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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Reprod Toxicol. 2019 Feb 14;85:42–50. doi: 10.1016/j.reprotox.2019.01.010

Dietary exposure to mycotoxin zearalenone (ZEA) during post-implantation adversely affects placental development in mice

Rong Li 1,2,3,#, Christian Lee Andersen 1,2,#, Lianmei Hu 1,4, Zidao Wang 1,2, Yuehuan Li 1,2, Tamas Nagy 5, Xiaoqin Ye 1,2,*
PMCID: PMC6443452  NIHMSID: NIHMS1522495  PMID: 30772436

Abstract

Zearalenone (ZEA) is a common food contaminant (ppb~ppm) derived from Fusarium fungi. With its estrogenicity and potential chronic exposure, ZEA poses a risk to pregnancy. Our previous studies implied post-implantational lethality by ZEA. Since a functional placenta is essential for fetal development and survival, it was hypothesized that ZEA may have adverse effects on placental development leading to post-implantational lethality. Exposure of young mice to 0, 0.8, 4, 10, and 40 ppm ZEA diets from gestation day 5.5 (D5.5) to D13.5 led to increased resorption of implantation sites, increased placental hemorrhage, decreased placental and fetal weights, proportionally reduced placental layers, and disorganized placental labyrinth vascular spaces in the 40 ppm ZEA group, as well as lipid accumulation in the labyrinth layer of all four ZEA treatment groups examined on D13.5. These data demonstrate adverse effects of ZEA on placental development.

1. Introduction

Zearalenone (ZEA) is a major mycotoxin derived from Fusarium fungi and a common food contaminant in the levels of parts per billion (ppb) ~ parts per million (ppm), with 600 ppm being the highest reported in contaminated food [1–4]. Contaminated food is the primary source of ZEA exposure for mammals. ZEA is quickly absorbed and mainly metabolized in the liver to form α- and β-zearalenol, from which α-zearalanol (zeranol), β-zearalanol (teranol), and zearalanone can be derived [1, 5–8]. Unconjugated ZEA has an elimination half-life of 16.8 hours after oral administration in male rats [4]. Because of their structural similarity with 17β-estradiol (E2) and their interactions with estrogen receptors (ERα, ERβ) [9–11], ZEA and its metabolites are also called mycoestrogens, and among them, α-zearalenol is the most potent [5, 12, 13]. Estrogen receptor(s)-modified animal models have revealed the essential in vivo roles of estrogen signaling in pregnancy [14–18]. The estrogenicity of ZEA gives it the potential to interfere with mammalian pregnancy. Indeed, studies have shown that ZEA and its metabolites can disrupt pregnancy in different species (e.g., pig and mouse) by affecting different pregnancy events, such as preimplantation embryo development and transport, embryo implantation, and potentially placental development (reviewed in [19]).

The placenta is a transient organ bridging the mother and fetus during eutherian pregnancy. It is an important organ for toxicological evaluations but is seriously understudied. This situation is also implicated in studying the effect of ZEA on placental development. Several studies have alluded to the placenta as a target of ZEA: for example, ZEA can be bioactivated in placental cells [20]; ZEA and its metabolites can pass through placentas [21, 22]; ZEA (2.76 ppm diet from D35 to D70 of pregnancy) can be accumulated in pig placentas and reduce placental and fetal weights [23], which may indicate impaired placental function that was not further investigated in the study; ZEA (20 ppm diet, from D0.5 to D20.5 of pregnancy) causes reduced birth weight of rats and reduced Esr1 (ERα) mRNA levels in the placentas but its effects on placental weight and placental morphology were not reported in the study [24]; rats exposed to 8 mg/kg body weight ZEA via gavage on D6–D19 had increased resorption of implantation sites and decreased fetal viability examined on D20, but the placentas were not examined in the study [25]. Since the placenta is the sole source of nutrients for supporting fetal development, the fetal toxicity from ZEA treatment could be a secondary effect of placental toxicity.

