Ethanol consumption during gestation promotes placental alterations in IGF-1 deficient mouse placentas

2.1 Animals and experimental design

As previously reported, IGF-1 heterozygous mice were obtained by cross-breeding transgenic mice, line 129SV and Igf1^tm1Ts/ImJ (003258, Jackson Laboratory, Maine, USA) and CD1 (non-consanguineous, Circulo A.D.N S. A de C.V., Ciudad de Mexico, Mexico).²⁰

Animals were housed in cages in a room with 12-hour light/dark cycle, constant humidity (50–55%) and temperature (20–22°C). Food (PicoLab^® Rodent Diet 20 5053*, Missouri, USA) and water were given ad libitum. All experimental procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978), the NORMA Oficial Mexicana (NOM-062-ZOO-1999) for technical specifications to produce, care and use of laboratory animals and the ARRIVE guidelines. Also, all experimental procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals of Tecnologico de Monterrey (Protocol 2018-006). All efforts were made to ameliorate any suffering of animals, such as minimal handling, adequate space for living and no other experimentation or pain inducing procedures.

The sample size was calculated using the resource equation method,²¹ where the value of E is calculated and should lie between 10 and 20 to be considered adequate: E = Total number of animals − Total number of groups = (7 × 2) − 2 = 12.

Wild-type (WT, Igf1^+/+) and heterozygous (HZ, Igf1^+/−) female mice 16 ± 8 weeks old were randomly included into four experimental groups: two control groups given water (WT-Control, n = 6; and HZ-Control, n = 6), and two other groups given food grade ethanol in water (WT-Ethanol, n = 4; and HZ-Ethanol, n = 9) (purity: 96%; La Fe, Nuevo Leon, Mexico).

An adaptation period to ethanol (14 days) was performed, where WT and HZ female mice were given increasing concentrations of 2%, 5% and 10% of ethanol in water, each introduced after a 48-hour acclimation to the previous concentration, according to Kleiber et al.²² During this period, control WT and HZ female mice were provided water (Figure 1).

Figure 1. Diagram of the experimental procedure.

D: day; GD: gestational day.

After the acclimation period to ethanol, WT and HZ male mice were mated overnight with WT and HZ female mice. To avoid ethanol consumption from males during mating, water was given to the ethanol groups. Once the vaginal plug was observed, it was considered gestational day one and males were removed. Throughout gestation, 10% ethanol or water was given to pregnant dams (WT-Ethanol or HZ-Ethanol, and WT-Control or HZ-Control; respectively), food and beverage consumption and weight gain were monitored throughout the experimental protocol. Nonpregnant females from WT-Ethanol and HZ-Ethanol groups were given 10% ethanol during 24 hours and then mated again with male mice. If vaginal plug was not observed after this second mating, ethanol exposed female mice were sacrificed by cervical dislocation. On the other hand, nonpregnant females from WT-Control and HZ-Control groups were mated again until the presence of the vaginal plug was observed.

At gestational day 19 (before the end of the gestational period in mice), pregnant females were sacrificed by cervical dislocation.^23,24 Subsequently, blood was collected by cardiac puncture and a caesarean section was performed to obtain fetuses and placentas (from HZ x HZ matings), which were measured and weighted.

Tails from fetuses were cut for genotype determinations. Fetuses and placentas were stored randomly in either paraformaldehyde 4%, for histology and immunohistochemistry analyses, or liquid nitrogen, for reverse transcription coupled to polymerase chain reaction (RT-qPCR) determinations.

2.2 Genotyping of animals

DNA was extracted from tails of fetuses using the Wizard Genomic DNA Purification Kit (A1125, Promega, Wisconsin, USA) following manufacturer’s instructions, and stored at −20°C until analysis. End-point polymerase chain reaction (PCR) (Veriti 96 well Thermal Cycler, Applied Biosystems, California, USA) was carried out for genotyping using the following set of primers for Igf1 gene: WT forward 5′-TTCATGCCACACTGCTCTTC-3′; common 5′-AGAGGGGATGGGAGAGCTAC-3′; and mutant forward 5′-GCCAGAGGCCACTTGTGTAG-3′. All primers were acquired from IDT (Iowa, USA). Secondly, conventional PCR analysis was achieved using the GoTaq Green Master Mix (M712C, Promega, Wisconsin, USA) following manufacturer’s instructions.

