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Abstract

Consumption of ergot alkaloids during the second half of gestation has been shown to decrease umbilical artery vasoactivity resulting in decreased birth weights. Negative vascular effects of ergot alkaloids are mediated predominantly through serotonergic and adrenergic receptors in other tissues. Vasoactivity of serotonin (5-HT) receptors 5-HT_2A and 5-HT_1B/1D in umbilical artery and vein from ewes receiving endophyte-infected seed (E + 1.77 mg ergovaline/hd/d) or a control total mixed ration (CON; 0 mg ergovaline/hd/d) tall fescue seed at d-110 and d-133 of gestation was evaluated. Gravid reproduction tracts were collected from ewes. Two-mm sections of umbilical artery and vein were exposed to increasing concentrations of a 5-HT_1B/1D agonist and 5-HT_2A agonist. The 5-HT_1B/1D agonist did not stimulate a contractile response in artery or vein or either gestation time point. 5-HT_2A agonist caused large responses in artery with greatest occurring at d-110 and decreasing in magnitude as days of gestation increased (p < 0.05). On d-110 and 133 of gestation, arteries from CON ewes had greater contractile response than arteries collected from E+ ewes (p < 0.05). Veins responded to increasing concentrations of the 5-HT_2A agonist. Maximal d-110 vein response was greater than d-133 when exposed to 5-HT_2A agonist (p < 0.05). Unlike the artery, veins from E+ ewes had greater d-133 contractile response than CON (p < 0.05). Vascular contractions of umbilical artery and vein are induced by 5-HT_2A receptor activity and not 5-HT_1B/1D. Umbilical artery 5-HT_2A receptor activity was more sensitive to seed treatment and could be responsible for ergot alkaloid-induced intra-uterine growth restriction.

Keywords

Ergot alkaloids Ewe gestation serotonin vasoconstriction

Introduction

Ergot alkaloids are a group of toxins that can be produced by a variety of fungi and are a continual source of livestock production and human health challenges around the world.¹ Ergot alkaloids such as those produced by the endophyte Epichloë coenophiala in tall fescue (Lolium arundinaceum) interact with biogenic amine receptors to elicit their negative effects.² While the mycotoxicosis associated with ergot alkaloids in livestock has been shown to interact with adrenergic.^3,4 and dopaminergic receptors,^5,6 serotonergic receptor interactions have been implicated in the associated vasoconstriction.^7–9 Specifically, serotonin (5-hydroxytryptamine, 5-HT) receptors 5HT_1B/1D and 5HT_2A have been associated with the ergot alkaloid-induced vasoconstriction in various animal models.^9–11 Work looking at 5HT_2A receptor vasoactivity in bovine vasculature demonstrated that ergovaline, the predominantly produced ergot alkaloid of E. coenophiala, can act as an agonist stimulating a contractile response and then as a strong antagonist of this receptor effectively preventing subsequent stimulation by 5-HT.¹² This ergot alkaloid-receptor complex results in a change from normal biological function and homeostasis that is similar to cases of prolonged 5-HT receptor stimulation and blunted signaling.¹³

Ewes exposed to ergot alkaloids during gestation have lambs with decreased birth weights¹⁴ and this intra-uterine growth restriction (IUGR) occurs in the second half of gestation.¹⁵ Ergot alkaloids have also been associated with vasoconstriction of umbilical vasculature that was associated with decreased fetal weights.¹⁶ Decreased serotonin-induced contractility in ewes consuming ergot alkaloids during gestation¹⁶ led to the hypothesis that serotonin receptors associated with vascular smooth muscle contraction would also be affected. Thus, the objectives of this experiment were to characterize the vasoactivity of serotonin receptors 5-HT_2A and 5-HT_1B/1D in the umbilical artery and vein during the second half of gestation and determine if vasoactivity and expression of genes associated with these receptors is altered in ewes consuming endophyte-infected tall fescue seed.

Material and methods

All experimental procedures involving live ewes and perinatal lambs were approved by the Clemson University Institutional Animal Care and Use Committee (protocol # 2018-055).

