Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: J Histotechnol. 2021 Apr 29;44(4):173–181. doi: 10.1080/01478885.2021.1902670

The effect of ergot alkaloid exposure during gestation on the microscopic morphology and vasculature of the ovine placenta

J L Britt 1, R R Powell 2, C McMahan 3, T F Bruce 2, S K Duckett 1
PMCID: PMC8553796  NIHMSID: NIHMS1687485  PMID: 33913402

Abstract

Ergot alkaloids, a class of mycotoxins associated with ergotism, act as agonists on serotonin (5HT) receptors, specifically 5HT2a, which mediate smooth muscle contraction and vasoconstriction. The objective of this study was to examine the impact of ergot alkaloid exposure during mid and late gestation on microscopic placental structure and vascular development. Ewes were fed endophyte-infected tall fescue seed containing ergot alkaloids (E+/E+, 1.77 mg ewe−1 d−1) or endophyte-free tall fescue seed (E-/E-, 0 mg ergot alkaloids) during both mid (d35 to 85) and late gestation (d 86 to 133). On d 133 of gestation, a terminal surgery was performed and two placentomes of the type B morphology were collected for microscopic analyses. Amorphous connective tissue regions were larger (p < 0.0001) and more numerous (p = 0.025) in the placentome of ergot alkaloid exposed ewes. Staining showed no difference (p = 0.83) in the number of vessels present, but luminal area of maternal vasculature was 117% greater (p < 0.0001) in ergot alkaloid exposed ewes. Results showed that exposure to ergot alkaloids during gestation slowed maturation of the fetal villi as indicated by greater amorphous connective tissue regions, and altered size and shape of blood vessels to counteract reductions in blood flow caused by vasoconstriction.

Keywords: Sheep, ergot alkaloids, placental structure, vascular development

INTRODUCTION

Adaption of the uterine vasculature during gestation supports a 20-fold increase in uterine artery blood flow which is necessary for proper placental development and fetal growth [1]. In humans, maternal cardiac output increases between 30 – 40% during pregnancy due to increased heart rate and stroke volume. This increase in cardiac output is necessary for adequate perfusion of the uterus and works to supply oxygen and nutrients to both the placenta and fetus [2, 3]. The uterine artery diameter doubles by mid-gestation leading to a four-fold increase in cross-sectional area [4, 5]. This works to maintain an adequate oxygen and nutrient supply to the placenta and fetus for both humans and sheep [6, 7]. Inadequate maternal vascular remodeling and uteroplacental malperfusion are prominent causes of intrauterine growth restriction (IUGR) and can also result in improper placental development, placental insufficiency, and preeclampsia in both human and animal models [8–11].

Ergot alkaloids, a class of mycotoxins associated with ergotism, act as agonists on serotonin (5HT) receptors, specifically 5HT2a [12–14]. Both vascular smooth muscle and uterine smooth muscle contain 5HT2a receptors which work to mediate smooth muscle contractions and vasoconstriction [15]. The consumption of ergot alkaloids results in systemic vasoconstriction which has been detected as a reduction in luminal vessel area of the carotid, auricular, and caudal arteries in various livestock species [12, 16–18]. Reductions in placental perfusion through occlusion of the uterine artery leads to impaired placental development and IUGR with a reduction of 25 – 34% in placental mass depending on the level of occlusion [9]. Ergot alkaloids are known to impair fetoplacental development to a similar degree, most likely due to their vasoconstrictive activity which has been reported in the ovarian, uterine, and umbilical arteries [14, 19, 20]. A 23% reduction in placental mass was reported for ewes consuming E+ tall fescue seed containing ergot alkaloids [21]. Furthermore, the maternal consumption of E+ tall fescue seed lead to a 15% reduction in total fetal lamb weights at d 133 of gestation and a 36% reduction in lamb birth weight [22]. Uterine artery samples from the same study exhibited a decrease in the inside and outside diameter as well as artery thickness for ewes exposed to ergot alkaloids between d 35 – 133 of gestation [20]. Volumetric blood flow depends largely on vessel diameter. Klotz et al. [20] reported a 9% reduction in the uterine artery diameter for ewes on E+/E+ fescue seed treatment. Because uteroplacental malperfusion is linked to altered placental development, the objective of this study was to examine the impact of gestational ergot alkaloid exposure on microscopic placental structure and vascular development.

MATERIALS AND METHODS

All animal experimental procedures were reviewed and approved by the Clemson University Institutional Animal Care and Use Committee (AUP 2014-081).

Sample Collection.

