % nutritional recommendations; CON (n = 11): 2 mL ethanol as placebo; MEL (n = 11): 20 mg/d of melatonin diluted in ethanol; AM (n = 12): 0500 hours (morning); PM (n = 10): 1300 hours (afternoon). Significant differences declared at (P ≤ 0.05).

Cotyledonary mRNA transcript abundance

In the fall trial, a nutrition by time interaction was observed for NR3C1 mRNA transcript abundance (P = 0.0004; Figure 5A) where it increased from morning to afternoon in ADQ animals, conversely, it decreased in the placentomes from RES dams. Moreover, during the morning, the NR3C1 mRNA transcript abundance in RES animals was increased compared to ADQ (P = 0.008). Conversely, the expression of NR3C1 in the afternoon was decreased in the RES compared to the ADQ (P = 0.006). A main effect of nutrition was found for PPARɣ where RES placentomes displayed a reduced mRNA transcript abundance compared to the ADQ (P = 0.029). A main effect of treatment was found in the RORA mRNA transcript abundance where MEL placentomes exhibited increased expression compared to the CON (P = 0.044). Lastly, the time of the day placentomes were collected influenced the mRNA relative expression of ARNT, ARNTL, CLOCK, NR1D1, PPARɣ, and RORA where all decreased from morning to afternoon (P < 0.05; Table 4).

Figure 5.

Cotyledonary mRNA transcript abundance of clock genes and angiogenic factors for fall and summer trials. (A) A nutrition by time interaction was observed NR3C1in the fall trial; while (B) a nutrition by time interaction was observed for CLOCK, CRY1, and VEGFA. The cotyledonary mRNA transcript abundance was normalized using SF3A1 and RPL19 as housekeeping genes. Least squares mean with different letters represent a difference (P ≤ 0.05).

Table 4.

Fall trial mRNA transcript abundance of target genes in cotyledonary tissue

	Nutritional plane			Treatment			Time			P-value
	ADQ	RES	SE	CON	MEL	SE	AM	PM	SE	Nut	Trt	Time
AKT1	1.30	1.30	0.14	1.17	1.43	0.14	1.41	1.20	0.15	0.978	0.199	0.326
ARNT	0.82	0.80	0.06	0.80	0.82	0.06	0.91	0.71	0.06	0.821	0.741	0.038
ARNTL	0.84	0.72	0.07	0.82	0.74	0.07	0.93	0.63	0.07	0.226	0.382	0.007
CITED2	1.14	1.04	0.06	1.13	1.05	0.06	1.16	1.02	0.06	0.231	0.350	0.125
CLOCK	0.59	0.46	0.06	0.51	0.53	0.06	0.62	0.43	0.06	0.106	0.762	0.031
CRY1	1.02	0.87	0.09	0.94	0.95	0.09	1.03	0.87	0.10	0.279	0.930	0.248
HIF1α	0.67	0.68	0.05	0.64	0.72	0.05	0.71	0.64	0.05	0.898	0.280	0.337
MTNR1	0.45	0.43	0.09	0.50	0.38	0.09	0.37	0.51	0.10	0.845	0.359	0.327
NAMPT	0.73	0.77	0.05	0.77	0.73	0.05	0.71	0.79	0.05	0.509	0.642	0.308
NOS3	1.71	1.85	0.27	1.42	2.14	0.28	1.92	1.64	0.28	0.722	0.072	0.481
NR1D1	0.73	0.57	0.10	0.56	0.74	0.10	0.84	0.46	0.10	0.281	0.213	0.018
NR3C1	–	–	–	0.60	0.54	0.03	–	–	–	–	0.223	–
PER2	1.85	1.69	0.32	1.37	2.17	0.32	2.02	1.52	0.34	0.720	0.097	0.299
PF4	0.18	0.13	0.02	0.15	0.17	0.03	0.16	0.15	0.03	0.139	0.584	0.842
PGF	0.54	0.67	0.12	0.54	0.67	0.12	0.56	0.65	0.13	0.461	0.475	0.610
PPARα	0.86	0.83	0.08	0.81	0.88	0.08	0.91	0.78	0.08	0.750	0.518	0.273
PPARɣ	0.82	0.64	0.06	0.78	0.67	0.06	0.84	0.62	0.06	0.029	0.183	0.015
PTGS2	1.00	0.87	0.11	0.82	1.04	0.11	0.78	1.08	0.11	0.380	0.159	0.083
RORA	0.95	0.65	0.13	0.60	0.99	0.13	1.08	0.51	0.13	0.114	0.044	0.007
RPBS6K1	0.60	0.62	0.05	0.67	0.55	0.05	0.64	0.58	0.05	0.722	0.079	0.403
VEGFA	0.56	0.56	0.06	0.56	0.55	0.06	0.49	0.62	0.06	0.997	0.933	0.171

ADQ (n = 14): 100% nutritional recommendations; RES (n = 15): 60 % nutritional recommendations; CON (n = 14): 2 mL ethanol as placebo; MEL (n = 15): 20 mg/d of melatonin diluted in ethanol; AM (n = 15): 0500 hours (morning); PM (n = 14): 1300 hours (afternoon). Significant differences declared at (P ≤ 0.05).