Animals and treatments

The initial group of mature ewes (n = 21; 81.2 kg ± 7.7) were synchronized, bred, and selected for twin pregnancies as described by Britt et al.¹⁷ Ewes estimated to be carrying twins by ultrasound were divided into treatment groups that began receiving either a TMR basal diet formulated for gestating ewes (NRC¹⁸; CON; 0 µg ergovaline + ergovalinine/g DM) or the basal diet plus endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g DM) on d 86. Seed was analyzed for ergot alkaloid content as described by Carter et al.¹⁹ and modified by Klotz et al.¹⁰ Ewes receiving the E+ treatment were fed seed that resulted in a final dosage of 1.77 mg of total ergovaline hd⁻¹ d⁻¹. Ewes were further divided into different time points for termination of pregnancy. The ewes were necropsied on d 110 (n = 4/group) and on d 133 (n = 3/group) for a total of n = 14 ewes. Two ewes required veterinary care and were removed from the study prior to necropsy due to severely reduced intake and weight gain (1 CON d 133 and 1 E+ d 133).

Umbilical artery and vein collection

Ewes were harvested via captive bolt and exsanguination on d 110 or 133 of gestation at the Clemson University Meats Laboratory as described by Britt et al.¹⁷ Following removal of the full reproductive tract, an incision was made in the uterus, and the fetuses were removed. The umbilical cord from the first exteriorized fetus was ligated and cut. The umbilical arteries and veins were separated from surrounding connective and adipose tissues and stored in modified Krebs-Henseleit oxygenated buffer solution (95% O₂/5% CO₂; pH = 7.4; mM composition = D-glucose, 11.1; MgSO₄, 1.2; KH₂PO₄, 1.2; KCl, 4.7; NaCl, 118.1; CaCl₂, 3.4; and NaHCO₃, 24.9; Sigma Chemical Co., St Louis, MO) on ice. All blood vessels from a given collection day were transported on ice to the Forage-Animal Production Unit in Lexington, KY where contractility assays were run the following day. Sections of blood vessels that were not used for contractility assays were snap frozen in liquid nitrogen and stored at −80°C until isolation of RNA was completed.

Contractility experiments

Cleaned blood vessels were cut into 2- to 3-mm cross sections using an adjustable acrylic channel tissue matrix (Braintree Scientific, Braintree, MA). Prior to mounting in a myograph, cross-sections were examined using a dissecting microscope (Stemi 2000-C, Carl Zeiss Inc., Oberkochen, Germany) at 12.5x magnification to measure dimensions for assurance of consistent segment size and to verify physical integrity of tissue. Cross-sections were suspended horizontally in a 5-mL tissue bath (DMT610 M multichamber myographs, Danish Myo Technologies, Atlanta, GA) containing continuously oxygenated modified-Krebs Henseleit buffer (95% O₂/5% CO₂; pH = 7.4; 37°C), with 3 × 10⁻⁵ M desipramine and 1 × 10⁻⁶ M propranolol (Sigma Chemical Co.) to inactivate catecholamine-neuronal uptake and β-adrenergic receptors, respectively. After equilibration to 1 g of tension (∼1.5 h), umbilical arteries and veins were exposed to an addition of 120 mM KCl for 15 min to verify tissue viability and for subsequent use as a reference for normalization of resultant contractile response data.

Umbilical artery and vein cross-sections were run in duplicate from each ewe for each treatment. Following recovery from the 120 mM KCl addition and reestablishment of the 1-g baseline tension, serotonin receptor agonist additions occurred in 15-min intervals. Each 15-min interval consisted of a 9-min treatment incubation period, followed by a washout period during which 5-mL aliquots of buffer without treatment were incubated with a blood vessel segment for two consecutive 2.5-min periods, followed by a final buffer replacement and 1-min incubation prior to the next addition.