To determine the effect of ZEA on placental development, we use mouse as a model that shares the same hemochorial structure as humans. Mice have a definitive chorioallantoic placenta that develops from the extraembryonic lineages originated from the trophoblasts in gestation day 3.5 (D3.5) blastocysts. Embryo implantation in mice initiates ~D4.0 and trophoblasts penetrate through uterine luminal epithelium into the uterine stromal layer by D5.0 for the subsequent establishment of a placenta [26]. The mural trophectoderm cells (not in contact with the inner cell mass) become trophoblast giant cells. The polar trophectoderm cells (adjacent to the inner cell mass) form the extraembryonic ectoderm and ectoplacental cone that will give rise to the spongiotrophoblast layer and labyrinth layer of the placenta. Structurally, a mouse placenta has three major layers: the outer maternal layer that includes decidual cells and maternal vasculature; the middle “junctional” zone that includes spongiotrophoblast layer and parietal trophoblast giant cell layer; and the inner labyrinth layer [27]. The labyrinth is the closest to the fetus for nutrient and gas exchanges between maternal and fetal blood as well as for disposal of waste from the fetus [28]. The mouse labyrinth consists of two separate, highly branched, and tortuously intertwined vascular networks, the maternal blood spaces and the fetal capillaries. They are separated by an interhemal membrane that consists of three layers of trophoblasts, which includes a layer of mononuclear sinusoidal trophoblast giant cells lining the maternal blood spaces and two layers of multiple nuclear syncytiotrophoblasts, as well as a layer of fetal blood vessel endothelial cells lining the fetal capillaries [28]. The flexuous network of maternal and fetal vessels in the labyrinth starts to develop following chorioallantoic attachment that occurs ~D8.5 [29], is established ~D10.5, and subsequently undergoes extensive modifications to accommodate fetal growth. The three major layers in the mouse placenta can be clearly defined by D12.5 [30].

Our previous study demonstrated that ZEA diet at 40 ppm blocked embryo implantation in mice [31]. To avoid the adverse effect of ZEA on embryo implantation [31] and to cover the main placental development period [27], in this study, we treated the mice during post-implantation period from D5.5 to D13.5 to determine the effect of ZEA on placental development. Indeed, ZEA at environmental relevant levels can adversely affect placental development.

2. Materials and methods

2.1. Animals

Wild type mice with C57BL/6 and129 mixed background, which were derived from Atp6v0d2+/− mice in C57BL/6 and129 mixed background [32, 33], were used in this study. Prior to treatment, all the mice were fed with regular chow 5053 (Labdiet, St. Louis, MO, USA) and housed in polypropylene cages with free access to food and water from water sip tubes in a reverse osmosis system. The Coverdell animal facility at the University of Georgia is on a 12-hour light/dark cycle (6:00 AM to 6:00 PM) at 23±1°C with 30–50% relative humidity. All methods used in this study were approved by the University of Georgia IACUC Committee (Institutional Animal Care and Use Committee) and conform to National Institutes of Health guidelines and public law.

2.2. Dose selection, treatment, and tissue collection

ZEA doses of 0 ppm (control), 0.8 ppm, 4 ppm, 10 ppm, and 40 ppm in the diets were used in our previous postweaning, premating, postmating, and/or multigenerational studies in mice [31, 34]. These ZEA levels have been reported in the contaminated food [1–4], which must also have contamination of other mycotoxins, including other mycoestrogens. The homemade ZEA diets were prepared by mixing ZEA (Cayman chemical, Ann Arbor, Michigan, USA) in casein-based phytoestrogen-free AIN-93G diet (Bio-Serv, Frenchtown, NJ) as we previously described [31, 34]. Our previous studies established 0.8 ppm, 4 ppm, 10 ppm, and 40 ppm ZEA diets to corresponding ZEA doses of about 0.1, 0.5, 1.25, and 5 mg/kg body weight per day, respectively, in mice [31, 34].

Female mice at 2–3 months old were mated with stud males (3–4 females with one male in a cage) and checked for a vaginal plug the next morning. The day of vaginal plug presence was designated as gestation day 0.5 (D0.5). The plugged mice were randomly assigned into 5 groups (N=6–9 pregnant mice/group, no littermates in the same treatment group). The weights of the females in different groups were comparable at the beginning of treatment on D5.5. The treatments started on D5.5 and ended on D13.5 when the mice were dissected. Food and water consumptions in each cage were monitored. Since the mice were plugged on different days and treated with different ZEA diets, to reduce the housing cost, up to 5 mice on different gestation days in the same treatment group could be housed in the same cage during treatment. Therefore, food and water consumptions were rough estimations without considering gestation days. Body weights were recorded to determine pregnancy status as we reported previously [35].

