**3. Underreplication and amplification of some genes and clasters regulate the giant trophoblast cells differentiation**

The cells undergone endoreduplication and formation of the classic polytene chromosomes are known to underreplicate a significant amounts of DNA [43, 44].

Recently it has been found underreplication (UR) of some chromosome regions and genes in the murine giant trophoblast cells. TGCs of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented [45]. UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. Interestingly, both size and degree of depletion of UR domains gradually progresses during early gestation. Thus, all UR domains at 9.5th day are also present at 8.0th day, and UR domains at 9.5 gestation day are also more numerous, larger and more depleted. However, unlike between 8.0th and 9.5th day, where the degree of depletion expanded, there were no significant change from 9.5th and 16.5th day.

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

Recently, a notion dominates that the multifold genome multiplication is achieved by modified cell cycles. Among them the shortest one leading to the highest levels of ploidy is characterized by alternating DNA synthesis (S) and Gap (G) phases in the absence of intervening mitoses, karyokinesis, and cytokinesis; a series of these shortened cycles allows cells to achieve high level of ploidy that may exceed

The trophoblast cells that form a barrier between semiallogenic fetal (trophoblastic) and maternal (decidua) tissues probably require mechanism(s) to sustain maternal-fetal tolerance achieved by different mechanisms. For example, the trophoblast cells secrete a range of cytokines and chemokines thereby contributing to the process of immune regulation at the placental–maternal interface [14, 15]. On the other hand, as we stated previously, the TGC multifold genome multiplication also may protect their genome from mutagenic effect of the DNA of the phagocytosed maternal cells [16, 17]. Besides, some of the TGC functions of a barrier may be performed due to their giantism. TGC produce enormous keratin-positive sprouts that allow them to phagocytose accumulations of decidual cells and simultaneously to sustain the continuous TGC layer at the border with decidua [18]. Destruction of the cytokeratin 8 and 19 results in disruption of integrity of the murine giant

In distinct from the primary and secondary TGC, the low-ploid trophoblast cells

in rat and mouse placenta show high proliferative activity and, being protected by a TGC barrier, accumulate a great bulk of cells that differentiate into a range of trophoblast cell subtypes, some of them form placental barrier supplying embryo by nutrition and oxygen; other subtypes are involved in glycogen storage, hormone

The lifespan of the endoreduplicated TGC ends in depolyploidization via non-mitotic division of the giant nucleus or nuclear whole-genome fragmentation. In this case, division is achieved without complete chromosome condensation and their arrangement in metaphase plate, spindle formation and poleward chromosome movement. DNA content as well as nucleoli, heterochromatin and gonosomal chromatin bodies distributed into "subnuclei" according to their ploidy levels [3, 27–30]. By now, it is possible to consider it as variant of so called "polyploidy cycle" [31–33] that implies alternation of diploid and polyploid state in a cell lineage. It should be noted that such a phenomenon is fairly rare encountered in the cell lifespan and may be found in the "ancient" organisms like Protists [31–33] and some Invertebrates [34]. In the multicellular Invertebrates and Plants a wast majority of the differentiated cell types are endopolyploid [35, 36]. In Vertebrates, most cells are diploid, and the mammalian trophoblast cells, probably, represent an example of a recapitulation to some ancient forms of cell cycle and cell lifespan

As to depolyploidization in TGC of rodent placenta, it should be emphasized that they do not belong to the complete polyploid cycle because they do not give rise to the cells capable of self-reproduction because they cease DNA replication shortly before the birth that probably prevents a massive proliferation of semiallogenic

As we stated in our previous paper, depolyploidization probably may result in aneuploidy in the trophoblast cells [5] because such a way of cell dividion, most probably does not ensure precise distribution of all chromosomes into dauther cells. Surprisingly, aneuploidy combined with polyploidy were recently reported as a factor of adaptation to the stressful conditions [37, 38]. Hepatocytes represent a cell

**2. Poly- and aneuploidy, their origin and significance**

trophoblast cell layer [19], which result in embryo death.

production and deep intrauterine invasion [20–26].

similar to protists and Invertebrates.

embryonic cells inside the maternal tissues.

1000c [10–13].

**62**

Notably, 8-10th day of gestation in mice corresponds to the placenta formation whereas at 10-16th days well-developed placenta functioning takes place. The authors [45] note that the increase in UR domain size and degree of underrepresentation from 8.0th to 9.5th day is linked to the "robust" endocycles of early gestation [46].

Besides, it should be mentionned that, during the late stages of TGC lifespan, new underreplicated regions are also formed but they are more stochastic, less reproducible, and significantly smaller than those conserved between all stages [45]. Notably, underreplication of TGC coincide with period of the most significant stages of TGC invasion and anchoring to endometrium and is integrated in its developmental program.

The above-mentioned data also show that UR domains are formed from a specific class of late-replicating heterochromatic regions that contain mainly non-coding DNA, suggesting that UR domains are not simply a byproduct of latereplicating heterochromatin, but are a precisely regulated subset of DNA sequences. The authors come to conclusion that presence of UR domains in *Drosophila* endoreduplicated cells and in murine TGC is an example of convergent evolution. In this case UR contributes to accelerating the cell cycles that makes possible fast rate of development both in flies and in mice [45].