In the summer group, a nutrition by time interaction was found for CLOCK and CRY1 where the mRNA expression in the ADQ caruncle was increased from morning to afternoon, whereas no temporal difference was observed between the transcript abundance of RES animals (P < 0.05; Figure 5B). Additionally, the expression of VEGFA in ADQ animals remained constant throughout the day (P > 0.05), while it decreased in the afternoon in the RES counterparts (P = 0.0117; Figure 5B). A nutrition by treatment interaction was observed for CLOCK and CITED2 (P < 0.05; data not shown). Table 5 illustrates the main effects of nutrition, treatment, and time. A main effect of nutrition was observed for the mRNA transcript abundance of ARNT, HIF1α, and NR3C1 where it was increased in RES dams compared to the control (P < 0.05), whereas the expression of NOS3 was decreased in RES placentomes (P = 0.004). In addition, a main effect of treatment was found where the mRNA transcript abundance of AKT1, CRY1, RORA, and VEGFA was increased in MEL animals compared to CON counterparts (P < 0.05). Lastly, a main effect of time was found where the mRNA relative expression of ARNTL, PER2, and RORA was increased in the afternoon compared to the morning (P < 0.05); while the mRNA transcript abundance of NOS3 was decreased in the afternoon (P = 0.005).

Table 5.

Summer trial mRNA transcript abundance of target genes in cotyledonary tissue

	Nutritional plane			Treatment			Time			P-value
	ADQ	RES	SE	CON	MEL	SE	AM	PM	SE	Nut	Trt	Time
AKT1	1.29	1.01	0.18	0.84	1.45	0.18	1.01	1.28	0.20	0.296	0.030	0.351
ARNT	0.74	0.98	0.08	0.89	0.83	0.08	0.82	0.90	0.09	0.038	0.580	0.516
ARNTL	0.84	1.21	0.13	0.92	1.14	0.13	0.69	1.36	0.16	0.057	0.276	0.011
CITED2	0.55	0.74	0.15	0.57	0.72	0.15	0.56	0.73	0.17	0.392	0.478	0.492
CLOCK	–	–	–	1.53	1.76	0.22	–	–	–	–	0.446	–
CRY1	–	–	–	1.18	1.68	0.17	–	–	–	–	0.052	–
HIF1α	0.71	1.01	0.08	0.82	0.89	0.08	0.86	0.85	0.10	0.021	0.573	0.968
MTNR1	1.23	0.99	0.24	0.79	1.42	0.23	1.02	1.19	0.26	0.466	0.067	0.645
NAMPT	0.84	1.36	0.29	1.10	1.09	0.29	1.28	0.92	0.30	0.221	0.979	0.379
NOS3	1.50	0.72	0.16	1.07	1.15	0.15	1.51	0.71	0.16	0.004	0.693	0.005
NR1D1	1.65	1.28	0.20	1.37	1.56	0.20	1.43	1.50	0.21	0.221	0.518	0.799
NR3C1	0.63	1.22	0.19	0.92	0.93	0.19	0.88	0.96	0.19	0.042	0.962	0.765
PER2	1.37	1.36	0.22	1.48	1.25	0.22	0.88	1.85	0.23	0.960	0.437	0.005
PF4	3.00	2.93	1.05	1.59	4.35	1.12	4.35	1.59	1.13	0.963	0.083	0.110
PGF	0.74	0.59	0.14	0.55	0.77	0.14	0.80	0.53	0.15	0.461	0.297	0.217
PPARα	0.96	0.90	0.13	0.90	0.96	0.13	0.75	1.11	0.14	0.777	0.751	0.070
PPARɣ	0.34	0.67	0.16	0.50	0.51	0.16	0.34	0.67	0.18	0.157	0.974	0.217
PTGS2	0.30	0.39	0.13	0.37	0.31	0.13	0.34	0.35	0.14	0.639	0.743	0.938
RORA	1.41	1.39	0.15	0.96	1.84	0.15	1.16	1.64	0.16	0.939	0.001	0.039
RPS6KB1	0.54	0.81	0.12	0.74	0.61	0.12	0.57	0.78	0.13	0.130	0.436	0.259
VEGFA	–	–	–	0.80	1.56	0.14	–	–	–	–	0.003	–

ADQ (n = 11): 100% nutritional recommendations; RES (n = 11): 60 % nutritional recommendations; CON (n = 11): 2 mL ethanol as placebo; MEL (n = 11): 20 mg/d of melatonin diluted in ethanol; AM (n = 12): 0500 hours (morning); PM (n = 10): 1300 hours (afternoon). Significant differences declared at (P ≤ 0.05).

