Exposure to ergot alkaloids during mid and late gestation reduced placentome and fetal weights at near-term, d 133 of an average 145 d gestation length. The reductions in the placentome weight were a direct result of reductions in the cotyledon, the fetal side of the placentome, mass and not of the caruncle, the maternal side. Estimates of placental sufficiency showed no difference in the fetal to cotyledon weight ratio, which suggests that the reduction in cotyledon weight limited growth of the fetus. During the first half of gestation, the ovine placenta experiences rapid proliferative growth and peaks in weight around d 80 after which a period of remodeling begins where connective tissue in the core of the fetal villi is replaced by vascular beds (31, 32). This angiogenic vascular development in the cotyledon results in a 12-fold increase in capillary density, a decrease in capillary size, and continues throughout late gestation to support rapid fetal growth (33). Previous research documents the extensive list of vascular and growth factors necessary during this process throughout mid and late gestation (15, 32-35) and differences in gene expression have been reported in cases of undernutrition (36), hyperthermia (37), and hypoxia (38). Ergot alkaloids are known vasoconstrictors (39, 40) that bind to 5HT2A serotonergic receptors. Previous examinations of known vascular factors involved in placental growth did not reveal any differences due to ergot alkaloid exposure (6) and therefore this RNA-sequencing project was developed to identify differentially expressed genes associated with ergot alkaloid exposure and discover potential adaptive mechanisms that alter nutrient supply to fetus.
The RNA-Seq results identified 95 genes that were up-regulated and 16 down-regulated in response to feeding mycotoxins. Interestingly, LECT2 was the most upregulated gene with a 7-fold increase in the cotyledon samples of E+/E+ treated ewes. It is described as an energy-sensing hepatokine that is preferentially expressed in human adult and fetal liver cells but is secreted into the bloodstream (41). Several studies have associated LECT2 with glucose metabolism, insulin resistance and inflammation (42-44). Knockout mouse studies show that loss of LECT2 increases insulin signaling in skeletal muscle (41). To our knowledge, LECT2 expression has not been previously identified in placental tissues nor associated with IUGR. Therefore, further research is warranted to investigate the role of LECT2 in cases of mycotoxin exposure and irregular placental development.
Ewes on E+/E+ fescue seed treatment during gestation experienced an upregulation in genes associated with responses to carbohydrates, glucose and carbohydrate biosynthesis. The main carbohydrate utilized by the placenta is glucose which is transported by GLUT transporters through the process of facilitated diffusion (45). Under normal conditions the majority of glucose is derived from maternal circulation, but glucose can be derived from fetal blood if maternal concentrations are lacking (46). Because of dependence on the concentration gradient, changes in the placental uptake and consumption of glucose is directly related to changes in maternal concentrations (47). The upregulation in genes associated with responses to carbohydrates and glucose may indicate an increase in maternal concentrations of glucose reaching the placenta. However, plasma glucose concentrations were not different based on fescue seed treatment in the associated study (6). During late gestation, placental consumption of glucose is up to 10 times higher than the fetus for the production of ATP and other sugars and carbohydrates including polyols (48). The polyol pathways are highly active in the early placenta and are closely associated with the pentose phosphate pathway, which supports rapid cell proliferation (49, 50). In sheep, additional carbohydrates such as lactate and fructose are produced and shuttled to umbilical and uterine circulations. Sheep with growth restricted placentae exhibit a reduction in overall glucose consumption on a placental weight basis, but an increase in the conversion of glucose (or other substrates) to lactate, much of which is shuttled to the fetus (51). A similar scenario is seen in humans in which up to 22% of glucose is converted to lactate under normoxic conditions (52). Because lactate is unable to be used by the placenta, it is speculated that this may be an attempt to set aside resources for the fetus (49). The increase in carbohydrate biosynthesis suggests a similar scenario in which glucose or other substrates are converted to lactate or fructose at an increased rate in ewes on E+/E+ fescue seed treatment.
