J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from

Review Article

J. med. Genet. (i964), I, 50. Sex Chromatin* URSULA MITTWOCH From Galton Laboratory, University College, London The term 'sex chromatin', in present-day usage, body, which stained with nuclear dyes, was inti- denotes a body in the nuclei of cells, which indicates mately associated with the nucleolus and was at the sex of the organism from which the cell origi- first called a 'nucleolar satellite'. This term became nated. The sex chromatin body is present in the inappropriate when Barr (I95I) found that in nucleus only during interphase and disappears neuroglial cells ofthe cat the sex differentiating body during mitosis. The term 'sex-chromatin' had lies against the inner side of the nuclear membrane. previously been used in a different sense by Wilson Henceforth, the term 'sex chromatin' was employed. (1925) to describe the (hypothetical) part of the The finding of the sex chromatin in the cat was an sex chromosomes that acted as a sex differentiator unexpected result of experimental neurocytology. in development. Barr and Bertram (1949) wrote: 'It appears not to Although the subject of sex chromatin has be- be generally known that the sex of a somatic cell as come a part of human genetics only within the last highly differentiated as a neurone may be detected decade or so, it has attracted very considerable with no more elaborate equipment than a compound interest and today provides one of the most active microscope following staining of the tissue with thecopyright. growing points. One reason for this is practical. It routine Nissl method'. Sections of brain, spinal has been estimated that about 2 or 3 persons per cord, or sympathetic ganglia could now be readily i,ooo suffer from an abnormality of their sexual attributed to the correct sex of the animal from development (Overzier, i963), and it appears that which they originated. the incidence may be even higher at birth (see p. A few years before the appearance of the paper by 70). The sex chromatin test provides a simple and Barr and Bertram, S. G. Smith (I944, I945a, b), in often indispensable aid in the diagnosis of such Ottawa, Ontario, found that in cells of the larvae patients. Moreover, the technique has made it of the spruce budworm, Choristoneura (Archips) http://jmg.bmj.com/ possible to screen large populations and thus obtain fumiferana (Clem.), a darkly staining body was estimates regarding the incidence of these abnor- present in the nuclei of the female and was absent malities. On a more theoretical level, the origin of from the male (Fig. 2). This body could be used for the sex chromatin and its relation to the sex chromo- sexing larvae prior to the development of the somes has given rise to speculations, which are gonads, and it was suggested that this method might further stimulating experimental work, as to the be applied to other organisms, including mammals.

way in which the X chromosome functions, both in These findings are of importance not only because on September 27, 2021 by guest. Protected sex determination and in general. they provide the first indication that a nuclear body present in interphase can be used as an indicator of Some Landmarks in the Development of the sex, but also because the spruce budworm is a Sex Chromatin Concept member of the Lepidoptera, and in this order the The discovery of sex chromatin is generally female is the heterogametic sex. A female spruce placed in I949, when Barr and Bertram, in London, budworm has only one X chromosome, and Smith Ontario, described a morphological distinction postulated that the single X remained condensed between neurones of male and female cats. The (heteropycnotic) in interphase nuclei, in contrast sex difference consisted of a small body which was to the two X chromosomes of the male, which be- present in the nuclei of nerve cells from female cats came invisible. Thus, the situation appears to be but was absent in those of males (Fig. i). The precisely the opposite from that found in mammals. Sex chromatin in heterogametic females will be *Received February 13, I964 further discussed later (p. 68). 50 J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 5I

_lra'E.....Xies |.wls I....@ .....S,S .wNlX, ,l|w. l,| ! -! || |*i.eWj| :4w- a F _ ..FF....s x_i..iI.SfflXW.0.E_tisw ..x_|EX ...... _ .. F ..

xwa_ : ..

| .,.

FIG. x. Sex difference in neurones of the cat. (a) 'nucleolar satellite' adjacent to nucleolus sn female; (b) no nucleolar satellite in malc. (By courtesy of Professor M. L. Barr.) copyright.

F 2. .

r * .: _ t .a:S: ...... *_

x,P' http://jmg.bmj.com/ ' .

_¢ e X; .a;J'.*S.;.:-Wf_^o.::: on September 27, 2021 by guest. Protected

X.

Im M-A

FIG. 2. Sex difference in the spruce budworm, Chorinstoneura (Archips) fumiferana (Clem.). (a) cells from female; (b) cells from male. (By courtesy of Dr. S. G. Smith.) J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 52 Ursula Mittwoch Some still earlier works, which are relevant to the difference could be demonstrated in the polymor- problem of sex chromosomes in interphase nuclei, phonuclear leucocytes of peripheral blood (David- are those of Geitler (I937, I938, I939), who worked son and Smith, 1954). Techniques for demonstrat- on insects belonging to the bugs (Hemiptera) and ing sex chromatin in vaginal smears were described flies (Diptera), in both of which the female is homo- by Carpentier, Stolte, and Visschers (I956) and gametic and the male heterogametic. Geitler found Guard (i959). that in somatic interphase nuclei the X chromosome The development of these new techniques pro- was heteropycnotic in both sexes, i.e. the female vided an enormous impetus to sex chromatin had two heteropycnotic bodies and the male one. studies, and in I956 at least 6 groups ofworkers were This situation seems to differ from that both in able to show that sex chromatin was present in a spruce budworms and in cats. The differences in majority of patients with Klinefelter's syndrome these findings have not yet been resolved, and it is (Riis, Johnsen, and Mosbech, I956; Jackson, clear that for the time being any theories based on Shapiro, Uys, and Hoffenberg, I956; Plunkett and data from the sex chromatin of mammals will have Barr, I956; Bradbury, Bunge, and Buccabella, I956; to be confined to this class. Nelson, I956; Sohval, Gabrilove, Gaines, and In I952, Graham and Barr were able to show that Soffer, I956). in the cat sex chromatin could be demonstrated in When techniques for studying human chromo- tissues other than neurones. The tissues examined somes became available from I959 onwards, a included smooth, skeletal, and cardiac muscle, striking correlation was revealed between the sex cartilage, epidermis, endothelium, gastric epithe- chromatin status and the number ofX chromosomes lium, liver, pancreas, adenohypophysis, thyroid, in an individual. The presence of sex chromatin was adrenal, cortex and medulla, interstitial cells of found to be associated with the presence of two ovary and testis, kidney, uterine epithelium, and X chromosomes (or, more accurately, i6 chromo- prostate. Sex chromatin was found in all tissues somes in the 6-12 group, since these cannot be except in liver and pancreatic acinar cells. The sex distinguished unequivocally from the X on morpho- chromatin was present in one-half to two-thirds of logical grounds), while the absence of sex chromatin the cells from female cats and only very rarely in was found to be associated with the presence of a those from male cats. single X chromosome (iS chromosomes in the 6-12copyright. Just a year after the discovery of sex chromatin in group) (Barr and Carr, I960; Stewart, I960). Thus, the cat, a sex difference was shown to exist in sex chromatin studies have become indispensable human sympathetic ganglion cells (Barr, Bertram, in the elucidation ofabnormalities in sexual develop- and Lindsay, I950; Bertram, Bertram, and Lindsay, ment, for the following reasons: (i) The simplicity 1950). Sex chromatin became part of human and speed of the techniques alows one to forecast genetics in I953 when Moore, Graham, and Barr the number of X chromosomes present in patients