On D13.5, the pregnant females were sacrificed by cervical dislocation. The numbers of total implantation sites, absorbed implantation site(s), placentas with live fetus, and weights of placentas and fetuses were recorded. One representative placenta from each mouse was fixed in Bouin’s solution and the rest of the placentas were snapped frozen in liquid N2 and kept at −80°C. The average weight of placentas with a live fetus from each dam was counted as one data point, so as the average weight of all live fetuses from each dam. The body weight gain per implantation site for each dam was calculated as the weight difference between D13.5 and D5.5 divided by the total number of implantation sites in each dam on D13.5. The implantation site resorption rate was counted as the percentage of resorbed implantation sites, which were smaller, could be darker, and without a live fetus compared to the unabsorbed ones, in each pregnant D13.5 mouse. The resorbed implantation sites were classified into two groups in the 40 ppm ZEA group: type I resorption with the implantation site completely dark; and type II resorption with a small and partially pinkish implantation site.

2.3. Histology and quantification of placental layers

After fixation in Bouin’s solution for 48 hrs, the placentas were dehydrated in 50%, 70%, 80%, 90% 100% (twice) alcohol for 1 hr each, and subsequently, cleared in xylene for 5–10 min until the tissue becomes transparent, and incubated in paraffin overnight before embedding. The processed placentas were then embedded in the orientation for cross sections. Every 10th cross placental sections in the largest middle part were collected. Haemotoxylin and Eosin staining (H&E) staining was done as previously described [35, 36]. The middle three sections with the largest areas were selected for area quantification. The total area of each placental section and the areas of labyrinth layer, junctional zone, and decidua, which were outlined manually and quantified using Image J (National Institutes of Health, Bethesda, MD, USA) [35–38]. The average of each area from three sections in the same placenta was counted as one data point for statistical analysis. A total of 5–6 placentas from different mice in each group were processed for layer quantification.

2.4. Proliferating cell nuclear antigen (PCNA) immunofluorescence and quantification of PCNA positive cells in the labyrinth layer

Cross sections (10 μm) of the middle part of frozen placentas from 0 ppm and 40 ppm ZEA-treated groups were collected on the same slides for immunofluorescence. The sections were fixed in 4% paraformaldehyde for 15 min, and blocked with 3% hydrogen peroxide in methanol for 10 min at 25°C, and 10% goat serum in 1x phosphate buffered saline (PBS) for 1 hour at 25°C, incubated with rabbit anti-proliferating cell nuclear antigen (PCNA (D3H8P)1:250, 13110XP, Cell Signaling Technology, Danvers, MA, USA) overnight [35, 38, 39]. On the second day, the slides were incubated with goat anti-rabbit secondary antibody (Alexa Fluor 288, 1:200, A-11034, ThermoFisher, Rockford, IL, USA). The sections were counterstained with DAPI. Quantification of PCNA positive cells was done as following: At least 4 representative images at 40x in the labyrinth layer of each placental section were taken for PCNA staining (green) and DAPI (blue). They were adjusted to the same exposure level on the background. The number of nuclei for each image was counted by ImageJ. The number of PCNA-positive cells in each image was counted manually by three people (two of them were blind to the identity of each image) or by ImageJ with the same criteria for all the images. The ratio of PCNA-positive cells in each image = the average counts of PCNA-positive cells (manually or ImageJ) /the number of nuclei (by DAPI staining). The average ratio of all images for the same section represented one mouse (N=6 mice/group).

2.5. Cytokeratin 19 immunohistochemistry & laminin immunofluorescence

Placental sections were prepared and processed as described in 2.4. For Cytokeratin 19 immunohistochemistry, the slides were incubated with rat anti-Cytokeratin 19 (CK19, 1:200, TROM-III, DSHB, Iowa city, Iowa, USA) at 4°C overnight. On the second day, the slides were incubated with biotinylated goat anti-rat IgG Antibody (1:200, BA-9400, Vector laboratories, Burlingame, CA, USA) for 1 hour at RT, and the signals were developed by DAB substrate kit (SK-4100, Vector lab, Burlingame, CA, USA). The nuclear were counter stained by Hematoxylin Solution, Harris Modified (HHS32–1L, Sigma-Aldrich, St. Louis, MO, USA). For laminin immunofluorescence, the slides were incubated with rat anti-Laminin (1:200, Lam-B, DSHB, Iowa city, Iowa, USA) at 4°C overnight. On the second day, the slides were incubated with goat anti-rat Alexa Fluor 594 (1:200, A11007, Fisher Scientific, Pittsburgh, PA, USA) at RT for 1 hour and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (H-1200, Vector laboratories, Burlingame, CA, USA).

