polyploidy , the state of having an increase in the number of chromosomes sets , is a widespread physiological phenomenon observed particularly in plants , fungi , insects , fishes , and amphibians . the additional set ( or sets ) of chromosomes may originate from the same individual (  autopolyploid  ) or from the hybridization of two different species (  allopolyploid  ) . although diploid is the normal status for mammalian cells , various studies have demonstrated during last decades a major role of  diploid - polyploid conversion  during physiopathological processes in several tissues . indeed , polyploidy seems to be part of a developmental program resulting in the formation of highly differentiated cells , as it has been reported for megacaryocytes ( 16n128n ) , cardiomyocytes ( 4n ) , trophoblast giant cells ( 8n64n ) , purkinje neurons ,   and retinal ganglion cells ( both 4n )   or hepatocytes ( 4n8n ) in the liver parenchyma . furthermore , in response to stress or injury , genesis of polyploid contingent can be also observed . uterine smooth muscle during pregnancy , heart muscle and vascular smooth muscle cells ( vsmc ) during hypertension [ 9 , 10 ] , and thyroid cells in hyperthyroidism   are prone to switch to polyploid state . finally , genesis of polyploid cells by unscheduled whole - genome duplications can also participate to carcinogenesis process , by inducing establishment of chromosomal instability ( cin ) . indeed , in many human carcinomas ( breast , lung , colon , pancreas , oesophagus ) emergence of tetraploid cells has been observed in early steps of tumorigenesis and precede the genesis of cells with intermediate dna content values ( aneuploid cells ) [ 12 , 13 ] . several mechanisms have been involved in the physiopathological emergence of polyploid cells in mammals . during cell - cell fusion , genesis of polyploid cells may occur independently of cell proliferation , as it has been observed during physiological development in osteoclasts   and skeletal muscle cells   or after pathological viral infection . in this process , cells fuse their nuclei and/or membranes , leading to the genesis of mononuclear or multinucleate cells , respectively . other mechanisms are directly associated with proliferative state of the cells . ( 1 ) endoreduplicationduring this process , cells alternate s ( dna replication ) and g phases , without performing mitosis and give rise to the genesis of autopolyploid cells ( i.e. , trophoblastic giant cells ) . 
  during this process , cells alternate s ( dna replication ) and g phases , without performing mitosis and give rise to the genesis of autopolyploid cells ( i.e. , trophoblastic giant cells ) . ( 2 ) endomitosiscells can reach metaphase or anaphase a , but nuclear ( karyokinesis ) and cytoplasmic ( cytokinesis ) divisions are never observed ; the best - studied example being polyploid megakaryocytes . these cells enter mitosis but never fully separate sister chromatids or undergo cytokinesis , resulting in globulated polyploid nuclei [ 18 , 19 ] . the regulatory mechanisms that control megakaryocytes polyploidization have been explored by different groups with a major focus on the regulation of mitotic phase and cytokinesis . studies of different megakaryoblastic cell lines suggest that endomitosis is promoted by the downregulation of cyclin b / cdk1 mitotic kinase activity [ 21 , 22 ] ; differently , in primary polyploid megakaryocytes , levels of cyclin b are reported to be upregulated [ 18 , 23 , 24 ] . moreover , other studies have reported a reduction in the duration of the g1 phase correlated with overexpression of cyclin e [ 21 , 25 , 26 ] . recent data have shown that cyclin e mediates its effect by promoting the expression of components of the prereplication complex ( cdc6 and mcm2 ) . overexpression of cyclin e can favor progression to s phase and cell cycling , thus promoting endomitosis and polyploidization of megakaryocytes . cells can reach metaphase or anaphase a , but nuclear ( karyokinesis ) and cytoplasmic ( cytokinesis ) divisions are never observed ; the best - studied example being polyploid megakaryocytes . these cells enter mitosis but never fully separate sister chromatids or undergo cytokinesis , resulting in globulated polyploid nuclei [ 18 , 19 ] . the regulatory mechanisms that control megakaryocytes polyploidization have been explored by different groups with a major focus on the regulation of mitotic phase and cytokinesis . studies of different megakaryoblastic cell lines suggest that endomitosis is promoted by the downregulation of cyclin b / cdk1 mitotic kinase activity [ 21 , 22 ] ; differently , in primary polyploid megakaryocytes , levels of cyclin b are reported to be upregulated [ 18 , 23 , 24 ] . moreover , other studies have reported a reduction in the duration of the g1 phase correlated with overexpression of cyclin e [ 21 , 25 , 26 ] . recent data have shown that cyclin e mediates its effect by promoting the expression of components of the prereplication complex ( cdc6 and mcm2 ) . overexpression of cyclin e can favor progression to s phase and cell cycling , thus promoting endomitosis and polyploidization of megakaryocytes . ( 3 ) mitotic slippageduring this pathological process , cells present an altered spindle - assembly - checkpoint ( sac ) . the sac prolongs mitosis until all kinetochores are stably attached to spindle microtubules ; when the sac can not be satisfied , cells exit mitosis without undergoing anaphase or cytokinesis ( genesis of mononucleated tetraploid cells ) . mitotic slippage has been observed for example in cells after prolonged mitotic arrest in response to spindle toxins   or in apc - deficient cells ( adenomatous polyposis coli , gene frequently mutated in colon cancers ) . 
