In some species, sex determination is genetic: males and females have different  alleles  or even different  genes  that specify their sexual  morphology . In animals this is often accompanied by  chromosomal  differences, generally through combinations of  XY ,  ZW ,  XO ,  ZO  chromosomes, or  haplodiploidy . The sexual differentiation is generally triggered by a main gene (a "sex locus"), with a multitude of other genes following in a  domino effect .


In other cases, sex of a fetus is determined by  environmental  variables (such as  temperature ). The details of some sex-determination systems are not yet fully understood. Hopes  [ who? ]  for future fetal biological system analysis include complete-reproduction-system initialized signals that can be measured during pregnancies to more accurately determine whether a determined sex of a fetus is male, or female. [ citation needed ]  Such analysis of biological systems could also signal whether the fetus is hermaphrodite, which includes total or partial of both male and female reproduction organs.


Some species such as various plants and fish do not have a fixed sex, and instead go through life cycles and  change sex  based on genetic cues during corresponding life stages of their type. This could be due to environmental factors such as seasons and temperature. In some  gonochoric  species, a few individuals may have  conditions  that cause a mix of different  sex characteristics . [ 3 ]


Sex determination was discovered in the  mealworm  by the American geneticist  Nettie Stevens  in 1903. [ 4 ] [ 5 ] [ 6 ]


In 1694, J.R. Camerarius, conducted early experiments on pollination and reported the existence of male and female characteristics in plants(Maize).


In  1866,  Gregor Mendel  published on inheritance of genetic traits. This is known as  Mendelian inheritance  and it eventually established the modern understanding of inheritance from two  gametes .


In 1902, C.E. McClung identified sex chromosomes in bugs.


In 1917, C.E. Allen, discovered sex determination mechanisms in plants.


In 1922, C.B. Bridges, put forth the Genic Balance Theory of sex determination.


Among animals, the most common chromosomal sex determination systems are XY, XO, ZW, ZO, but with numerous exceptions.


According to the Tree of Sex database [ 7 ]  (as of 2023), the known sex determination systems are: [ 8 ]


1. complex sex chromosomes, homomorphic sex chromosomes, or others


The  XX/XY sex-determination system  is the most familiar, as it is found in humans. The XX/XY system is found in most other  mammals , as well as some insects. In this system, females have two of the same kind of sex chromosome (XX), while males have two distinct sex chromosomes (XY). The X and Y sex chromosomes are different in shape and size from each other, unlike the rest of the chromosomes ( autosomes ), and are sometimes called  allosomes . In some species, such as humans, organisms remain sex indifferent for a time during development ( embryogenesis ); in others, however, such as fruit flies, sexual differentiation occurs as soon as the egg is fertilized. [ 9 ]


Some species (including humans) have a gene  SRY  on the Y chromosome that determines  maleness . Members of SRY-reliant species can have uncommon XY chromosomal combinations such as  XXY  and still live. [ 9 ]
Human sex is determined by the presence or absence of a Y chromosome with a functional SRY gene. Once the SRY gene is activated, cells create  testosterone  and  anti-müllerian hormone  which typically ensures the development of a single, male reproductive system. [ 9 ]  In typical XX embryos, cells secrete  estrogen , which drives the body toward the female pathway.


In Y-centered sex determination, the SRY gene is the main gene in determining male characteristics, but multiple genes are required to develop testes. In XY mice, lack of the gene  DAX1  on the X chromosome results in sterility, but in humans it causes  adrenal hypoplasia congenita . [ 10 ]  However, when an extra DAX1 gene is placed on the X chromosome, the result is a female, despite the existence of SRY, since it overrides the effects of SRY. [ 11 ]  Even when there are normal sex chromosomes in XX females, duplication or expression of  SOX9  causes testes to develop. [ 12 ] [ 13 ]  Gradual  sex reversal  in developed mice can also occur when the gene  FOXL2  is removed from females. [ 14 ]  Even though the gene  DMRT1  is used by birds as their sex locus, species who have XY chromosomes also rely upon DMRT1, contained on chromosome 9, for sexual differentiation at some point in their formation. [ 9 ]


Some species, such as  fruit flies , use the presence of two X chromosomes to determine  femaleness . [ 15 ]  Species that use the number of Xs to determine sex are nonviable with an extra X chromosome.


Some fish have variants of the  XY sex-determination system , as well as the regular system. For example, while having an XY format,  Xiphophorus nezahualcoyotl  and  X. milleri  also have a second Y chromosome, known as Y', that creates XY' females and YY' males. [ 16 ]


At least one  monotreme , the  platypus , presents a particular sex determination scheme that in some ways resembles that of the  ZW sex chromosomes  of birds and lacks the SRY gene. The  platypus  has sex chromosomes

 X

 1

 ,

 X

 2

 ,

 X

 3

 ,

 X

 4

 ,

 X

 5

 ,

 Y

 1

 ,

 Y

 2

 ,

 Y

 3

 ,

 Y

 4

 ,

 Y

 5

 {\displaystyle X_{1},X_{2},X_{3},X_{4},X_{5},Y_{1},Y_{2},Y_{3},Y_{4},Y_{5}}

 . The males have

 X

 1

 Y

 1

 /

 X

 2

 Y

 2

 /

 X

 3

 Y

 3

 /

 X

 4

 Y

 4

 /

 X

 5

 Y

 5

 {\displaystyle X_{1}Y_{1}/X_{2}Y_{2}/X_{3}Y_{3}/X_{4}Y_{4}/X_{5}Y_{5}}

 , while females have

 X

 1

 X

 1

 /

 X

 2

 X

 2

 /

 X

 3

 X

 3

 /

 X

 4

 X

 4

 /

 X

 5

 X

 5

 {\displaystyle X_{1}X_{1}/X_{2}X_{2}/X_{3}X_{3}/X_{4}X_{4}/X_{5}X_{5}}

 . During meiosis, 5 of X form one chain, and 5 of Y form another chain. Thus, they behave effectively as a typical XY chromosomal system, except each of X and Y is broken into 5 parts, with the effect at  recombinations  occur very frequently at 4 particular points. [ 17 ]  One of the X chromosomes is homologous to the human X chromosome, and another is homologous to the bird Z chromosome. [ 18 ]


Although it is an XY system, the platypus' sex chromosomes share no homologues with  eutherian  sex chromosomes. [ 19 ]  Instead, homologues with eutherian sex chromosomes lie on the platypus chromosome 6, which means that the eutherian sex chromosomes were  autosomes  at the time that the monotremes diverged from the therian mammals (marsupials and eutherian mammals). However, homologues to the avian  DMRT1  gene on platypus sex chromosomes X3 and X5 suggest that it is possible the sex-determining gene for the platypus is the same one that is involved in bird sex-determination. More research must be conducted in order to determine the exact sex determining gene of the platypus. [ 20 ]


In this variant of the XY system, females have two copies of the sex chromosome (XX) but males have only one (X0). The  0  denotes the absence of a second sex chromosome. Generally in this method, the sex is determined by amount of genes expressed across the two chromosomes. This system is observed in a number of insects, including the grasshoppers and crickets of order  Orthoptera  and in cockroaches (order  Blattodea ). A small number of mammals also lack a Y chromosome. These include the Amami spiny rat ( Tokudaia osimensis ) and the Tokunoshima spiny rat ( Tokudaia tokunoshimensis ) and  Sorex araneus , a  shrew  species. Transcaucasian mole voles ( Ellobius lutescens ) also have a form of XO determination, in which both sexes lack a second sex chromosome. [ 11 ]  The mechanism of sex determination is not yet understood. [ 21 ]


The  nematode   C. elegans  is male with one sex chromosome (X0); with a pair of chromosomes (XX) it is a hermaphrodite. [ 22 ]  Its main sex gene is XOL, which encodes  XOL-1  and also controls the expression of the genes TRA-2 and HER-1. These genes reduce male gene activation and increase it, respectively. [ 23 ]


The  ZW sex-determination system  is found in birds, some reptiles, and some insects and other organisms. The ZW sex-determination system is reversed compared to the XY system: females have two different kinds of  chromosomes  (ZW), and males have two of the same kind of chromosomes (ZZ). In the chicken, this was found to be dependent on the expression of DMRT1. [ 24 ]  In birds, the genes FET1 and ASW are found on the W chromosome for females, similar to how the Y chromosome contains SRY. [ 9 ]  However, not all species depend upon the W for their sex. For example, there are moths and butterflies that are ZW, but some have been found female with ZO, as well as female with ZZW. [ 22 ]  Also, while mammals deactivate one of their extra X chromosomes when female, it appears that in the case of  Lepidoptera , the males produce double the normal amount of enzymes, due to having two Z's. [ 22 ]  Because the use of ZW sex determination is varied, it is still unknown how exactly most species determine their sex. [ 22 ]  However, reportedly, the silkworm  Bombyx mori  uses a single female-specific  piRNA  as the primary determiner of sex. [ 25 ]  Despite the similarities between the ZW and XY systems, these sex chromosomes evolved separately. In the case of the chicken, their Z chromosome is more similar to humans' autosome 9. [ 26 ]  The chicken's Z chromosome also seems to be related to the X chromosome of the platypus. [ 27 ]  When a ZW species, such as the  Komodo dragon , reproduces  parthenogenetically , usually only males are produced. This is due to the fact that the haploid eggs double their chromosomes, resulting in ZZ or WW. The ZZ become males, but the WW are not viable and are not brought to term. [ 28 ]


In both XY and ZW sex determination systems, the sex chromosome carrying the critical factors is often significantly smaller, carrying little more than the genes necessary for triggering the development of a given sex. [ 29 ] [ better source needed ]


The  ZZ/Z0 sex-determination system  is found in some moths. In these insects there is one sex chromosome, Z. Males have two Z chromosomes, whereas females have one Z. Males are ZZ, while females are Z0. [ 30 ] [ 31 ] [ 32 ]


In some  bryophyte  and some  algae  species, the  gametophyte  stage of the life cycle, rather than being hermaphrodite, occurs as separate male or female individuals that produce male and female gametes respectively. When meiosis occurs in the  sporophyte  generation of the life cycle, the sex chromosomes known as U and V assort in spores that carry either the U chromosome and give rise to female gametophytes, or the V chromosome and give rise to male gametophytes. [ 33 ] [ 34 ]


The  mating type  in  microorganisms  is analogous to sex in multi-cellular organisms, and is sometimes described using those terms, though they are not necessarily correlated with physical body structures. Some species have more than two mating types.  Tetrahymena ,  a type of  ciliate , has 7 mating types


Mating types are extensively studied in fungi. Among fungi, mating type is determined by chromosomal regions called  mating-type loci . Furthermore, it is not as simple as "two different mating types can mate", but rather, a matter of combinatorics. As a simple example, most  basidiomycete  have a "tetrapolar  heterothallism " mating system: there are two loci, and mating between two individuals is possible if the alleles on  both  loci are different. For example, if there are 3 alleles per locus, then there would be 9 mating types, each of which can mate with 4 other mating types. [ 35 ]  By multiplicative combination, it generates a vast number of mating types. For example,  Schizophyllum commune ,  a type of fungus, has

 9
 ×
 32
 ×
 9
 ×
 9
 =
 23328

 {\displaystyle 9\times 32\times 9\times 9=23328}

  mating types.