Placental samples were collected from ewes fed endophyte-infected tall fescue containing ergot alkaloids (E+/E+, 1.77 mg ewe−1 d−1 of ergovaline and ergovalinine) or endophyte-free tall fescue (E-/E-, 0 mg ergovaline and ergovalinine) during mid (d 35 to 85) and late (d 86 to 133) gestation [21]. For this study, a subsample of ewes from ewes carrying twins and fed E-/E- (n = 3) or E+/E+ (n = 3) seed during mid/late gestation were selected for microscopic analyses. Seed treatments were mixed into total mixed ration to meet National Research Council (NRC ) requirements for pregnant ewes with twins during early and late gestation [23] and fed individually to ewes. On d 133 ± 1 of gestation, ewes were transported to Godley-Snell Research Facility (14.3 km) for terminal surgery. Ewes were anesthetized and subjected to a midventral laparotomy. Immediately following removal of the first fetus, two placentomes of the type B morphology according to Vatnick et al. [24] were selected adjacent to the initial incision. Sheep are ruminant animals with a cotyledonary placenta that has points of attachment between the maternal (caruncle) and fetal (cotyledon) side of the placenta called placentomes. Any remaining endometrial and fetal membranes were removed and placentomes were dissected with a medial incision. A sample was collected from the center of the intact placentome adjacent to the medial incision and was embedded in optimal cutting temperature (O.C.T.; Tissue-Tek 4583, VWR, USA) compound before flash freezing in liquid nitrogen for histology. Samples were stored at -80°C prior to cryomicrotomy.

Hematoxylin and eosin Staining.

Samples were cryosectioned at 8 μm thickness, fixed in 95% ethanol (#111000200; Pharmco), and stained immediately following fixation cryosectioning using a routine hematoxylin and eosin (H&E) method with Mayer’s hematoxylin (#26043-05; VWR) and eosin Y (yellowish) solution (#95057-848; VWR) in order to evaluate placental histomorphology. The imaging of H&E stained cryosections was conducted using a Leica LMD6500 equipped with a Leica DFC7000 color camera, Leica Application Suite X Version 3.4.1.17822 (Leica Microsystems, USA) and a 20x objective.

Double Immunofluorescence assay with SMCA and Collagen IV.

Frozen sections for immunofluorescence (IF) were fixed in 4°C acetone (#BDH1101-1LP VWR) for 3 min then allowed to air dry for 1 h. Slides were stored at −20°C until the assay was performed. Smooth muscle cell actin (SMCA) was used to detect smooth muscle cells in vessel walls in order to label maternal vasculature in the placenta and measure luminal area. The use of SMCA with ovine placental samples works to label almost exclusively the maternal vasculature due to the lack of pericytes in fetal capillaries [25]. Type IV collagen (Collagen IV), a major structural component of basement membranes, is highly expressed in the placenta. Fixed, dried cryosections were rehydrated in phosphate buffered saline (PBS) diluted from 10X PBS stock (#SH30258.02, HyClone, GE Life Science, USA). All IF assays were conducted manually and slides were incubated in a dark, humidity chamber on a rotating platform (60 rotation/min) starting at the blocking step until coverslipping. Blocking was done with 10% goat serum (#092939149, MP Biomedicals, USA) in 0.1% Tween 20 (#BP337500, Fisher Scientific, USA) in PBS for 1 h at room temperature (RT). Next, sections were incubated overnight at 4°C with primary antibodies diluted in 10% goat serum blocking solution. Antibodies were SMCA (4 ug/ml) mouse anti-α-smooth muscle Actin (1A4), monoclonal, IgG2a, RRID:AB 262054, lot #GR309548-7, (ab7817, Abcam, USA) and Rabbit anti-α-collagen IV, polyclonal, IgG, RRID:AB 262054, lot #GR309548-7 (#ab6586, Abcam), working concentration (1/400 dilution). Primary antibodies were applied in a cocktail. Sections were washed in blocking buffer 3 X, 5 min each, and incubated with a secondary antibody cocktail: Goat anti-Mouse IgG2a, Alexa Fluor 546, RRIS:AB 2535772, (#A-21133, Thermo Fisher Scientific) and Goat anti-Rabbit IgG, Alexa Fluor 488, RRID:AB 143165 (# A-11008, Thermo Fisher Scientific) at a working concentration of 10 ug/ml for 1 h at RT, then washed twice, 5 min each in PBS. Nuclei were counterstained with DAPI (D3571, Invitrogen) according to product instructions, washed 2X, 5 min each in PBS, and #1.5 coverslip mounted with ProLong Gold Antifade Mountant (#P36930, Thermo Fisher Scientific) and kept in the dark at RT until mountant cured then sealed semi-permanently with clear nail polish. Sections were imaged using a IN Cell Analyzer 2500 HS, Version 7.2-16735, Cytiva, USA) equipped with a Nikon 20X objective, N.A. 0.75, (Nikon Instruments, USA).