  during this pathological process , cells present an altered spindle - assembly - checkpoint ( sac ) . the sac prolongs mitosis until all kinetochores are stably attached to spindle microtubules ; when the sac can not be satisfied , cells exit mitosis without undergoing anaphase or cytokinesis ( genesis of mononucleated tetraploid cells ) . mitotic slippage has been observed for example in cells after prolonged mitotic arrest in response to spindle toxins   or in apc - deficient cells ( adenomatous polyposis coli , gene frequently mutated in colon cancers ) . ( 4 ) incomplete cytokinesisthis process has been extensively described during pathological division and leads to the genesis of binucleated tetraploid cells . these cells can appear following dysfunction of any of a large number of different proteins controlling cytokinesis process . in addition , bulk chromatin or even a single lagging chromosome trapped in the cleavage furrow can induce cytokinesis failure and tetraploidization [ 30 , 31 ] . remarkably , recent studies demonstrated that cytokinesis failure process is also a programmed step in normal development ( as example : liver and heart tissues ; see section 3.1 for more details ) producing differentiated binucleated tetraploid progenies [ 3235 ] . finally , it is important to note that whatever the mechanism of polyploidization , the increase in cellular dna content will be associated with centrosomes amplification , which in certain cases could lead to the genesis of aneuploid progenies and cin ( see [ 13 , 36 ] for reviews ) . 
  this process has been extensively described during pathological division and leads to the genesis of binucleated tetraploid cells . these cells can appear following dysfunction of any of a large number of different proteins controlling cytokinesis process . in addition , bulk chromatin or even a single lagging chromosome trapped in the cleavage furrow can induce cytokinesis failure and tetraploidization [ 30 , 31 ] . remarkably , recent studies demonstrated that cytokinesis failure process is also a programmed step in normal development ( as example : liver and heart tissues ; see section 3.1 for more details ) producing differentiated binucleated tetraploid progenies [ 3235 ] . finally , it is important to note that whatever the mechanism of polyploidization , the increase in cellular dna content will be associated with centrosomes amplification , which in certain cases could lead to the genesis of aneuploid progenies and cin ( see [ 13 , 36 ] for reviews ) . hepatic polyploidy is a characteristic feature of mammalian liver and accompanies late fetal development and postnatal maturation [ 7 , 37 ] . in rodents , through 14th embryonic development day ( e.g. , e14 ) , most hepatoblasts are bipotent with the ability to differentiate into hepatocytes or into biliary cells ; by e15 most hepatoblasts are committed to the hepatocyte lineage [ 38 , 39 ] . during the remaining period of gestation and the first four postnatal weeks , hepatoblasts acquire functions of differentiated hepatocytes , and this period is correlated with a severe decline in proliferative state [ 40 , 41 ] . during previous studies , we have observed that the liver is almost exclusively made up of diploid hepatocytes for the first three weeks after birth . after weaning ( day 21 ) , the proportion of diploid hepatocytes started to fall significantly , with the successive appearance of binucleated tetraploid ( 2  2n ) and mononucleated tetraploid ( 4n ) hepatocytes [ 32 , 33 ] ( see figure 1 ) . the hepatocyte ploidy level effectively reaches a plateau at maturity , octoploid ( binucleated 2  4n and mononucleated 8n ) hepatocytes appearing in significant numbers during the second and third months after birth . interestingly , a second wave of high ploidization has been also observed at senescence in different species . for example , in humans , polyploid hepatocytes begin to appear during postnatal liver development ; their accumulation rate stays stable during the maturity period , and finally a significant increase of polyploid cells is observed during ageing process . in adults , 70% of all hepatocytes in rodents and 40% in humans it has to be noticed that a negative correlation exists between mitotic index in the liver and the level of hepatocyte polyploidization found in different species [ 46 , 47 ] . as example , mouse liver has a much lower mitotic index than rat liver and accordingly the highest level of hepatocytes polyploidization was found in the mouse liver . interestingly , in the adult liver , the genesis of polyploid cells can be reinduced following a variety of signals ( see figure 1 ) . indeed , after two - thirds hepatectomy , mitogenic signals ( cytokines and growth factors ; for review , see ) induce exit of quiescence (  priming  ) of hepatocytes . these hepatocytes undergo one or two rounds of one or two rounds of cell division