2.6. Oil red O staining and quantification

Oil Red O (O0625, Sigma-Aldrich) is a lysochrome diazo dye that stains neutral lipids including triglycerides on frozen sections. Cross sections (10 μm) of the middle part of frozen placentas from different treatment groups were placed on the same slides, air dry for 10 min. The slides were fixed in 4% PFA for 15 min, washed in 1xPBS for 10 min, and stained with Oil red O working solution, which was freshly prepared as described [40], for 10 min. The slides were then rinsed in ddH2O (30 s), 60% isopropyl (30 s), and ddH2O (30s). One set of slides with all sections was counterstained with Hematoxylin (HHS16–550ML, Sigma Aldrich, St. Louis, MO, USA), washed in tap water for 10 min, and mounted with glycerol (BP229–1, Fisher Scientific, Pittsburgh, PA, USA). Another set of slides with serial sections were mounted with glycerol without further counterstaining for quantification of Oil red O staining using Image J [35–38]. Briefly, three 0.0359 mm2 representative areas in the labyrinth layer of each section were selected. After converting the pictures into 8 bit images, a threshold of 0–174, 55–255, 45–255 was cut off to calculate the density of staining which is the percentage of positively labeled area in the whole pictures. The average of the density of three areas in one section was used to represent one sample. N=4 mice/group.

2.7. Statistical analyses

Two-tailed Fisher’s exact test was used for comparing “% mice w/placental hemorrhage”. Two-tailed unequal variance student’s t-test was used to compare other variables between two groups. The significance level was set at P<0.05.

3. Results

3.1. Implantation site on D13.5

Exposure to 0.8 ppm to 40 ppm ZEA diets from D5.5 to D13.5 did not have an obvious effect on food consumption and water consumption of mice (data not shown) as we previously reported [31, 34]. All the mice appeared healthy, indicating no general toxicity caused by the treatment regimens. Since exposure to ZEA diets started on D5.5 after embryo implantation initiation (~D4.0 in mice [26]), ZEA treatments were not expected to affect embryo implantation. Exposure to 40 ppm ZEA diet from D5.5 affected several post-implantation parameters detected on D13.5. Body weight gain: The absolute body weight gain (data not shown) or the body weight gain per implantation site during the treatment were comparable among 0 ppm, 0.8 ppm, 4 ppm, and 10 ppm ZEA groups, but were significantly reduced in the 40 ppm ZEA group compared to the other four groups (Fig. 1A, 1C). Resorption of embryo implantation site: Although there were mice with resorbed implantation sites in each group and the individual resorption rate, which was the percentage of resorbed implantation sites in each mouse, varied among individual mice, all 9 mice in the 40 ppm ZEA group had resorbed implantation sites and one of them had all implantation sites reabsorbed. The average resorption rate in the 40 ppm ZEA group (54%) was significantly higher than those in the rest of the four groups (<15%), which did not have significant difference among them (Fig. 1B, 1C). The resorbed implantation sites had different appearances in the 40 ppm ZEA group compared to control and other ZEA-treated groups. All the resorbed implantation sites in the control and the majority of those in the 0.8 ppm, 4 ppm, and 10 ppm ZEA groups were small and with part of the implantation site dark and the other part still pinkish (Fig. 1D upper middle panel), and without detectable fetal tissue (data not shown). While in the 40 ppm ZEA group, all 9 mice had resorbed implantation site(s) that was (were) small and completely dark (type I), and without any recognizable structure; and 3 of them also had resorbed implantation site(s) that was (were) small but still partially pinkish (type II) (Table 1 and Fig. 1D lower middle panel), and the degenerating placental and fetal tissues could still be identifiable (data not shown). Type II resorption most likely occurred later than type I resorption. The decreased weight gain in the 40 ppm ZEA group (Fig. 1A) most likely reflected the increased resorption of implantation sites (Fig. 1B, 1C). These data demonstrate the toxicity of 40 ppm ZEA diet on the post-implantation pregnancy in mice.

Figure 1. Effects of ZEA diets on fetal and placental growth detected on D13.5.

Figure 1.

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A. Body weight gain from gestation day 5.5 (D5.5) to D13.5. B. Resorption rate of implantation site in individual mouse. Each diamond represents one mouse. Red line indicates average. C. Percentage of mice with placental hemorrhage. D. From left to right: images of one D13.5 uterus with implantation sites each from 0 ppm (upper panel) and 40 ppm (lower panel) ZEA groups (see Table 1), enlarged view of resorbed implantation sites, and a fetus with its placenta from an unabsorbed implantation site on the left. . Black arrow, unabsorbed implantation site; blue arrow, a small implantation site being absorbed with part dark tissue on the left side and part pinkish tissue on the right side in 0 ppm ZEA group; blue dotted arrow, an implantation site with complete dark tissue (type I) in 40 ppm ZEA group; red dotted arrow, a small implantation site being absorbed (type II) in 40 ppm ZEA group; red arrow, placental hemorrhage in 40 ppm ZEA group. E. Placental weight. F. Fetal weight. A, E, and F: Error bar, standard deviation; N=6–9 (A) and 6–8 (C, E, F); * P<0.05, compared to the rest four groups.