showed that sex chromatin could be demonstrated suspected ofan abnormality ofthe sex chromosomes; http://jmg.bmj.com/ in preparations of skin biopsies, including that of a (2) since X chromosomes in metaphase cannot be hermaphrodite. Cells in the stratum spinosum are recognized unequivocaUy, the sex chromatin pro- particularly favourable in showing the sex chromatin. vides a check as to the number of X chromosomes In an intensive survey of human tissues, Moore present; (3) the method can be used in surveys of and Barr (I954) found sex chromatin in all the cell large groups of people. types examined, which included cartilage and The early assumption that the drumsticks of the adrenal cortex, smooth muscle, bladder, and polymorphonuclear leucocytes are equivalent to thyroid cells. The material was mainly derived from the sex chromotin bodies of epithelial and other on September 27, 2021 by guest. Protected necropsies. tissue cells has been substantiated by later work As a result of using the technique of sexing skin (see p. 66). Therefore, the term 'sex chromatin' biopsies, it was shown that most patients with now includes drumsticks as well as sex chromatin Turner's syndrome lacked sex chromatin (Polani, bodies in cells other than leucocytes. The latter Hunter, and Lennox, I954; Wilkins, Grumbach, are now often called 'Barr bodies'. and van Wyk, 1954). In I955 the sex chromatin test became further simplified when it was found that Barr Bodies: General Properties the presence or absence of sex chromatin could be A Barr body is a small well-defined body which demonstrated in cells scraped off the buccal mucosa stains intensely with nuclear dyes (Fig. 3-5). It is (Marberger, Boccabella, and Nelson, 1955; Moore present in a large proportion of nuclei of female and Barr, I955). Yet another finding of great origin and absent in male nuclei. The size of a Barr potential importance was the discovery that a sex body is about i V± in diameter. The average size has J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 53

k4.. d .. .?. ... ,V:.

Pi: ,. ... W.

^ *.i : 5.... nF. s I i FIG. 3 FIG. 4 FIG. 5

FIG. 3. Sex chromatin in buccal mucosa FIG. 4 aand 5. Sex chromatin in fibroblasts cultured from skin of cell of a woman. (Cresyl fast violet. x female donor. (Thionin. x 2,200.) 2,200.) been estimated as 0o7 X I 2 V., both in nuclei of particularly the rectangular ones, appear to be buccal mucosa and in sections of several human bipartite, but, as pointed out by Klinger (I958), the tissues (Moore and Barr, I954, I955). Interphase distance separating the 2 components is near the nuclei are rarely quite homogeneous, and they may limit of the resolving power of the light microscope. contain a number of more or less darkly staining Some investigators, when scoring Barr bodies, bodies, which are known as chromocentres (Fig. 5). include only those that are situated at the periphery Such non-specific chromocentres are generally of the nuclear membrane. This practice almost copyright. smaller than Barr bodies and have a less well-defined certainly underestimates the true incidence of the outline. In cases where a small percentage of Barr Barr bodies. On the other hand, since it is sometimes bodies have been reported in male nuclei, this is more difficult to distinguish central Barr bodies almost certainly due to non-sex-specific chromo- from non-specific chromocentres (Miles, i960), it centres, which are sufficiently similar to Barr bodies may be wise to exclude all centrally placed bodies to have been mistaken for them.