2.3 Serum IGF-1 circulating levels at gestational day 19

In order to evaluate serum IGF-1 circulating levels in HZ dams at gestational day 19, sera from control (WT) mice treated both with water (n = 5) or ethanol (n = 5) were also used. Serum IGF-1 levels were determined by enzyme-linked immunosorbent assay (ELISA) using the Mouse/Rat IGF-1 commercial kit (22-IG1MS-E01, Alpco, New Hampshire, USA), following manufacturer’s instructions. The signal was measured using a spectrophotometer Synergy HT (Biotek, Vermont, USA) and data were interpreted using Gen5 Data Analysis Software (Biotek, Vermont, USA).

2.4 Placental histological study

For histopathological analysis, placental samples from all experimental groups were fixed in 4% paraformaldehyde diluted in 10 mM phosphate buffered saline (PBS) solution for 24 hours. Once samples were properly fixed, they were dehydrated in increasing ethanol concentrations and were embedded in liquid paraffin using the automated equipment Leica TP 1020 (Leica, Wetzlar, Germany). Sections 4 μm thick were cut using a Reichert Jung Leica BC2030 Histocut Rotary Microtome (Leica, Hesse, Germany) and subsequently stained with hematoxylin-eosin (HHS32 and 2853, Sigma-Aldrich, Missouri, USA). Finally, all histological preparations were evaluated independently by three observers (double-blind) in a Zeiss Axio Imager M2 microscope (Zeiss, Baden-Württemberg, Germany) to establish morphology alterations in the placenta due to ethanol consumption throughout gestation.

All images were analysed using the processing package ImageJ (National Institutes of Health, Maryland, USA) (RRID:SCR_003070). The area of the junctional zone of the placenta was evaluated according to the presence of trophoblasts in this placental zone. Three images were taken per sample of at least three individuals from each group and genotype.

2.5 Immunohistochemical staining

Paraffin-embedded placental tissues were deparaffinized and rehydrated in decreasing ethanol concentrations. Sample sections were incubated with 3% hydrogen peroxide at room temperature in darkness for 30 minutes to inactivate endogenous peroxidases. Retrieval of antigen was induced with 2 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0 and 50 mM Tris–HCl pH 9.0 by microwave heating at 100°C for 8 minutes. Sections were incubated overnight at 4°C with specific primary antibodies: rabbit anti-ASPH (362200, USBiological Life Sciences, Massachusetts, USA) diluted at 1:200; or rabbit anti-β actin (ab8227, Abcam, Cambridgeshire, United Kingdom) diluted at 1:1,000, the latter being an endogenous control. Afterwards, primary antibodies sections were incubated with goat anti-rabbit IgG H&L (horseradish peroxidase, HRP) (ab205718, Abcam, Cambridgeshire, United Kingdom) diluted at 1:5,000 for one hour at room temperature. Staining was developed using Steady Diaminobenzidine (DAB)/Plus kit (ab103723, Abcam, Cambridgeshire, United Kingdom) following manufacturer’s instructions. Negative controls were conducted by omitting the primary antibody.

Digital images of tissue sections (five images per placenta) were captured and analyzed by three observers (double-blind) using a Zeiss Axio Imager M2 microscope. All images were analysed using the processing package ImageJ (National Institutes of Health, Maryland, USA).

2.6 Gene expression studies in placentas via RT-qPCR

RNA was isolated from placentas using Trizol reagent (15596026, Invitrogen, California, USA). Quality was checked by the A₂₆₀/A₂₈₀ ratio using the Nanodrop 2000 Spectrophotometer (Thermo Scientific, Massachusetts, USA), whereas integrity was determined by electrophoresis in 1% agarose gel. Samples were stored at −80°C until analysis. Subsequently, the SuperScript III First-Strand Synthesis System (Invitrogen, California, USA) was utilized to prepare complementary DNA (cDNA) from 500 ng of the total RNA, according to the manufacturer’s instructions.