Discussion

In the current study, temporal changes in placentome vascularity and gene expression were evaluated during the fall and summer months in a compromised pregnancy model. In addition, pregnant heifers that were nutrient restricted received dietary melatonin supplementation as a therapeutic and we aimed to examine changes in bovine placental circadian rhythms. In the fall trial, when we compared the placentomes obtained in the morning (before feeding time) to the placentomes collected in the afternoon (after feeding time), the latter ones displayed greater cotyledonary macroscopic blood vessel density. These results indicate an increase in blood vessel size and blood distribution across the cotyledon, which better facilitates nutrient transport after feeding. In addition, the placentomes collected in the summer from RES-CON dams exhibited increased macroscopic blood vessel perfusion in the afternoon compared to the morning. Moreover, the fluorescent signal in the afternoon was increased in the aforementioned placentomes compared to the ADQ-CON and RES-MEL suggesting that underfed dams experienced a compensatory response to better facilitate nutrient transport across the cotyledon. Interestingly, in both seasons, there was a concomitant temporal increase of cotyledonary blood vessel perfusion and the caruncular melatonin receptor 1A transcript abundance (Figure 6A and B). Although physiological melatonin concentrations are low during the day in diurnal animals (3.40 to 14.73 pg/mL), its receptor can still be upregulated. Preeclamptic placentas have been found to have reduced expression of melatonin receptors in the placenta and preeclamptic pregnancies exhibit decreased blood levels of maternal melatonin, increased placental vascular resistance and increased oxidative stress (Lanoix et al., 2012). Therefore, melatonin supplementation could mitigate compromised pregnancies directly through melatonergic receptor-dependent pathways or indirectly by decreasing oxidative stress in placental vascular beds (Lemley and Vonnahme, 2017). The melatonin receptor 1A is a G-protein coupled receptor that when upregulated, generates contractile effects on vascular smooth muscle cells (Doolen et al., 1998). Therefore, it is possible that upregulation of the melatonin receptor 1A in the caruncle mediates maternal uterine vasoconstriction, signaling a greater need for fetal cotyledon blood perfusion, which was observed in the afternoon during both seasons. It is important to note that melatonin supplementation did not directly alter melatonin receptor 1A transcript abundance or the placentome vascularity, rather, it seems that time of feeding might be a controlling factor. Vollmers et al. (2009) reported that by manipulating timing of food intake the circadian gene expression in peripheral tissues is altered. Certainly, the timing of feed intake is a potent stimulus for peripheral circadian entrainment (Pickel and Sung, 2020) and from the current study, it is suggested that feeding time and/or nutritional plane plays a crucial role i in regulating the bovine placental circadian clock.

Figure 6.

Seasonal differences in placental circadian rhythms during melatonin supplementation in a bovine compromised pregnancy model. (A) Late gestational maternal nutrient restriction during the fall reduced uteroplacental blood flow and fetal weight, while melatonin supplementation did not rescue the aforementioned variables. (B) Melatonin supplementation in the summer increased uteroplacental blood flow and rescued fetal growth restriction. In both seasons there is a concomitant increased in MTNR1A and placentome perfusion. The AM sampling represents 0500 hours and PM represents 1300 hours. Feeding and melatonin treatments were offered daily at 0900 hours. (↑) represent increased and (↓) represent decreased.