Double immunofluorescence assays with MMP9, CD45 antibodies and BSLI/GSLI lectin.

The matrix metalloprotease (MMP) family works to breakdown the extracellular matrix during instances of tissue remodeling. Matrix metalloprotease 9 (MMP9) is active in the bovine placenta and known to target Collagen IV. CD45, a leukocyte common antigen, was utilized as an immune cell marker and Griffonia (Bandeiraea) Simplicifolia lectin I (GSL1/BSL1) was used to detect the fetal trophoblast. CD45 is ubiquitously expressed on ovine lymphocytes, macrophages and granulocytes. Cryosections for MMP9 and CD45 were manually processed according to the same protocol as outlined for SMCA and Collagen IV assays with a modification to the blocking buffer. The blocking buffer included 5% BSA (Bovine Serum Albumin, # A2153, Millipore Sigma, USA), 10% goat serum, 0.1% Tween20 in PBS for 1 h at RT. Primary antibodies to MMP9 Goat anti-rabbit α-MMP9, polyclonal, IgG, RRID: AB_2144889, lot #QC49618-43062 (#RP33090_T100, Aviva Systems Biology, USA) at 20 ug/ml, and CD45 mouse anti-sheep IgG1 α-CD45, RRID:AB_1.11.32, lot #1701 (#MCA2220GA, BioRad, USA) at 50 ug/ml were cocktailed, and used to detect the respective marker proteins. The following secondary antibodies were used at 10 ug/ml and applied as a cocktail for 1 h at RT: Goat anti-Rabbit IgG, Alexa Fluor 635, RRID:AB 10374303 (# A31576, Invitrogen) and Goat anti-Mouse IgG1, Alexa Fluor 488, RRID:AB 10374303 (#A-21121, Invitrogen). Finally, a cocktail of DAPI and rhodamine-BSL1/GSL1 (Griffonia (Bandeiraea) Simplicifolia Lectin I at 20 ug/ml (#RL-1102, Vector Laboratories, USA) was applied to the sections to label nuclei andfetal trophoblast. Sections were washed in PBS, cover slipped with ProLong Gold and imaged using a White Light Laser Confocal Microscope Leica TCS SP8 X confocal microscope (Leica Microsystems) equipped with a tunable white light laser, HyD detectors and time gating, a 63X objective (N.A. = 1.4) and 1.3X digital zoom and the Leica Application Suite X Version 3.5.5.19976). Five regions from each section were selected for imaging and each image represents a field of view of 180 μm by 180 μm.

Data Recording and Statistical Analysis.

Ten images were randomly selected per placental section from each ewe for H&E analysis with each image representing a field of view of 620 μm x 464 μm. Data was collected using the region of interest function in ImageJ (NIH, https://imagej.nih.gov/ij/). and included size, perimeter, and number of amorphous connective tissue (ACT) deposits in H&E stained sections. Data from maternal vascular tissues detected by SMCA was collected using five images and the region of interest function in ImageJ. Data included luminal area, luminal perimeter, and number of vessels. Additionally, roundness, maximum diameter, and minimum diameter were used to measure irregularity of both ACT and maternal vessel shape. Calculation for circularity measurement is: circularity = 4 π x area (μm2)/perimeter (μm)2 or inverse of the aspect ratio was utilized to determine if there was a difference in ACT or maternal vessel shape based on fescue seed treatment wherein a value of 1.0 indicates a perfect circle while 0.0 indicates an elongated shape. Vessels were only counted if 75% of the vessel perimeter was highlighted by SMCA and the analysis of SMCA was conducted on merged immunofluorescent images which allowed for selection of high quality and consistent regions of tissue for analysis. Data were analyzed using Mixed Procedure of SAS (SAS Inst. Inc., NC) using fescue seed treatment (E-/E- or E+/E+) during gestation in the model. Significance was determined at p < 0.05 with trends at p ≤ 0.10.

RESULTS

Hematoxylin and Eosin (H&E) Staining.