Barr bodies are most commonly situated at the http://jmg.bmj.com/ periphery of the nucleus (Fig. 3-5). However, a minority of Barr bodies are found in other parts of the nucleus, and many of these are situated in proximity of a nucleolus (Fig. 6). Klinger (I957) found that in amnion and connective tissue, 6i -8 % of the Barr bodies were at the nuclear periphery, 2322% were lying against a nucleolus, and 9-2% were apparently free in the cytoplasm (although it on September 27, 2021 by guest. Protected cannot be exlcuded that a Barr body, which is seen in the centre of a nuclear profile is not, in fact, lying against the nuclear membrane in the three-dimen- sional nucleus). Barr bodies have one of several distinct shapes. Many appear to be plano-convex or wedge-shaped, with the plane side resting against the nuclear membrane and the convex part pointing towards the cytoplasm. Barr bodies in the centre of the nucleus appear to be rectangular, and some rect- angular Barr bodies may also be observed at the FIG. 6. Sex chromatin adjacent to nucleolus in fibroblast of female origin. (Thionin, X 2,200.) (By courtesy of the Editor of Cyto- periphery of the nucleus. Some Barr bodies, genetics.) J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 54 Ursula Mittwoch if preparations have to be used that are not of the cat, and by Miles (I960) who cultured cells from highest technical standard. This happens not in- human amnion. DeMars (I962), using fibroblast frequently in preparations of buccal mucosa which cultures obtained from human skin biopsies, may contain a high proportion of moribund cells. observed the sex chromatin in the same cells both Since the majority of Barr bodies are at the peri- in the living condition and after fixation and Feul- phery of the nucleus, whichever method of scoring gen staining. The sex chromatin after staining was is used should not affect the end-result of whether found to be in the same position where it had been sex chromatin is present or absent. It goes without in the living cell. Schwarzacher (I963) reported saying that the criteria used in scoring the Barr that in living human cells grown in vitro, sex bodies should be explicitly stated. chromatin is visible in 46 % of the nuclei, whereas Barr bodies can be demonstrated particularly well after fixation and staining it can be seen in about in cells grown in culture. Thus Serr, Ferguson- 90% of the cells. In the remaining io% of the Smith, Lennox, and Paul (I958) found sex chro- nuclei sex chromatin could not be seen by any matin in go % of the cells grown from human method. thyroid, while Fraccaro and Lindsten (i959), who There is as yet no conclusive explanation as to cultured a large number of organs from human why the sex chromatin does not appear to be foetuses, reported incidences of sex chromatin present in all the cells of female origin. Mittwoch varying between 34 % and 88 % in the cultures (I963, I964b), as a result of measuring nuclear areas originating from female foetuses. Miles (I960) in short-term human fibroblast cultures, found that found a negative correlation between the incidence nuclei containing a Barr body were on an average of Barr bodies and the frequency of mitoses in about 6 % smaller than the nuclei in which no Barr cultured amnion cells, and this relation was con- body can be seen. The smaller size of nuclei firmed by Therkelsen and Petersen (I962) in fibro- containing a Barr body might be due to two reasons: blast cultures originating from bone marrow and (i) the condensation of one X chromosome to form embryonic skin. A technique for demonstrating a Barr body (see p. 58), and (2) the possibility that sex chromatin in tissue culture has been described the Barr bodies disappear at some stage during by Ross (I962). Sex chromatin may no longer be DNA synthesis, when the nucleus might be ex- visible in tissue culture if large-scale chromosomal pected to reach its maximum size. The lattercopyright. changes have taken place, i.e. the cells are 'trans- possibility would be in agreement with the observa- formed' (Orsi and Ritter, I958; Miles, I959). tion of an inverse relation between the incidenc Apart from this, cultured cells are particularly of sex chromatin and the rate of mitosis. favourable for the study of sex chromatin, since the Barr bodies can be seen in female embryos, but cells are present in a single layer and the nuclei not in the earliest ones. Glenister (I956) examined flatten themselves on the glass of the cover slip. For 22 male and 12 female human foetuses, of I8-I50 the same reasons, excellent preparations of sex mm. crown-rump lengths: in all of them, the http://jmg.bmj.com/ chromatin may also be obtained from whole mounts presence or absence of sex chromatin corresponded of embryonic membranes, as demonstrated by to the histological state of the gonads. In the female Klinger (I957) and Klinger and Schwarzacher embryos, 30-50 % of the nuclei had sex chromatin. (I962). Klinger (1957) found that in 2I embryos the sex Barr bodies can be stained with a large number of chromatin state was in accordance with the external nuclear dyes (for a review see Barr, I963). Staining genitalia and the morphology of the gonads; of 3 techniques in common use employ cresyl echt further embryos, which were too young to be sexed violet (Moore and Barr, i955), or thionin (Klinger in this way, i contained sex chromatin and 2 lacked on September 27, 2021 by guest. Protected and Ludwig, 1957), or haematoxylin for sectioned it. Park (1957) studied serial sections from 33 material, such as skin biopsies (Barr, I955). The human and i8 macaque embryos, belonging to the fact that Barr bodies will stain by the Feulgen Carnegie Institute, Washington. The ages of the technique following acid hydrolysis, and have an human embryos ranged between 36 hours and 24 affinity for methyl green rather than pyronin days (the youngest was in the two-cell stage), and (Lindsay and Barr, I955), indicate that they are the macaque embryos were between 9 and 34 days. composed of material similar to that of the chromo- In the human embryos, sex chromatin was first seen somes and contain DNA. in the trophoblast at about the twelfth day of ges- Barr bodies can also be seen in unstained and in tation and in the embryo itself after about I6 days. living cells by means of phase-contrast microscopy. In the macaque embryos, sex chromatin began to This was described by James (I960), who observed be seen on the tenth day in the trophoblast, while sex chromatin in living cells cultured from a female larger numbers of Barr bodies appeared in the J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 55 embryo on the nineteenth day. Other embryos of equal age had no sex chromatin. These results are in accordance with observations on embryos of the cat, made by Graham (i954a, b) and by Austin and Amoroso (I957) who suggested that the absence of sex chromatin in early embryos was connected with the large size of their cell nuclei. Sex chromatin findings in tumour cells have recently been reviewed by Lennox (I963). In the majority of tumours, the sex chromatin status appears to be the same as that of the host. However, the teratomata seem to offer an interesting exception, as was first shown by Hunter and Lennox (1954). All the teratomata in females contain sex chromatin, and sex chromatin is also found in the teratomata of FIG. 7. Two Barr bodies in buccal mucosa cell of triple X female. half the males, while the other half lack it. Lennox (Cresyl fast violet, x 2,200.) explains this on the assumption that teratomata arise by the fusion of haploid cells; thus, two hap- loid male cells, each containing an X chromosome, having either one or none. Double Barr bodies are could give rise to a cell with two X chromosomes, an invariable finding in these patients (Johnston, which would account for the Barr body. On the Ferguson-Smith, Handmaker, Jones, and Jones, other hand, Taylor (I963a), who reported aber- I96I). Such cells can be well demonstrated in rant sex chromatin findings in a proportion of buccal mucosa or cultured fibroblasts employed for embryonic teratomata and some other embryonic chromosome analysis (Fig. 7 and 8). tumours suggests that the most likely explanation Carr, Barr, and Plunkett (I96I) described 2 is the presence of an abnormal sex chromosome patients with 4 X chromosomes, and these had a constitution, such as XO, XXY, etc. proportion of cells with 3 Barr bodies in their Atkin (I960), working on uterine and other buccal mucosa. Finally, Kesaree and Woolley copyright. tumours in women, found that when sex chromatin (I963) described a female patient with five X was present, those with a near diploid number of chromosomes, and she had 4 Barr bodies in many chromosomes had i sex chromatin body in the cells of the buccal mucosa. majority of nuclei, while tumours with a near- The Y chromosome appears to have no effect on tetraploid number of chromosomes had 2 sex the formation of Barr bodies, even though it results bodies in most nuclei. The significance in a male phenotype. All patients with 2 or more chromatin Y the of this finding will be discussed in the following X chromosomes in addition to a carry stig- http://jmg.bmj.com/ section. mata of Klinefelter's