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Introduction
Genome imprinting is the latest technique that has genetically modified the face of infertility; it is an epigenetic phenomenon through which the parental origin of a gene is determined based on its expression. 1 There are a vast number of genes in mammals that are marked with their parental origin, and the result of this phenomenon is a single parental allele. These marked genes are known as “imprinted.” They rely on the epigenetic machinery determining their parental identity and maintaining their origin-specific gene expression. There are 100 imprinted genes estimated in human and 200 in mice. 1, 2 The imprinted gene Igf2 is expressed by the paternal chromosome and H19 is expressed by maternal chromosomes. Imprinted genes are mostly clustered throughout the genome that indicates their mutual involvement in gene regulation at a specific chromosomal site. The two key features of imprinted genes are their distinct dominance of CPG islands and existence of clustered direct repeats adjacent or inside the CPG islands. An epigenetic programming process takes place in genome that assures an appropriate distribution of sex-specific imprints to every generation during gametogenesis and the pre-implantation stage. Imprinted genes are managed by cis-acting regulatory factors known as imprinting control regions (ICRs) that contain the distinct parental epigenetic modifications, such as DNA methylation. DNA methylation is the first epigenetic procedure linked to imprinting, which directly uses a DNA strand for modification. DNA Methyltransferases (DNMT) belongs to a highly conserved class of enzymes are engaged in translocating the methyl groups on the cytosine-C5 which is crucial for the stability of genomes. During the development of primordial germ cells (PGC) the imprinting methylation patterns are eliminated that is re-established during the preimplantation development. This process may occur multiple times in germ lines. This stage is achieved at haploid or meiotic stage of spermatogenesis in males. Epigenetic reprogramming is responsible for precise development through controlling the early embryonic gene expressions, cell division and determination in the early stage of embryonic developments. Further reprogramming proceeds with imposing the epigenetic changes during the embryonic preimplantation stage via demethylation of non-imprinted genes of both origins. 1,2,3,4
It is hypothesized in this proposal that the in vitro protocol recapitulates (at a molecular level) the normal in vivo changes at imprinted loci that occur in the male germ line. The purpose of this in vitro research is to fill the gap of the imprinting field. The experimental approach of in vitro will help to analyze every molecular and cellular modification of machinery at the very minute level of genetic imprinting in the male germ line during spermatogenesis. Secondly, it will help in designing an undifferentiated germ cell extracted from an infertile subject and manipulate and induce to develop further meiotically to produce spermatozoa with the capability of fertilizing an oocyte. The research protocol may further assist in identifying the unknown imprinted loci that are paternally methylated.3,4,5
Research Methodology
Analysis of the expression patterns of the Igf2 and Igf2r genes in the four cell types
The PI research group has already developed a protocol to study the development of sperm in vitro using iPS cells. For the proposed protocol the induced pluripotent stem cells (iPS cells) are established using the skin fibroblasts of a young adult mouse. These stem cells are induced to divide further into the embryoid bodies (EB) using suspension cultures of ES cell medium. On inducing, the atmosphere of the culture changes (day zero) and they start to differentiate and after three days they start to develop like primordial germ cells (PG cells) originated from the epiblast of the early post-implantation embryo (embryonic day 7.5, E7.5). These cells are called early-stage PG cells. After seven days the early-stage PG cells start resembling PG cells found in the genital ridge at the E12.5 stage of embryonic development. These newly formed cells are called late-stage PG cells. The third and last stage are very crucial that facilitates further differentiation of the PG cells and by the 20th day, these cells reach metaphase I stage of meiosis. This stage is a critical stage to recognize the conditions allowing the cells to reach the final stage of meiosis and develop into mature spermatozoa. 6
Under the PI’s specific laboratory conditions we propose to analyze the genomic imprinting in the cultured cell types, specifically to examine the expression patterns of the Igf2 and Igf2r genes in the four cell types namely iPS cells, early-stage PG cells, late-stage PG cells and metaphase stage-1 spermatocyte.