Treatment additions consisted of increasing concentrations of the potent agonists rizatriptan benzoate (RTB; 5-HT_1B/1D agonist; Cat. No. 5136; Tocris Bioscience, Minneapolis, MN, USA) and (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide, (TCB-2; 5-HT_2A agonist; Cat. No. 2592, Tocris Bioscience). Agonists were diluted to corresponding concentrations for final working concentrations in myograph wells of 5 × 10⁻⁹ to 1 × 10⁻⁴ M with water for a total of 10 standard additions (5 × 10⁻⁹, 1 × 10⁻⁸, 5 × 10⁻⁸, 1 × 10⁻⁷, 5 × 10⁻⁷, 1 × 10⁻⁶, 5 × 10⁻⁶, 1 × 10⁻⁵, 5 × 10⁻⁴, and 1 × 10⁻⁴ M).

Isometric contractions of umbilical artery and vein preparations were recorded as grams of tension in response to exposure to the KCl reference and the 5-HT_1B/1D and 5-HT_2A agonists. Data were digitally recorded using a Powerlab 16/35 (ADInstruments, Colorado Springs, CO) and Chart software (Version 8, ADInstruments). Contractile response was recorded as the maximum response, in grams, during the 9-min incubation following a treatment addition and corrected by baseline tension recorded just prior to the addition of 120 mM KCl. Treatment response data were normalized as a percentage of the maximal contraction produced by KCl addition. Concentration-response curves were constructed by plotting the normalized data using GraphPad Prism (version 8; San Diego, CA). This data presentation employed a nonlinear regression (sigmoidal concentration-response curve) to fit a line to contractile response data points for a treatment using the 3-parameter equation:

y = b o t t o m + \frac{(t o p - b o t t o m)}{{1 + 10^{[(\log {E C}_{50} - x)]}}},

Where the top and bottom are plateaus in the units of the y-axis. This calculation permitted the calculation of a compound’s potency for umbilical arteries and veins expressed as the concentration the compound required to produce 50% of the observed contractile response (EC₅₀).

RNA isolation and real-time PCR

Genes selected for analysis were specific for each serotonin receptor subtype (HTR1B, HTR1D, and HTR2A), their associated intracellular signaling G proteins (GNAS, GNAQ, and GNA11) and the gene for endothelial nitric oxide synthase (NOS3) that could influence the vasoactivity results. Frozen artery and vein tissue was ground under liquid nitrogen and total RNA was isolated using a phenol-chloroform extraction using Trizol (Thermo) following the manufacturer’s instructions with the addition of a second precipitation with isopropanol to remove contaminants. All RNA samples were treated with DNase (TURBO DNA-free kit; Thermo) to remove genomic DNA contamination. A spectrophotometer (Nanodrop One, Thermo) was used to quantify RNA and to confirm A260/A280 rations of ∼2.0. The RNA integrity was assessed using a bioanalyzer (Agilent) and all samples were confirmed to have an RNA Integrity Number of at least 7. Two micrograms of RNA were reverse transcribed (High Capacity cDNA Reverse Transcription Kit; Thermo) and the resulting cDNA was diluted to a final concentration of 10 ng/µL with nuclease-free water. Primers for housekeeping and target genes were designed using Primer3 software ²⁰ and based on refseq sequence data (National Center for Biotechnology Information; Bethesda MD). Where possible, primers for genes of interest were designed to span exon-exon junctions (Table 1). The HTR1D gene was originally included as a gene of interest, however expression was low and not consistently expressed at detectable levels across all ewes. Thus, HTR1D was omitted from analysis. Primer efficiencies were confirmed to be between 90 and 110% using a five-point serial dilution of pooled cDNA. Quantitative real-time PCR was performed with 20 ng cDNA, run in duplicate, using Fast SYBR Green Master Mix (Thermo) with a StepOnePlus Real-Time PCR system (Thermo). The qPCR reaction was performed in a volume of 15 μL under the following conditions: hold for 20 s at 95°C followed by 3 s at 95°C then 30 s at 60°C for 39 cycles. A subsequent melt curve was conducted for verification of a single amplicon product. Reference genes were assessed for stability using the statistical model by verifying mean Ct did not differ with treatments.²¹ For artery, GAPDH, HPRT, ACTB and RPLPO were found to be stable, whereas for vein, only HPRT and RPLPO were used. Expression of target genes was normalized to the geometric mean of the stable reference genes within tissue type, and statistical analysis was conducted on the ΔCt. Results are presented as 2^−ΔΔCt, ²² with E+ treatment held relative to the CON treatment, within tissue and within each collection time point.