to restore the hepatic liver mass and this process is associated with a pronounced increase of polyploid hepatocytes [ 37 , 46 , 49 , 50 ] . several reports indicated that liver regeneration depends mainly on the proliferation of hepatocytes [ 48 , 51 ] . however , it has to be noticed that hypertrophy of hepatocytes in the regenerated liver has also been described [ 5254 ] . a recent study has conciliated with these two pathways by revealing that hypertrophy precedes proliferation in the regenerating liver . furthermore , this work also established that preexisting diploid and tetraploid mononucleated hepatocytes generate cells with tetraploid and octoploid nuclei , respectively , by an unconventional cell cycle , probably by endoreplication as hepatocytes entering into s phase and skipping mitosis . finally , they also described that binuclear hepatocytes undergo reductive divisions to generate two mononuclear daughter hepatocytes of higher ploidy . all these processes lead to an increase in both size and ploidy of hepatocytes during liver regeneration . it is interesting to note , that sigal and coll have also observed that 16n populations are found in the hepatic tissue , during the second day of liver regeneration . at the end of the regenerative process , 16n contingent is no more present in liver parenchyma . the authors suggested that the disappearance of these highly polyploid hepatocytes is associated with the establishment of apoptotic mechanisms that target preferentially hepatocytes of advanced ploidy . in adult rodents , dna synthesis induced by chemical compounds adjunction of hepatic mitogens such as sodium phenobarbitone , 1 , 4-dichlorobenzene , or peroxisome proliferators   are known to favor octoploid mononucleated hepatocytes genesis . different studies have described that liver of long - evans cinnamon ( lec ) rat ( animal model of human wilson 's disease ) , which exhibits abnormal hepatic copper and iron concentration due to the deletion of the p - type copper transport atpase gene ( atp7b ) , possesses a feature of increase in polyploidy ( enlarged hepatocytes with huge nuclei ) and a delay in mitotic progression . interestingly , injection of irondextran in normal mice induces liver polyploidization ; this effect is inhibited by the oral intake of iron chelator [ 60 , 61 ] . have demonstrated that subsequent to radiation , hepatocytes exhibit evidence for oxidative injury with deletion of intracellular antioxidants ( as glutathione and catalase ) and for increase of polyploidy . furthermore , a study on rats indicates that the rate of reactive oxygen species generation exceeds the induced antioxidant ability with aging , generating a situation that favors oxidative stress and peroxidation . this state is correlated with changes in the proliferative potential of hepatocytes and an increase in the genesis of octoploid contingent . further evidence for the role of oxidative injury in polyploidy is provided by studies showing that in transgenic mice overexpressing copper - zinc - superoxide dismutase and glutathione peroxidase , which are antioxidants , ph - induced hepatic polyploidization is decreased . similarly , it is interesting to note that in others polyploid cell types , such as vsmc , a crucial role of oxidative stress in polyploidization process has been underlined . indeed , mccrann and coll have described that increased expression of an ros - producing enzyme , nox4 ( member of the nadph oxidase family ) results in vsmc polyploidy . treatment of mouse bone - marrow cultures with nox inhibitors resulted in accumulation of mks with low dna content levels and significant reduction of higher ploidy mks . further examination indicated that nox - inhibited mks showed a notable decrease in the level of the g1 phase cyclin e , a cyclin associated with mk polyploidy , and its upregulation restored most of the effect of nox inhibitors . all together , these results underline an extensive correlation between the generation of polyploid hepatocytes and a variety of cellular stress in the adult liver ; however , cellular and molecular mechanisms involved in ploidization modification during pathological state are not well characterized . we previously unveiled that during postnatal development and more precisely after weaning , diploid hepatocytes ( mononucleated 2n ) can engage either into a normal cell cycle and give rise to two diploid hepatocytes or follow an adaptive cell cycle with incomplete cytokinesis and give rise to one tetraploid hepatocyte ( binucleated 2  2n ) [ 32 , 33 ] . in these hepatocytes , several studies have revealed that rhoa gtpase is a key player to ensure a successful cytokinesis , by regulating the organization of the actin cytoskeleton and myosin ii activity at the cleavage plane [ 68 , 69 ] . we revealed that in hepatocytes , deficiencies in cytoskeleton reorganization inhibit rho - a gtpase recruitment to the cleavage plane ; consequently the cytokinesis ring is never formed . the genesis of such binucleated tetraploid cells is the crucial step for the establishment of gradual polyploidization during postnatal liver growth . indeed these cells are capable to proliferate and to give rise to two mononucleated 4n cells , which , if they divide , can generate 2  4n binucleated or 8n mononucleated hepatocytes .   