The underreplication in the endoreduplicated trophoblast cells not only may fasten the cell cycles but also be important for the TGC specific functions. Thus, Hannibal et al. [45] also note that UR domains are enriched for specific classes of genes involved in cell adhesion and neurogenesis. It is still difficult to find an explanation for the UR of specific genes and gene clusters. It can only be assumed that a certain number of gene copies is optimal in a given cell type. UR of separate genome regions at the background of its multiple duplication makes it possible to fine-tune the number of functioning copies necessary for performing specific functions.

In some cases, significance of UR was clearly demonstrated. Thus, downregulation of genes that regulate cell adhesion, junction and related cytoskeleton rearrangements is necessary for trophoblast EMT transition and invasion in both mice and humans [47, 48, 58]. Upregulation of genes in the SLIT/ROBO neuronal guidance system in the human placenta has been found to be bound with preeclampsia [49]. It is possible, placenta oxygenation requires precise specific function of SLIT/ ROBO signaling achieved by UR of its genes.

Therefore, significance of endoreduplication is not only multifold genome duplication itself and enlargement of the cell that may be of significance for TGC barrier function but also a possibility of a fine regulation of a number of functional gene copies to provide cells capabilities to accomplish some functions necessary at the precise stages of development.

## **4. Amplification of some genes significant for the pregnancy also takes place in TGC**

The mammalian polytene chromosomes may also undergo amplification of specific gene cluster. In the murine placenta TGC, five amplified regions were found using whole-genome sequencing and digital droplet PCR [50]. All the gene clusters are known to play key roles in mammalian placenta development and maintenance: the prolactins that regulate trophoblast cell lineage differentiations [51], serpins [52] and cathepsins [53] that promote trophoblast invasion, as well as (NK)/C-type lectin complex that play a crucial role in the feto-maternal cross-talk [54–56].

Therefore, amplification at selective genomic regions is another important mode of genome regulation in placental TGCs.

**65**

tion in TGC.

*Genome Modifications Involved in Developmental Programs of the Placental Trophoblast*

Besides the non-classic polytene chromosomes in rodent placenta TGC [5, 26], some details of unusual chromosome structure have been revealed recently in the endoreduplicated TGC of mice. In the course of differentiation of TSC into TGC, expression of most genes encoding canonical histone were downregulated [1] By contrast, genes encoding non-canonical histones - H2AX, H2AZ and H3.3 did not show downregulation. The micrococcal nuclease digesion assay as well as nucleosome stability assay using a microfluidic devise showed that chromatin progressive loosening of chromatin in the course of TSC differentiated. Experiments combining H3.3 knockdown and overexpression showed that variant H3.3 resulted in forma-

The presence of H2AZ and H3.3 in the genome potentially correlated with actively transcribed genes, indicating that H2AZ and H3.3 were necessary for creating relaxed and transcriptionally active chromatin structures [57–59]. Therefore, H2AX, H2AZ, and H3.3 histone variant may be responsible for the formation of a

Interestingly, knockdown of H3.3 variant in the differentiationg TSCs significantly decreased the number of cells containing more than 4n DNA content compared to the control cells [1]. Therefore, switch to the non-canonic histone variants seems to be a prereqisite of the trophoblast cell endoreduplication. On the other hand, loose chromatin organization may, like underreplication, promote fastening the modified cell cycle that allow reach multifold (up to 512c and higher) genome multiplication and formation a giant trophoblast cell layer at the border with

The unusual chromatin status is revealed, in particular, in the organization of the inactive X-chromosome of the murine TGC [60]. Thus, investigation of the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos showed stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type - TGCs. It has been found that the *Xist* RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs. However, some key loci seem to require dosage compensation in TGC that probably points out to combination of the relaxed and silenced gene expression as a specific mode of gene activity regulation in a condition of chromatin unusual organiza-

Methylation status provides some new insight in the understanding of the trophoblast cell organization that underly their unique features. The human placental trophoblast shows general global hypomethylation [61]. It is possible that the loose-nucleosome structure of TGC in murine placenta and global hypomethylation in human placenta are similar phenomena. In human placenta, genome-wide hypomethylation, coupled with gene-specific hypermethylation of tumor-suppressor genes, is a common feature of human cancers [64]. Interestingly, the placenta parallels human cancers in both the overall decreased level of genomic DNA methylation and the specific hypermethylation of several tumor suppressor genes [61–64].

*DOI: http://dx.doi.org/10.5772/intechopen.97247*

**5. Unusual chromatin structure of TGC**

tion of the loose nucleosomes in the murine TGC [1].

loose nucleosome structure that was unique to TGCs [1].

**6. Hypomethylation of human and rodent placenta**

semiallogenic maternal tissue.

*Genome Modifications Involved in Developmental Programs of the Placental Trophoblast DOI: http://dx.doi.org/10.5772/intechopen.97247*