Table 1.

Gene names, Ovis aires accession numbers (National Center for Biotechnology Information), and forward and reverse primer sequences for genes assessed with real-time PCR.

Gene^a	Symbol	Accession number	Primer sequence 5’ → 3’ (forward; reverse)	Amplicon (bp)
β -Actin	ACTB	NM_001009784.1	TCC ACC GCA AAT GCT TCT	99
β -Actin	ACTB	NM_001009784.1	CAC GAG GCC AAT CTC ATC TC	99
Glyceraldehyde 3-phosphate dehydrogenase	GAPDH	NM_001190390.1	AGT TCC ACG GCA CAG TCA AG	83
Glyceraldehyde 3-phosphate dehydrogenase	GAPDH	NM_001190390.1	AGG ATC TCG CTC CTG GAA GAT	83
Ribosomal protein, larger, P0	RPLP0	XM_004017413.2	CTC TGG AGA AAC TGT TGC CT	96
Ribosomal protein, larger, P0	RPLP0	XM_004017413.2	TAT TGG CCA GCA GCA TGT	96
Hypoxanthine phosphoribosyltransferase 1	HPRT1	XM_015105023.3	TGC CGA CCT GTT GGA TTA	103
Hypoxanthine phosphoribosyltransferase 1	HPRT1	XM_015105023.3	CTG GTC GTT ACA GTA GCT CTT C	103
5-Hydroxytryptamine receptor 1B	HTR1B	XM_015097299.1	CCA CGG AAT TGA CTG AGA TAG G	138
5-Hydroxytryptamine receptor 1B	HTR1B	XM_015097299.1	GGG AAG GAG AGT GCA AAT GA	138
5-Hydroxytryptamine receptor 2A	HTR2A	XM_004012066.3	GCA GAA TGC CAC CAA CTA TTT C	107
5-Hydroxytryptamine receptor 2A	HTR2A	XM_004012066.3	ACC GGT ACC CAT AGA GGA TG	107
Guanine nucleotide-binding protein G(s) subunit alpha	GNAS	XM_012188911.1	GCA TGC TGT TTC TAG GTG CT	143
Guanine nucleotide-binding protein G(s) subunit alpha	GNAS	XM_012188911.1	CTT CTC ACT ACC ATC GCT GTT G	143
G protein subunit alpha q	GNAQ	XM_015093177.1	GTA CGA GCA CAA TAA GGC TCA	143
G protein subunit alpha q	GNAQ	XM_015093177.1	CTC GTC GTC TGT CAT AGC ATT C	143
G protein subunit alpha 11	GNA11	XM_015095900.1	CGA GCA GAA CAA GGC CAA	104
G protein subunit alpha 11	GNA11	XM_015095900.1	CGT TCC ACA GGG TCT TGA TG	104
Nitric oxide synthase 3	NOS3	NM_001129901.1	TCC CTG TAC TAT CTC ATC CTC TC	101
Nitric oxide synthase 3	NOS3	NM_001129901.1	CTG ACT GTA GGC TCT GGA ATA C	101

^aβ-Actin, GAPDH, HPRT1, and RPLP0 are reference genes used to normalize gene expression of target genes.

Statistical analyses

All statistical analyses were conducted using the Mixed models of SAS (SAS 9.4; SAS Inst. Inc., Cary, NC) and tests of fixed effects used the Satterthwaite approximation of denominator degrees of freedom. Umbilical artery and vein contractile response data to serotonin receptor agonist treatments were analyzed as a completely randomized design with a split-plot treatment design. Whole plot experimental unit was ewe with a 2 × 2 factorial of gestational day and seed treatment as the treatment factors. The blood vessels collected were the subplot experimental units and the concentration of serotonin receptor agonists were used as the subplot treatment factor. The model included fixed effects of gestational day, seed treatment, myograph treatment concentration, and the interaction. For the variables EC₅₀, gene expression and vessel dimensions (inside and outside diameter), analysis of variance was conducted as a completely randomized design for the main effects of seed treatment and gestational day.