syndrome. Barr, Shaver, Carr, and Plunkett (i959) described 3 such patients, in Numbers of Barr Bodies per Nucleus whom cells with 2 Barr bodies were found and all In general, the maximum number of Barr bodies per nucleus in any organism or tissue is either o or i, corresponding to a karyotype containing one or two X chromosomes respectively. However, cells with multiple Barr bodies may be encountered, and this on September 27, 2021 by guest. Protected invariably means that more than two X chromosomes are present in the karyotype. The excess of X chromosomes may be due either to aneuploidy (e.g. if three X chromosomes are present in a diploid nucleus) or to polyploidy (e.g. as a result of doubling a female chromosome complement, resulting in a tetraploid nucleus with four X chromosomes). A female patient with three X chromosomes was described by Jacobs, Baikie, Court Brown, Mac- Gregor, Maclean, and Harnden (I959). On exam- 14 % ining the buccal mucosa it was found that of FIG. 8. Two Barr bodies in fibroblast nucleus of triple X female. the nuclei contained two Barr bodies, the remainder (Thionin, x 2,200.) J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 56 Ursula Mittwoch were found to have an XXXY sex chromosome constitution. Two further male patients with double Barr bodies and XXXY sex chromosome constitu- tion were described by Ferguson-Smith, Johnston, and Handmaker (I960). A patient with XXXXY sex chromosomes was first described by Fraccaro, Kaijser, and Lindsten, (ig6ob), Fraccaro and Lindsten (I960), and Fraccaro, Klinger, and Schutt (I962). By now, more than I2 such patients are known (Atkins, Book, Gustavson, Hansson, and Hjelm, I963); all have cells with 3 Barr bodies in their buccal mucosae. It is clear from the sex chromatin findings in these patients, as well as in normal male and female subjects, that in diploid cells the maximum number of Barr bodies is one less than the number of X chromosomes. That polyploidy also affects the formation of Barr bodies became apparent from the work of Reitalu (1957) who observed that in liver obtained from female embryos, some of the larger cells which were assumed to be tetraploid contained 2 sex chromatin masses; from Klinger and Schwar- zacher (I960) who found 2 Barr bodies in cells from human amnion, in which DNA measurements indicated the presence of a large proportion of polyploid cells; and from the finding ofAtkin (I960) FIG. 9. Large fibroblast cell, presumed to be at least tetraploid with that tumours of female origin with a near-tetraploid 2 Barr bodies. (Thionin, x 2,200.) number of chromosomes had two sex chromatincopyright. bodies in many nuclei. In order to explain the relation between the number of Barr bodies, the number of X chromo- somes, and the degree of ploidy of the cell, Harnden (I96I) proposed the formula, B =X - Pl2, = where B = number of Barr bodies, X number http://jmg.bmj.com/ of X chromosomes, and P = degree of ploidy. This formula fits in well with observation, providing (i) B is taken as the maximum number (which is not present in all cells) and (2) the ploidy number is even. Thus, in diploid cells the maximum number of Barr bodies is one less than the number of X chromosomes, in tetraploid cells it is two less than the number of X chromosomes, while in octoploid on September 27, 2021 by guest. Protected cells it is four less than the number of X chromo- somes (Fig. 9 and io). However, the formula can- not be meaningfully applied to cells with uneven ploidy numbers, for in such cases the result would be in half Barr bodies, which is not a reasonable answer. There are 6 reports on the presence of Barr bodies in triploid cells. The first is by Book and Santesson (I96I) who described a patient with triploid and diploid cells. The triploid cells had no sex chromatin was FIG. IO. Large fibroblast cell, presumed to be at least octoploid, with XXY sex chromosomes, and 4 Barr bodies. (Thionin, x 2,200.) found in any of the tissues. Similarly, Carr (I963) J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 57 found no sex chromatin in triploid cells with XXY was confirmed by measurements of the area of the chromosomes, derived from an aborted foetus, while sex chromatin in patients with iso-X chromosomes, Ferrier, Ferrier, Stalder, Buhler, Bamatter, and and in normal women (Lindsten, I963), which Klein (I964) found no sex chromatin in a patient showed that the area of the sex chromatin in the who was a mosaic with diploid triploid cells with patients with iso-X chromosomes was significantly XXY sex chromosomes; in the latter case, however, larger than in the normal women. DNA measure- the proportion of triploid cells was less than I0 %. ments on the Barr bodies were also undertaken and By contrast, in a similar culture derived from an indicated that the Barr bodies in iso-X patients XXY triploid foetus, Mittwoch and Delhanty contained on an average more DNA than those in (I96I) found sex chromatin to be present. Atkin normal women, while the Barr bodies in a patient and Klinger (I962) observed that in a culture grown with a deleted X chromosome contained less DNA from a hydatifidorm mole, in which the modal than those of the normal women (Lindsten, I963). number of chromosomes was 69, a considerable number of nuclei had 2 Barr bodies. Similar results were obtained by Mittwoch, Atkin, and Ellis (I963) Late Replicating X Chromosomes in fibroblast cultures obtained from a diploid/ The findings on Barr bodies in normal women triploid female patient. The diploid cells had XX, and in patients with numerically or structurally and the triploid cells XXX sex chromosomes. abnormal X chromosomes have recently found Double Barr bodies were found in 6 % of the cells, striking confirmation as a result of experiments and DNA measurements made on individual inter- involving autoradiography. It was shown by Taylor, phase nuclei, using an integrating microdensito- Woods, and Hughes in I957 that if growing cells meter, indicated that some of these double Barr were exposed to a solution of thymidine labelled bodies were present in triploid cells, the rest being with tritium for a briefperiod during DNA synthesis, in more highly polyploid cells. the radioactive substance would be taken up by the No explanation can as yet be advanced as to the chromosomes and could be revealed by applying a reason for these apparently conflicting reports. radio-sensitive film. Using this technique, Taylor Nevertheless it may be concluded that, under cer- (I960) has shown that in the Chinese hamster not all tain conditions, triploid nuclei may have the same chromosomes take up thymidine and duplicate their copyright. maximum number of Barr bodies as diploid ones. DNA at the same time. When tritiated thymidine is applied to cultures of human cells just before the end of the period during which DNA is synthesized, Barr Bodies of Abnormal Size most of the chromosomes have almost completed Although the majority of patients with Turner's their DNA synthesis; however, autoradiography syndrome have an XO sex chromosome constitu- reveals that in cells from normal females one chro- tion, a large number of cases have now been des- mosome in the 6-I2 is heavily labelled (Grumbach http://jmg.bmj.com/ cribed where a second X chromosome was present and Morishima, I962; German, i962a, b; Gilbert, but was structurally abnormal. The first such Muldal, Lajtha, and Rowley, I962; Morishima, patient to be described (Jacobs, Harnden, Court Grumbach, and Taylor, I962; Moorhead and Brown, Goldstein, Close, MacGregor, Maclean, and Defendi, I963) (Fig. ii). Since this chromosome Strong, I960) had an X chromosome from which a does not occur in normal males, the chromosome part of the long arm had been deleted, and this which duplicates its DNA later is assumed to be the patient was noted to have Barr bodies that were second X chromosome. In patients with 3 X smaller than usual. Small Barr bodies were also chromosomes, 2 such late replicating chromosomes on September 27, 2021 by guest. Protected noted in another patient who had a deletion of the are found in many cells (Grumbach, Morishima, and short arm of the X chromosome (Jacobs, Harnden, Taylor, I963; Giannelli, I963) and in patients with Buckton, Court Brown, King, McBride, Mac- XXXXY sex chromosomes, 3 X chromosomes are Gregor, and Maclean, I96I). A relatively frequent late labelling (Rowley, Muldal, Gilbert, Lajtha, abnormality of the X chromosome is the so-called Lindsten, Fraccaro, and Kaijser, I963; Giannelli, presumptive iso-chromosome for the long arm of I963) (Fig. I2); while in a female infant with 5 the X, which was first described by Fraccaro, X chromosomes, Grumbach et al. (I963) found 4 Ikkos, Lindsten, Luft, and Kaijser (ii6oa). This late replicating X chromosomes! It is apparent that chromosome is larger than the normal X, and all the number of late-labelling X chromosomes is, like such patients have sex chromatin that appears to that of Barr bodies, one less than the total number be larger than normal (Lindsten, Fraccaro, Ikkos, of X chromosomes, and there can be little doubt Kaijser, Klinger, and Luft, I963). This impression that these late-labelling chromosomes do, in fact, J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Ursula Mittwoch