Table 1.

Implantation site resorption rate on D13.5.

ZEA
treatment group		 Mouse No. No. of total implantation sites Unabsorbed (%) Resorption (%)
0 ppm 0–1 5 80 20
0–2* 8 87.5 12.5
0–3 9 100 0
0–4 8 100 0
0–5 6 83.3 16.7
0–6 4 75 25
Type I resorption
(%)		 Type II resorption
(%)
40 ppm 40–1 9 55.6 33.3 11.1
40–2 11 72.8 27.2 0
40–3 6 50 50 0
40–4 6 83.3 16.7 0
40–5 6 50 16.7 33.3
40–6* 7 28.57 42.86 28.57
40–7 10 20 80 0
40–8 9 0 100 0
40–9 7 57.1 42.9 0
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*

Uterine images of 0–2 and 40–6 were shown in Fig. 1D. Type I resorption: The implantation site was completely dark and without identifiable structure. Type II resorption: The implantation site was partially pinkish and the degenerating placental and fetal tissues could still be identifiable.

3.2. Placental weight and fetus weight on D13.5

Accompanying with the increased resorption of implantation sites (Fig. 1B, 1C), ZEA also affected the placenta. Placental hemorrhage: All the 8 mice with unabsorbed implantation site(s) in the 40 ppm ZEA group had placental hemorrhage (100%), which appeared as a dark patch(s), most located within the rim of the placental disk (Fig. 1D), while only one in six mice (16.7%) had obvious placental hemorrhage in the 10 ppm ZEA group and none was observed in the other three groups (Fig. 1C). Placental weight and fetus weight: Significantly reduced weights of placentas and fetuses were observed in the 40 ppm ZEA group compared to the rest 4 groups, which were comparable among them (Fig. 1E, 1F). These data demonstrate the toxicity of 40 ppm ZEA diet on placental development in mice.

3.3. Histology of placenta

Quantification of three placental layers, labyrinth, junctional zone, and decidua (Fig. 2A), indicated comparable areas for all these three parameters in the 0 ppm, 0.8 ppm, 4 ppm, and 10 ppm ZEA groups, while those three areas were significant reduced in the 40 ppm group compared to all the other four groups (Fig. 2B). Correspondingly, the same pattern was seen in the total placental area (Fig. 2C). These data were consistent with the reduced placental weight only seen in the 40 ppm ZEA group (Fig. 1E). They also indicated that all placental layers were proportionally reduced in the 40 ppm ZEA group, suggesting overall suppressed placental development in this group.

Figure 2. Quantification of D13.5 placental layers.

Figure 2.

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A. Outlines of three layers in a placenta in 0 ppm ZEA group and two placentas in 40 ppm ZEA group. Yellow lined area, labyrinth; blue lined area, junctional zone; green lined area, decidua; black line, width of a 5x image. B. Areas of placental layers. C. Total area of the placentas. B & C: Error bar, standard deviation; N=5–6; * P<0.05, compared to the rest four groups.

Histology revealed morphological changes in the ZEA-treated placentas. In the decidual layer, focal necrosis in the superficial decidual cells was observed in multiple placentas of each ZEA-treated group without an obvious dose-response relationship, while none was observed in the five control placentas (data not shown). The junctional zone did not show obvious structural abnormalities in the ZEA-treated groups. The labyrinth layer had the most abnormalities among the three main placental layers upon ZEA treatment. Focal necrosis in the labyrinth layer was observed in 1/5 placenta of 4 ppm ZEA group (data not shown) and 3/7 placentas of the 40 ppm ZEA group (Fig. 3B–3B2), but none in the control (Fig. 3A–3A2), 0.8 ppm and 10 ppm ZEA groups that were examined (data not shown). The following abnormalities of vascular spaces were observed in the 40 ppm ZEA-treated group. Dilated lacunae with accumulation of red blood cells: Although normally there were nucleated fetal blood cells in the fetal capillaries and enucleated maternal blood cells in the maternal blood spaces of the labyrinth layer, dilated lacunae with abnormal accumulation of red blood cells were observed in the labyrinth layer of 100% of the placentas in the 40 ppm ZEA group (Fig. 3C–3C2 and data not shown). These observations were consistent with the placental hemorrhage (Fig. 1C, 1D). Such abnormal accumulation of blood cells in the labyrinth layer was also occasionally observed in the 0.8 ppm, 4 ppm, and 10 ppm ZEA groups (data not shown). Reduced vascular spaces: Accompanying with the dilated blood spaces, there were areas in the labyrinth layer of 40 ppm ZEA group with reduced blood spaces (Fig. 3C1, 3C2). The histology data revealed disrupted vascular spaces in the labyrinth layer of placentas in the 40 ppm ZEA group (Fig. 3B–3C2).