Triglyceride and cellular lipid metabolism were also upregulated in E+/E+ cotyledon samples compared to controls. Lipids and free fatty acids are required for growth and development of both the fetus and placenta but transport across the placenta appears minimal for most livestock species (53). In sheep, the placenta may hydrolyze esterified lipids and then desaturate and/or elongate them to supply essential fatty acids to the fetus (53). These processes, along with the synthesis of lipids from glucose and ketoacids, may provide necessary fats for both placental and fetal tissues during gestation (45). Research also shows that volatile fatty acids (VFAs) produced through the process of rumen fermentation are utilized by the placenta and transported to the fetus in both cows and sheep (53). It is hypothesized that the placental utilization of VFAs may work to generate ATP or synthesize fatty acids and, in adverse conditions, lipids could be utilized as an alternate energy source if insufficient glucose is available (48,54). In cases of maternal undernutrition in sheep, there is an increase in placental fatty acid transporters (55) and placental lipid metabolism is known to be altered in cases of IUGR (56). Additionally, KEGG pathway analysis determined that PPAR signaling was upregulated in the cotyledon of E+/E+ treated ewes. Recent research has elucidated critical roles for PPARs in placental development and the pathophysiology of IUGR. PPARs are ligand-activated transcription factors that regulate gene expression in a variety of tissues and play a critical role in placental lipid metabolism (57). Human placentae were found to have increased PPAR expression levels in cases of preeclampsia and IUGR when compared to controls and it has been suggested this may be an adaptive response to compensate for insufficient placental development (58). APOC3 is found within the PPAR pathway and had a 5-fold greater expression in cotyledon samples from E+/E+ treated ewes. APOC3 is associated with hypertriglyceridemia and elevates plasma triglyceride levels by preventing clearance of very-low-density lipoproteins (VLDLs) and high-density lipoproteins (HDLs) but limited research is available denoting its presence or role in placental function.
A primary function of the placenta is to facilitate nutrient exchange to the fetus through the use of transport systems. Transporters are in place to mediate the transfer of endogenous compounds across the placental barrier but, in some cases, transporters accept specific exogenous substrates such as drugs or xenobiotics if they are present (59). Such transporters are identified as drug transporters and include monocarboxylate transporters (MCTs) and anion transporters (59). MCTs generally work to transport lactate, much of which is produced by placental metabolism when glucose is in short supply (60). The placenta also uses anion transporters, such as organic anion transporters (OATs), which have been discovered in almost all barrier epithelia within the body (61). Both MCTs and OATs also function to transport a wide range of drugs and toxins in addition to their endogenous substrates (59). Ewes on E+/E+ fescue seed treatment experienced a consistent downregulation in a variety of transport systems, including monocarboxylic acid and anion transport, within the cotyledon compared to ewes on the control treatment. In contrast, solute carrier family 22 member 9 (SLC22A9; also known as OAT7) experienced a 5-fold increase in the cotyledon from E+/E+ ewes and was previously thought to be liver-specific (62). It has been hypothesized that ergot alkaloids or metabolites may cross the placental barrier (7) but no research is available documenting the presence of these compounds in the fetus or which transport systems may be involved if placental transport exists. It was previously proposed that the downregulation of certain transport systems may augment fetal exposure to environmental toxicants during pregnancy (63). A similar mechanism may be at work to limit fetal exposure to ergot alkaloids if placental transport occurs.