in the heart , incomplete cytokinesis has been also implicated in the genesis of binucleated tetraploid cardiomyocytes . in mammals , the growth of embryonic heart results in proliferation of cardiomyocytes ( hyperplasia ) . after birth , ventricular cardiomyocytes respond to an amplification of blood flow by an adaptive increase in volume ( hypertrophy ) . this transition from hyperplasia to hypertrophy is correlated to a tetraploidization process [ 35 , 71 ] . in this system , a drastic reduction of rhoa and its effector rock after birth could account for defects in the process of cytokinesis . indeed , in some diploid cardiomyocytes , a cytokinetic ring is formed but as it is not at all functional , cytokinesis is never achieved , and tetraploid binucleated cell is consequently generated . differently from hepatocytes , adult 4n cardiomyocytes are in a postmitotic state and display a low proliferative potential ( for review , see ) . recently , gao et al . have shown that rhoa regulation is also a key target in mks polyploidization and differentiation . indeed , they have demonstrated that downregulation of the guanine exchange factor ect2 prevents rhoa activation and cleavage furrow ingression during endomitosis cycle , allowing the formation of 4n mks . interestingly , even if failed cytokinesis is the major event for liver polyploidization during postnatal development , some studies reveal that under certain circumstances , cell fusion can also contribute to this process . experiments on stem cells and therapeutic applications have discovered that polyploid hepatocytes can be generated following cell fusion between exogenous bone marrow cells and mature hepatocytes [ 75 , 76 ] . furthermore , faggioli and coll have shown that in adult liver , genesis of binucleated hepatocyte could be directly promoted by homotypic fusion but with a rare occurrence . recently , we discovered that the suckling - to - weaning transition strictly controls the establishment of the cytokinesis failure process in the liver . using a specific immunocytochemistry approach to detect mitotic events in liver tissue , we showed that cytokinesis failure events never occurred in 19-day - old suckling rat ( < 3% ) ; whereas in 19-day - old rats weaned early ( at 15 days ) , such events were frequent ( > 35% ) , and numerous binucleated tetraploid hepatocytes were generated . it is interesting to note that while suckling is prolonged to 25 days , hepatocytes mostly enter into complete cytokinesis events ( > 95%data not published ) . moreover , we reported there was a new wave of proliferation in the liver associated with the establishment of these specific adaptive cell cycles [ 41 , 78 ] . if the physiological rise in insulin after weaning was inhibited in rats ( by destroying pancreatic beta cells with streptozotocin drug ) , hepatocytes did not undergo cytokinesis failure , whereas if this rise was further accentuated ( by injecting insulin ) , cytokinesis failure was even more frequent and an increase in the genesis of binucleated tetraploid hepatocytes was observed . by investigating how insulin controls polyploidy program , we discovered that pi3  k / akt pathway ( signaling pathway regulating cellular homeostasis through its role in regulation of apoptosis , cell growth , cell cycle , cytoskeleton organization and angiogenesis ; see   for , review ) is a key regulator of cytokinesis through the control of cytoskeleton networks . indeed , direct inhibition ok akt by chemical compound ( iakt ) in hepatocytes primary culture prevents the appearance of incomplete cytokinesis process . we also examined the cytoskeleton organization in treated cells ; iakt - treated cells that completed cytokinesis reorganized the actin cytoskeleton and recruited rhoa to the equatorial cortex by contrast to cells that did not complete cytokinesis . in the past , several studies in yeast , metazoans , and mammals underlined a role of insulin in the regulation of cell proliferation and growth , by controlling g1/s - and g2/m - specific checkpoints [ 8082 ] . however , we have demonstrated for the first time in mammals that this hormone , through the pi3  k - akt pathway , can also regulate late mitosis progression and tightly control physiological polyploidization process during liver development . interestingly , a role of pi3  k - akt pathway has been also described during a pathological polyploidization process . by overexpressing akt1 , hixon et al . have demonstrated that vsmcs are able