Figure 3. Histology of D13.5 placentas.

Figure 3.

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A-A2. A representative labyrinth layer in 0 ppm ZEA group. BB2. Focal necrosis in the labyrinth layer of a placenta in 40 ppm ZEA group. C-C2. Dilation of blood space in the labyrinth layer of a placenta in 40 ppm ZEA group. A1-C1, enlarged from the boxed area in A-C, respectively; A2-C2, enlarged from the boxed area in A1-C1, respectively; laby, labyrinth layer; yellow star in B2, focal necrosis; blue arrow in C2, dilated maternal blood space filled with enucleated red blood cells; black arrow in C2, nucleated fetal blood cells in fetal capillary; scale bar, 400 μm (A-C), 100 μm (A1-C1), or 25 μm (A2-C2).

3.4. Immunostaining of cytokeratin 19 (CK19) and laminin in placental labyrinth layer

CK19 labels the sinusoidal trophoblast giant cells that line the maternal blood space in the labyrinth [28]. Immunohistochemistry of CK19 showed relatively uniform distribution of maternal blood spaces in the control labyrinth (Fig. 4A), but enlarged or unexpanded maternal blood sinusoids in the 40 ppm ZEA-treated labyrinth associated with thickened intrahemal membrane (Fig. 4B, 4C). Laminin is a marker of basal laminin surrounding the fetal capillary in the labyrinth [41]. Immunofluorescence revealed laminin staining as stretches of lines, which are supposed to be lining the fetal capillaries, in the control labyrinth (Fig. 4D). In the 40 ppm ZEA group, there were enlarged areas outlined by discontinuous laminin staining (Fig. 4E), most likely indicative of the dilated areas in the labyrinth layer; there were small patches of laminin staining (Fig. 4F), which did not appear to have adjacent lines of laminin staining to form fetal blood space and might be areas with unexpanded fetal capillaries; there were also lines with laminin staining that appeared to be fragmented in the labyrinth layer of 40 ppm ZEA-treated placenta (Fig. 3E). Fig. 3 and Fig. 4 consistently demonstrated disrupted labyrinth structure in the 40 ppm ZEA group.

Figure 4. Immunostaining of cytokeratin 19 (CK19) and laminin in labyrinth layer.

Figure 4.

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A-C. Immunohistochemistry of CK19 in control (A) and 40 ppm ZEA (B, C) labyrinths. CK19 (brown staining) labels sinusoidal trophoblast giant cells that line the maternal blood space in the labyrinth. Blue arrow, expanded maternal blood space; black dotted arrow, unexpanded maternal blood space; scale bar, 50 μm. D-F. Immunofluorescence of laminin in control (D) and 40 ppm ZEA (E, F) labyrinths. Laminin (red staining) is a marker of basal laminin surrounding the fetal capillary in the labyrinth. White arrow in D, fetal capillary lined up by laminin staining; yellow arrow in E, dilated fetal capillary; yellow dotted arrow in E, discontinuous laminin staining; white dotted arrow in F, focal cluster of laminin staining; scale bar, 25 μm.

3.5. PCNA staining of D13.5 placentas

PCNA is involved in DNA synthesis. Many trophoblast cells undergo endoreplication without going through mitosis, resulting in large nuclei. In our study, we found PCNA that was highly expressed in many cells in the placenta. PCNA staining was mainly detected in the labyrinth layer and appeared less in the junctional zone (data not shown). Although disrupted labyrinth structure was found in the 40 ppm ZEA group (Figs. 3, 4), the percentage of PCNA positive cells in the 40 ppm ZEA-treated labyrinth was not significantly different from that of the control, both by manual counting and by ImageJ automatic counting (data not shown). These data indicate that DNA replication in the labyrinth layer was not affected by 40 ppm ZEA treatment.