For the PCR analyses, TaqMan Universal PCR Master Mix (Thermofisher, Massachusetts, USA) was used in a total volume of 20 μL containing 400 nM of each oligonucleotide and 200 mM of specific Taqman^® probes for particular genes, supplied by Applied Biosystems (California, USA) (Supplementary table 1). The assays were performed in a 96-well reaction plate on a Quant Studio 3.0 thermocycler (Applied Biosystems, California, USA). Ribosomal 18S RNA expression was used as an endogenous control (Applied Biosystems, California, USA).

Each sample was run in triplicate, and negative controls were included in the same plate. Results were analyzed using the comparative threshold cycle method for relative gene expression.²⁵

2.7 Statistical analysis

All data are represented by mean±standard deviation (SD). Significance was estimated with non parametric (Mann–Whitney U test and Kruskal–Wallis test) or parametric (Student T test and ANOVA) statistical tests, using Mann–Whitney U test or Student T test to compare between two groups (for example between HZ-Control and HZ-Ethanol groups), whereas comparisons among more than two groups were carried out using the Kruskal-Wallis test or ANOVA (for example, comparisons between WT, HZ and knock-out (KO) placentas). To evaluate the effect of the two independent variables in the present study (ethanol consumption and IGF-1 deficiency) a general linear model was performed. Differences were considered significant at a level of p < 0.05. Statistical analyses were performed on SPSS 26 (IBM, New York, USA) (RRID:SCR_019096) and graphs were generated on Prism 8.2.1 (GraphPad Software, California, USA) (RRID:SCR_002798).

3.1 IGF-1 serum levels in pregnant dams chronically exposed to ethanol at gestational day 19

To validate IGF-1 serum levels in the present experimental model, control (WT) pregnant female mice treated with water or ethanol were used. At gestational day 19, as expected, IGF-1 serum levels in HZ pregnant dams (HZ-Control, 454 ± 58 ng/mL, and HZ-Ethanol, 422 ± 43 ng/mL) were decreased compared to WT female mice (WT-Control, 739 ± 48 ng/mL, and WT-Ethanol, 795 ± 147 ng/mL) (p < 0.001). HZ-Control and HZ-Ethanol dams had significant lower IGF-1 serum levels than WT-Control female mice (p < 0.05 and p < 0.01, respectively). Additionally, ethanol consumption decreased even more IGF-1 serum levels than WT-Control (p < 0.01) (Table 2).

3.2 Alterations in placental morphology due to ethanol consumption during gestation

The placenta is conformed of several strata, such as myometrium, decidua, junctional zone and labyrinth (Extended data: figure 1). In the WT placentas from the control group (from HZ x HZ matings) all morphology and structure that characterizes these layers were well defined (Figure 2A). It was observed that IGF-1 deficiency promoted a disorganization in placental strata, especially in the junctional zone, where trophoblast cells were observed (Figure 2B). Remarkably, an abnormal placental development was observed in KO placentas from the control group, where trophoblasts were arrested in islets (Figure 2C). Ethanol consumption throughout gestation exacerbated placental layer disorganization and promoted throphoblast arrest in islets in all groups (WT, HZ and KO) (Figures 2D, 2E and 2F). Additionally, ethanol use altered trophoblast migration (Figure 2D) particularly in HZ and KO placentas (Figures 2E and 2F). Due to the variability between all experimental groups, no significant differences were observed in the area of the junctional zone of the placenta (data not shown); in this area a clear disorganization is detected with both IGF-1 deficiency and ethanol consumption.

Figure 2. Effect of gestational ethanol consumption on placental morphology on gestational day 19.

Placentas from WT, HZ and KO fetuses from pregnant dams given water (A, n = 5; B, n = 6; and C, n = 6) or ethanol (D, n = 7; E, n = 6; and F, n = 5) were stained with hematoxylin & eosin and examined by light microscopy. Representative images from one placenta of fetuses from each group are included. Black arrows indicate trophoblast islets, suggesting an abnormal migration. IGF-1 deficiency promoted placental disorganization, especially in the junctional zone; ethanol consumption during gestation exacerbated such disorganization. Original magnification 100×. Scale bar = 100 μm.