Haplodiploidy  is found in insects belonging to  Hymenoptera , such as  ants  and  bees . Sex determination is controlled by the  zygosity  of a complementary sex determiner ( csd ) locus. Unfertilized eggs develop into  haploid  individuals which have a single, hemizygous copy of the  csd  locus and are therefore males. Fertilized eggs develop into  diploid  individuals which, due to high variability in the  csd  locus, are generally heterozygous females. In rare instances diploid individuals may be homozygous, these develop into sterile males.
The gene acting as a  csd  locus has been identified in the  honeybee  and several candidate genes have been proposed as a  csd  locus for other Hymenopterans. [ 36 ] [ 37 ] [ 38 ]
Most females in the Hymenoptera order can decide the sex of their offspring by holding received sperm in their  spermatheca  and either releasing it into their oviduct or not. This allows them to create more workers, depending on the status of the colony. [ 39 ]


Polygenic sex determination is when the sex is primarily determined by genes that occur on multiple non- homologous chromosomes . The environment may have a limited, minor influence on sex determination. Examples include African cichlid fish ( Metriaclima  spp. ), lemmings ( Myopus schisticolor ),  green swordtail , [ 16 ]   medaka , [ 16 ]  etc. In such systems, there is typically a dominance hierarchy, where one system is dominant over another if in conflict. For example, in some species of cichlid fish from Lake Malawi, if an individual has both the XY locus (on one chromosome pair) and the WZ locus (on another chromosome pair), then the W is dominant and the individual has a female phenotype. [ 40 ]


The sex-determination system of  zebrafish  is polygenic. Juvenile zebrafishes (0–30 days after hatching) have both ovary-like tissue to testis tissue. They then develop into male or female adults, with the determination based on a complex interaction genes on multiple chromosomes, but not affected by environmental variations. [ 41 ] [ 42 ]


In systems with two sex chromosomes, they can be heteromorphic or homomorphic. Homomorphic sex chromosomes are almost identical in size and gene content. The two familiar kinds of sex chromosome pairs (XY and ZW) are heteromorphic. Homomorphic sex chromosomes exist among pufferfish, ratite birds, pythons, and European tree frogs. Some are quite old, meaning that there is some evolutionary force that resists their differentiation. [ 43 ]  For example, three species of  European tree frogs  have homologous, homomorphic sex chromosomes, and this homomorphism was maintained for at least 5.4 million years by occasional recombination. [ 44 ]


The  Nematocera , particularly the  Simuliids  and  Chironomus , have sex determination regions that are labile, meaning that one species may have the sex determination region in one chromosome, but a closely related species might have the same region moved to a different non- homologous chromosome . Some species even have the sex determination region different among individuals  within   the same species  ( intraspecific variation ). [ 45 ] [ 46 ] [ 47 ]  In some species, some populations have homomorphic sex chromosomes while other populations have heteromorphic sex chromosomes.


The New Zealand frog,  Leiopelma hochstetteri , uses a  supernumerary sex chromosome . With zero of that chromosome, the frog develops into a male. With one or more, the frog develops into a female. One female had as many as 16 of that chromosome. [ 48 ]


Different populations of the  Japanese frog  Rana rugosa  uses different systems. Two use homomorphic male heterogamety, one uses XX/XY, one uses ZZ/ZW. Remarkably, the X and Z chromosomes are homologous, and the Y and W as well.  Dmrt1  is on autosome 1 and not sex-linked. This means that an XX female individual is genetically similar to a ZZ male individual, and an XY male individual is to a ZW female individual. The mechanism behind this is yet unclear, but it is hypothesized that during its recent evolution, the XY-to-ZW transition occurred twice. [ 49 ] [ 50 ]


Clarias gariepinus  uses both XX/XY and ZW/ZZ system within the species, with some populations using homomorphic XX/XY while others using heteromorphic ZW/ZZ. A population in Thailand appears to use both systems simultaneously, possibly because  C. gariepinus  were not native to Thailand, and were introduced from different source populations which resulted in a mixture. [ 51 ]


Multiple sex chromosomes like those of platypus also occurs in bony fish. [ 52 ]  Some moths and butterflies have

 W

 1

 W

 2

 Z

 ♀

 /

 Z
 Z

 ♂

 {\displaystyle W_{1}W_{2}Z{\text{♀}}/ZZ{\text{♂}}}

  or

 W

 Z

 1

 Z

 2

 ♀

 /

 Z

 1

 Z

 1

 Z

 2

 Z

 2

 ♂

 {\displaystyle WZ_{1}Z_{2}{\text{♀}}/Z_{1}Z_{1}Z_{2}Z_{2}{\text{♂}}}

 . [ 53 ]


The  Southern platyfish  has a complex sex determination system involving 3 sex chromosomes and 4 autosomal alleles. [ 54 ] [ 55 ]


Gastrotheca pseustes  has

 X

 Y

 b

 ♀

 /

 X

 Y

 a

 ♂

 {\displaystyle XY_{b}{\text{♀}}/XY_{a}{\text{♂}}}

   C-banding  heteromorphism, meaning that both males and females have XY chromosomes, but their Y chromosomes are different on one or more C-bands.  Eleutherodactylus maussi  has a

 X

 1

 X

 1

 X

 2

 X

 2

 ♀

 /

 X

 1

 X

 2

 Y

 ♂

 {\displaystyle \mathrm {X} _{1}\mathrm {X} _{1}\mathrm {X} _{2}\mathrm {X} _{2}{\text{♀}}/\mathrm {X} _{1}\mathrm {X} _{2}\mathrm {Y} {\text{♂}}}

  system. [ 56 ] [ 57 ]


See  [ 58 ]  for a review.


Sexual chromosome pairs can arise from an autosomal pair that, for various reasons, stopped recombination, allowing for their divergence. The rate at which recombination is suppressed, and therefore the rate of sex chromosome divergence, is very different across  clades . [ 43 ]


In analogy with  geological strata , historical events in the evolution of sex chromosomes are called evolutionary strata. The human Y-chromosome has had about 5 strata since the origin of the X and Y chromosomes about 300 Mya from a pair of autosomes. Each stratum was formed when a  pseudoautosomal region  (PAR) of the Y chromosome is  inverted , stopping it from recombination with the X chromosome. Over time, each inverted region decays, possibly due to  Muller's ratchet . [ 59 ] [ 60 ]  Primate Y-chromosome evolution was rapid, with multiple inversions and shifts of the boundary of PAR. [ 61 ]


Among many species of the  salamanders , the two chromosomes are only distinguished by a  pericentric inversion , so that the banding pattern of the X chromosome is the same as that of Y, but with a region near the centromere reversed. (fig 7  [ 62 ] ) In some species, the X is pericentrically inverted and the Y is ancestral. In other species it is the opposite. (p. 15  [ 62 ] )


The gene content of the X chromosome is almost identical among placental mammals. This is hypothesized to be because the X inactivation means any change would cause serious disruption, thus subjecting it to strong purifying selection. Similarly, birds have highly conserved Z chromosomes. [ 50 ]


Neo-sex chromosomes  are currently existing sex chromosomes that formed when an autosome pair fused to the previously existing sex chromosome pair. Following this fusion, the autosomal portion undergoes recombination suppression, allowing them to differentiate. Such systems have been observed in insects, reptiles, birds, and mammals. They are useful to the study of the evolution of Y chromosome degeneration and dosage compensation. [ 63 ] [ 64 ]


The  sex-chromosome turnover  is an evolutionary phenomenon where sex chromosomes disappear, or becomes autosomal, and autosomal chromosomes become sexual, repeatedly over evolutionary time. Some lineages have extensive turnover, but others don't. Generally, in an XY system, if the Y chromosome is degenerate, mostly different from the X chromosome, and has X  dosage compensation , then turnover is unlikely. In particular, this applies to humans. [ 65 ] [ 58 ] [ 66 ]


The ZW and XY systems can evolve into to each other due to  sexual conflict . [ 67 ]


It is an evolutionary puzzle why certain sex chromosomes remain homomorphic over millions of years, especially among lineages of fishes, amphibians, and nonavian reptiles. The fountain-of-youth model states that heteromorphy results from recombination suppression, and recombination suppression results from the male phenotype, not the sex chromosomes themselves. Therefore, if some XY sex-reversed females are fertile and adaptive under some circumstances, then the X and Y chromosomes would recombine in these individuals, preventing Y chromosome decay and maintaining long-term homomorphism. [ 68 ]


Sex reversal  denotes a situation where the phenotypic sex is different from the genotypic sex. While in humans,  sex reversal  (such as the  XX male syndrome ) are often infertile, sex-reversed individuals of some species are fertile under some conditions. For example, some XY-individuals in population of  Chinook salmon  in the  Columbia River  became fertile females, producing YY sons. Since Chinook salmons have homomorphic sex chromosomes, such YY sons are healthy. When YY males mate with XX females, all their progeny would be XY male if grown under normal conditions. [ 69 ]


Support for the hypothesis is found in the  common frog , for which XX males and XY males both suppresses sex chromosome recombination, but XX and XY females both recombine at the same rate. [ 70 ]


Many other sex-determination systems exist. In some species of reptiles, including  alligators , some  turtles , and the  tuatara , sex is determined by the temperature at which the egg is incubated during a temperature-sensitive period. There are no examples of temperature-dependent sex determination (TSD) in birds.  Megapodes  had formerly been thought to exhibit this phenomenon, but were found to actually have different temperature-dependent embryo mortality rates for each sex. [ 71 ]  For some species with TSD, sex determination is achieved by exposure to hotter temperatures resulting in the offspring being one sex and cooler temperatures resulting in the other. This type of TSD is called  Pattern I . For others species using TSD, it is exposure to temperatures on both extremes that results in offspring of one sex, and exposure to moderate temperatures that results in offspring of the opposite sex, called  Pattern II  TSD. The specific temperatures required to produce each sex are known as the female-promoting temperature and the male-promoting temperature. [ 72 ]  When the temperature stays near the threshold during the temperature sensitive period, the  sex ratio  is varied between the two sexes. [ 73 ]  Some species' temperature standards are based on when a particular enzyme is created. These species that rely upon temperature for their sex determination do not have the  SRY gene , but have other genes such as  DAX1 ,  DMRT1 , and  SOX9  that are expressed or not expressed depending on the temperature. [ 72 ]  The sex of some species, such as the  Nile tilapia ,  Australian skink lizard , and  Australian dragon lizard , has an initial bias, set by chromosomes, but can later be changed by the temperature of incubation. [ 16 ]


It is unknown how exactly temperature-dependent sex determination evolved. [ 74 ]  It could have evolved through certain sexes being more suited to certain areas that fit the temperature requirements. For example, a warmer area could be more suitable for nesting, so more females are produced to increase the amount that nest next season. [ 74 ]
In amniotes, environmental sex determination preceded the genetically determined systems of birds and mammals; it is thought that a temperature-dependent  amniote  was the  common ancestor  of amniotes with sex chromosomes. [ 75 ]


There are other  environmental sex determination  systems including location-dependent determination systems as seen in the marine worm  Bonellia viridis  – larvae become males if they make physical contact with a female, and females if they end up on the bare sea floor. This is triggered by the presence of a chemical produced by the females,  bonellin . [ 76 ]  Some species, such as some  snails , practice  sex change : adults start out male, then become female. In tropical  clownfish , the dominant individual in a group becomes female while the other ones are male, and bluehead wrasses ( Thalassoma bifasciatum ) are the reverse.


Clownfish live in colonies of several small undifferentiated fish and two large fish (male and female). The male and female are the only sexually mature fish to reproduce. Clownfish are protandrous hermaphrodites, which means after they mature into males, they eventually can transform into females. They develop undifferentiated until they are needed to fill a certain role in their environment, i.e., if they receive the social and environmental cues to do so.  [ 77 ]


Some species, however, have no sex-determination system. Hermaphrodite species include the common earthworm and certain species of snails. A few species of fish, reptiles, and insects reproduce by  parthenogenesis  and are female altogether. There are some reptiles, such as the  boa constrictor  and  Komodo dragon  that can reproduce both sexually and asexually, depending on whether a mate is available. [ 78 ]


There are exceptional sex-determination systems, neither genetic nor environmental.


The  Wolbachia  genus of parasitic bacteria lives inside the cytoplasm of its host, and is  vertically transmitted  from parents to children. They primarily infect arthropods and nematodes. Different  Wolbachia  can  determine the sex of its host  by a variety of means. [ 79 ]


In some species, there is  paternal genome elimination , where sons lose the entire genome from the father. [ 80 ]


Mitochondrial male sterility : In many flowering plants, the mitochondria can cause hermaphrodite individuals to be unable to father offsprings, effectively turning them into exclusive females. This is a form of  mother’s curse . It is an evolutionarily adaptive strategy for mitochondria as mitochondrial inheritance is exclusively from mother to child. [ 80 ]  The first published case of mitochondrial male sterility among metazoan was reported in 2022 in the hermaphroditic snail  Physa acuta . [ 81 ]


In some flies and crustaceans, all offspring of a particular individual female are either exclusively male or exclusively female ( monogeny ). [ 80 ]


Sex determination systems may have evolved from  mating type , which is a feature of  microorganisms .


Chromosomal sex determination may have evolved early in the history of eukaryotes. [ 82 ]  But in plants it has been suggested to have evolved recently. [ 83 ]


The accepted hypothesis of XY and ZW sex chromosome evolution in amniotes is that they evolved at the same time, in two different branches. [ 84 ] [ 85 ]


No genes are shared between the avian ZW and mammal XY chromosomes [ 26 ]  and the chicken Z chromosome is similar to the human  autosomal  chromosome 9, rather than X or Y. This suggests not that the ZW and XY sex-determination systems share an origin but that the sex chromosomes are derived from autosomal chromosomes of the  common ancestor  of birds and mammals. In the  platypus , a  monotreme , the X 1  chromosome shares homology with  therian  mammals, while the X 5  chromosome contains an avian sex-determination gene, further suggesting an evolutionary link. [ 86 ]


However, there is some evidence to suggest that there could have been transitions between ZW and XY, such as in  Xiphophorus maculatus , which have both ZW and XY systems in the same population, despite the fact that ZW and XY have different gene locations. [ 87 ] [ 88 ]  A recent theoretical model raises the possibility of both transitions between the XY/XX and ZZ/ZW system and environmental sex determination [ 89 ]  The platypus' genes also back up the possible evolutionary link between XY and ZW, because they have the  DMRT1  gene possessed by birds on their X chromosomes. [ 90 ]  Regardless, XY and ZW follow a similar route. All sex chromosomes started out as an original autosome of an original amniote that relied upon temperature to determine the sex of offspring. After the mammals separated, the reptile branch further split into  Lepidosauria  and  Archosauromorpha . These two groups both evolved the ZW system separately, as evidenced by the existence of different sex chromosomal locations. [ 85 ]  In mammals, one of the autosome pair, now Y, mutated its  SOX3  gene into the  SRY  gene, causing that chromosome to designate sex. [ 85 ] [ 90 ] [ 91 ]  After this mutation, the SRY-containing chromosome  inverted  and was no longer completely  homologous  with its partner. The regions of the  X  and  Y chromosomes  that are still homologous to one another are known as the  pseudoautosomal region . [ 92 ]  Once it inverted, the Y chromosome became unable to remedy deleterious mutations, and thus  degenerated . [ 85 ]  There is some concern that the Y chromosome will shrink further and stop functioning in ten million years: but the Y chromosome has been strictly conserved after its initial rapid gene loss. [ 93 ] [ 94 ]


There are some vertebrate species, such as the  medaka  fish, that evolved sex chromosomes separately; their Y chromosome never inverted and can still swap genes with the X. These species' sex chromosomes are relatively primitive and unspecialized. Because the Y does not have male-specific genes and can interact with the X, XY and YY females can be formed as well as XX males. [ 16 ]  Non-inverted Y chromosomes with long histories are found in  pythons  and  emus , each system being more than 120 million years old, suggesting that inversions are not necessarily an eventuality. [ 80 ]  XO sex determination can evolve from XY sex determination with about 2 million years. [ clarification needed ] [ 95 ]


1HRY ,  1HRZ ,  1J46 ,  1J47 ,  2GZK


Sex-determining region Y protein  ( SRY ), or  testis-determining factor  ( TDF ), is a  DNA-binding protein  (also known as gene-regulatory protein/ transcription factor ) encoded by the  SRY   gene  that is responsible for the initiation of male  sex determination  in  therian   mammals  ( placentals  and  marsupials ). [ 5 ]  SRY is an  intronless   sex -determining gene on the  Y chromosome . [ 6 ]   Mutations  in this gene lead to a range of  disorders of sex development  with varying effects on an individual's  phenotype  and  genotype .


SRY is a member of the  SOX (SRY-like box) gene  family of  DNA -binding proteins. When complexed with the  (SF-1) protein , SRY acts as a transcription factor that causes  upregulation  of other transcription factors, most importantly  SOX9 . [ 7 ]  Its  expression  causes the development of primary  sex cords , which later develop into  seminiferous tubules . These cords form in the central part of the yet-undifferentiated  gonad , turning it into a  testis . The now-induced  Leydig cells  of the testis then start secreting  testosterone , while the  Sertoli cells  produce  anti-Müllerian hormone . [ 8 ]  SRY gene effects normally take place 6–8 weeks after fetus formation which inhibits the female anatomical structural growth in males. It also works towards developing the  secondary sexual characteristics  of males.


SRY  may have arisen from a  gene duplication  of the  X chromosome  bound gene  SOX3 , a member of the  SOX family . [ 9 ] [ 10 ]  This duplication occurred after the split between  monotremes  and  therians . Monotremes lack SRY and some of their sex chromosomes share homology with bird sex chromosomes. [ 11 ]   SRY  is a quickly evolving gene, and its regulation has been difficult to study because sex determination is not a highly conserved phenomenon within the animal kingdom. [ 12 ]  Even within  marsupials  and  placentals , which use  SRY  in their sex determination process, the action of  SRY  differs between species. [ 10 ]  The gene sequence also changes; while the core of the gene, the  high-mobility group   (HMG) box , is conserved between species, other regions of the gene are not. [ 10 ]   SRY  is one of only four genes on the human Y chromosome that have been shown to have arisen from the original Y chromosome. [ 13 ]  The other genes on the human Y chromosome arose from an  autosome  that fused with the original Y chromosome. [ 13 ]


SRY  has little in common with sex determination genes of other model organisms, therefore, mice are the main model research organisms that can be utilized for its study. Understanding its regulation is further complicated because even between mammalian species, there is little protein  sequence conservation . The only conserved group in mice and other mammals is the HMG box region that is responsible for DNA binding. Mutations in this region result in  sex reversal , where the opposite sex is produced. [ 14 ]  Because there is little conservation, the  SRY   promoter , regulatory elements and regulation are not well understood. Within related mammalian groups there are homologies within the first 400–600  base pairs  (bp) upstream from the  translational start site . In vitro studies of human  SRY  promoter have shown that a region of at least 310 bp upstream to translational start site are required for  SRY  promoter function. It has been shown that binding of three transcription factors, steroidogenic factor 1 ( SF1 ), specificity protein 1 ( Sp1 transcription factor ) and Wilms tumor protein 1 ( WT1 ), to the human promoter sequence, influence expression of  SRY . [ 14 ]


The promoter region has two Sp1 binding sites, at -150 and -13 that function as regulatory sites. Sp1 is a transcription factor that binds GC-rich consensus sequences, and mutation of the  SRY  binding sites leads to a 90% reduction in gene transcription. Studies of SF1 have resulted in less definite results. Mutations of SF1 can lead to sex reversal, and deletion can lead to incomplete gonad development. However, it is not clear how SF1 interacts with the  SR1  promoter directly. [ 15 ]  The promoter region also has two WT1 binding sites at -78 and -87 bp from the ATG codon. WT1 is transcription factor that has four C-terminal  zinc fingers  and an N-terminal Pro/Glu-rich region and primarily functions as an activator. Mutation of the zinc fingers or inactivation of WT1 results in reduced male gonad size. Deletion of the gene resulted in complete sex reversal. It is not clear how WT1 functions to up-regulate  SRY , but some research suggests that it helps stabilize message processing. [ 15 ]  However, there are complications to this hypothesis, because WT1 also is responsible for expression of an antagonist of male development,  DAX1 , which stands for dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. An additional copy of DAX1 in mice leads to sex reversal. It is not clear how DAX1 functions, and many different pathways have been suggested, including  SRY  transcriptional destabilization and RNA binding. There is evidence from work on suppression of male development that DAX1 can interfere with function of SF1, and in turn transcription of  SRY  by recruiting corepressors. [ 14 ]


There is also evidence that GATA binding protein 4 ( GATA4 ) and FOG2 contribute to activation of  SRY  by associating with its promoter. How these proteins regulate  SRY  transcription is not clear, but FOG2 and GATA4 mutants have significantly lower levels of  SRY  transcription. [ 16 ]  FOGs have zinc finger motifs that can bind DNA, but there is no evidence of FOG2 interaction with  SRY . Studies suggest that FOG2 and GATA4 associate with nucleosome remodeling proteins that could lead to its activation. [ 17 ]


During gestation, the cells of the primordial gonad that lie along the  urogenital ridge  are in a bipotential state, meaning they possess the ability to become either male cells ( Sertoli  and  Leydig  cells) or female cells ( follicle  cells and  theca  cells). SRY initiates testis differentiation by activating male-specific transcription factors that allow these bipotential cells to differentiate and proliferate. SRY accomplishes this by upregulating  SOX9 , a transcription factor with a DNA-binding site very similar to SRY's. SOX9 leads to the upregulation of fibroblast growth factor 9 ( Fgf9 ), which in turn leads to further upregulation of SOX9. Once proper SOX9 levels are reached, the bipotential cells of the gonad begin to differentiate into Sertoli cells. Additionally, cells expressing SRY will continue to proliferate to form the primordial testis. This brief review constitutes the basic series of events, but there are many more factors that influence sex differentiation.


The SRY protein consists of three main regions. The central region encompasses the  high-mobility group  (HMG) domain, which contains  nuclear localization sequences  and acts as the DNA-binding domain. The  C-terminal  domain has no conserved structure, and the  N-terminal  domain can be phosphorylated to enhance DNA-binding. [ 15 ]  The process begins with  nuclear localization  of SRY by  acetylation  of the nuclear localization signal regions, which allows for the binding of  importin β  and  calmodulin  to SRY, facilitating its import into the nucleus. Once in the nucleus, SRY and SF1 ( steroidogenic factor 1 , another transcriptional regulator) complex and bind to TESCO (testis-specific enhancer of Sox9 core), the testes-specific enhancer element of the Sox9 gene in Sertoli cell precursors, located upstream of the Sox9 gene transcription start site. [ 7 ]  Specifically, it is the HMG region of SRY that binds to the minor groove of the DNA target sequence, causing the DNA to bend and unwind. The establishment of this particular DNA "architecture" facilitates the transcription of the Sox9 gene. [ 15 ]  In the nucleus of Sertoli cells, SOX9 directly targets the  Amh  gene as well as the  prostaglandin D synthase  ( Ptgds)  gene. SOX9 binding to the enhancer near the  Amh  promoter allows for the synthesis of  Amh  while SOX9 binding to the  Ptgds  gene allows for the production of  prostaglandin D2  (PGD 2 ). The reentry of SOX9 into the nucleus is facilitated by autocrine or paracrine signaling conducted by PGD 2 . [ 18 ]  SOX9 protein then initiates a  positive feedback  loop, involving SOX9 acting as its own transcription factor and resulting in the synthesis of large amounts of SOX9. [ 15 ]


The SF-1 protein, on its own, leads to minimal transcription of the SOX9 gene in both the XX and XY bipotential gonadal cells along the urogenital ridge. However, binding of the SRY-SF1 complex to the testis-specific enhancer (TESCO) on SOX9 leads to significant up-regulation of the gene in only the XY gonad, while transcription in the XX gonad remains negligible. Part of this up-regulation is accomplished by SOX9 itself through a positive feedback loop; like SRY, SOX9 complexes with SF1 and binds to the TESCO enhancer, leading to further expression of SOX9 in the XY gonad. Two other proteins,  FGF9  (fibroblast growth factor 9) and PDG2 (prostaglandin D2), also maintain this up-regulation. Although their exact pathways are not fully understood, they have been proven to be essential for the continued expression of SOX9 at the levels necessary for testes development. [ 7 ]


SOX9 and SRY are believed to be responsible for the cell-autonomous differentiation of supporting cell precursors in the gonads into Sertoli cells, the beginning of testes development. These initial Sertoli cells, in the center of the gonad, are hypothesized to be the starting point for a wave of FGF9 that spreads throughout the developing XY gonad, leading to further differentiation of Sertoli cells via the up-regulation of SOX9. [ 19 ]  SOX9 and SRY are also believed to be responsible for many of the later processes of testis development (such as Leydig cell differentiation, sex cord formation, and formation of testis-specific vasculature), although exact mechanisms remain unclear. [ 20 ]  It has been shown, however, that SOX9, in the presence of PDG2, acts directly on Amh (encoding anti-Müllerian hormone) and is capable of inducing testis formation in XX mice gonads, indicating it is vital to testes development. [ 19 ]


Embryos are gonadally identical, regardless of genetic sex, until a certain point in development when the testis-determining factor causes male sex organs to develop. A typical male  karyotype  is XY, whereas a female's is XX. There are exceptions, however, in which SRY plays a major role. Individuals with  Klinefelter syndrome  inherit a normal Y chromosome and multiple X chromosomes, giving them a karyotype of XXY. Atypical  genetic recombination  during  crossover , when a sperm cell is developing, can result in karyotypes that are not typical for their phenotypic expression.


Most of the time, when a developing sperm cell undergoes crossover during meiosis, the SRY gene stays on the Y chromosome. If the SRY gene is transferred to the X chromosome instead of staying on the Y chromosome, testis development will no longer occur. This is known as  Swyer syndrome , characterized by an XY karyotype and a female phenotype. Individuals who have this syndrome have normally formed uteri and fallopian tubes, but the gonads are not functional. Swyer syndrome individuals are usually considered as females. [ 21 ]  On the other spectrum,  XX male syndrome  occurs when a body has 46:XX Karyotype and SRY attaches to one of them through translocation. People with XX male syndrome have a XX Karyotype but are male. [ 22 ]  Individuals with either of these syndromes can experience delayed puberty, infertility, and growth features of the opposite sex they identify with. XX male syndrome expressers may develop breasts, and those with Swyer syndrome may have facial hair. [ 21 ] [ 23 ]


While the presence or absence of SRY has generally determined whether or not testis development occurs, it has been suggested that there are other factors that affect the functionality of SRY. [ 24 ]  Therefore, there are individuals who have the SRY gene, but still develop as females, either because the gene itself is defective or mutated, or because one of the contributing factors is defective. [ 25 ]  This can happen in individuals exhibiting a XY, XXY, or XX SRY-positive karyotype.


Additionally, other sex determining systems that rely on SRY beyond XY are the processes that come after SRY is present or absent in the development of an embryo. In a normal system, if SRY is present for XY, SRY will activate the medulla to develop gonads into testes. Testosterone will then be produced and initiate the development of other male sexual characteristics. Comparably, if SRY is not present for XX, there will be a lack of the SRY based on no Y chromosome. The lack of SRY will allow the cortex of embryonic gonads to develop into ovaries, which will then produce estrogen, and lead to the development of other female sexual characteristics. [ 26 ]


SRY has been shown to  interact  with the  androgen receptor  and individuals with XY karyotype and a functional SRY gene can have an outwardly female phenotype due to an underlying  androgen insensitivity syndrome  (AIS). [ 27 ]  Individuals with AIS are unable to respond to androgens properly due to a defect in their androgen receptor gene, and affected individuals can have complete or partial AIS. [ 28 ]  SRY has also been linked to the fact that males are more likely than females to develop  dopamine -related diseases such as  schizophrenia  and  Parkinson's disease . SRY encodes a protein that controls the concentration of dopamine, the neurotransmitter that carries signals from the brain that control movement and coordination. [ 29 ]  Research in mice has shown that a mutation in SOX10, an SRY encoded transcription factor, is linked to the condition of Dominant megacolon in mice. [ 30 ]  This mouse model is being used to investigate the link between SRY and  Hirschsprung disease , or congenital megacolon in humans. [ 30 ]  There is also a link between SRY encoded transcription factor SOX9 and  campomelic dysplasia  (CD). [ 31 ]  This missense mutation causes defective  chondrogenesis , or the process of cartilage formation, and manifests as skeletal CD. [ 32 ]  Two thirds of 46,XY individuals diagnosed with CD have fluctuating amounts of male-to-female sex reversal. [ 31 ]


One of the most controversial uses of this discovery was as a means for sex verification at the  Olympic Games , under a system implemented by the  International Olympic Committee  in 1992. Athletes with an SRY gene were not permitted to participate as females, although all athletes in whom this was "detected" at the  1996 Summer Olympics  were ruled  false positives  and were not disqualified. Specifically, eight female participants (out of a total of 3387) at these games were found to have the SRY gene. However, after further investigation of their genetic conditions, all these athletes were verified as female and allowed to compete. These athletes were found to have either partial or full  androgen insensitivity , despite having an SRY gene, making them externally phenotypically female. [ 33 ]  In the late 1990s, a number of relevant professional societies in United States called for elimination of gender verification, including the  American Medical Association , stating that the method used was uncertain and ineffective. [ 34 ]  Chromosomal screening was eliminated as of the  2000 Summer Olympics , [ 34 ] [ 35 ] [ 36 ]  but this was later followed by other forms of testing based on hormone levels. [ 37 ]


Despite the progress made during the past several decades in the study of sex determination, the SRY gene, and its protein, work is still being conducted to further understanding in these areas. There remain factors that need to be identified in the sex-determining molecular network, and the chromosomal changes involved in many other human sex-reversal cases are still unknown. Scientists continue to search for additional sex-determining genes, using techniques such as  microarray  screening of the genital ridge genes at varying developmental stages, mutagenesis screens in mice for sex-reversal phenotypes, and identifying the genes that transcription factors act on using  chromatin immunoprecipitation . [ 15 ]


One of the knockout models for the SRY gene was done in pigs. Through the use of CRISPR technology the SRY gene was knocked out in male pigs. The target for the CRISPR technology is the high mobility group located on the SRY gene. The research showed that with the absence of SRY, both the internal and external genitalia were reversed. When the piglets were born they were phenotypically male but expressed female genitalia. [ 38 ]  Another study done on mice used TALEN technology to produce an SRY knockout model. These mice expressed external and internal genitalia as well as a normal female level of circulating testosterone. [ 39 ]  These mice, despite having XY chromosomes, expressed a normal estrus cycle albeit with reduced fertility. Both of these studies highlighted the role that SRY plays in the development of the testes and other male reproductive organs.


Sexual differentiation in humans  is the process of development of  sex differences in humans . It is defined as the development of  phenotypic  structures consequent to the action of hormones produced following gonadal determination. [ 1 ]   Sexual differentiation  includes development of different genitalia and the internal genital tracts and body hair plays a role in sex identification. [ 2 ]


The development of sexual differences begins with the  XY sex-determination system  that is present in humans, and complex mechanisms are responsible for the development of the phenotypic differences between  male  and  female   humans  from an undifferentiated  zygote . [ 3 ]  Females typically have two  X chromosomes , and males typically have a  Y chromosome  and an X chromosome. At an early stage in  embryonic  development, both sexes possess equivalent internal structures. These are the  mesonephric ducts  and  paramesonephric ducts . The presence of the  SRY gene  on the Y chromosome causes the development of the testes in males, and the subsequent release of hormones which cause the  paramesonephric ducts  to regress. In females, the mesonephric ducts regress.


Disorders of sexual development (DSD), encompassing conditions characterized by  the appearance of undeveloped genitals that may be ambiguous, or look like those typical for the opposite sex, sometimes known as  intersex , can be a result of genetic and hormonal factors. [ 4 ]


Most  mammals , including humans, have an  XY sex-determination system : the  Y chromosome  carries factors responsible for triggering male development. In the absence of a Y chromosome, the fetus will undergo female development. This is because of the presence of the sex-determining region of the Y chromosome, also known as the  SRY  gene. [ 5 ]  Thus, male mammals typically have an X and a Y chromosome (XY), while female mammals typically have two  X chromosomes  (XX).


Chromosomal sex is determined at the time of  fertilization ; a  chromosome  from the  sperm  cell, either X or Y, fuses with the X chromosome in the egg cell.  Gonadal sex  refers to the gonads, that is the testicles or ovaries, depending on which genes are expressed.  Phenotypic sex  refers to the structures of the external and internal genitalia. [ 6 ]


Six weeks elapse after fertilization before the first signs of sex differentiation can be observed in human embryos. [ 5 ]  The embryo and subsequent early fetus appear to be sexually indifferent, looking neither like a male or a female. Over the next several weeks, hormones are produced that cause undifferentiated tissue to transform into either male or female reproductive organs. This process is called sexual differentiation. The precursor of the internal female sex organs is called the  Müllerian system .


Differentiation between the sexes of the sex organs occurs throughout embryological, fetal and later life. In both males and females, the sex organs consist of two structures: the internal genitalia and the external genitalia. In males, the gonads are the  testicles  and in females, they are the  ovaries . These are the organs that produce  gametes  (egg and sperm), the reproductive cells that will eventually meet to form the fertilized egg ( zygote ).


As the zygote divides, it first becomes the  embryo  (which means 'growing within'), typically between zero and eight weeks, then from the eighth week until birth, it is considered the fetus (which means 'unborn offspring'). The internal genitalia are all the accessory glands and ducts that connect the gonads to the outside environment. The external genitalia consist of all the external reproductive structures. The sex of an early embryo cannot be determined because the reproductive structures do not differentiate until the seventh week. Prior to this, the child is considered bipotential because it cannot be identified as male or female.


The internal genitalia consist of two accessory ducts:  mesonephric ducts  (male) and  paramesonephric ducts  (female). The mesonephric system is the precursor to the male genitalia and the paramesonephric to the female reproductive system. [ 8 ]  As development proceeds, one of the pairs of ducts develops while the other regresses. This depends on the presence or absence of the sex determining region of the Y chromosome, also known as the SRY  gene . [ 5 ]  In the presence of a functional SRY gene, the bipotential gonads develop into testes.  Gonads  are histologically distinguishable by 6–8 weeks of gestation.


Subsequent development of one set and degeneration of the other depends on the presence or absence of two testicular hormones:  testosterone  and  anti-Müllerian hormone (AMH) . Disruption of typical development may result in the development of both, or neither, duct system, which may produce morphologically  intersex  individuals.


Males:  The SRY gene when transcribed and processed produces SRY protein that binds to DNA and directs the development of the gonad into testes. Male development can only occur when the fetal testis secretes key hormones at a critical period in early gestation. The testes begin to secrete three hormones that influence the male internal and external genitalia: they secrete anti-Müllerian hormone (AMH),  testosterone , and  dihydrotestosterone  (DHT). Anti-Müllerian hormone causes the paramesonephric ducts to regress. Testosterone converts the mesonephric ducts into male accessory structures, including the  epididymides ,  vasa deferentia , and  seminal vesicles . Testosterone will also control the descending of the testes from the abdomen. [ 1 ]  Many other genes found on other  autosomes , including  WT1 ,  SOX9  and  SF1  also play a role in gonadal development. [ 9 ]


Females:  Without testosterone and AMH, the mesonephric ducts degenerate and disappear. The paramesonephric ducts develop into the  uterus ,  fallopian tubes , and upper  vagina  (the lower vagina develops from the urogenital sinus). [ 9 ]  There still remains a broad lack of information about the genetic controls of female development, and much remains unknown about the female embryonic process. [ 10 ]


By 7 weeks, a fetus has a  genital tubercle ,  urogenital sinus ,  urogenital folds  and  labioscrotal swellings . In females, without excess androgens, these become the  vulva  ( clitoris ,  vestibule ,  labia minora  and  labia majora  respectively). Males become externally distinct between 8 and 12 weeks, as androgens enlarge the genital tubercle and cause the urogenital groove and sinus to fuse in the midline, producing an unambiguous  penis  with a phallic urethra, and the labioscrotal swellings become a thinned, rugate  scrotum  where the testicles are situated. Dihydrotestosterone will differentiate the remaining male characteristics of the external genitalia. [ 1 ]


A sufficient amount of any androgen can cause external  masculinization . The most potent is  dihydrotestosterone  (DHT), generated from testosterone in skin and genital tissue by the action of 5α-reductase. A male fetus may be incompletely masculinized if this enzyme is  deficient . In some  diseases  and circumstances, other androgens may be present in high enough concentrations to cause partial or (rarely) complete masculinization of the external genitalia of a genetically female fetus. The testes begin to secrete three hormones that influence the male internal and external genitalia. They secrete anti-Müllerian hormone, testosterone, and Dihydrotestosterone. Anti-Müllerian hormone (AMH) causes the paramesonephric ducts to regress. Testosterone, which is secreted and converts the mesonephric ducts into male accessory structures, such as epididymis, vas deferens and seminal vesicle. Testosterone will also control the descending of the testes from the abdomen into the scrotum. Dihydrotestosterone, also known as (DHT) will differentiate the remaining male characteristics of the external genitalia. [ 11 ]


Further sex differentiation of the external genitalia occurs at  puberty , when androgen levels again become disparate. Male levels of testosterone directly induce growth of the penis, and indirectly (via DHT) the  prostate .


Alfred Jost  observed that while testosterone was required for mesonephric duct development, the regression of the  paramesonephric duct  was due to another substance. This was later determined to be paramesonephric inhibiting substance (MIS), a 140 kD dimeric glycoprotein that is produced by  Sertoli cells . MIS blocks the development of  paramesonephric ducts , promoting their regression. [ 12 ]


Visible differentiation occurs at  puberty , when  estradiol  and other hormones cause  breasts  to develop in typical females.


Human adults and children show many psychological and behavioral sex differences. Some (e.g. dress) are learned and cultural. Others are demonstrable across cultures and have both biological and learned determinants. For example, some studies claim girls are, on average, more verbally fluent than boys, but boys are, on average, better at spatial calculation. [ 13 ] [ 14 ]  Some have observed that this may be due to two different patterns in parental communication with infants, noting that parents are more likely to talk to girls and more likely to engage in physical play with boys. [ 10 ]


The following are some of the variations associated with atypical determination and differentiation process: [ 15 ]


Silent   Information   Regulator  ( SIR ) proteins are involved in regulating gene expression.  SIR proteins organize  heterochromatin  near  telomeres , [ 1 ]   ribosomal DNA (rDNA) , [ 2 ]  and at silent loci including  hidden mating type loci  in yeast. [ 3 ] [ 4 ]  The SIR family of genes encodes catalytic and non-catalytic proteins that are involved in de-acetylation of  histone  tails and the subsequent condensation of chromatin around a SIR protein scaffold. [ 5 ]  Some SIR family members are conserved from yeast to humans.


SIR proteins have been identified in many  screens , and have historically been known as SIR [ 3 ]   ( s ilent  i nformation  r egulator), MAR [ 6 ]   ( ma ting-type  r egulator), STE [ 7 ]   ( ste rile), CMT [ 8 ]   ( c hange of  m ating  t ype) or SSP [ 9 ]   ( s terile  s u p pressor) according to which screen led to their identification. Ultimately, the name SIR had the most staying power, because it most accurately describes the function of the encoded proteins. [ citation needed ]


One of the early yeast screens to identify SIR genes was performed by  Anita Hopper  and Benjamin Hall, who screened with mutagenesis for alleles that allow sporulation in a normally sporulation-deficient heterothallic α/α ( ho/ho MATα/MATα ). Their screen identified a mutation in a novel gene that was not linked to  HO  that allowed the α/α diploid to sporulate, as if it were an α/a diploid, and inferred that the mutation affected a change in mating type by an  HO -independent mechanism. [ 8 ]  Later, it was discovered at the CMT allele identified by Hopper & Hall did not cause a mating type conversion at the MAT locus, but rather allowed the expression of cryptic mating type genes that are silenced in wild-type yeast. [ 4 ]  In their paper clarifying the mechanism of the CMT mutation, Haber and  acknowledge the contribution of  Amar Klar , who presented his MAR mutant strains that had similar properties as the CMT mutants at the  Cold Spring Harbor Laboratory  yeast genetics meeting, which led Haber and  to consider the hypothesis that the  cmt  mutants may act by de-repressing silent information. [ 10 ]


In the same year that Haber &  demonstrated that the  cmt  mutant restores sporulation by de-repressing hidden mating type loci, two other groups published screens for genes involved in the regulation of silent mating type cassettes. [ 6 ]  The first study, performed by Amar Klar, Seymour Fogel and Kathy Macleod, identified a mutation in a spontaneous a/a diploid that caused the products of sporulation to be haploids with an apparent diploid phenotype, as assayed by ability to mate. [ 6 ]  The authors reasoned that the mutation caused the de-repression of then-recently appreciated silent mating type loci HMa and HMα, which would allow an a/a diploid to sporulate and would cause haploid segregants inheriting the mutant allele to behave as a/α diploids despite being haploid. [ 6 ]  The authors named the mutation MAR for its apparent role in mating type regulation, and were able to map the mutation to chromosome IV, and determined that it was located 27.3 cM from a commonly used  trp1  marker. [ 6 ]


A few months later,  Jasper Rine  and  Ira Herskowitz  published a different screen for genes that affect the ability of yeast to  mate , and ultimate discovered the gene family that they called SIR, a name that remains in the modern parlance. [ 3 ]  Unlike the Klar et al. screen that identified a mutant by its inability to mate, Rine & Herskowitz took a more directed approach towards discovering factors responsible for mating type silencing. Specifically, Rine & Herskowitz reasoned that a haploid yeast cell with a recessive mutation in matα1 could be complemented if the silent copy of MATα were de-repressed. Starting in a  ho matα1  haploid strain, Rine & Herskowitz screened mutants arising from mutagenesis and identified five mutants that restored a MATα phenotype in matα cells, but were not linked to the MAT locus and did not cause a gene conversion between the HMα locus and matα. [ 3 ]   These mutants, they reasoned, were specifically defective in silencing the cryptic mating type genes.


Eventually, all of the mutants resulting from the original Hopper & Hall screen as well as the later Rine & Herskowitz screen and the Klar et al. screen were characterized and mapped, and it was shown that the causative genes were the same. [ 11 ]  In fact, the genes that are now referred to as SIR1-4 have at one time been referred to as MAR, CMT or STE according to the screen that identified the mutants.


Although Klar, Hartwell and Hopper identified mutations in SIR genes and applied other names to the genes before Rine performed his screen, the SIR name was eventually adopted because Rine eventually identified the most complete set of functionally related genes (SIR1-4), and because the work by Rine and Herskowitz most accurately described the function of the SIR family genes. [ 11 ]  Later it would be shown that in yeast and in higher organisms, SIR proteins are important for transcriptional regulation of many chromatin domains.


In budding yeast, SIR proteins are found at the silent mating type loci, telomeres, and at the rDNA locus. At the silent mating type loci and at the telomeres, SIR proteins participate in transcriptional silencing of genes within their domain of localization. At the rDNA locus, SIR proteins are thought to primarily be important for repressing recombination between rDNA repeats rather than for suppressing transcription. [ 12 ]


In transcriptional silencing, SIR2,3,4 are required in stoichiometric amounts to silence specific chromosomal regions. In yeast, SIR proteins bind sites on  nucleosome  tails and form a multimeric compound of SIR2,3,4 that condenses chromatin and is thought to physically occlude promoters in the silenced interval, preventing their interaction with transcription machinery. [ 12 ]  The establishment of SIR-repressed heterochromatin domains is a complicated process that involves different subsets of proteins and regulatory proteins depending on the locus in the genome. [ 12 ]  At the silent mating type loci and at yeast telomeres, the transcription factors Abf1 ( A RS  b inding  f actor) and Rap1 ( r epressor- a ctivator  p rotein) associate with specific nucleotide sequences in the silencers that flank heterochromatic regions. [ 13 ]  Rap1 contains a Sir3-binding domain that recruits SIR3 to the silencers. [ 14 ]  Once at the silencers, Sir3 recruits Sir4-Sir2 dimers to the chromatin nucleation site. Sir2 then deacetylates histone H3 and H4 tails, and free Sir3 binds the now-deacetylated lysine residues H4K16,79, and recruits additional Sir4-Sir2 dimers to promote the further spreading of the heterochromatin domain. [ 12 ]


Once it has spread to cover a genomic locus, the SIR2,3,4 effectively prevents transcription from the region it occupies, in a process that is thought to depend on the physical occlusion of DNA by SIR proteins. Recently, it has been shown that certain promoters are capable of directing transcription inside regions that are otherwise silenced by SIR proteins. [ 15 ]  Specifically, if an inducible promoter is induced inside a silent chromatin domain, it can achieve ~200x increase in expression levels with little detectable change in covalent  histone modifications . [ 15 ]


SIR2 is an NAD-dependent lysine deacetylase. [ 12 ]  It was the first-discovered member of the Sirtuin protein family and it is highly conserved, with homologs found in organisms ranging from humans to bacteria [ 16 ]  and archaea. [ 12 ]  It interacts with a variety of protein substrates, but does not exhibit strong affinity for DNA, chromatin, or other silencer-binding factors. [ 12 ]  Instead, it relies on other SIR proteins to find its appropriate silencing target. [ 12 ]


In the SIR protein complex, SIR2 removes acetyl groups from the lysine on histone tails H3 and H4, [ 17 ]  'priming' the nucleosome for chromatin packaging by the SIR3 component of the complex. [ 18 ]


Beyond its canonical role in the SIR complex, SIR2 also plays a role in rDNA repression. [ 19 ]  As part of the cell's regulation mechanism, rDNA repeats are excised from the chromosome so they cannot be expressed. SIR2 forms a complex with NET1 (a nuclear protein) and CDC14 (a phosphatase) to form the  re gulator of  n ucleolar silencing and  t elophase (RENT) complex. [ 19 ]  The RENT complex sequesters excised rDNA in 'extrachromosomal circles,' preventing recombination. Accumulation of these circles has been linked to premature aging. [ 12 ]   Sirtuin 2 (SIRT2) , SIR2's human analog, has also been linked to age-related disease. [ 16 ]


SIR3 is principally involved in heterochromatin spreading, the silencing activity of the SIR protein complex. [ 12 ]  When overexpressed, SIR3 leads to spreading beyond the normal nucleation site. [ 12 ]  SIR3 can continue to operate at very low levels of SIR2 and SIR4, but not without them. [ 17 ] [ 18 ]  It preferentially binds to unmodified nucleosomes (no acetylation at H4K16 or methylation at H3K79), and relies on SIR2's deacetylation of H4K16 to enhance silencing. [ 18 ]  H3K79 methylation by DOT1 methyltransferase inhibits SIR3, resulting in an unsilenced chromatin region. [ 17 ] [ 18 ]  SIR3 is recruited to target sequence by the transcription factors RAP1 or ABF1. [ 12 ] [ 17 ]


SIR4 is involved in scaffolding the assembly of silenced chromatin. [ 12 ] [ 19 ]  It binds to DNA with high affinity, but low specificity. [ 19 ]  It is most stable when co-expressed with SIR2, but neither SIR2 nor SIR3 are required for it to operate at the telomeres. [ 12 ]  Each half of the SIR4 protein has distinct responsibilities in heterochromatin spreading. SIR4's  N-terminus  is required for telomeric silencing, but not for homothallic mating-type (HM) silencing. [ 12 ]  Conversely, its  C-terminus  supports HM but not telomeric repression. [ 12 ]  The N-terminus is positively charged and can be recruited to the telomeric repression site by SIR1 and YKU80. [ 12 ]  The C-terminus contains the coiled-coil region, which interacts with SIR3 in the heterotrimeric SIR complex and can also interact with RAP1 and YKU70 for recruitment to the telomeric region of the chromosome. [ 17 ]  The C-terminus also contains the SIR2-interacting domain (SID), where SIR4 can bind to the extended N-terminus of SIR2. [ 12 ]  SIR2 can catalyze reactions without being bound to SIR4, but SIR2's catalytic activity is enhanced when interacting with SIR4. [ 12 ]


SIR proteins are conserved from yeast to humans, and lend their name to a class of mammalian  histone deacetylases  ( Sirtuins , homologs of Sir2). Sirtuins have been implicated in myriad human traits including Alzheimer's and diabetes, and have been proposed to regulate of lifespan. [ 16 ]


Skewed X-chromosome inactivation  ( skewed X-inactivation ) occurs when the  X-inactivation  of one  X chromosome  is favored over the other, leading to an uneven number of cells with each  chromosome  inactivated. It is usually defined as one  allele  being found on the active X chromosome in over 75% of cells, and extreme skewing is when over 90% of cells have inactivated the same X chromosome. [ 1 ] [ 2 ]  It can be caused by primary nonrandom inactivation, either by chance due to a small cell pool or directed by genes, or by secondary nonrandom inactivation, which occurs by  selection . [ citation needed ]


X-chromosome inactivation occurs in females to provide  dosage compensation  between the sexes. If females kept both X chromosomes active, they would have twice the number of active X  genes  than males, who only have one copy of the X chromosome. At approximately the time of embryonic  implantation , one of the two X chromosomes in each cell of the female embryo is randomly selected for inactivation. Cells then undergo  transcriptional  and  epigenetic  changes to ensure this inactivation is permanent (such as  methylation  and being modified into  Barr bodies ). All progeny from these initial cells will maintain the inactivation of the same chromosome, resulting in a phenotypic mosaic pattern of cells in females [ 1 ]  although not a  genotypic mosaic .


Most females will have some levels of skewing. It is relatively common in adult females; around 35% of women have a skewed ratio over 70:30, and 7% of women have an extreme skewed ratio of over 90:10. [ 3 ]  This is of medical significance, due to the potential for the expression of disease genes present on the X chromosome that are normally not expressed due to random X-inactivation.


Nonrandom X-inactivation leads to skewed X-inactivation. Nonrandom X-inactivation can be caused by chance or directed by genes. If the initial pool of cells in which X-inactivation occurs is small, chance can cause skewing to occur in some individuals by causing a bigger proportion of the initial cell pool to inactivate one X chromosome. A reduction in the size of this initial cell pool would increase the likelihood of skewing occurring. [ 1 ] [ 4 ]  This skewing can then be inherited by progeny cells, or increased by secondary selection.


The X-chromosome controlling element (Xce) gene in mice has been found to influence genetically mediated skewing. It is unknown whether a similar gene plays a role in human X-inactivation, although a 2008 study found that skewing in humans is mostly caused by secondary events rather than a genetic tendency. [ 1 ]


There is a much higher concordance rate in genetically identical (monozygotic) twins compared to non-identical (dizygotic) twins, which suggests a strong genetic input. A 10% difference in the skewing of genetically identical twins did exist however, so there are other contributing factors outside of genetics alone. It is difficult to identify primary nonrandom inactivation in humans, as early cell selection occurs in the embryo. Mutation and imprinting of the  XIST gene , a part of the X-inactivation centre, can result in skewing. This is rare in humans. [ citation needed ]


Skewed X-inactivation in mice is controlled by the Xce gene on the X chromosome. Xce acts in  cis , which means that it acts upon the chromosome from which it was transcribed. [ 4 ]  There are four alleles of Xce, labeled  a ,  b ,  c , and  d . Each allele has a different likelihood of inactivation, with  a  <  b  <  c  <  d , where  d  is the most likely to remain active and  a  is the least likely. The strength differences between the four alleles are likely due to variations in the number of binding sites for a crucial actor in inactivation. The specific transfactor is not known currently. [ citation needed ]


Homozygotic mouse cells will have roughly even levels of inactivation due to both alleles having equal chance of being inactivated. For example, a mouse with the genotype  dd  will have an inactivation ratio very close to 50:50. Heterozygotes, will experience greater levels of skewing due to the differing inactivation likelihood of the two alleles. A mouse cell with the Xce genotype  ad  will have a greater number of the  a -carrying than  d -carrying X chromosomes inactivated, because the  d -carrying X chromosome is less likely to be inactivated. [ 5 ]


There are two theories on the mechanism Xce uses to affect inactivation. The first is that genomic differences in the Xce alleles alter the sequence of the long non-coding RNA that is an integral part of X chromosome inactivation. The second is that Xce acts as a binding site for dosage factors that will affect  XIST gene  and  Tsix  expression (long non-coding RNAs involved in X chromosome inactivation). [ 5 ]


Skewing can also be influenced by the parent-of-origin effect, in which skewing becomes biased towards either the maternal or paternal X chromosome. [ 5 ]  Studies have suggested an X-linked gene or genes that control this effect, but the exact gene has not yet been identified. [ 6 ]


A 2010 study found a small but significant under-expression of the paternal X chromosome in mice. Extra-embryonic tissues are found to preferentially inactivate the paternal X chromosome. [ 2 ]   Marsupials  will always inactivate the paternal X chromosome, in a process named  imprinting . [ 7 ]  Researchers hypothesized a link between the slight preference for inactivation of the paternal X in mice tissue, and the preference in extra-embryonic tissue and Marsupials. There may be a conserved epigenetic mark that drives this preference. [ 2 ]


Skewed inactivation patterns can also emerge due to mutations that change the quantity of guanine on the Xist promoter. The Xist gene is responsible for inactivating the X chromosome from which it is transcribed. X-chromosome inactivation in general is influenced by the number of guanine-containing nucleotides on the Xist promoter, although generally inactivation still follows a random pattern. A rare mutation can occur, however, in which a cytosine residue is converted to guanine on the Xist promoter. It has been hypothesized that the mutation causes a change in the Xist transcript or in the levels of transcript produced, which causes the cell to differentiate between the two X chromosomes and causes the chromosome with the mutation to become preferentially inactivated. The mechanism has not been fully elucidated at this time, although research does point towards decreased promoter activity as a result of the mutation being a major part of the process. [ 8 ]


Secondary skewing occurs when an X-linked mutation affects cell proliferation or survival. If a mutation on one X chromosome negatively affects a cell's ability to proliferate or survive, there will end up being a larger proportion of cells with the other X chromosome active. This selection of one X chromosome can vary between tissue types, as it depends on the specific gene and its activity in the tissue, with rapidly dividing cells giving selection processes more time to work.  Blood cells , for example, tend to have the highest rates of skewing due to the extremely high dividing and replacement rate within the human body. [ 9 ]  The strength of selection can also vary depending on the gene under selection, and so skewing can occur at different rates and to different extents. [ 1 ] [ 4 ]


Secondary selection tends to cause an increase in skewing with age. This is primarily due to a longer span over which selective pressure has room in which to act. [ 9 ]  Skewing is still seen in young children, but with a lower frequency and at less extreme levels in most cases. [ 2 ]


Skewed X-inactivation has medical significance due to its impacts on X-linked diseases. X-chromosome skewing has an ability to amplify diseases on the X chromosome. In wildtype women, recessive diseases on the X chromosome are often unexpressed due to the roughly even inactivation process, which prevents mutated alleles from becoming heavily expressed. However, skewed inactivation can lead to a more severe expression of the disease.


The diseased X-linked allele can also cause strong selection in a heterozygote for the cells with the diseased allele on the inactive chromosome. Therefore, strong skewing in female members of a family can suggest they are carriers of an X-linked disease.


Skewed X-inactivation has also been found to correlate with a higher rate of  ovarian cancer , although the mechanism behind this is unknown. A 2013 study also found skewed X-inactivation to be a factor that predisposes individuals to  esophageal carcinomas . [ 10 ]  It has been postulated that skewed X-inactivation might lead to a decrease in the expression of X-linked  tumor suppressor genes  in an individual who also has a  germline mutation  in the expressed chromosome. This would cause the gene on that chromosome to become under-expressed, making it more difficult for cells to regulate themselves properly. Other researchers have contended that such a mutation would lead to higher rates of cancer among wild type females, as approximately half the cells would not express the gene due to random inactivation. One would also see a higher rate of cancer in males with the mutation. Instead, the researchers proposed that the cause of cancer and skewed inactivation could potentially be separate events, or both be caused by an unknown source. [ 4 ]


Rett syndrome  is a genetic disorder caused by a mutation of the  MECP2  gene on the X chromosome. The disease occurs mostly in females and involves repetitive hand movements, frequent seizures and a loss of vocal skills and sometimes motor skills. Females with one copy of the mutated allele show symptoms of severe mental retardation. Asymptomatic carriers and patients with very mild symptoms have been described, who can show skewed X-inactivation that favors the inactivation of the mutated allele. Asymptomatic carriers can pass on the mutated allele to their daughters, who can show full symptoms if skewing does not occur. Most Rett syndrome cases show no skewing. [ 11 ]


Skewed X-inactivation has been correlated with several  autoimmune diseases , including  autoimmune thyroid disease  (ATD) and  scleroderma . Autoimmune thyroid disease is a disease involving the  thyroid gland . The immune system of those who have the condition recognize the thyroid as foreign and attack it, causing it to  atrophy . Women have a predisposition towards the condition, and research indicates that this might in part be due to skewed X-inactivation. It was discovered that when twins with the disease were examined, the prevalence of skewing was above 30% as compared to 11% in the control group of wild type women, indicating that X-chromosome skewing could possibly be involved in the cause of the condition. [ 12 ] [ 13 ]  Similar results have also been witnessed in scleroderma, which involves hardening of the skin and inner organs. Skewing levels were found in 64% of informative patients, as compared to only 8% of the control group, also indicating a strong correlation and possible cause. The mechanism behind both conditions is unclear at this time. [ 14 ]


Higher levels of skewed X chromosome inactivation have been correlated with cases of  autism  in women. 33% of autistic women in a study had extreme levels of skewing, with only 11% of the wildtype control having extreme levels of skewing. The study also revealed that the mothers of the autistic daughters with skewing also had significant levels of skewing, indicating a higher level of heritability as compared to the wild type population. The reason behind this is currently unknown, as no mutations in the  Xist  promoter were detected. [ 15 ]


Klinefelter 47,XXY  and  48,XXYY  patients were found to have significantly skewed X-chromosome levels in 31% of the patients examined, with researchers predicting that this skewing might be responsible for the mental deficiencies and abnormalities present. Different forms of the disease also showed preferential activation towards either the maternal or paternal X chromosome. This might indicate that parent-of-origin effects such as  imprinting  might be involved with the X-chromosome skewing. [ 16 ]


X-linked  glycogen storage disease  (GSD IXa) is a  metabolic disorder  typically only seen in males because of the X-linked inheritance pattern. Since women are  mosaic  models when it comes to gene expression, they tend to mask X-linked mutations by using the other X to compensate. Skewed X-inactivation resulting in the expression of the defective X chromosome can cause X-linked mutations to be expressed in women. [ 17 ]


The problem occurring in IXa is a defect in  phosphorylase b kinase  (PHK). PHK activates  glycogen phosphorylase , which is a key enzyme to mobilize glucose from stored glycogen, through  phosphorylation .  Glycogen  is the polymer storage unit of glucose in the body. When the body requires energy it can use enzymes such as PHK to break down the glycogen into glucose for the body to use. Some symptoms of the disease are altered blood glucose levels,  ketoacidosis , growth retardation, or liver distention. [ 17 ]


Skewed X-chromosome inactivation has been implicated in  miscarriages  in the past. Recurrent pregnancy loss can be defined as either two or three consecutive lost pregnancies within five months. In most cases, the loss of pregnancy can be attributed to genetic, hormonal, anatomical and immunological problems. However, there are still about 50% of cases without a known cause. [ 18 ]  A study hypothesized that skewed X-inactivation may play a role in these miscarriages. However, a 2003 study found that there was no significant correlation between miscarriages and skewed X-inactivation, with only 6.6% of the patients showing significant skewing as compared to the 3.9% rate in the control group. It also stated that there had been a lack of controlling for age-related skewing in similar studies and concluded that it is unlikely for skewed X-inactivation to influence recurrent miscarriages. [ 19 ]


To study skewed X chromosome inactivation, there must be a detectable difference between the two parental chromosomes. This difference, or  polymorphism , will allow detection of which chromosome is active in the cell, so an inactivation ratio can be determined. Often, the  methylation  levels of the inactive DNA are detected in order to identify the inactive chromosome.  Loci  that are known to be polymorphic within the human population are selected. Assays that detect the methylation level of the highly polymorphic CAG trinucleotide at the 5' end of the androgen receptor gene are often used in skewed X-inactivation studies. Other loci used include phosphoglycerate kinase,  hypoxanthine phosphoribosyl transferase  and the DXS255 locus. [ 1 ]  If these loci contain heavy methylation, it indicates the chromosome is inactive.


At the turn of the 21st century, ratio detection moved to more direct methods by using  mRNA  or protein levels, and  whole exome sequencing . With the exception of escaped genes, only the active X chromosome will transcribe mRNA and produce protein. [ 9 ]  The exome sequencing provides a dataset that shows target sequences, giving an indication of disease-related protein coding regions. mRNA sequencing is then used on these regions to focus on the X chromosome and find  single nucleotide polymorphisms  (SNP) that are associated with the disease. These SNPs are genotyped and traced to parental contributor to calculate the ratio of inactivation, based on how much genetic information each parent donated and how much of each parental allele is expressed. These levels of expression may give greater insight to the fundamental cause of the diseases seen from skewed X-inactivation. [ 20 ]


There are several factors which must be taken into account when studying skewed X-inactivation. Escaped genes are ones found on the inactive X chromosome but are still expressed; this particular gene will be expressed from both chromosomes. It is estimated that 25% of the genes escape inactivation. [ 2 ]  Genes used to study skewing must be carefully selected to ensure they do not escape inactivation, as they will not show any skewed pattern.


A skewed pattern might be more common in affected females than unaffected. [ 2 ]  This must be considered when studying X-linked diseases. Due to the random nature of inactivation, women can have skewed inactivation due to simple statistical probability. This makes it difficult to determine when the ratio is abnormally skewed. Additionally, skewed activation can also be localized to specific cell lineages. For example, one woman might have skewed activation in her T cells but not the B cells, which in turn necessitates deep analysis work and adequate control of cell lines to ensure proper diagnosis. [ 21 ]


Sleep epigenetics  is the field of how  epigenetics  (heritable characteristics that do not involve changes in DNA sequence) affects  sleep .


Research in the field of epigenetics has proven the significance of various environmental experiences. Changes in sleep can cause critical changes to the epigenome, while changes to the epigenome can, in turn, have a crucial influence on experiences related to sleep. Early life experiences with stress can produce lifelong changes in the number of glucocorticoid receptors and impair sleep. Additionally, sleep deprivation alters individuals’ epigenome via changes to methylation, histones, and non-coding RNA. The development of sleep disorders such as insomnia, sleep apnea, and narcolepsy can be greatly impacted by epigenetic changes. Additionally, various methods have been discovered that can reverse, prevent, and treat sleep-related issues by causing epigenetic changes.


Exposure to stress, particularly during early life, has significant impacts on an individual's response to future experiences of stress. Research on epigenetic changes caused by differences in the amount of time rats were nurtured by their mother is one example of these significant impacts.  When examining the epigenome of rat offspring, it is noted that alterations to the epigenome occur at the  glucocorticoid receptor  (GR) gene promoter in the hippocampus. This happens when the offspring experience a significant amount of licking and grooming from their mother at an early age . [ 1 ]  The offspring that experience a significant amount of licking and grooming develop differences in  DNA methylation  compared to the offspring that receive an insignificant amount of licking and grooming. The offspring that are inadequately nurtured by their mother become highly susceptible to stressful environments. These epigenetic differences can be seen as early as the first week of life and persist into adulthood. [ 1 ]  However, evidence shows that when offspring are cross-fostered to receive a good amount of licking and grooming, the epigenetic differences are reversed, supporting a causal relationship between the maternal effect and the epigenetic stress responses in offspring. This proposes that the offspring's epigenome can be altered and established through early life experiences.


The effect of stress on sleep can be predicted long before a baby is born. It is hypothesized that increasing cortisol levels in mothers reduces the amount of  glucocorticoid receptors  (GRs) in an infant's hippocampus, lowering the physiological role of the negative feedback loop on the  hypothalamic-pituitary-adrenal  (HPA) axis. The HPA axis is important for regulating the wake-sleep cycle but works with other factors that help modulate sleep as well. When the negative feedback loop is disrupted due to stress, the HPA axis in newborns becomes hyperactive and the amount of cortisol in circulation elevates. [ 2 ]  However, the hyperactivity of the HPA axis and the elevated levels of cortisol in the hippocampus can be reversed or lowered to normal levels after demethylation of the hippocampal GR promoter, further providing evidence of the involvement of epigenetic mechanisms in HPA axis modifications.


Glucocorticoids are a necessity for life. They play a large role in a majority of physiological functions involving metabolism, blood pressure, breathing, the immune system, and behavior. Either acute or chronic stress can alter the response of the HPA axis. However, the stage of life at which an individual is exposed to stress will determine the magnitude of the consequences they will face in the future. Early life exposure to stress  during the critical period of childhood development can result in permanent changes to adult response systems.


Sleep deprivation is a significant societal problem. It is estimated that around 35.2% of all adults in the US sleep less than 7 hours. [ 3 ]  Lifestyle choices, health conditions, and the use of stimulants are examples of some of the causes that underlie  sleep deprivation  in humans. Sleep deprivation is known to cause metabolic changes, such as altered expression of metabolic genes and hormones. These changes in expression lead to a higher risk of obesity, heart disease, stroke, and high blood pressure. These impacts are also seen at the cognitive level, causing deficits in synaptic plasticity, decreased synapse strength, an inhibited ability to form and maintain memories, and changes in mood. In the long term, these cognitive changes may develop into psychiatric disorders and neurodegenerative diseases like  Alzheimer's disease . Epigenomic changes such as changes in  DNA methylation , histone modifications, and changes in non-coding RNA caused by sleep deprivation may be the cause of these phenotypic presentations. [ 4 ]


CpG methylation  is the addition of a methyl group to a cytosine-guanine dinucleotide. It is the most common epigenetic modification and is correlated with decreased gene expression. Past studies have suggested that sleep deprivation may increase CpG methylation. One such study in mice showed that sleep deprivation increases gene expression of  Dnmt3a1  and  Dnmt3a2,  which are genes that code for enzymes that establish and maintain CpG methylation. Another study found that one night of sleep deprivation causes the hypermethylation of circadian clock genes– CLOCK  and CRY1. [ 4 ]  The circadian  clock  is a biochemical oscillator in organisms that is in sync with the 24 hour cycle of the Earth's rotation and has been found to impact gene expression and behavior. [ 5 ]  A different study showed that the gene encoding for the enzyme SCD1 was hypermethylated following sleep deprivation; this enzyme is crucial in fatty acid desaturation, which is involved in metabolism. The disruption of metabolic pathways may affect hippocampal memory as well. [ 4 ]  A study of differentially methylated positions caused by experiencing insufficient sleep found that 78% of the differentially methylated positions were hypomethylated, yet it is noted that the effect of hypomethylation on transcription depends on the location of the CpG. Concluding the exact effect on the level of gene expression is difficult. [ 6 ]


One post-translational modification that alters histone conformation is histone acetylation which is the transferring of an acetyl moiety from acetyl-CoA to lysine residues. This modification promotes the assembly of transcription factors via the loosening of chromatin and the ability of acetyl groups to act as binding sites. The binding of transcription factors promotes gene expression. Some evidence suggests that sleep deprivation causes a decrease in histone acetylation. Studies in sleep-deprived rats have found a decrease in acetylated histones at the BDNF promoter IV, which is crucial for learning and memory. Models disrupting the sleep-wake cycle in flies and models of neurodegenerative diseases suggest that decreased histone acetylation is associated with neuronal dysfunction. [ 4 ]


Long  non-coding RNAs  and microRNAs have important roles in a majority of biological pathways. The roles of LncRNAs include regulation of splicing and translation and recruitment of epigenetic and regulatory components to target genomic loci. The fact that a large portion of tissue-specific RNA exists in the brain is evidence that LncRNAs might play a role in neuronal function. Studies in sleep deprived mice observed an impact in the levels of expression of several LncRNAs, yet no function has been found for them. MicroRNAs have a role in many neurological processes, such as synaptic plasticity. Several studies have associated them with circadian expression and sleep. Some studies have found altered microRNA expression following sleep deprivation such as altered let-7b and miR-125a expression, yet the ability to draw concrete conclusions on this is limited because there is an abundance of both microRNAs and their targets. [ 4 ]


There are epigenetic changes that may contribute to the development of various sleep disorders.


The epigenetics of insomnia has a large literature. There are arguments that insomnia may be influenced by epigenetic phenomena, and insomnia has both sleep mechanisms and stress-response-related gene environment interactions that affect  brain plasticity . [ 7 ]  There has been growing evidence of the role heritability has on insomnia development but the main factor that determines if a person develops insomnia is stress. Insomnia may be the result of an epigenetic control process of sleep mechanics and may be influenced by changes in brain plasticity caused by exposure to stress. [ 7 ]  There has been a link to the development of insomnia to the  circadian clock . It is believed that humans’ intrinsic time-tracking system, the circadian clock, may be affected by dynamic changes in  chromatin  transitions. [ 8 ]  It has been found that there are links to circadian regulators, the remodeling of chromatins, and cellular metabolism. [ 8 ]  The protein of interest is known as the central clock protein CLOCK, and this protein has HAT enzymatic properties. The central clock protein CLOCK allows histone H3 to be acetylated, and it also allows its dimerization partner BMAL1 at K537 to be acetylated. [ 9 ]  This acetylation is important for the function of the circadian clock. The HDAC activity of the NAD(+) - dependent SIRT1 enzyme's regulation is also related to the circadian clock. The SIRT1 enzyme is thought to work as an enzymatic rheostat of circadian function, and it does this by sending transduced signals from the cellular metabolites to the circadian clock. [ 9 ]  Therefore, it is believed that at the core of the circadian machinery there is a method of chromatin remodeling responsible for insomnia.


Sleep apnea is a condition commonly found in the general population. This condition causes an individual to stop and restart breathing on multiple occasions while sleeping. This condition negatively impacts the well-being of the individual because it can prevent the body from receiving enough oxygen. [ 10 ]  Current evidence indicates that  histone modifications ,  non-coding RNAs , and  DNA methylation  are epigenetic mechanisms that are involved in sleep apnea. [ 11 ]  Having untreated obstructive sleep apnea can lead to developmental deficits such as  cognitive impairments ,  hyperactivity disorder , and poor performance in academic endeavors. [ 11 ]  There are various  hypoxia -mediated perturbations of  gene expression  that are attributed to obstructive sleep apnea comorbidities due to epigenetic mechanisms. [ 11 ]  They are however, heritable and reversible epigenetic alterations which include DNA methylation, histone modifications, and the activation of short and long non-coding RNAs that are known to be the response leading to hypoxia and chronic  intermittent hypoxia . [ 11 ]  As of now, there is not much known about the associations of pediatric obstructive sleep apnea and epigenetic alterations. [ 11 ]


There are instances where a person may feel fatigued in the daytime, causing experiences of drowsiness and sudden instances of going to sleep. These moments are characterized as a chronic  sleep disorder  known as narcolepsy. [ 12 ]  There are several genetic factors such as HLA-DQB*6:02 that have been identified as possibly playing a role in the development of narcolepsy. [ 13 ]  It is found that top-ranked narcolepsy-associated differentially methylated positions are more common in non- CpG islands . [ 13 ]  It was reported that patients experiencing narcolepsy in the study had these sites hypomethylated 95% of the time. [ 13 ]  There is a gene that has been identified that may contribute to the development of this disease known as the  CCR3  gene. [ 13 ]  It appears that this gene shows that both methylation and  single-nucleotide polymorphism  (SNP) had a close association with narcolepsy. This gene is not necessarily the main cause of this disease and the research suggests that there are other unidentified genetic factors linked to the disease. [ 13 ]


Epigenetic Treatment


Abnormal functioning of dopaminergic pathways has been linked to various disorders, including PTSD. Research has been conducted to investigate an epigenetic means by which various symptoms of PTSD, including lucid nightmares, can be reduced. Given that possession of alleles such as the D2A1 allele which cause low dopamine function make an individual susceptible to PTSD, treating the modulation of  dopaminergic  signals may serve as a way to decrease  lucid nightmares  in individuals suffering from PTSD. [ 14 ]  Additionally, the critical role dopamine plays in the synaptic signals of  thalamic  and  neocortical  neurons which cause the visual and motor hallucinations in dreams is worth considering. In one study, the use of KB200Z, a dopamine agonist, alleviated the negative symptoms of lucid nightmares in participants with histories of abuse, addiction and PTSD. [ 14 ]  Epigenetic alteration of  neuroplasticity  may be significantly inhibited via targeting dopamine pathways and trauma-induced deficiencies in  functional connectivity . [ 14 ]  The use of neurotransmitters to increase potentiation of synaptic pathways specifically at CA1 and CA3  pyramidal cells  is a promising way to treat nightmares.


Sleep deprivation increases the expression of  HDAC2  which in turn leads to a significant deficiency in histone acetylation. [ 4 ]  This decreased histone acetylation has been linked to learning and memory deficits and abnormalities in individuals' circadian clock. Trichostatin A  has proven to be beneficial when treating sleep deprivation. [ 4 ]  Since it is an HDAC inhibitor, Trichostatin A has been shown to specifically alleviate spatial memory loss tied to the  hippocampus  via specifically targeting HDAC2. [ 4 ]  A decrease in HDAC2 works to reverse the cognitive deficiencies associated with epigenetic changes from chronic sleep deprivation.


Aside from the use of epigenetic drugs to treat the epigenetic effects of sleep disorders, lifestyle changes that can induce epigenetic changes have also been considered. In one study, mice that experienced gestational sleep fragmentation were found to have  Foxo1  misregulation. [ 15 ]  Foxo1 plays a critical role in monitoring  insulin  signaling in the liver. Various histone modifications and an increase in the epigenetic mark of 5-hydroxy-methyl-CpG at Foxo1 was found to occur in mice which experienced gestational sleep fragmentation. However, when the mice participated in early-life exercise, these epigenetic effects were reversed. The research suggests that taking measures to reverse the epigenetic changes caused by gestational sleep fragmentation decreases the susceptibility of individuals to metabolic disorders such as  diabetes  and  preeclampsia .