Hematoxylin and eosin staining revealed large regions of amorphous connective tissue (ACT), sometimes referred to as Wharton’s Jelly, which comprises the core of fetal villi during late gestation. (Figure 1 d,e,f). These regions were 172% larger (p < 0.0001) and 111% more numerous (p = 0.025) in the placentome of E+/E+ ewes compared to E- treatment (Figure 2). The maximum diameter of ACT regions was 54% greater (p = 0.0006) in ergot alkaloid exposed (E+/E+) ewes versus controls (E-/E-). ACT regions from E+/E+ also had an increased (p < 0.0001) minimum diameter. The distribution of ACT regions by size and treatment is seen in Figure 3. While all placentome samples contained small ACT regions, 75% of these regions were < 5 x 103 μm2 in size for E-/E- samples (Figure 3). Comparatively, only 39% of ACT regions were < 5 x 103 μm2 for E+/E+ ewes (Figure 3). Placentomes from E+/E+ treated ewes contained regions as large as 43 x 103 μm2 in size while ACT regions in E-/E- samples rarely exceeded 10 x 103 μm2 (Figure 3).

Figure 1.

Figure 1.

Open in a new tab

H&E staining of the placentome from ewes fed endophyte-free (E-/E-; a, b, c) or endophyte infected (E+/E+; d, e, f) tall fescue seed from d 35 – d 133 of gestation. (a,b,d,e) Thick black arrows denote regions of amorphous connective tissue (ACT). In c and f, the zoomed images provide easier visualization of maternal capillaries (thin black arrows) and fetal capillaries (black arrowheads). (d,e) E+/E+ treatment exhibits larger, more numerous ACT regions (thick black arrows) as compared to E-/E-. (f) E+/E+ treatment shows larger maternal capillaries (thin black arrows) compared to (c) E-/E-. Scale bar = 20 μm for a, b, d, e and Scale bar = 100 μm for c , f.

Figure 2:

Figure 2:

Open in a new tab

The average area of amorphous connective tissue (ACT) regions. Average area is 0 – 14 X 103 μm2 in the placentomes of E+/E+ fescue treated ewes versus control ewes (E-/E-).

Figure 3:

Figure 3:

Open in a new tab

The number of ACT deposits and distribution of amorphous connective tissue (ACT) regions measuring 0 to >25 x 103 μm in the placentomes of E-/E - and E+/E+ fescue treated ewes.

SMCA and Collagen IV.

Collagen IV expression revealed overall placental structure as well as areas that were devoid of nuclei; whereas SMCA outlined maternal vasculature of the caruncle (Figure 4 a–f). Luminal area, luminal perimeter, and vessel circularity of maternal vasculature is presented in Figure 5 a – c. SMCA labelling showed no difference (p= 0.83) in the number of vessels present, but did show a 117% increase (p < 0.0001) in luminal area for the maternal vasculature of E+/E+ ewes as compared to E-/E- ewes (Figure 5a). Both the maximum and minimum luminal diameter of vessels was increased (p < 0.0001) for ewes on E+/E+ treatment. Ewes on E+/E+ treatment showed a decrease (p = 0.003) in circularity of the maternal vessels as compared to E-/E- ewes (Figure 5c). On basement membranes, Collagen IV expression with SMCA appeared to be more prominent in E+/E+ treated sample (Figure 4 d,e,f).

Figure 4.

Figure 4.

Open in a new tab

Double immunofluorescence assays for SMCA and Collagen IV of the placentomes from (a,b,c) ewes fed endophyte-free (E-) and (d,e,f) ewes fed endophyte-infected (E+) tall fescue seed from d 35 – d 133 of gestation. Collagen IV served as a marker for basement membranes (green), while SMCA served as a marker for smooth muscle cells of the vessels (orange). The placentomes of E+/E+ treated ewes show an increased luminal vessel area and more irregular vessel shape. DAPI was used to counterstain the nuclei (blue). Scale bar = 100 μm.

Figure 5:

Figure 5:

Open in a new tab

Measurements for (a) Luminal vessel area, (b) vessel perimeter, and (c) vessel circularity for SMCA immunofluorescent staining of the placentomes from ewes fed endophyte-free (E-) or endophyte-infected (E+) tall fescue seed from d 35 – d 133 of gestation.

CD45, MMP9 and BSL1/GSL1 Lectin.

Expression of CD45 was variable, but minimal across samples with few positive cells and appeared to be non-specific to treatment (Figure 6a–f). Some staining from CD45 appears to be punctate and potentially non-specific in the background of these images; however, no such patterning was observed in the negative control (data not shown); Therefore, despite the patterning, we presume that CD45 staining occurs at a baseline level throughout the tissue. BSL1/GSL1 lectin detected the fetal trophoblast (Figure 6 a – f). Within the trophoblast layer, there are regions mostly devoid of nuclei that are consistent with ACT as seen in the H&E images which appears specific to placentomes from E+/E+ treated ewes (Figure 6 d,e,f). These regions appear larger in placentomes of E+/E+ ewes which is also consistent with H&E findings. MMP9 expression was pronounced in the placentomes of E-/E- treated ewes and appeared to colocalize mostly with maternal tissue as there was minimal expression in regions with the fetal trophoblast as indicated by BSL1/GSL1 lectin staining (Figure 6 a,b,c).