Pairwise comparisons of least squares means (±SEM) were conducted only if the probability of a greater F-statistic in the ANOVA was significant for the tested effect. If significant, means separation was conducted using least significant difference (LSD) feature in SAS and comparisons were considered significant at p ≤ 0.05 and a tendency for significance at p < 0.1, unless reported otherwise.

Results

Blood vessels dimensions

The mean blood vessel lengths for umbilical artery and vein were not different across seed treatments at d 110 (Table 2). However, the length of umbilical artery and vein cross sections were different on d 133 with longer umbilical artery sections and shorter umbilical vein sections for the E+ vessels (p < 0.05). There were no differences in the umbilical artery outside diameter across treatments. Unlike the CON umbilical vein that increased in outside diameter with days of gestation (p < 0.05), the umbilical vein from ewes treated with E+ did not increase in size from d110 to d133.

Table 2.

Vascular dimensions of umbilical artery and vein collected from ewes receiving endophyte-infected tall fescue seed (E+) or an ergot alkaloid free total mixed ration (CON) at day 110 or day 133 of gestation.

Variable^a	Day 110		Day 133		p-value
Variable^a	CON	E+	CON	E+	SEM	Seed	Day	Seed x day
Umbilical artery
Outside diameter, mm	3.23	3.14	3.49	3.19	0.07	<0.01	0.02	0.12
Length, mm	2.44*	2.31*	2.34*	2.68*	0.06	0.07	0.01	<0.01
Umbilical vein
Outside diameter, mm	2.48*	2.56*	2.82*	2.59*	0.07	0.31	<0.01	0.02
Length, mm	2.19*	2.25*	2.24*	2.04*	0.06	0.27	0.19	0.03

Unable to collect umbilical artery and vein inside diameter.

Values not sharing like superscripts within a row are different (p < 0.05).

5-HT_1B/1D receptor

The 5-HT_1B/1D agonist rizatriptan benzoate (RTB) did not stimulate a contractile response in either the umbilical artery (Figure 1(a)) or vein (Figure 1(b)) for either timepoint in gestation evaluated. The main effect of RTB concentration differed across concentrations evaluated (p < 0.01), but as the concentrations of RTB increased the vascular response decreased below baseline tension resulting in a negative contractile response for both the umbilical artery and vein.

Figure 1.

Assessment of vasoactivity to increasing concentrations of rizatriptan benzoate (RTB), a selective agonist for serotonin (5-HT) receptor 1B and 1D. Sections of A.) umbilical artery (Seed Effect: p = 0.09; Day Effect: p = 0.06; and Concentration Effect: p < 0.01; all interactions were not significant) and B.) umbilical vein (Seed Effect: p < 0.05; Day Effect: p < 0.01; and Concentration Effect: p < 0.01; Day × Concentration interaction: p < 0.01; all remaining interactions were not significant) were collected from ewes at d 110 and 133 of gestation that were receiving endophyte-infected (E+) tall fescue seed, or an ergot alkaloid free total mixed ration (CON).

5-HT_2A receptor

The 5-HT_2A agonist TCB-2 stimulated a contractile response in both the umbilical artery (Figure 2(a)) and vein (Figure 2(b)). The response became different from baseline at 1 × 10⁻⁶ M TCB-2 for both vessels and continued to increase through 1 × 10⁻⁴ M TCB-2. For the umbilical artery, the lines began to separate as the concentrations of TCB-2 increased due to a significant effect of the E+ seed at both d 110 and d 133. The umbilical arteries collected from ewes dosed with E+ seed had decreased contractile responses to the 5-HT_2A agonist at both gestational time points (p < 0.05). The umbilical artery contractile response to TCB-2 was also less at d 133 of gestation compared to d 110 for both seed treatments. The umbilical vein did not differ between seed treatments at d 110 of gestation, but did differ on d 133 with the E+ umbilical veins having greater contractile responses to increasing concentrations of TCB-2 than the CON veins (p < 0.05). Like the artery, the umbilical veins response to the 5-HT_2A agonist was less on d 133 than on d 110 of gestation.

Figure 2.

Assessment of vasoactivity to increasing concentrations of (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2), a selective agonist for serotonin (5-HT) receptor 2A. Sections of A.) umbilical artery (Seed Effect: p < 0.05; Day Effect: p < 0.01; and Concentration Effect: p < 0.01; Day × Concentration interaction: p < 0.01; Seed × Concentration interaction: p < 0.01; all remaining interactions were not significant) and B.) umbilical vein (Day Effect: p = 0.07; and Concentration Effect: p < 0.01; Day × Concentration interaction: p < 0.01; all remaining interactions were not significant)were collected from ewes at d 110 and 133 of gestation that were receiving endophyte-infected (E+) tall fescue seed, or an ergot alkaloid free total mixed ration (CON). The negative log 50% effective concentration (-log EC₅₀) of TCB-2 for A.) umbilical artery at d110 was 5.10 and 5.05 M and at d133 was 5.23 and 5.23 M for CON and E+, respectively. The -log EC₅₀ of TCB-2 for B.) umbilical vein at d110 was 5.24 and 5.37 M and at d133 was 5.29 and 5.40 M for CON and E+, respectively.

Gene expression

There were no significant seed treatment x gestational day interactions for any of the genes evaluated that were associated with the 5-HT_2A and 5-HT_1B/1D receptors. There were significant main effects associated with days of gestation. Expression of HTR2A receptor gene tended to be decreased on d 133 (p = 0.06) for umbilical artery (Figure 3(a)) and was less for umbilical vein (p = 0.009; Figure 3(b)). GNA11 was different across days of gestation for both umbilical artery and vein (Figure 3(c) and (d)), whereas GNAQ was different across days of gestation for umbilical vein, but not artery (Figure 3(e) and 3(f)). The gene for endothelial nitric oxide synthase, NOS3 was expressed at decreased levels on d 133 of gestation compared to d 110 (p < 0.05) in the umbilical artery (Figure 3(g)), but not the umbilical vein (Figure 3(h)). Differential expression was not detected in the remaining genes evaluated across day of gestation within umbilical artery and vein (data not shown).

Figure 3.

Expression levels of genes associated with vascular smooth muscle contraction and relaxation in the ovine umbilical artery (UA) and vein (UV) collected from ewes at d 110 and 133 of gestation that were receiving endophyte-infected (E+) tall fescue seed, or an ergot alkaloid free total mixed ration (CON). Serotonin receptor (HTR) (A) HTR2A_UA; (B) HTR2A_UV; G proteins (C) GNA11_UA; (D) GNA11_UV; (E) GNAQ_UA; (F) GNAQ_UV; and endothelial nitric oxide synthase (G) NOS3_UA; and (H) NOS3_UV.

Discussion

A major premise of this experiment is that ergot alkaloids disrupt the normal physiology of gestation in sheep. Ergot alkaloids applied intravenously have been shown to cross the placenta and can influence the fetus. Leist and Grauwiler ²³ intravenously administered tritium-labeled ergotamine to rats 2 weeks post-breeding. There was three times the label in the uterus and placental tissues, than in the blood and radioactivity was detected in the amniotic fluid and fetal tissues. Indänpään-Heikkilä and Schoolar ²⁴ demonstrated transplacental passage of 14C-labeled lysergic acid diethylamide in the final week of gestation in mice. The mice were intravenously infused with 14C-LSD and after 5 min the label was detectable in the fetus. If ergot alkaloids found in wildtype endophyte-infected tall fescue can cross the ruminant placenta, the fetus could be negatively affected. The dosage of ergovaline selected for use in the current study was based on previous work validating the induction of ergot alkaloid-induced IUGR in sheep.^14,15,25

Serum prolactin is often used as an experimental indicator of successful induction of fescue toxicosis in livestock. Britt et al.¹⁷ reported decreased serum prolactin concentrations associated with the E+ seed treatment at both d 110 and d 133 of gestation and fetal body weights were decreased for the E+ treatment on d 110 of gestation. This is evidence that the ergot alkaloids associated with E+ tall fescue successfully induced toxicosis in the ewes and an ergot alkaloid-induced intra-uterine growth restriction in the corresponding fetuses. This agrees with similar previous studies that employed the comparable treatment models. Duckett et al.¹⁴ and Britt et al.¹⁵ also reported similar outcomes using the same E+ tall fescue seed model to induce intra-uterine growth restriction in sheep. While there was no confirmation that ergot alkaloids are crossing over to the fetal side of the placenta in the current study nor was there confirmation of decreased umbilical blood flow, there were significant effects on maternal and fetal physiology¹⁷ when ergot alkaloids are present in the maternal diet. This could be extended to the observation that the umbilical vein in the E+ treatment did not increase in size from d 110 to d 133 of gestation and contributing to a reduced blood flow during late gestation. While there were also differences in length, these were inadvertently introduced during the processing of the blood vessel and would be corrected for as part of the data normalization.

Serotonin is a biogenic amine neurotransmitter that is involved in the regulation of numerous physiological processes, one of which is the regulation of vascular tone.²⁶ Depending on the animal species and tissue bed, 5-HT can stimulate relaxation and contraction of arteries and veins. The umbilical cord is a link between the fetal and maternal bodies. In sheep it consists of two umbilical arteries, two umbilical veins, and an allantoid duct that diminishes in size as gestation progresses.²⁷ The regulation of the contractile state of these vessels is critical for gas and nutrient exchange between maternal and fetal tissues. Due to a lack of innervation of umbilical blood vessels, the reliance on localized vasoactive substances like 5-HT are critical to the control of umbilical blood flow.^28,29 Serotonin has been shown to be a potent agonist of contractility in sheep umbilical blood vessels ³⁰ as well as human umbilical vessels ³¹ as it is responsible for the postpartum closure of the umbilical artery. Thus, dysregulation of serotonin could have profound effects on the trajectory of gestation.

Serotonin dysregulation during pregnancy in humans is associated with pathologies like preeclampsia and umbilical artery contractility in humans has been shown to be regulated through activation of the 5-HT_2A and 5-HT_1B/1D receptors.^32–34 In the current study, TCB-2 the selective agonist for 5-HT_2A was able to stimulate large contractile responses in both umbilical arteries and veins. Unlike in human umbilical vessels, RTB the selective agonist for 5-HT_1B/1D receptors did not stimulate a contractile response of either the umbilical artery or vein. While this does not rule out the presence of these receptor subtypes, it does indicate that the 5-HT_1B/1D receptors do not play a role in 5-HT-induced contractility in sheep umbilical vasculature. Although this study was not designed to quantify relaxation, it did appear as though the agonist caused vasorelaxation for both gestation lengths. Future work should investigate the role of these serotonin receptor subtypes in vasorelaxation as it did appear that the umbilical vessels relaxed in response to the 5-HT1B/1D receptor agonist. Lovren, Li ³⁵ reported expression 5-HT_1B and 5-HT_2A genes in human umbilical artery, but not 5-HT_1D. This agrees with the findings of current study as levels of 5-HT_1D receptor were below the threshold for detection with qPCR for several of the ewes in the study and the remaining ewes had very low levels of expression. There was also an observed decrease in expression of HTR2A receptor gene from d 110 to d 133 that coincided with a significant decrease in vasoactivity to increasing concentrations of TCB-2 from d 110 to d 133. While significant main effect differences were detected, the level of animal-to-animal variation and small fold changes suggest some caution when interpreting these data and changes in vasoactivity are not necessarily directly related to regulation of target gene transcription. Future research should confirm if this downregulation is responsible or associated with the observed decrease in 5-HT_2A vasoactivity.