We hypothesize that the Mouse iPSCs and other PG cells generated through the proposed method will protect it from the loss of methylation in genome imprinting. Moreover, The expressions of Igf2 and Igf2r will be analyzed using Southern Blot offers an advantage of greater specificity and visible bands with clear splice variants’ detection in methylation patterns that affect restriction sites.5,6,9¸19
Expression pattern of Igf2r and Igf2 genes
Previous studies have verified the repression of Igf2r on the paternally passed on allele in mid-gestation mouse embryos. 9 In the proposed experiment I expect to retrieve the similar results. The expression patterns of Igf2r gene turn monoallelic only post-implantation. Thus, I believe that Igf2r gene retrieved from iPS cells would show biallelic expression. It has been verified by previous studies that all cells at an early pre-implantation stage (E4.5) are biallelic, and the monoallelic expression noticed at the E6.5 stage, and later cells happens due to implantation.8,9 Thus, the early PG cells and late PG cells should exhibit monoallelic expressions. The imprinted expression is sustained by the genes advanced embryonic developments up to E13.5 stage of development.10 Moreover, it is seen that these late embryonic cells maintain the paternal repression till the late stages. 8 We expect that Igf2r will show complete methylation on the maternal alleles and Igf2 will demonstrate the methylation on paternal alleles in PG cells.8, 9 The expected gene expression by Igf2 is similar to Igf2r; that means a biallelic expression in pre-implantation stage development and monoallelic expressions in post implantation stage developments. 9,10
DNA methylation pattern at ICRs
A study using mouse iPSc stated that DNA methylation imprint at most ICRs is transported on the maternal chromosome. The parentally inherited DNA methylation imprints were mostly mislaid at multiple regions on iPS cells. 6, 7, 8, 11
According to the Tucker and co-worker, the methylation pattern of the Igf2r gene has exhibited an intronic CpG rich region with a monoallelic methylation prototype. The maternally originated alleles are methylated but the paternally derived alleles are hypomethylated at the HpaII sites within that particular segment. On the other hand in the mutant cells, both alleles were in a demethylated state showing a complete loss of methylation. 5, 8, 9
In the region of 1.5-kb upstream at the Igf2 promoter, the paternally attained allele is highly methylated, in comparison of Igf2r. It is assumed that the similar results will be retrieved in this experiment. 7, 12, 13, 14,15
Examination of DNA methylation patterns at ICRs
Identification of full imprinted complement genes in the mouse
Imprinting regions of mice have been mapped using partial uniparental disomy in mice which are also referred as called maternal (MatDp) or paternal duplication (PatDp). 19 Uniparental-partial disomy genotype is generated in mice through crossing the heterozygous that yields the reciprocal translocations identifiable by visible marker genes. 19
There are various methods to isolate imprinted genes. In this research, we propose Me-RDA method for identifying the full imprinted complement genes in the mouse iPS cells that uses frequent-cutting methylation-sensitive restriction enzyme.19 This method is highly successful in DMR isolation from the imprinted regions located at distal chromosome as well as valuable for the genome-wide screen. Me-RDA method is based on differential methylation screening and enables to monitor a larger extent of DMRs (differential methylation regions) in each experiment. For experimental purpose, gDNA from PatDps and MatDps is digested with HpaII enzymes that cut it into unmethylated smaller HpaII allele fragments which are further amplified by PCR to produce amplicons. The larger methylated fragments are not suitable for amplification. The amplicons go through subtractive hybridization to augment the unmethylated fragments in one source (MatDp or PatDp) but methylated in the other. 19
Loss-of-function or gain-of-function experiments
Imprinted genes perform various cellular mechanisms. Several of them act as transporters, and they are involved in cell cycle control, G-protein-coupled receptor signaling, ubiquitin linked pathways, creatine formation, transcription, and intracellular signalling pathways. A genome data contains the information of mouse genomes that was retrieved by sequencing specific imprinted regions. This data can be comparatively evaluated to study the loss or gain of function in the four cells but preferably in late PG cells taken from the culture. 19 The imprinted genes clusters are analyzed using various methods such as hybridization, transcriptome sequencing, SNP genotyping arrays, and in silico prediction pipelines. 19
In this research, we propose to use Allele-Specific Expression using SNP.20 In this method, the first chromosome taken from late PG cell will be marked for its parental origin which should be firmly maintained after the cellular divisions. This marked imprint may continue alike to the original one or become a secondary derivative of it. Transcriptional Machinery should be able to acknowledge the parent-of-origin mark if a monoallelic expression emerges. Finally, in the PG cell, the mark should be erased and reorganized. This step is very crucial because a little failure can result in a loss of imprinting (LOI) mutation. 20
Mutations of allelic genes impact the pre and postnatal growth which is verified in result in MatDp and PatDp mice case. The genetic mutations in associated with loss-of-function in paternally articulated genes negatively impact the growth while the loss of function mutations in maternally articulated genes induce the growth phenomenon. We propose that the present experiment will deliver the similar results. Igf2 has shown growth reduction while Igf2r is associated with lethal growth enhancement.19
Value of the Research
Epidemiological researches have demonstrated a higher occurrence of birth and growth associated disorders among the progeny originated from in vitro fertilization (IVF). The proposed concept in this research will help to identify the problems at genetic levels at various embryonic stages as well as the initial cell of sperm development. Analysis at this initial stage will facilitate to remove the probable genes causing disorders. This research is conducted on the mouse model, though the expected results will help on human subjects. The study proposes that the in vitro development of iPSc cells developed from mouse fibroblast will develop into similar early and late PG cells and spermatocytes as they progress in vivo, or they will recapitulate the in vivo process.
This well-designed study will help in understanding the effects of inducing pluripotency in mouse ES cells and modifications in expressions in post-implantation and metaphase stages. The methylation status of Igf2 and Igf2r in mice cells could be applied as a sensor for the further epigenetic studies for improved fertility results.
References
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