Figure 6.

Figure 6.

Open in a new tab

Immunofluorescence assay for MMP-9, CD45 and BSL1/GSL1 lectin of the placentome from ewes fed endophyte-free (E-/E-;a,b,c)) or endophyte-infected (E+/E+; d,e,f) tall fescue seed from d 35 – d 133 of gestation. CD45 positive immune cells (green) were identified with immunofluorescence, and were found to be minimal for all samples, indicating that immune infiltration and/or inflammation was likely not a major contributor to the difference in the placentome appearance. (a,b,c) BSL1/GSL1 lectin staining for fetal trophoblast (red) was more prominent in the placentomes of ewes from the E-/E- treatment group. Immunostaining using an antibody MMP-9 (magenta), a gelatinase/collagenase, was used to highlight regions of the tissue that might be undergoing collagen restructuring. (d,e,f) Placentomes of E+/E+ treated ewes exhibited large, empty regions with few nuclei (DAPI, blue) and less MMP-9 staining overall compared to (a,b,c) E-/E- . Scale bar = 100 μm.

DISCUSSION

Although the sheep and human placenta differ in morphology, the villous structure within the ovine placentome is similar to that seen in the discoid placenta of humans [26, 27]. Fetal vasculature within the placenta is also comparable making sheep a suitable model for studying placental insufficiency [27]. Normal villous maturation has been studied extensively in humans and is linked to optimal placental function and fetal growth [28]. Additionally, delayed maturation has been associated with maternal obesity, hypoxia, intrauterine growth restriction (IUGR), and fetal death [28, 29]. In the case of sheep, the placenta experiences a rapid increase in growth during the first half of gestation in which the fetal villi invade the maternal stroma and are filled with an amorphous connective tissue similar to that of Wharton’s Jelly [30, 31]. Beginning around d 90 of gestation, the placenta enters a period of remodeling which works to increase vascularity. The plump fetal villi which have invaded the caruncle lose their ACT and begin branching extensively [31, 32]. As parturition approaches, the ACT within the fetal villi is either reduced to thin strands or disappear altogether. The disappearance of the ACT coincides with increased vascularization of the villi which brings the blood vessels into close proximity with each other and is indicative of appropriate villous maturation in sheep [32]. In the present study, this ACT within the fetal villi exists across all samples. However, the placentome of ewes on E+/E+ fescue seed treatment exhibited ACT regions which were larger and more numerous compared to their E-/E- counterparts. The ACT regions in E+/E+ placentomes also appear rounder as opposed to the thin strands of ACT seen in E-/E- tissues. Stegeman [25] reported the complete loss of fetal ACT by approximately day 110 of gestation in placentome cross sections but, our research suggests that small pockets of ACT still remain within the fetal villi of all samples. The increased size and number as well as the rounded shape of ACT deposits in the placentomes of E+/E+ treated ewes may indicate slowed villous maturation of the placenta. Additionally, residual ACT represents regions of undeveloped vascular tissue which likely limits nutrient exchange to the fetus.

The remodeling phase of ovine placental development focuses on substantially increasing cotyledonary capillary area density, surface density, and number density [33]. A similar pattern is also seen over time in the caruncular vasculature though not to the same degree [34]. The maternal vasculature primarily works as a low velocity delivery system of nutrients consisting of larger capillaries some of which exceed 20 μm in diameter. In contrast, the fetal vasculature facilitates rapid transport for nutrient exchange through smaller, more abundant capillary system with an average capillary diameter of 4.5 μm [25]. Penninga and Longo reported a 31% increase in luminal size of maternal vessels in sheep placenta exposed to high elevation induced-hypoxia [35]. Our findings align with previous reports of maternal and fetal vessel size, but indicate a much larger increase in maternal luminal area of 117% for ewes exposed to E+/E+ fescue seed treatment compared to those exposed to elevation induced hypoxia. It is speculated that an increase in vessel size without an increase in number may be a placental adaption to increase vascular surface area and slow the blood flow rate for a more efficient exchange process [36]. In the current study, vessels showed a decrease in roundness and vasculature was visibly irregular in shape. In the case of environmental heat stress, uterine and umbilical blood flows are reduced leading to a reduction in placental weights and IUGR.