Circadian clocks are known to influence embryonic development and the embryo interaction with its prenatal environment (Astiz and Oster, 2021). Even though the fetus is dependent on the maternal cues to entrain its own biological clock (Torres-Farfan et al., 2006), based on our previous placental explant study, the bovine maternal and the fetal clock appeared to be two independent systems (Contreras-Correa et al., 2020). The mammalian clock gene network exhibits a complex oscillatory feedback loop regulating thousands of transcripts related to physiology and metabolism. In both years, we observed a temporal alteration of the core clock genes in the cotyledonary tissue in a season-dependent manner, where in the fall the ARNTL, CLOCK, NR1D1, and RORA transcript abundance was downregulated in the afternoon compared to the morning, whereas in the summer where the ARNTL, PER2, and RORA expression was upregulated in the afternoon. The timing of feed remained the same in both seasons, but two factors that differed were daily light length and ambient temperature, which are reliable cues to synchronize the animal’s circadian clock to the external time (George and Stanewsky, 2021). In addition, melatonin supplemented dams in both seasons exhibited increased cotyledonary RORA mRNA expression compared to their control counterparts. Interestingly, Slominski et al. (2016) proposed that melatonin or its metabolites stimulate the transcription and translation activity of RORA, thus further implications on the placenta should be examined.

In models of compromised pregnancies, researchers have constantly observed decreased uteroplacental blood flow, which is vital for the maternal system to support the exponential fetal growth observed in the last third of gestation (Reynolds et al., 2010). There is strong evidence indicating that low birth weight due to fetal undernutrition is the origin of various metabolic diseases including cardiovascular diseases, hypertension, and diabetes (Barker, 1998). The nutrient-sensing gene, nicotinamide phosphorybosyltransferase (NAMPT), is responsible for the biosynthesis of NAD⁺which is needed for fuel oxidation and metabolism (Xu et al., 2020). It has been proposed that NAMPT modulates daily cycles of energy storage and utilization (Ramsey et al., 2009) along with lipid, glucose, and insulin metabolism (Reverchon et al., 2016). Morgan et al. (2008) observed an increase in NAMPT expression in the human placenta during pregnancy, particularly in the syncytiotrophoblast and fetal capillary endothelium which led the authors to suggest that NAMPT is associated with glucose transport between the maternal and fetal systems. In the present study, a significant temporal variation was observed in both seasons where the NAMPT transcript abundance was increased in the caruncle 4 h after feeding time. Similarly, using bovine placental explants, our group previously reported temporal variations of NAMPT transcript abundance in the maternal caruncle and fetal cotyledonary villi (Contreras-Correa et al., 2020). Besides NAMPT function in cellular status, it has been shown to have anti-apoptotic, pro-inflammatory, and pro-angiogenic properties (Dalamaga et al., 2018). Based on our fall and summer findings, it is suggested that NAMPT has an important role in the bovine placenta and further research should evaluate its contribution to nutrient transport and angiogenic regulation.

Using immunofluorescent techniques, during fall, the capillary size and capillary area from RES-CON dams were reduced in the afternoon compared to the morning. This may be a nutrient partitioning mechanism exerted by the underfed dams to retain more nutrients to maintain their central nervous system functioning along with their own growth and metabolism leading to fetal IUGR. Moreover, in the afternoon, the capillary size and capillary area from the RES-CON dams were reduced when compared to ADQ-CON and RES-MEL. A decreased capillary size and area may be reasonably interpreted as decreased blood flow to the placenta (Vonnahme et al., 2008). During the fall trial, we observed a decreased peroxisome proliferator-activated receptors-ɣ (PPARɣ) expression in the cotyledon from RES dams (Figure 6A). The PPARɣ plays a crucial role in lipid regulation and metabolism. It has been proposed that PPARɣ promotes placental growth and vascularization by increasing trophoblast differentiation and angiogenesis of fetal blood vessels (Hewitt et al., 2006). Moreover, recent research indicates that PPARɣ plays a pivotal role in regulating uteroplacental vascular development and that reduced placental PPARɣ expression results in pregnancy disorders such as IUGR and preeclampsia (Lane et al., 2019). Thus, it is possible that the decreased uteroplacental blood flow and the downregulation of cotyledonary PPARɣ expression might be an underlying mechanism by which maternal undernutrition resulted in placental insufficiency and IUGR during the fall. Additionally, during the fall, the RES dams exhibited limited temporal variation of caruncular VEGFA gene expression compared to ADQ dams suggesting that a disruption of the VEGFA circadian rhythm can negatively affect the placentome vascularity. Vascular endothelial growth factor is known to increase the number of blood vessels in the placental endometrial interface, facilitating blood flow and nutrient transfer capacity between the maternal and fetal systems (Wu et al., 2012). Thereby, alterations in the VEGFA gene expression can significantly disrupt the placental functional capacity.