3.6. Oil red O staining of D13.5 placentas

Fetal growth depends on the supply of nutrients from the placenta. One important class of nutrients is lipid. To obtain a general picture of lipid supply in the placenta, we used a widely used method, Oil red O staining, which reveals neutral lipid droplets in the cell. In the D13.5 placentas, the Oil red O staining was mainly detected in the labyrinth layer, detectable in trophoblast giant cells and spongiotrophoblast cells in the junctional zones, and to a lesser extent, in some stromal cells in the decidual layer (Fig. 5A, 5B). Since nutrient transfer to the fetus occurs in the labyrinth layer, we quantified Oil red O staining areas in the labyrinth layer using ImageJ. All four ZEA-treated groups had increased Oil red O staining compared to the vehicle control 0 ppm ZEA group (Fig. 5). These data indicate that there was no lack of lipid in the labyrinth layer to support the fetus in the ZEA-treated groups.

Figure 5. ZEA diets increase oil red staining in labyrinth layer (Laby) of D13.5 placentas.

Figure 5.

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A-D. Oil red staining counter-stained with Hematoxylin. C & D: Enlarged from A & B, respectively. E-F. Oil red staining only for quantification using ImageJ. A-F: scale bar, 400 μm (A,B) and 25 μm (C-F). G. Oil red staining as % of area. N=4; error bar, standard deviation; * P<0.05, compared to 0 ppm group.

4. Discussion

The dose-response relationship between ZEA diets and placental weight is not linear (Fig. 1). It seems that there is a dose threshold between 10 ppm and 40 ppm ZEA to affect placental weight. Such threshold may reflect placental adaptation. Our previous multigenerational study showed that exposure to 20 ppm ZEA diet in F0 females from weaning to the end of pregnancy did not adversely affect their pregnancy rate, litter size, or offspring body weight measured at one week old [34], suggesting that 20 ppm ZEA diet did not compromise placental function in supporting fetal development and survival in the F0 females. There could be potential cellular and molecular changes in the 20 ppm ZEA-treated placentas of F0 females that were not investigated in the study. However, life time exposure to 20 ppm ZEA diet in F1 and F2 females impaired early pregnancy event(s) leading to reduced pregnancy rates [34]. Interestingly, the fertile F1 and F2 females treated with 20 ppm ZEA diet had comparable numbers of implantation sites to controls on D4.5 but significantly reduced litter sizes at birth, indicating increased post-implantational lethality [34], which could result from defective placentas and/or fetuses that were not further examined. These observations imply that the placenta has certain capacity to handle up to 20 ppm ZEA diet in the F0 mice. However, 40 ppm ZEA diet impairs multiple pregnancy events in F0 mice, such as fertilization, embryo transport and preimplantation embryo development, and embryo implantation [31], as well as placental development and fetal development demonstrated in this study. These adverse effects of 40 ppm ZEA diet prevent further study on F1 and F2 females.

There are three potential possibilities for the reduced weight of both the placentas and the fetuses in the 40 ppm ZEA group. 1. Since ZEA and its metabolites can pass through the placenta to reach the fetus [21, 22], ZEA may have a direct adverse effect on the fetus to reduce fetal development and the reduced placental weight is an indirect effect to adapt to the slow fetal development; 2) Since ZEA can be bioactivated in placental cells [20] and accumulated in placentas [23], the placenta can be a direct target of ZEA and the reduced fetal weight is the consequence of comprised placental function in transferring adequate nutrients for fetal growth; and 3) ZEA directly targets both the placenta and the fetus to cause their reduced weights. Although the available information cannot exclude any of these three possibilities, our data support that the placenta is a direct target for ZEA. In the event of fetal death, the placenta continues to function for days in rodents [42, 43] or weeks in primates [44, 45]. For example, fetectomy on D13.5 in mice does not affect placental weight, placental area, or labyrinth volume, but significantly decreases the volume of fetoplacental capillaries on D17.5 [42]. ZEA treatment started on D5.5 in our study that covered the entire period of placental formation, from the initial choriovitelline pattern to the subsequent chorioallantoic pattern [30]. In mice, chorioallantoic attachment occurs ~D8.5 [27] that marks the initiation of transition to chorioallantoic placentation. Most of the absorbed implantation sites in the 40 ppm ZEA group were small and completely dark, which were most likely indicative of early resorption. Since ZEA treatment started on D5.5, the increased presence of such small and dark resorbed implantation sites in the 40 ppm ZEA group could be a result from disrupted decidualization, and/or early nutritional disruption of the visceral yolk sac and its interaction with the antimesometrium during implantation chamber remodeling, or possibly from ZEA toxicity on the embryo. Because the treatment started on D5.5 and decidualization starts by D4.5 in mice [26], it is expected that the embryos were alive beyond D5.5 when ZEA treatment started. If ZEA only targeted the embryo/fetus, the placenta is expected to continue developing for certain period of time. And for the fetuses that were alive on D13.5, it is expected that the associated placentas would be normal if the fetus is the only target. However, we observed placentas with disrupted labyrinth layer, especially in the 40 ppm ZEA group. These observations support the placenta being a target of ZEA.