This becomes especially evident during late gestation and most likely occurs due to thermoregulation in the dam [33, 37]. During heat stress, the body attempts to facilitate the loss of body heat by directing blood flow to the skin surface. When heat stress continues for extended periods of time, it redirects crucial blood flow away from the reproductive tract and uterus thereby reducing the overall blood supply to the placenta and the fetus [38]. Both hyperthermia and hypoxia have also resulted in increased irregularity and coiling of maternal and fetal vasculature [35–37]. Additionally, hyperthermia leads to reduced umbilical oxygen uptake which leads to a state of hypoxia [39]. Although the mechanism varies, resulting hypoxia causes altered vasculature of the ovine placentome likely as an attempt to rescue placental development and subsequent fetal growth. While uterine blood flow was not measured in the present study, the vasoconstrictive action of ergot alkaloids has been repeatedly documented on both non-reproductive [13, 17, 18] and reproductive [14, 19, 20] vasculature. In addition, the reduction in luminal vessel area of the uterine artery by Klotz et al. [20] indicated that samples in the present study were experiencing a reduction in uteroplacental blood flow when compared to E-/E- samples at the time of surgical collection. Results presented here suggests that ergot alkaloid exposure alters placental development similar to what is reported in cases of hypoxia and hyperthermia, relating to reduced blood flow.

Collagen IV accounts for approximately 50% of basement membrane proteins and self assembles into organized networks crucial for stability [40]. MMP-9, one of two gelatinases, contains a collagen binding site and works to cleave several macromolecules found in the extracellular matrix, including collagen IV [41]. This process, which also activates growth factors and chemokines, stimulates the release of vascular endothelial growth factor (VEGF) and blood vessel formation [42]. MMPs are mediators of tissue remodeling and angiogenesis in the placentae of humans and mice and function as an angiogenic switch [43, 44]. In humans, it has been hypothesized that reductions in MMP9 may result in insufficient VEGF stimulation and reduced extracellular matrix degradation and remodeling during the later stages of pregnancy [45]. Though not quantified in the present study, placentomes from E+/E+ treated ewes appear to have more collagen IV and less MMP-9 staining. As parturition approaches, the detachment of the placenta from the uterine wall requires degradation of the extracellular matrix which is regulated by MMPs [46]. Exposure to ergot alkaloids during gestation has been shown to reduce gestation length by approximately four days in ewes [22]. While it was hypothesized that this might be due in part to premature degradation of collagen IV by MMP-9, this does not appear to be the case. However, a reduction in MMP-9 in the placentome of E+/E+ treated ewes may indicate reduced remodeling.

The present study suggests that exposure to ergot alkaloids from d 35 – d 133 of gestation (E+/E+ treatment) results in slowed and/or stunted maturation of the fetal villi and greater regions of ACT. Ergot alkaloid exposure also alter blood vessel shape and luminal area, which indicate changes to counteract reduced blood flow due to vasoconstriction.

Acknowledgments

Appreciation is expressed to: M. C. Miller for animal handling, R. Smith for laboratory assistance, and C. Piatak and B. Pinckney for assistance with image analysis.

Funding:

Technical Contribution No. 6579 of the Clemson University Experiment Station. This research was supported, in part, by USDA Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23218. The research reported in this publication was conducted using a Leica 6500 Laser Microdissection system and a Leica SP8X Confocal multiphoton/HyVolution microscope system, both housed in the Clemson Light Imaging Facility (CLIF). CLIF is supported, in part, by the Clemson University Division of Research, NIH Award #P20GM109094, and NIH Award #5P20RR021949-03; Equipment funding was provided in part by NSF MRI Award #0722841, NSF Award #IOS-1444461, NSF MRI Award #1126407. The content of this material and any opinions, findings, conclusions, or recommendations expressed in this material is solely the responsibility of the author(s) and does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation.

Footnotes

Declaration of Interest

None.