Compelling evidence from the current study shows that melatonin supplementation in the summer increased the VEGFA transcript abundance in the cotyledon. In contrast, previous studies have reported that melatonin has antitumoral effects reducing the expression of HIF1α and VEGFA (Alvarez-Garcia et al., 2013; Cheng et al., 2019) and suppressing angiogenesis in tumor cells. However, existing data on melatonin inducing angiogenesis in reproductive organs have been contradictory. Thereby, melatonin exerts different functions of neovascularization and anti-angiogenesis depending on physiological and pathological conditions (Ma et al., 2020). In addition, in the summer, the average capillary size and the HIF1α mRNA transcript abundance were increased in RES placentomes compared to ADQ. This increase in the aforementioned variable at the placentome level could be a compensatory response to allow blood redistribution to the placenta and it is possible that this mechanism prevented us to observe differences in UBF between nutritional planes in the summer trial (Contreras-Correa et al., 2021). Moreover, these results are similar to Lemley et al. (2018) where placentomes from nutrient restricted dams collected at day 180 of gestation showed an increase in cotyledonary macroscopic blood vessel density combined with increased placentome capillary size, area, and perimeter. These observations may indicate a cotyledonary vascular compensatory response to reduced blood flow, oxygen, and nutrient availability in the nutrient-restricted model. Under hypoxic conditions, cellular mechanisms are triggered to maintain cell survival for oxygen delivery (Soares et al., 2017), and the hypoxia inducible factors (HIF1α and HIF2α), are upregulated targeting approximately 200 genes regulating angiogenesis, erythropoiesis, apoptosis, cell proliferation, glucose metabolism, pH regulation, and proteolysis (Patel et al., 2010). As reviewed by Sferruzzi-Perri and Camm, (2016), research shows that under adverse intrauterine conditions, the placenta can alter its morphology and functionality to optimize nutrient and oxygen supply to the fetus.

A major limitation of the current study was the reduced sample size of three animals per group at the higher-level interactions. Nevertheless, concomitant differences in fetal weights and placental phenotypes in our previously published study (Contreras-Correa et al., 2021) in conjunction with the biological role of the transcripts evaluated in this study provide an adequate interpretation for guiding future research in placental clock pathways. In conclusion, the placentome vascularity and gene expression results presented in this study provide insight into the impact of nutrient restriction on placental circadian clock leading to placental insufficiency and impaired fetal growth. In addition, this research shows seasonal changes in the expression of the clock gene network in the bovine placenta and overall, these findings indicate that maternal nutritional plane plays an important role in controlling placental circadian clocks. Moreover, the significant changes in gene expression and cotyledonary fluorescent signaling within a day could directly inform the development of guidelines for the proper temporal administration of placental blood flow therapeutics such as melatonin.

Glossary

Abbreviations

ADQ

adequately fed; 100% nutritional requirements

AKT1

AKT serine/threonine kinase 1

AM

morning, 0500 hours

ARNT

aryl hydrocarbon receptor nuclear translocator

ARNTL

aryl hydrocarbon receptor nuclear

CITED2

Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2

VEGFA

vascular endothelial growth factor A

CLOCK

circadian locomotor output cycles kaput

CON

control group; no melatonin supplementation

CRY1

cryptochrome circadian regulator 1

HIF1α

hypoxia inducible factor 1 subunit alpha

IUGR

intrauterine growth restriction

IVIS

in vivo imaging system

MEL

melatonin supplemented group; received 20 mg/d of dietary melatonin

MTNR1A

melatonin receptor 1A

MTNR1B

melatonin receptor 1B

NAMPT

nicotinamide phosphoribosyltransferase

NOS3

nitric oxide synthase 3

NR1D1

nuclear receptor subfamily 1, group D, member 1

NR3C1

nuclear receptor subfamily 3 group C

PER2

period circadian regulator 2

PF4

platelet factor 4

PGF

placental growth factor

PM

afternoon, 1300 hours

PPARɣ

peroxisome proliferator activated receptor gamma

PPARα

peroxisome proliferator activated receptor alpha

PTGS2

prostaglandin-endoperoxide synthase 2

RES

nutrient restricted group; 60% of nutritional requirements

RORA

RAR-related orphan receptor alpha

RPL19

ribosomal protein L19

RPS6KB1

ribosomal protein S6 kinase B1

SF3A1

splicing factor 3a subunit 1

TMR

total mixed ration

UBF

uterine blood flow

Funding

This publication is a contribution of the Mississippi Agricultural and Forestry Experiment Station. This work was supported by the Agriculture and Food Research Initiative competitive grant number 2018-67016-27580 from the U.S. Department of Agriculture National Institute of Food and Agriculture. This research was supported in part by the U.S. Department of Agriculture, Agricultural Research Service, project 6066-31000-015-00D.

Conflicts of Interest Statement

The authors declare no real or perceived conflicts of interest.