The epigenetic role of breastfeeding in mammary differentiation

Maternal milk consumption can cause changes in the mammary epithelium of the offspring that result in the expression of molecules involved in the induction of differentiation, reducing the risk of developing mammary cancer later in life. We previously showed that animals that maintained a higher intake of maternal milk had a lower incidence of mammary cancer. In the present study, we evaluated one of the possible mechanisms by which the consumption of maternal milk could modify the susceptibility to mammary carcinogenesis. We used Sprague Dawley rats reared in litters of 3 (L3), 8 (L8), or 12 (L12) pups per mother in order to generate a differential consumption of milk. Whole mounts of mammary glands were performed to analyze the changes in morphology. Using real-time polymerase chain reaction (PCR), we analyzed the expression of mammary Pinc, Tbx3, Stat6, and Gata3 genes. We use the real-time methylation-specific polymerase chain reaction method to assess the methylation status of Stat6 and Gata3 CpG sites. Our findings show an increase in the size of the epithelial tree and a smaller number of ducts called terminal end buds in L3 vs. L12. We observed an increased expression of mRNA of Stat6, Gata3, Tbx3, and a lower expression of Pinc in L3 with respect to L12. Stat6 and Gata3 are more methylated in the CpG islands of the promoter analyzed in L12 vs. L3. In conclusion, the increased consumption of maternal milk during the postnatal stage generates epigenetic and morphological changes associated with the differentiation of the mammary gland.

Métodos

Postnatal litter size adjustment Female Sprague Dawley rats bred in our laboratory were used. The animals were kept in a light (lights on 06:00–20:00 h) and temperature (22–24°C) controlled room. One day-old pups (n = 72) born on the same day were distributed at random in litters of different sizes: 3 (L3), 8 (L8), or 12 (L12) pups per dam, to induce a differential consumption of maternal milk as previously described.32 Fostering of 1-d-old pups of the Sprague Dawley strain does not lead to any adverse effect.33 Body weight of pups was monitored every 3 d. On Day 21, the litters were weaned and fed with rat chow (Cargill, Argentina) and tap water ad libitum until the end of the experiment. They were housed in cages containing approximately six rats from the same group per cage. In this way, we obtained three groups of rat’s growth with different levels of lactation, as we have previously demonstrated32: L3 (n = 24), L8 (n = 24), and L12 (n = 24). Sample collection Six animals from each group were sacrificed on Day 21 by decapitation, and the right inguinal mammary glands were removed for the complete assembly of the organ (Whole Mount technique). Day 21 of age was chosen because it is the usual day of weaning in rats and it is the earliest pre-pubertal stage of the mammary gland. The remaining animals (n = 54) were sacrificed at 55 d of age by decapitation. The two inguinal mammary glands were removed, one for the whole mount, and the other as a sample for genetic and epigenetic studies. Day 55 of age represents a post-pubertal stage of the mammary gland and the rats show the maximal sensitivity to carcinogenic transformation as we have previously described.32 All the animals were decapitated between 10:00 and 12:00 h. Hormone determinations Animals were sacrificed on Day 55 of life regardless of the phase of the estrus cycle. At the time of sacrifice, the trunk blood was collected and allowed to clot at room temperature. Serum was separated by centrifugation and stored at −20°C until assayed for hormone determinations. Serum levels of estradiol and progesterone were determined by electrochemiluminescence in Roche’s Cobas e411 system following the manufacturer instructions. Mammary gland whole mounts Whole mounts of the mammary glands were performed on Days 21 and 55 of life, in animals that maintained differential lactation. Once the animal was euthanized, we separated the mammary gland from the skin using sharp scissors, and immediately it was spread onto a slide. Then, the slide was placed in a container carrying Carnoy’s fixative (75% glacial acetic acid and 25% absolute ethanol) at room temperature for 2 d. Subsequently, they were dipped into 70% ethanol at room temperature for 1 h, rinsed with distilled water for 30 min and placed in a Carmine Alum coloring solution (1 g of carmine and 2.5 g of aluminum potassium sulfate – Sigma Aldrich, USA – in 500 ml of distilled water, the solution was boiled 20 min, filtered and refrigerated) for 2 d, until the lymph nodes were found to be stained. Then we proceeded to dehydrate in increasing concentrations of ethanol: 70%, 95%, and 100%. Finally, we placed the glands in xylene at room temperature for 2 d.34 Then, we proceeded to mount the glasses with synthetic Canada balsam (Biopack, Argentina). Images were captured with an Eclipse E200 microscope coupled to a digital camera with 5.0M resolution, and were analyzed with Micrometrics SE Premium (both from Nikon Corp., Japan) under magnification of 100×. In order to evaluate mammary epithelial growth on Day 21 (pre-pubertal age), we measured the length of the epithelial tree considered as the distance from the nipple to the end of the epithelial tree.34 We also measure the width of the epithelial tree considered from one end of the tree to the other, in the widest region. On Day 55 (post-pubertal age), the growth of the epithelial tree was evaluated by measuring the distance from the lymph node to the end of the epithelial tree and the distance from the tip of the epithelial tree to the end of the fat pad.34 All measurements were considered in millimeters using a ruler. In addition, we evaluated the potential for malignant transformation through the count of TEBs under the microscope. Between 10 and 18 fields per mammary gland were analyzed, with an area of 2.36mm2 per field. The number of fields analyzed per sample was necessary to cover the entire mammary surface. Subsequently, the density of TEBs per field was determined using the following formula: Total number of TEBs/ number of fields analyzed. The analysis of TEBs was performed in double-blind by two independent observers. The number of TEBs and the measurements of the mammary tree of each animal were normalized by the respective body weight and they are expressed per 100 g of the animal. RNA isolation and real-time RT-PCR analysis Total RNA was isolated from 150/200mg of mammary tissue using TRI Reagent® (SigmaAldrich,Argentina) according to the manufacturer’s instructions. Tenmicrograms of totalRNAwere reverse transcribed (RT) at 37°C using random hexamer primers and Moloney Murine Leukemia Virus Retrotranscriptase (MMLV, Invitrogen- Life Technologies, Argentina) in a 20 μL reaction mixture. The primers were designed with the Primer-BLAST tool (www. ncbi.nlm.nih.gov) and used for genes determination (Table 1). The PCR reactions were performed using a Corbett Rotor-Gene 6000® Real-Time Thermocycler (Corbett Research Pty Ltd, Australia) and Eva-Green™ (Biotium, Hayward, USA) in a final volume of 10 μL. The reaction mixture consisted of 1 μL of 10× PCR Buffer, 0.5 μL of 50mM MgCl2, 0.2 μL of 10mM dNTP Mix (Invitrogen, Argentina), 0.5 μL of 20× Eva Green, 0.125 μL of 5U/μL Taq DNA Polymerase (Invitrogen, Argentina), 0.05 μL of each 50 pM primer (forward and reverse primers), and 5 μL of diluted cDNA. The PCR reactions were initiated with 5-min incubation at 95°C, followed by 40 cycles of 95°C for 30 s, 30 s at the annealing temperatures shown in Table 1 and 72°C for 30 s. A melt curve analysis was used to check that a single specific amplified product was generated. Real-time quantification was monitored by measuring the increase in fluorescence caused by the binding of EvaGreen™ dye to double-strand DNA at the end of each amplification cycle. Relative expression was determined using the Comparative Quantitation method of normalized samples in relation to the expression of a calibrator sample, according to the manufacturer’s protocol.35 Each PCR run included a notemplate control and a sample without retrotranscriptase. All measurements were performed in duplicate. The reaction conditions and quantities of cDNA added were calibrated so that the assay response was linear with respect to the amount of input cDNA for each pair of primers. RNA samples were assayed for DNA contamination by performing the different PCR reactions without prior RT. Relative levels of mRNA were normalized to the Hypoxanthine Guanine Phosphoribosyltransferase reference gene. The RT-real-time PCR products were analyzed on 2% agarose gels containing 0.5 μg/ml ethidium bromide and a unique band of the approximately correct molecular weight corresponded with a unique peak in the melt curve analysis. Extraction of genomic DNA Mammary tissue samples were resuspended in T10 E10 buffer (10mM Tris, 10mM ethylenediaminetetraacetic acid (EDTA)) and subjected to a mechanical break through the use of a homogenizer (Ultraturrax) until completely homogenized. They were then centrifuged for 15 min at 5000 g and the supernatant was discarded. Next, 3 ml of a solution of hexadecyltrimethylammonium bromide (CTAB, 1.4MNaCl, 20mMEDTA, 100mMTris, 0.2% β- mercaptoethanol, CTAB 2%) were added. The tubes were stirred vigorously for 10 min and incubated 1 h at 60°C until complete disintegration of the precipitates. In order to purify the DNA, one volume of IAC (chloroform: isoamyl alcohol, 24:1 v/v) saturated inH2Owas added. The supernatant was recovered and transferred to a clean tube. Finally, the DNA was precipitated with two volumes of 100% cold ethanol. The visible DNA was recovered, allowed to dry in an oven at 37 °C and resuspended in 200 μL of T10 E1 buffer. Design of primers With the Methyl Primer Express® Version 1.0 program, the CpG islands of the promoter region of the Stat6 and Gata3 genes of the rat were detected using the reference sequences: ENSRNOG00000025026 and ENSRNOG00000019336 from ENSEMBL, respectively. Through the Promoter 2.0 program, the chosen regions were analyzed to verify the existence of regulatory elements of the transcription and the binding sites. The primers designed for the determination of the expression of each gene are detailed in Table 2. The primers are specific to fully methylated sequences, and recognize unconverted cytosine during bisulfite treatment. Treatment with bisulfite The DNA samples were treated with sodium bisulfite using the EZ DNA Methylation-Gold™ kit (Zymo Research, USA) according to the manufacturer’s instructions. The conversion was made from a total DNA amount between 50 and 450 ng/μL. To do this, the total DNA of the samples was quantified in a NanoDrop spectrophotometer (LNS-101 Labocon Scientific Limited, UK). Real-time methylation-specific polymerase chain reaction (real-time MSP) For real-time MSP, 1 μL of bisulfite-modified DNA from each sample was used. Five samples from each group were pooled to use for amplification. The amplification of the fragments was carried out using the enzyme Go Taq DNA polymerase (Promega, Argentina) following the manufacturer’s instructions. The final volume of the PCR reaction was adjusted with sterile H2O to 25 μL. The PCR conditions were: 5 min at 95°C, 35 cycles for 20 s at 95°C, 30 s at the temperature of annealing and 40 s at 72°C, followed by a final step at 72°C for 10 min. All of the procedures were done in duplicate to confirm repeatability and they were averaged following analysis. Gel-based fragmentation analysis To verify the correct amplification of the DNA product of PCR and the state of methylation, 10 μL of the PCR product was loaded, with 2 μL of SYBR Gold at a concentration of 10 pg/μL in a 12% polyacrylamide gel. Three microliters of 100 bp molecular weight marker (Bio-Logical Products, Argentina) was loaded along with the DNA samples. Electrophoresis was performed at 110 V for 40 min. The DNA was visualized, and a digital image of the gel was obtained using a ChemiDoc XRS þ Image Lab Software from BIO-RAD to detect specific bands that were quantified by densitometry using a FIJI Image processing package.

Palabras clave: POSTNATAL DEVELOPMENT, LACTATION, MAMMARY GLAND, EPIGENETICS

Alcance geográfico

.

Identificador del recurso

URI: http://hdl.handle.net/11336/190467

Colecciones

Datos de Investigación(IMBECU)
Datos de Investigación de INST. DE MEDICINA Y BIO. EXP. DE CUYO

Citación

Santiano, Flavia Eliana; Campo Verde Arbocco, Fiorella; Pistone Creydt, Virginia; López Fontana, Constanza Matilde; Caron, Ruben Walter; (2023): The epigenetic role of breastfeeding in mammary differentiation. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/190467

Condiciones de uso

Las buenas prácticas científicas esperan que se otorgue el crédito adecuado mediante una citación. Utilice un formato de citación y aplique estas normas de reutilización.

Archivos del conjunto de datos

Archivo

Notas de uso

Tamaño

ANALISIS_EN_PRISMA_PCR_REAL_TIME__pesos_dia_21__55__200_y___masa_grasa_.pzf

Más

21.67Mb

Consultar

Descargar solo metadatos (JSON) Descargar solo metadatos (XML)

Ver el registro completo