A recent study highlights the association of placentation defects with embryonic lethality in 103 mutant mouse models [46]. It finds that almost every line that died before embryonic/gestation day 14.5 (E14.5/D14.5) exhibited placental abnormalities, while only 35% of lines that were viable beyond E14.5/D14.5 had placental abnormalities and these lines were associated with a younger developmental stage (delayed fetal development). Although placental defects and embryonic lethality are linked [46], they can be differentiated [47].

Since ZEA treatment (especially at 40 ppm) disrupted the structure of labyrinth layer, it is expected that the maternal-fetal material exchange is impaired. Consequently, it will affect fetal survival and growth. Indeed, the fetuses in the 40 ppm ZEA group had significantly reduced weight. ZEA treatment increases oil red staining in the labyrinth layer of all the ZEA treatment groups from 0.8 ppm to 40 ppm ZEA (Fig. 5), indicating that ZEA has an effect in the placenta at levels as low as 0.8 ppm. The effect of ZEA on fetal survival and growth was only manifested in the 40 ppm ZEA group would suggest that the placenta has certain capacity to accommodate the insult from ZEA in order to protect the developing fetus.

The accumulation of lipid in the ZEA-treated labyrinth layer indicates that there is no lack of lipid in the labyrinth layer. It is possible that the disrupted labyrinth structure in 40 ppm ZEA group would impede the transfer of lipid to fetus leading to lipid accumulation and compromised fetal growth. However, in the lower doses, the morphological changes in the labyrinth layer were not severe or not obvious (e.g., 0.8 ppm ZEA group) and the fetal survival and growth were not affected, yet lipid accumulation still occurred, suggesting that ZEA may affect molecular changes without obvious effects on the cellular level to disrupt lipid homeostasis.

Estrogen has been generally associated with reduction of lipid, while loss of estrogen or estrogen receptor is associated with adiposity and hepatic lipogenesis [48]. Estrogen can reduce lipid accumulation in the liver through ERα-mediated pathways [49]. It may be responsible for the decreased saturate fat acid content in the female brain compared to male brain [50]. Its levels are oppositely related to mouse uterine lipid contents during estrous cycle, with minimum at estrus stage and maximum at diestrus stage [51]. Therefore, it is unlikely that ZEA-induced lipid accumulation observed in our study is directly caused by the estrogenicity of ZEA.

Increased neutral lipid accumulation in placenta has also been observed in women with preeclampsia, gestational diabetes, and fetal growth restriction [52, 53]. One potential cause that has been proposed is the hypoxia in the placenta which can be induced by defects in vasculature development [54]. Unexpanded or dilated blood spaces in the labyrinth layer of the placentas in the 40 ppm ZEA group indicated defective vasculature development, which could contribute to the increased lipid accumulation. The molecular mechanisms of ZEA in affecting the placenta (e.g., lipid homeostatis) and the time course of ZEA in affecting placental development remain to be investigated.

Highlights.

  • Dietary exposure to 40 ppm mycoestrogen zearalenone (ZEA) increases resorption of implantation sites;

  • Exposure to 40 ppm ZEA diet decreases placental and fetal weights;

  • Exposure to 40 ppm ZEA diet disrupts placental labyrinth vascular spaces;

  • Exposure to 0.8 ppm, 4 ppm, 10 ppm, and 40 ppm ZEA diets leads to lipid accumulation in the placental labyrinth layer;

  • ZEA diet at a contamination relevant level adversely affects placental development.

5. Acknowledgements

The authors thank the Office of the Vice President for Research, Interdisciplinary Toxicology Program, and Department of Physiology and Pharmacology at the University of Georgia, and the National Institutes of Health (NIH R01HD065939 (co-funded by ORWH and NICHD) to XY) for financial support.

Grant support

NIH R01HD065939

Footnotes

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6.

Competing interests

The authors declare no conflict of interest.

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