...... *. S...... 2 XA

k o.. *....,..o--N *. a :b:::w.. l: ., ,,, .<. ... ,,,;C.X.a *, ..:: . . .. 10.- E ..w:

*.:: ...... *.t __Ee:' .....4.

I,, q ..s

,",Sj:|.: My:: ,:.... :@l

; ...... :!:;. -' .$ .:e° ^.o,: :[email protected]. ,.L. .:;*, .S6, ... copyright.

... :\: ,:: :.. :. ILA .. 4..f wt!wx6E., .S.>e,>-_ W W :...

A...'A :'. ...:: .. o, w t ': ; c ..:l A~~~ p , Sx:.eco http://jmg.bmj.com/

FIG. II. Metaphase chromosomes of a human female, following application of radioactive thymidine and autoradiography. The late replicating chromosome is assumed to be one of the X chromosomes. (By courtesy of Dr S. Muldahl.) form the Barr bodies. This conclusion is further Also using tritiated thymidine, Bishop and strengthened as a result of autoradiographic studies Bishop (I963) succeeded in labelling the Barr body on structurally abnormal X chromosomes. In in the interphase nucleus. After removing the on September 27, 2021 by guest. Protected patients with iso-X chromosomes, this chromosome, grains, the most heavily labelled area of the cell was and not the normal X chromosome, is found to be found to correspond to the Barr body in the same labelled with the isotope (Muldal, Gilbert, Lajtha, cell. Lindsten, Rowley, and Fraccaro, I963; Giannelli, I963); in a patient who had one X chromosome in The Origin of the Barr Body the form of a ring, this ring chromosome was late The findings just described have greatly clarified labelling (Fig. I3; Lindsten, I963); and in a patient our ideas as to the origin of the Barr body. The with a deleted X chromosome, the deleted chromo- older hypothesis that a Barr body was formed from some was late labelling (London, Kemp, Ellis, and both members of a pair of X chromosomes (Moore Mittwoch, I964). These findings explain the fact and Barr, I953) became inadequate when the that the Barr bodies associated with these abnormal numerical relation between Barr bodies and X chromosomes are of abnormal size. chromosomes was discovered, and was replaced by J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 59 the idea, first formulated by Ohno, Kaplan, and I925; Schrader, I928). Conspicuous heteropycnosis Kinosita (I959), that the Barr body wasformedfrom of the sex chromosomes in mammalian spermato- a single X chromosome. The observation underlying cytes was also observed (Minouchi, I928). Recently this idea was that during prophase of mitosis in this contrary behaviour of the X chromosomes in liver cells of the rat, one chromosome in the female germ and somatic cells of males and females has appeared to be more condensed than the rest and been found to apply also to man (Ohno and Makino, that such a condensed chromosome was not seen in 196I; Ohno, Makino, Kaplan, and Kinosita, I96I): the male. Tetraploid cells in the female appeared to the single X chromosome of the male becomes have two such chromosomes. The numerical heteropycnotic in the spermatocyte, whereas both relation between Barr bodies and X chromosomes, X chromosomes in the oocyte remain isopycnotic; as well as knowledge gained from autoradiographic while in somatic cells, one of the X chromosomes of studies, leave no reasonable doubt that it originates the female becomes heteropycnotic and the X from a single X chromosome which is highly con- chromosome of the male does not. densed during interphase, while the rest of the Chromosomes, or parts of chromosomes, whose chromosomes are extended. The other X chromo- cycle of condensation differs from that of the some in females, as well as the single X chromosome majority of the chromosomes are said to exhibit in males, is similarly extended. Thus, the behaviour 'heteropycnosis' (Gutherz, I907). Heteropycnosis of the X chromosome in somatic cells is exactly is said to be positive if the chromosomes are more opposite to that which it shows in germ cells for, condensed, and negative if they are less condensed, as has been known for a long time, the single X than the rest. A Barr body, therefore, is the result chromosome of insects becomes condensed (hetero- of positive heteropycnosis of a single X chromosome pycnotic) during the later spermatogonial divisions, during interphase. Chromosomes or parts of and particularly during the prophase of meiosis in chromosomes exhibiting heteropycnosis are often the spermatocyte, whereas the paired chromosomes said to consist of 'heterochromatin' (Heitz, I928). of the female remain uncondensed (see Wilson, The remains of a Barr body can often be seen copyright.

0 0 AP Nj.. OIII 4 I *f

:.... http://jmg.bmj.com/

*~~~.2

* 4as on September 27, 2021 by guest. Protected

' ,

FIG. I2. Metaphase chromosomes of a patient with XXXXY sex chromosomes. (a) before applying radio-sensitive film, (b) after applying radio-sensitive film. (By courtesy of Drs B. B. Mukherjee, 0. J. Miller, S. Bader, and W. R. Breg, and the Editor of the Scientific American (I963), 209, 54.) J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 60 Ursula Mittwoch

.:.a to A) .*.

IJWX%1r.* *

.4~~~~~~~~~~~~~~~~~~~~~~~~.

-t o: b

i .4w* A/ ss. *:

...... :

'v**copyright.

a

*:

... x i.- '4. . .al '4 0. 0 .4.. -o 0.0 f .4 .%... * '. .0 -. i.W. iowlr 'O. A*. 44

I. http://jmg.bmj.com/ 400 .... lo * U- 9* 4 a: i to41 ...4 A0 * V

Jt dw d Q~~~~~~~`~A on September 27, 2021 by guest. Protected

FIG.I3. Autoradiography in patients with abnormal X chromosomes: (a) autoradiograph of a metaphase cell containing the ring X chromosome; (b) the same cell after removal of the grains; (c) autoradiograph of metaphase cell containing an X chromosome with a deletion of the short arm; (d) similar cell containing an X chromosome with a possible deletion of the long arm. (By courtesy of Drs M. Fraccaro and J. Lindsten, and the Editor of Proceedings of the Symposium of Cytogenetics of Cells in Culture, Academic Press.) during the prophase ofmitosis (Fig. I4). As prophase resembling a Barr body may sometimes be seen in proceeds, heteropycnosis of the X chromosome such nuclei (Fig. I5); though the fact that telophase disappears and at metaphase the two X chromosomes nuclei contain many chromocentres still leaves some are indistinguishable from each other, nor, indeed, doubt on this point. can they be distinguished with certainty from auto- There is now considerable evidence that during somes 6-12. It is possible that in the daughter nuclei mitotic metaphase the chromosomes do not distri- one of the X chromosomes may become heteropyc- bute themselves at random within the cell, but that notic again during telophase, since a structure certain chromosomes tend to be found in character- J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 6I preparations, the X chromosomes cannot be distin- guished with certainty, but the Barr body forming X chromosome is clearly labelled in cells treated with radioactive thymidine. In such preparations it is found that this chromosome is situated at the peri- phery of the nucleus in the majority of the cells. This observation doubtless throws some light on the hitherto puzzling fact (Miles, I96I) that the Barr body appears to be preferentially situated at this site. The conclusion that the formation of a Barr body is a function intrinsic in the X chromosome con- stitution is entirely consistent with certain other observations made over the past years. For instance, the sex chromatin of animals is not affected by castration (Moore and Barr, I953); the membranes of male foetuses do not contain sex chromatin in spite of their maternal environment (Klinger, 1957); and in organisms that are mosaics with respect to their X chromosome constitution, the sex chromatin FIG. 14. Fibroblast nucleus in prophase showing remains of Barr occurs only in tissues containing at least two X body. (Thionin, x 2,200.) chromosomes. A particularly striking example of the last phenomenon was furnished by Klinger and istic positions (Schneiderman and Smith, I962; Schwarzacher (I962) who described a foetus in Miller, Breg, Mukherjee, Gamble, and Christakos, which patches of cells containing sex chromatin I963a; Miller, Mukherjee, Breg, and Gamble, and an XXY sex chromosome constitution alter- i963b; Barton, David, and Merrington, I963; nated with areas that lacked sex chromatin and had Merrington and Penrose, I964). In ordinary an XY constitution. The fact that sex chromatin is intrinsic in the copyright. X chromosome constitution does not, of course, imply that its formation is entirely uninfluenced by external factors. The observation that the incidence of Barr bodies is low immediately after birth (Smith, Marden, McDonald, and Speckhard, I962; Taylor, I963b;) may suggest such a possibility. Sohval and Casselman (I96I) found that the effect of a number http://jmg.bmj.com/ of antibiotics was to reduce the size of Barr bodies without altering the incidence. Further work on the effect of external agents on Barr body formation q' would certainly be of value. 0-1: -.-% Drumsticks: General Characteristics and A 'W .:...:1 Incidence on September 27, 2021 by guest. Protected Following the discovery of the sex chromatin in i; a large variety of cells, Davidson and Smith (I954) searched for a similar sex difference among the leucocytes of the peripheral blood and found one among the granulocytes. The sex specific body which is present in the blood films from females but I"? not from males consists of a head, about 1-5 ,. in diameter, which is attached to the body of the nucleus by means of a filament of varying length (Fig. i6). This appendage is commonly called a FIG. I5. Fibroblast nuclei in telophase, showingpossible re-formation 'drumstick', though some other names such as 'sex of Barr bodies. (Thionin, X 2,200.) nodules' have sometimes been given to it. The J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 62 Ursula AAittwoch

...,.SS., ....: *: S. 9SE:.Sk .. ..-

_. .g x @o S. . y.e .::..: . * . ::. . : . wk::*2_ . FIG. i6a s <. ~~~~~~~FIG.I6b .:... :s, :Xz .-_

.... x 11...10 s ...... I .:: .:..::: :.: :.:. ..: .. ::. ..: .: :: :.::

::: :.:.... .:: :::i: : *.* :.:: : :.:: :.:: :: . : ::.::.:. :

.: :: :: ... *:: .. .::...: .. .. : :: . ....: .: :: r* .... ::::..:: ...... :.:..5L.. ::..:.. C f:.:.:

...... t: X; copyright...... : ,: :::. ::.,FIG. I7a ._ FIG. 17b FIG. I 7C ..... ,.,.,.O2.'.;.,...... _r FIG. I6, a, b. Polymorphonuclear neutrophil leucocytes from a female, each showing a drumstick. (May-Grunwald-Giemsa,

.. - . .. .. : ; X http://jmg.bmj.com/ 2,200.) s.e, FIG. of the nuclei of polymorpho- .. 0 Non-specific appendages 17. nuclear leucocytes, which must be distinguished from drumsticks. (a) and (b) Small appendages, shaped like a drumstick but smaller and sometimes less intensely stained; (c) small lobe, distinguished from a drumstick by irregular outlines and contours, in addition to larger size. FIG. I8 a, b. Polymorphonuclear leucocytes from females, showing a 'sessile nodule'. (May-Grunwald-Giemsa, x 2,200.) on September 27, 2021 by guest. Protected

F '.

FIG. I8b FIG. i8a J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 63 occurrence and sex specificity of drumsticks was origin generally contain another type of appendage, soon confirmed by numerous investigators (e.g. consisting of a head which is attached more or less Wiedemann, Romatowski, and Tolksdorf, 1956; directly to the nucleus (Fig. i8). Such a structure Liiers, I956; and see Davidson and Smith, I963). is known as a sessile nodule (according to Kosenow Like the Barr bodies, drumsticks are stained by and Scupin, I956, drumsticks are called 'type A' nuclear dyes, including basic fuchsin (Feulgen). and sessile nodules 'type B' appendages). A sessile Blood films stained with Romanowsky stains, such nodule may be regarded as an incompletely devel- as the May-Grunwald-Giemsa technique, are oped drumstick; and varying degrees of develop- suitable for the demonstration of drumsticks; but ment may be observed. Although sex specific, for a proper evaluation a high technical standard of sessile nodules are more easily confused with non- preparation is necessary. For this reason blood films specific appendages than are drumsticks, and the made by the cover slip technique (Wintrobe, I96I) error inherent in their determination is greater. are preferable to those made on slides, since in From a practical point of view, sessiles are of value addition to ensuring a more thorough distribution mainly in corroborating the evidence obtained from of the different cell types, the cover slip method drumstick data. The 2 types of appendages should results in better cytological preservation. be scored separately. Drumsticks may be demonstrated on all types of The incidence of drumsticks in individual polymorphonuclear leucocytes, but for practical women varies very considerably. Davidson and purposes only those present on neutrophils are Smith (I954) found that among 125 blood films usually considered. from women, the incidence of drumsticks varied The average incidence of drumsticks is about i between I per 98 neutrophils (i %) to i per 6 neutro- per 38 neutrophils in the blood films from females, phils (I7 %), with an average of I per 38 cells and they are absent in males. However, the nuclei (2-9 %). These frequencies were arrived at by of neutrophils may show a variety of appendages enumerating the number of neutrophils containing that are not sex specific, and care must be taken to 6 drumsticks, and it should be noted that this distinguish these from true drumsticks (Fig. I7, see method results in a somewhat higher frequency of also Davidson and Smith, I963). These non-specific drumsticks (since the last cell always contains a copyright. appendages are distinguished from drumsticks by drumstick) than if a constant number of cells is (i) their size and shape, and/or (2) their less intense counted; this discrepancy is more marked if the staining (drumsticks, like Barr bodies, are highly incidence of drumsticks is low. Following Davidson condensed deeply staining bodies). The majority and Smith, it has become an accepted rule among of such non-specific appendages (small clubs) are many investigators that a blood film from a normal smaller than drumsticks (less than i [i in diameter); female should contain at least 6 drumsticks per 500 they may be intensely stained, and in such cases neutrophils, and though this rule is obeyed in the can be distinguished from true drumsticks only by majority of instances, there is a tail end of the http://jmg.bmj.com/ size (but see p. 66). More rarely, an appendage of distribution that falls outside this range. Whether the size of a drumstick may have an unstained high or low, the drumstick counts of individual centre ('tennis racket', Davidson and Smith, 1954), women show considerable constancy on repeated or an apparently larger appendage shows uneven occasions (Briggs and Kupperman, 1956; Briggs, staining, and generally also a more or less uneven 1958; Mittwoch, 196I). outline (Fig. I7c, 'minor lobe', Davidson and Smith, One factor that influences the incidence of drum-