to override the activity of the mitotic spindle checkpoint , facilitating unscheduled degradation of cyclin b , cell - cycle reentry ( endoreduplication ) , and polyploidization process . the same results were obtained by incubating vsmcs isolated from normotensive animals with angiotensin ii ( regulator of hypertrophic signals during hypertension ) , which is a key activator of akt1 in vsmcs . these results demonstrate that akt1 regulates ploidy levels in vsmcs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension . many examples from the literature illustrate that the acquisition of a polyploid status confers specific biological properties of cells . in the yeast saccharomyces cerevisiae , polyploidization alters the expression profile of specific genes and regulates certain aspects of physiology and cell morphology . in plants , high polyploidy is correlated with epigenetic changes associated with hybrid vigor ( stronger and taller plants ) . in mammals , polyploidization of megakaryocytes is associated with terminal differentiation and regulation of platelets formation and function . indeed , polyploidization process increases the overall mk mass , resulting in an increase in platelet formation . furthermore , a study suggested that mks from different ploidy levels produce platelets with different functions : platelets originating from high - ploidy mks are thought to be more easily activated than platelets generated from mks with a lower ploidy . in the liver our understanding of the consequences for hepatocytes polyploidization still remains enigmatic . ( 1 ) polyploidy could protect hepatocytes of genotoxic damage by increasing the number of copies of functional genes ; this might be especially important for the liver that has a primary function in metabolizing and eliminating toxic compounds . ( 2 ) polyploidy could be an economical solution to growth problems that occur when an organ work within its capabilities , avoiding the great demand in energy that represents cell division . recently , two studies using multitest approach of modular biology underline alteration in a wide range of functional gene groups between diploid and polyploidy hepatocytes . the authors suggest a link between genome multiplication and emergence of specific pathways ( increase in metabolic plasticity and for the protection of replicating dna from oxidative damage ) that could promote hepatocyte cell survival and tissue regeneration under stressful conditions [ 88 , 89 ] . cellular polyploidization is now well known to be correlated to chromosomal instability appearance and carcinogenesis process development . indeed , in some tumor types , there is direct evidence for the development of aneuploidy from a transient 4n state . however , the impact of polyploid hepatocytes status on hepatocarcinoma ( hcc ) is still in debate . recently , grompe and coll have shown that hepatocytes can increase ( failed cytokinesis ) and reduce ( multipolar mitosis ) their ploidy , thus resulting in the concept of a in their works , authors showed that this dynamic mechanism can induce the genesis of  near - diploid / polyploid  , that is , aneuploid hepatocytes in rodents and humans livers [ 90 , 91 ] . given the high tumoral potential of aneuploid cells in tissue , these data are quite surprising as spontaneous tumor in the liver is rarely observed . the genesis of such  near polyploid  cells could then finally represent a source of genetic diversity , providing a strong selective advantage in response to multiple environmental stressors , as it has been demonstrated in yeast . further investigations on this topic promise to increase our understanding of the mechanisms and functional consequences of hepatocytes polyploidization and could offer insights into hepatic physiopathology .
most cells in mammalian tissues usually contain a diploid complement of chromosomes . 
 however , numerous studies have demonstrated a major role of  diploid - polyploid conversion  during physiopathological processes in several tissues . in the liver parenchyma , 
 progressive polyploidization of hepatocytes takes place during postnatal growth . 
 indeed , at the suckling - weaning transition , cytokinesis failure events induce the genesis of binucleated tetraploid liver cells . 
 insulin signalling , through regulation of the pi3k / akt signalling pathway , is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression . 
 liver cell polyploidy is generally considered to indicate terminal differentiation and senescence , and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity . although adult liver is a quiescent organ , it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression ( partial hepatectomy , metabolic overload ( i.e. , high copper and iron hepatic levels ) , oxidative stress , toxic insult , and chronic hepatitis etc . ) . 
 here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth .