Ovulation Induction Changes Epigenetic Marks of Imprinting Genes
in Mice Fetus Organs
Genomic imprinting is an epigenetic phenomenon that plays a critical role in normal development of embryo. Using exogenous hormones during assisted reproductive technology (ART) can change an organism hormonal profile and subsequently affect epigenetic events. Ovarian stimulation changes gene expression and epigenetic pattern of imprinted genes in the organs of mouse fetus.
Materials and Methods
For this experimental study, expression of three imprinted genes H19, Igf2 (Insulin-like growth factor 2) and Cdkn1c (Cyclin-dependent kinase inhibitor 1C), which have important roles in development of placenta and embryo, and the epigenetic profile of their regulatory region in some tissues of 19-days-old female fetuses, from female mice subjected to ovarian stimulation, were evaluated by quantitative reverse-transcription PCR (qRT-PCR) and Chromatin immunoprecipitation (ChIP) methods.
H19 gene was significantly lower in heart (P<0.05), liver (P<0.05), lung (P<0.01), placenta (P<0.01) and ovary (P<0.01). It was significantly higher in kidney of ovarian stimulation group compared to control fetuses (P<0.05). Igf2 expression was significantly higher in brain (P<0.05) and kidney (P<0.05), while it was significantly lower in lung of experimental group fetuses in comparison with control fetuses (P<0.05). Cdkn1c expression was significantly higher in lung (P<0.05). It was significantly decreased in placenta of experimental group fetuses rather than the control fetuses (P<0.05). Histone modification data and DNA methylation data were in accordance to the gene expression profiles.
Results showed altered gene expressions in line with changes in epigenetic pattern of their promoters in the ovarian stimulation group, compared to normal cycle.
During prenatal stages of development, specific parental gene or cluster of genes are
widely expressed monoallelically and termed "imprinted genes" (1). Expression of these genes
is down-regulated after birth (1). Although imprinted genes occupied a small subset of the
genome, they play critical roles for normal development of organisms (1).
Imprinted genes mainly are regulated by epigenetic mechanisms including DNA methylation, interfering RNAs (including miRNA, piRNA, siRNA) and histone modification to promote normal development of embryo. It has also been shown that some defects in genomic imprinting can cause infertility (6, 7).
The major epigenetic process that is recognized to be associated with imprinted genes in both gametes and developing embryos is DNA methylation (8). It is one of the most studied epigenetic mechanisms that can affect activity of DNA segment and gene expression without changing its sequence. There are three epigenetic mechanisms that control gene expression:
1. DNA methylation is a process in which the methyl group is added to specific dinucleotide CpG sites in the genome. Hypermethylation of these sites in the genome leads to gene suppression, while hypomethylation of them can cause gene over-expression. Sites of DNA methylation are engaged by various proteins, containing methyl-CpG binding domain (MBD) proteins which recruit enzymatic machinery to create silent chromatin (9). Among them, Methyl CpG binding protein 2 (MeCP2) as a DNA methylation "reader" protein specifically binds to methylated DNA regions and typically can be detected by chromatin immunoprecipitation techniques, as an epigenetic marker for DNA methylation (10). 2. interfering RNAs (including miRNA, piRNA, siRNA), which describes epigenetic and posttranscriptional regulation of transposons and genes (7). 3. histone modification which describes posttranslational modifications altering interaction of the histones with DNA and nuclear proteins (11).
H3K9 (lysine 9 of histone 3) is an important position in the genome, as balance between its acetylation (H3K9ac) and deacetylation can regulate gene expression. H3K9ac and H3K9 trimethylation (H3K9me3) could have critical roles in epigenetic regulation of gene expression. Acetylation of this position causes opening of chromatin and mediating gene transcriptional activity. In contrast, its deacetylation (which is usually simultaneous with methylation) results in gene transcriptional repression. These two situations cause chromatin structure to be accessible or inaccessible for transcription. In addition, "bivalent marks" of H3K4me3 and H3K27me3(trimethylated lysine 4 and 27 on histone H3) are respectively activating and repressing histone marks that regulate gene expression level (12).
Histone codes like H3K9ac and H3K4me3 cause gene up-regulation and others such as H3K9me2 and H3K27me3 lead to gene repression (11). Some studies have shown a link between transcription of imprinted differentially methylated regions and removal insertion of histone modifications (13).
Patients undergo ovarian stimulation through
Ovarian stimulation is the most important cause of multiple pregnancies and consequently
low birth weight, increased risk of miscarriage, growth retardation and preterm delivery
(14). Finally, ovarian stimulation has been shown to be the cause for imprinting defects.
For example, overexpression of gene
Manipulations in hormonal profile, reproductive system and gametes of organism during
assisted reproductive technology (ART) can affect epigenetic events of genome, e.g. genomic
imprinting (19). Assessment of the relationships between epigenetics, genomic imprinting and
ART offers new perspectives in the understanding of molecular bases of infertility and ART
failure. This study focuses on understanding expression changes of the important
developmental imprinted genes (
Materials and Methods
Ovarian stimulation of naval medical research institute mice and obtaining embryos
In this experimental study, assessment was performed on two groups of 19-days-old fetuses of Naval Medical Research Institute (NMRI) mice (Pasteur Institute, Iran). In the first group, 16 fetuses were collected from uterus of four female mice, subjected to ovarian stimulation before gestation. The second group consisted of the 16 fetuses obtained from female mice with natural pregnancy, as control. Four fetuses were excluded and 12 fetuses were included for gene expression assessments in this study. Female mice were kept in the animal house of Royan Institute (Tehran, Iran) at temperature of 19-23˚C and humidity of 40-50%, 12 hours light (6 am- 6 pm) and 12 hours darkness. For growth and maturation of ovarian follicles in 8-weeks-old female mice of the first group, 7.5 IU PMSG (pregnant mare serum gonadotropin) hormone (Folligon; Invert, Belgium) followed 48 hours later by 7.5 IU of hCG (Human chorionic gonadotropin) hormone (Organon, Netherlands) were administered. Female mice of the both groups were mated with NMRI male mice (20). After formation of vaginal plaque (mating indication) females were isolated and sacrificed on the 19th day of pregnancy. The fetuses were obtained, and seven different tissues of each fetus -including brain, lung, heart, liver, kidney, ovary and placenta- were collected. Few parts of tissues were preserved in RNA later (Ambion, USA) reagent at -70˚C for future RNA isolation and the rest of tissues were preserved at -70˚C for later epigenetic evaluations (20, 21). This study was approved by the Institutional Ethics Committee of Royan Institute (Tehran, Iran) on 2nd July 2014 (code: EC/93/1038).
RNA isolation and quantitative reverse-transcription PCR
RNA isolation and qRT-PCR quantitative reveres transcription PCR (qRT-PCR) were performed
on tissues using the RNeasy micro kit (Qiagen, USA) according manufacture’s instruction.
Quantification of mRNA levels of imprinted genes (
Chromatin immunoprecipitation real time polymerase Chain Reaction analysis
Chromatin immunoprecipitated (ChIP) PCR experiments were performed, using a histone ChIP kit according to manufacturer’s instruction (Diagenode, Belgium). Briefly, all tissues were suspended in PBS. Then formaldehyde (1% final concentration) was added to the samples and then incubated gently on a shaking platform for 10 minutes at room temperature. In the next step, glycine was added into the samples to reach final concentration of 125 mM to quench the cross-linking reaction of formaldehyde. After washing the samples with PBS, lysis buffer was added and sonicated for 10 minutes (30 "on/30" off; Bioruptor sonication system, Diagenode) to get soluble sheared chromatin. After 5 minutes centrifugation at 14000 g, the supernatant was divided into six parts (Each part 10 µl). One part was used as input control, and the other 5 parts were incubated with 1µl of anti-H3K9ac, anti-H3K9me2, anti-H3K4me3, anti-H3K27me3 and anti-MeCP2 antibodies (1µg/µl; Abcam, UK) overnight at 4˚C on rotator. Immune complexes were washed three times using 100μl ice-cold washing buffer and then incubated on a rotating wheel for 4 minutes at 4˚C. Using a magnetic rack the beads were captured and immediately treated with 100μl DNA isolation buffer. The recovered DNA from immunoprecipitated fractions and total chromatin input, were quantified by real-time PCR. Data were expressed as fold enrichment of DNA associated with different immunoprecipitated histone modifications. DNA methylation was expressed as relative to a 1/100 dilution of input chromatin. Quantitative real-time PCR was carried out on a step one plus Real-Time PCR System (Applied Biosystems) using SYBR Green PCR master mix (Applied Biosystems) and designed primers (Table 1,). The condition was 95˚C for 10 minutes; and 40 cycles of 95˚C for 15 seconds, 60˚C for 45 seconds. Results were normalized to input DNA and expressed as (%) input, which means percentage of enriched DNA associated with immunoprecipitated chromatin [mean ± SEM, (21)].
|Gene||Primer sequence (5´- 3´)||Product size (bp)||Location|
|Primers used in qRT-PCR|
|Primers used in ChIP real time PCR|
Data analysis was carried out using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., USA). In this study, continuous variables were expressed as mean ± SEM (standard error of mean). An independent t test was used to compare control and ovarian stimulation groups. All statistical tests were two-tailed and a P<0.05 was considered statistically significant.
Alterations of gene expression in ovarian stimulation group
Relative mRNA expression levels of
Histone modification profile of the studied genes in the experimental group embryos
Our histone modification analyses, based on the ChIP data, were in accordance with the
gene expression profile. Thus, higher incorporation of H3K9me2 gene repressing mark was
detected in the
Higher incorporation of H3K9me2 gene repressing mark was detected in
H3K9me2 gene repressing histone mark was expressed significantly lower in promoter
There was no significant difference between the fetus weight of ovarian stimulated and control groups, in our study (Table 2,).
|Ovulation stimulation group fetuses||Control group fetuses|
|Number of fetuses||Average weight of each fetus (g)||Number of fetuses||Average weight of each fetus (g)|
|79||1.53 ± 0.1||69||1.66 ± 0.07|
Exogenous gonadotropins, used in ART cycles, could have negative effect on gene expression and consequently embryo development and growth (14). Both of the animal and limited human studies showed high possibility of ovarian stimulation responsibility for modifications in maternal-affected gene products that are later required for imprinting maintenance in developing embryos (18).
Although most ART children do not show any abnormality, some studies have suggested the correlation between ART and increased incidences of low birth weight and also rare imprinting syndromes, such as BeckwithWiedemann syndrome (BWS), Angelman syndrome (AS) and etc. (22, 23).
Our histone modification analyses based on the ChIP data was in accordance to the gene
expression profile; in the way that higher incorporation of gene repressing marks of MeCP2,
H3K9me2 and H3K27me3 were detected in promoter region of
It is important to note that tissues derived from trophoblast, unlike ICM (inner cell mass) derived tissues, have no control mechanisms through gene expression and they are more susceptible to imprinting disorders. There are two hypotheses. In the first, environment affects more on extra-embryonic cells and this causes loss of imprinting in mid-gestation placentas. In the second, loss of imprinting may also occur in cells destined to form the embryo. Biallelic expression was occasionally observed in the embryo, suggesting mechanisms that safeguard imprinting might be more robust in the embryo, than the placenta. Probably a de novo lineage-restricted wave of methylation occurs in ICM, but not in trophectoderm lineages (28). This is consistent with the results of our study which showed extreme changes in gene expression of placenta.
IGF1, IGF2 and their receptors are expressed in the fetal lung of humans, rodents and other
species (30). There are increasing evidences suggesting that the IGF system plays a pivotal
role in the development and differentiation of the fetal lung (31). Our study showed
Some observations have shown that methylation changes can be a highly consistent feature of
carcinogenesis and methylation errors are perhaps common observations in cancer (36). Wilms’
tumor, a childhood cancer of the kidney, is often associated with defects in the
Low expression of
To summarize, the current study showed the impact of ovarian stimulation on the expression of genes and the epigenetic alterations- even at the end of gestation. Occurrence of these long lasting epigenetic changes may be a reason of growth and development disturbances, in future. Although many researchers believe that the fetus is able to eliminate and correct many of the problems created during its development, the present study showed that some of the problems could remain with fetus until birth and they can affect growth of the fetus.
This work was supported by the Royan institute for Reproductive Biomedicine (grant number 91000270). The authors would like to thank Royan institute for the financial support of this study. There is no conflict of interest in the process of this study.
B.M.; Was responsible for overall supervision and provided critical revision of the manuscript. A.O., A.V., M.S., R.F., S.M.; Participated in study design, data collection, evaluation and drafting. All authors read and approved the final manuscript.