3.3 Impairments in placental efficiency due to ethanol consumption and IGF-1 deficiency

Placental efficiency is frequently defined as the grams of fetus produced per gram of placenta, being an indicator of the ability of this organ to maintain an adequate nutrient supply to the fetus.^26–28 The chronic exposure to ethanol during gestation resulted in a significant reduction in placental efficiency (p < 0.001) compared to control groups. Subsequently, genotype offspring outcome was evaluated, showing that IGF-1 deficiency significantly decreased placental efficiency in KO placentas compared to WT placentas (p < 0.05) and HZ placentas (p < 0.01) from control group (Figure 3). Also, ethanol consumption during gestation significantly reduced placental efficiency in KO placentas compared to WT and HZ placentas from ethanol treated group (p < 0.001 in both cases) (Figure 3). Markedly, there was a significant diminution in 0.75-fold in placental efficiency in KO ethanol treated placentas compared to KO placentas from control group (p < 0.05) (Figure 3).

Figure 3. Placental efficiency represented as feto/placental weight ratio in all experimental groups (Control: WT, n = 9; HZ, n = 32; KO, n = 16; Ethanol: WT, n = 23; HZ, n = 44; KO, n = 16).

Data are represented as mean ± standard deviation. The statistical tests used were Student T test to compare between two groups, and general linear model to evaluate the effect of the two independent variables in the present study (ethanol consumption and IGF-1 deficiency). Differences were considered significant at a level of p < 0.05.

3.4 Immunohistochemical analysis of AAH placental expression

To determine ethanol’s harmful effects on placental development, protein expression levels of AAH were determined by immunohistochemistry. AAH was predominantly expressed in the junctional zone, as shown in WT placentas from control group (Figure 4A). IGF-1 deficiency revealed a decrease in AAH expression within the junctional zone in both HZ and KO placentas compared with WT placentas from control group (Figures 4B and 4C). Ethanol consumption during gestation also decreased AAH expression, especially in HZ and KO placentas, denoting the aforementioned alterations in placental morphology, particularly in the junctional zone, where trophoblasts are arrested in islets, avoiding their correct migration towards the junctional zone for an appropriate placental development (Figures 4D, 4E and 4F).

Figure 4. Effect of ethanol consumption during gestation in AAH levels analyzed by immunohistochemistry in placentas from WT, HZ and KO mice given water (A, n = 5; B, n = 6; and C, n = 6) or ethanol (D, n = 7; E, n = 6; and F, n = 5).

Original magnification 100x. Scale bar = 100 μm. Representative images from one mice of each group are included. Red arrows denote AAH expression in the junctional zone of the placenta, where trophoblast cells are expressed. Blue arrows indicate trophoblast islets. G) Effect of ethanol consumption during gestation in Asph gene expression in placentas from WT, HZ and KO fetuses from pregnant dams given water or ethanol (n = 4 per group). The statistical tests used were Mann-Whitney U to compare between two groups, ANOVA or Kruskal-Wallis test to compare between more than two groups, and general linear model to evaluate the effect of the two independent variables in the present study (ethanol consumption and IGF-1 deficiency). Differences were considered significant at a level of p < 0.05.

3.5 The effect of ethanol consumption during gestation in placental expression of the IGF-1 signaling pathway

The placental expression of components from the IGF-1 signaling pathway (Insulin-like growth factor 1 (Igf1), Insulin-like growth factor 2 (Igf2), Insulin-like growth factor 1 receptor (Igf1r), Insulin-like growth factor 2 receptor (Igf2r) and Aspartyl/asparaginyl β-hydroxylase (Asph)) was analyzed by RT-qPCR. No significant differences were found in Igf1 and Igf2 expression level between both treatments (control and ethanol). When grouped by genotype, no significant differences were found (Table 1).

Regarding Igf1r and Igf2r expression, a significant increase in Igf1r was observed with ethanol consumption throughout gestation (p < 0.05). When grouped by genotype, no significant differences were observed (Table 1).

When Asph expression levels were evaluated, no significant differences were shown between both treatments (water or control, and ethanol). When grouped by genotype, a significant increase in Asph expression was observed in KO placentas compared to WT (p < 0.05) and HZ (p < 0.05) placentas from control group (Figure 4G).