Zhang and Dyer ³⁶ demonstrated that 5-HT₂ receptor is responsible for regulation of constriction in ovine umbilical arteries. Klotz et al.¹⁶ demonstrated a reduction in serotonin-induced vasoactivity in the umbilical artery from ewes treated with E+ seed through d 133 of gestion. This is similar to reductions in serotonin reactivity in human umbilical arteries from women with preeclampsia.³⁷ The current study extends this observation of decreased vasoactivity associated with ergot alkaloid exposure to the 5-HT_2A receptor in the umbilical artery. A decreased vasoactivity at the 5-HT_2A receptor was observed in umbilical blood vessels collected from ewes receiving the E+ seed treatment at both gestational timepoints. However, effect of ergot alkaloids differed across blood vessel types at d 133. Specifically, 5-HT_2A-mediated vasoactivity was decreased by E+ in the umbilical artery and increased by E+ in the umbilical vein. This is the first instance that the authors have reported where 5-HT regulated vasoactivity was increased by ergot alkaloid exposure. Others have reported increased vasoactivity in response to ergot alkaloid treatments³ and this is an area that deserves additional research. Ergot alkaloids produced by E. coenophalia infected tall fescue have been shown to antagonize the 5-HT_2A receptor.^7–9 This interference by ergot alkaloids with the normal function of the 5-HT_2A receptor causes an impairment in the regulation of vascular tone and can result in decreased blood flow to and from the fetus. This can contribute to the IUGR observed in the ewes receiving E+ in the current study.

Conclusions

Ergot alkaloids have a significant effect on 5-HT-mediated vasoactivity and this could extend to altered fetal growth and development. This study used selective agonists to characterize the serotonin receptors involved in the contraction of ovine umbilical arteries and veins at two different timepoints in gestation. The serotonin receptor 5-HT_2A was the primary subtype responsible for mediating umbilical vascular contraction. The agonist for the 5-HT_1B/1D receptors did not stimulate a contractile response, but may have caused relaxation. Vasoactivity at the 5-HT_2A receptor decreased as gestational days increased. Exposure to ergot alkaloids caused a decrease in contractile response to 5-HT_2A agonist TCB-2 in umbilical arteries on d 133, but caused an increase in 5-HT_2A-induced contractile response in umbilical vein. Also, ergot alkaloids had no effect of the expression of associated genes. The ergot alkaloid disruption of the regulation of vascular tone by serotonin is a likely contributor to the ergot-alkaloid induced intra-uterine growth restriction that was also observed in this study. Future research should continue to investigate the mechanisms by which ergot alkaloids disrupt normal physiological fetal development and additionally the role that serotonin plays in relaxation of umbilical arteries and veins should be evaluated.

Footnotes

Acknowledgments

The authors would like to acknowledge with gratitude the technical support of Adam J. Barnes of the USDA-ARS Forage-Animal Production Research Unit. Mention of trade name, proprietary product of specified equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that may be available. USDA is an equal opportunity provider and employer.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the USDA-ARS National Program 101 – Food Animal Production, Project Plan Number: 5042-32630-004-000-D, was based upon work supported by NIFA/USDA under project # SC-1700537 of the Clemson University Experiment Station, and was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author’s and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE.

ORCID iDs

James L Klotz

Susan K Duckett

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Ergot alkaloid consumption alters serotonin receptor-induced vasoactivity in ovine umbilical vasculature

Abstract

Keywords

Introduction

Material and methods

Animals and treatments

Umbilical artery and vein collection

Contractility experiments

RNA isolation and real-time PCR

Statistical analyses

Results

Blood vessels dimensions

5-HT1B/1D receptor

5-HT2A receptor

Gene expression

Discussion

Conclusions

Footnotes

Acknowledgments

Declaration of conflicting interests

Funding

ORCID iDs

References

5-HT_1B/1D receptor

5-HT_2A receptor