During late gestation, the primary function of the placenta is to facilitate nutrient exchange to the fetus in order to keep pace with exponential fetal growth (33). REViGO revealed several upregulated GO clusters associated with macromolecule metabolism, including protein and lipid metabolism, in the cotyledon of ewes exposed to E+ fescue seed during gestation compared to those ewes on E- treatment. In addition to facilitating nutrient exchange to the fetus, the placenta is a metabolically active organ with specific energy and nutrient requirements. The placenta also utilizes, produces, and converts amino acids throughout gestation (53). The placental protein turnover rate was previously reported at 60% per day in sheep (64). Such high protein turnover rates are heavily dependent on the amino-acid availability in maternal circulation. During the initial period of placental growth in humans, the placenta is the primary recipient of maternally circulating amino acids. As fetal growth accelerates during late gestation, the fetus becomes the primary beneficiary of amino acids (65). In sheep, the placenta is a net consumer of glutamate, serine, valine, leucine and isoleucine and shuttles greater concentrations of glutamine, methionine and glycine towards the fetus compared to levels found in maternal uterine circulation suggesting significant utilization and/or conversion of amino acids (66). The placenta experiences a high level of protein synthesis during late gestation due to the changes in placental morphology. It was hypothesized that this period would result in a high rate of protein turnover (45, 67). Nutritional stress in sheep has been shown to alter fetoplacental amino acid metabolism and cycling and in some cases changes in metabolism appear permanent (68, 69). The upregulation in protein metabolism and proteolysis in the cotyledon of E+/E+ fescue treated ewes may indicate increased placental remodeling or a higher rate of protein turnover.
The KEGG pathway analysis determined the complement and coagulation cascade pathway was highly upregulated in the cotyledon of E+/E+ treated ewes. While the complement and coagulation networks are distinct, several key crossover points keep them linked and both must be tightly regulated during pregnancy. Increased thrombin production, which has been associated with preeclampsia and IUGR, may be a result of increased activation of the coagulation cascade beyond that of normal pregnancy (70). Additionally, several coagulation components are necessary for vascular differentiation which is often impaired in the placentae of preeclamptic women (70). Regulation of the innate and adaptive immune response through the complement system is necessary for successful placental and fetal development (71). While some degree of activation is required, early embryonic loss and IUGR are often associated with increased complement activation (72). The complement system has three routes of activation: the classical pathway, alternate pathway, and mannose-binding lectin pathway. The cotyledon samples of ewes exposed to E+/E+ fescue seed treatment had a 5-fold upregulation of MBL. Mannose binding lectin functions as a critical part of the innate immune system and works as a first line of defense against microorganisms (72). Under normal physiological conditions, MBL does not recognize an organism’s own tissues. However, in cases of cellular hypoxia, altered cell surface glycosylation can stimulate MBL expression and activate the complement system (73). In this study, ergot alkaloid induced vasoconstriction may result in placental hypoxia that stimulates MBL and the complement cascade.
Several genes presented with a high rate of expression that have not previously been identified in ovine cotyledonary tissues. TIMP2 and TIMP3 are members of the TIMP metallopeptidase inhibitor family that are involved in degradation of extracellular matrix and suppress endothelial cell proliferation. CPXM1 is a member of the carboxypeptidase X, M14 family and placental expression has not been reported previously but it has been shown to be a positive regulator of adipogenesis (74). CAPN6 is a member of the calpain family which are calcium-dependent cysteine proteases shown to be highly expressed in the placenta where they are involved in tumorigenesis by promoting angiogenesis (75). Placentally expressed transcript 1 (PLET1) has been characterized in the pig where it was shown to be involved in trophoblastic elongation of the conceptus (76). While these genes did not show differences based on treatment, their high level of expression not previously reported in sheep was worth noting.
The research presented here was our initial exploration to identify the genes associated with reduced cotyledon mass observed when feeding mycotoxins containing ergovaline and ergovalinine. Using RNA-Seq methods, we identified several genes (LECT2, SLC22A9, APOC3, MBL2) that were highly (log2FC > 5; FDR < 0.01) up-regulated in response to mycotoxin exposure and one (STRA6) that was down-regulated (log2FC > 3; FDR < 0.01). Comparisons between RNA-Seq and qPCR showed 86% agreement in fold-change but genes with lower fold-change were not significant for qPCR. These comparisons are similar to those reported by Everaert and co-workers (77) who reported 85% agreement between RNA-Seq and qPCR comparisons of gene expression fold-change but genes with lower expression had inconsistent expression measurements. We acknowledge that this study had a very small sample size (n = 3 biological replicates per treatment) and that this is a limitation of the study. These results identified novel genes that may be involved in mycotoxin exposure and further research with larger numbers is warranted to examine how the cotyledon tissue responds to ergot alkaloid induced vasoconstriction.