References

  • 1.Browne VA, Julian CG, Toledo-Jaldin L, et al. , Uterine artery blood flow, fetal hypoxia and fetal growth. Philos Trans R Soc Lond B Biol Sci, 2015. 370(1663): 20140068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ueland K, Novy MJ, Peterson EN, Metcalfe J. Maternal cardiovascular dynamics. IV. The influence of gestational age on the maternal cardiovascular response to posture and exercise. Am J Obstet Gynecol, 1969. 104(6): 856–864. [PubMed] [Google Scholar]
  • 3.Clapp JF 3rd, Maternal heart rate in pregnancy. Am J Obstet Gynecol, 1985. 152(6 Pt 1): 659–660. [DOI] [PubMed] [Google Scholar]
  • 4.Dickey RP, Hower JF, Ultrasonographic features of uterine blood flow during the first 16 weeks of pregnancy. Hum Reprod, 1995. 10(9): 2448–2452. [DOI] [PubMed] [Google Scholar]
  • 5.Thaler I, Manor D, Itskovitz J, et al. , Changes in uterine blood flow during human pregnancy. Am J Obstet Gynecol, 1990. 162(1): 121–125. [DOI] [PubMed] [Google Scholar]
  • 6.Reynolds LP, Caton JS, Redmer DA, et al. , Evidence for altered placental blood flow and vascularity in compromised pregnancies. J Physiol, 2006. 572(Pt 1): 51–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Reynolds LP, Boroqicz PP, Vonnahme KA, et al. , Animal models of placental angiogenesis. Placenta, 2005. 26(10): 689–708. [DOI] [PubMed] [Google Scholar]
  • 8.Lunell NO, Nylund LE, Lewander R, Sarby B., Uteroplacental blood flow in pre-eclampsia measurements with indium-113m and a computer-linked gamma camera. Clin Exp Hypertens B, 1982. 1(1): 105–117. [DOI] [PubMed] [Google Scholar]
  • 9.Lang U, Baker RS, Braems G, et al. , Uterine blood flow--a determinant of fetal growth. Eur J Obstet Gynecol Reprod Biol, 2003. 110 Suppl 1: S55–61. [DOI] [PubMed] [Google Scholar]
  • 10.Young BC, Levine RJ, Karumanchi SA, Pathogenesis of preeclampsia. Annu Rev Pathol, 2010. 5: 173–192. [DOI] [PubMed] [Google Scholar]
  • 11.Sharma D, Shastri S, Farahbakhsh N, Sharma P, Intrauterine growth restriction - part 1. J Matern Fetal Neonatal Med, 2016. 29(24): 3977–3987. [DOI] [PubMed] [Google Scholar]
  • 12.Strickland JR, Looper ML, Matthews JC, et al. , Board-invited review: St. Anthony's Fire in livestock: causes, mechanisms, and potential solutions. J Anim Sci, 2011. 89(5): 1603–1626. [DOI] [PubMed] [Google Scholar]
  • 13.Schoning C, Flieger M, Pertz HH, Complex interaction of ergovaline with 5-HT2A, 5HT1B/1D, and alpha1 receptors in isolated arteries of rat and guinea pig. J Anim Sci, 2001. 79(8): p. 2202–2209. [DOI] [PubMed] [Google Scholar]
  • 14.Dyer DC, Evidence that ergovaline acts on serotonin receptors. Life Sci, 1993. 53(14): PL2238. [DOI] [PubMed] [Google Scholar]
  • 15.Nagatomo T, Rashid M, Abul Muntasir H, Komiyama T. Functions of 5-HT2A receptor and its antagonists in the cardiovascular system. Pharmacol Ther, 2004. 104(1): 59–81. [DOI] [PubMed] [Google Scholar]
  • 16.Klotz JL, Activities and Effects of Ergot Alkaloids on Livestock Physiology and Production. Toxins (Basel), 2015. 7(8): 2801–2821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Aiken GE, Kirch BH, Strickland JR, et al. , Hemodynamic responses of the caudal artery to toxic tall fescue in beef heifers. J Anim Sci, 2007. 85(9): 2337–2345. [DOI] [PubMed] [Google Scholar]
  • 18.Aiken GE, Strickland JR, Looper ML, et al. , Hemodynamics are altered in the caudal artery of beef heifers fed different ergot alkaloid concentrations. J Anim Sci, 2009. 87(6): 2142–2150. [DOI] [PubMed] [Google Scholar]
  • 19.Poole DH, Lyons SE, Poole RK, Poore MH, Ergot alkaloids induce vasoconstriction of bovine uterine and ovarian blood vessels. J Anim Sci, 2018. 96(11): 4812–4822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Klotz JL, Britt JL, Miller MF, et al. , Ergot alkaloid exposure during gestation alters: II. Uterine and umbilical artery vasoactivity1. J Anim Sci, 2019. 97(4): 1891–1902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Britt JL, Greene MA, Bridges WC, et al. , Ergot alkaloid exposure during gestation alters. I. Maternal characteristics and placental development of pregnant ewes1. J Anim Sci, 2019. 97(4):1874–1890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Duckett SK, Andrae JG, Pratt SL, Exposure to ergot alkaloids during gestation reduces fetal growth in sheep. Front Chem, 2014; 2:68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.NRC National Research Council, Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids. 2007, Washington, DC: The National Academies Press. p. 244–270. [Google Scholar]
  • 24.Vatnick I, Schoknecht PA, Darrigrand R, Bell AW. Growth and metabolism of the placenta after unilateral fetectomy in twin pregnant ewes. J Dev Physiol, 1991. 15(6): 351–356. [PubMed] [Google Scholar]
  • 25.Reynolds LP, Biondini ME, Borowicz PP, et al. , Functional significance of developmental changes in placental microvascular architecture. Endothelium, 2005. 12(1–2):11–19. [DOI] [PubMed] [Google Scholar]
  • 26.Barry JS, Anthony RV, The pregnant sheep as a model for human pregnancy. Theriogenology, 2008. 69(1):55–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Leiser R, Krebs C, Ebert B, Dantzer V. Placental vascular corrosion cast studies: a comparison between ruminants and humans. Microsc Res Tech, 1997. 38(1–2): p. 76–87. [DOI] [PubMed] [Google Scholar]
  • 28.Turowski G and Vogel M, Re-view and view on maturation disorders in the placenta. APMIS, 2018. 126(7):602–612. [DOI] [PubMed] [Google Scholar]
  • 29.Fox H and Elston CW, Pathology of the placenta. Major Probl Pathol, 1978. 7: 1–491. [PubMed] [Google Scholar]
  • 30.Alexander G, Studies on the Placenta of the Sheep (Ovis Aries L.). Placental Size. J Reprod Fertil, 1964. 7:289–305. [DOI] [PubMed] [Google Scholar]
  • 31.Barcroft J, Barron DH, Observations upon the form and relations of the maternal and fetal vessels in the placenta of the sheep. Anat Rec, 1946. 94:569–595. [DOI] [PubMed] [Google Scholar]
  • 32.Stegeman JHJ. Placental development in the sheep and its relation to fetal development; A qualitative and quantitative anatomic and histologic study. Bijdragen Tot De Dierkunde Amsterdam, University of Amsterdam, 1974; 44(1):3–72 [Google Scholar]
  • 33.Reynolds LP, Borowicz PP, Caton JS, et al. , Uteroplacental vascular development and placental function: an update. Int J Dev Biol, 2010. 54(2–3):355–366. [DOI] [PubMed] [Google Scholar]
  • 34.Borowicz PP, Arnold DR, Johnson ML, et al. , Placental growth throughout the last two thirds of pregnancy in sheep: vascular development and angiogenic factor expression. Biol Reprod, 2007. 76(2):259–267. [DOI] [PubMed] [Google Scholar]
  • 35.Penninga LLD Longo, Ovine placentome morphology: effect of high altitude, long-term hypoxia. Placenta, 1998. 19(2–3): 187–193. [DOI] [PubMed] [Google Scholar]
  • 36.Krebs C, Longo LD, Leiser R, Term ovine placental vasculature: comparison of sea level and high altitude conditions by corrosion cast and histomorphometry. Placenta, 1997. 18(1): 43–51. [DOI] [PubMed] [Google Scholar]
  • 37.Regnault TR, Galan HL, Parker TA, Anthony RV. Placental development in normal and compromised pregnancies-- a review. Placenta, 2002. 23 Suppl A: S119–129. [DOI] [PubMed] [Google Scholar]
  • 38.Dreiling CE, Carman FS 3rd, and Brown DE, Maternal endocrine and fetal metabolic responses to heat stress. J Dairy Sci, 1991. 74(1): 312–327. [DOI] [PubMed] [Google Scholar]
  • 39.Regnault TR, de Vrijer B, Galan HL, et al. , Development and mechanisms of fetal hypoxia in severe fetal growth restriction. Placenta, 2007. 28(7):714–723. [DOI] [PubMed] [Google Scholar]
  • 40.Timpl R, Wiedemann H, van Delden V, et al. , A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem, 1981. 120(2):203–211. [DOI] [PubMed] [Google Scholar]
  • 41.Somerville RP, Oblander SA, Apte SS, Matrix metalloproteinases: old dogs with new tricks. Genome Biol, 2003; 4(6):216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kalluri R, Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer, 2003. 3(6): 422–433. [DOI] [PubMed] [Google Scholar]
  • 43.Alexander CM, Hansell EJ, Behrendtsen O, et al. , Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development, 1996. 122(6):1723–1736. [DOI] [PubMed] [Google Scholar]
  • 44.Anacker J, Segerer SE, Hagemann C, et al. , Human decidua and invasive trophoblasts are rich sources of nearly all human matrix metalloproteinases. Mol Hum Reprod, 2011. 17(10):637–652. [DOI] [PubMed] [Google Scholar]
  • 45.Plaks V, Rikenberger J, Dai J, et al. , Matrix metalloproteinase-9 deficiency phenocopies features of preeclampsia and intrauterine growth restriction. PNAS, 2013. 110(27):11109–11114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Xu P, Alfaidy N, and Challis JR, Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor. J Clin Endocrinol Metab, 2002. 87(3):1353–1361. [DOI] [PubMed] [Google Scholar]

RESOURCES