1954). Minor lobes may be connected to the rest of sticks is the degree of lobing of the polymorpho- on September 27, 2021 by guest. Protected the nucleus by one or two filaments. nuclear nucleus, the more highly lobed cells having In practice, no doubt, an occasional structure of a higher incidence of drumsticks (Davidson and this type may look sufficiently similar to a drumstick Smith, I954; Briggs, 1958). Drumsticks are rare to be scored as such. Mittwoch (I96I) scored 5 in the Pelger anomaly (Davidson and Smith, 1954; drumsticks among I5,ooo neutrophils from males Luers and Petzel, I958; Siebner, Klaus, and Heni and suggested that an error of this order of magni- I963). Thus, many but not all women with low tude was unavoidable in the method of scoring drumstick counts also have low nuclear lobe counts drumsticks. This agrees with the opinion by Sohval (Briggs and Kupperman, I956). Mittwoch (I964a) (I963) that the finding of a single drumstick in less found that in I2 apparently normal women, in whom than a i,ooo cells has no certain diagnostic value. a detailed analysis of drumsticks was undertaken, In addition to drumsticks proper, in which the the incidence varied between i 6 and 7 2 %, and the head is attached to the rest of the nucleus by a single correlation coefficient between the incidence of thin filament, a proportion of neutrophils of female drumsticks and the mean lobe count was o056. J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from

64 Ursula Mittwoch TABLE DRUMSTICKS PER IOO NEUTROPHILS, AND MEAN LOBE COUNTS IN PATIENTS WITH CHROMOSOMAL ABNORMALITIES AND IN CONTROLS (from Nature (I964), 201, p. 317) Groups % Patients No. of Drumsticks Mean Lobe Counts Patients I IIII7IIIIII IV v For~~~NumbersAll Lobe Non-mongol metally retarded girls 28 0-48 1 59 3-19 5-68 6-83 2.74 2-6I Mongol girls 28 0-22 0°79 1 53 2-39 3-22 0'97 2-9 Klinefelters 12 0-77 o-83 I-87 2-36 (3-45) II27 2-26 Triple X females 3 77 2-26 220 392 207 2-26 Triple X Klinefelters 3 0.45 I 23 2*07 (I *56) I.45 2-22

Groups I-III. 500 cells were investigated from each patient. Groups IV and V. i,ooo cells were investigated from each patient. Numbers in brackets are based on less than Ioo cells.

When cells with constant lobe numbers were con- variable, so that different investigators counting sidered, it could be shown that in all women the lobes on the same blood film may arrive at widely drumstick count rose as the lobe number increased differing counts. It is clearly essential that when- from I to 4 or 5, but for each given lobe number the ever the results of lobe counts are reported these drumstick count of the woman with the highest must be compared with the values obtained from a number was about 3 or 4 times that of the woman sample of apparently normal individuals counted with the lowest number. by the same investigators. There is, as yet, no explanation for the observed variation in drumstick counts. However, there is evidence that the frequency of drumsticks is some- Drumsticks in Patients with Chromosomal what lower during the menstrual cycle and that this Abnormalities copyright. is due to hormonal influences (Caratzali, Phleps, and In mongolism (Down's syndrome) it was shown Turpin, 1957; Caratzali, I963). Hormonal in- (Turpin and Bernyer, I947) that the nuclei of the fluences might also account for the increased inci- polymorphonuclear leucocytes have, on an average, dence of drumsticks in newborn infants, and of their fewer lobes than those of unaffected subjects, and mothers immediately before and after parturition, Mittwoch (I957) reported that the over-all incidence which has been reported (von Harnack and Striet- of drumsticks in i8 girls with mongolism was only a and Oehme, 1956). An increase fraction of that of I 8 non-mongol mentally defective zel, I956; Peiper http://jmg.bmj.com/ in the incidence of drumsticks as a result of adreno- girls. corticotrophin (ACTH) and insulin has also been In Klinefelter's syndrome, an incidence of drum- noted (Caratzali and Phleps, 1958). In this connex- sticks considerably below that of normal females ion, it is of interest that in the case of Barr bodies was noted by Davidson and Smith (I958). Mitt- a decrease has been observed both in the newborn woch (I96I) found the low incidence of drumsticks (Smith et al., I962; Taylor, I963b) and as a result in these patients to be associated with decreased of cortisone (A. I. Taylor, i964, personal com- polymorphonuclear lobing. A low drumstick count munication). A slight decrease in the incidence of coupled with a low lobe count has been noted in a on September 27, 2021 by guest. Protected drumsticks with increasing age of the individual has few individual patients with XXXXY Klinefelter's been described by Wiedemann (1958) and Tono- syndrome (Miller, Breg, Schmickel, and Tretter, mura, Toyofuko, and Matsunaga (I962). de Castro I96I; Fraccaro et al., I962; Barr, Carr, Pozsonyi, (I963) reported a low incidence of drumsticks in Wilson, Dunn, Jacobson, and Miller, I962). cachectic women, while fasting rabbits showed first The Table contains drumstick frequencies and a rise, and then a drop, in the drumstick count. mean lobe counts of patients with various chromo- Since the degree of lobulation of the neutrophil somal abnormalities and of mentally defective nucleus is an important factor in determining drum- children, the majority of whom may be assumed to stick formation, lobe counts must be carried out have normal chromosomes. The incidence of drum- whenever quantitative findings on drumsticks are of sticks in the mentally retarded controls is very close importance, or where the absence of drumsticks to that found in normal women (Davidson and requires to be evaluated. Unfortunately, the Smith, I954), but all groups of patients with criteria employed in enumerating lobes are very chromosomal abnormalities have a lower incidence J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin of drumsticks, coupled with a lower mean lobe high incidence of drumsticks in 3 patients, the count, than the controls. Thus the mean frequency incidence in 5 further patients quoted by them is of drumsticks for mongol girls actually falls below below normal. In 8 adult patients with XXX sex the value of 6 drumsticks per 500 neutrophils, often chromosomes studied by Maclean (I962) the average regarded as the lower limit for normal female blood incidence of drumsticks was only i-5%; though films. four newborn infants had an average incidence of One rather remarkable fact, which is borne out 5 %, this is below the average incidence of drum- by the data in the Table, is that the patients with sticks reported for newborn and premature infants chromosomal abnormalities not only have lower (von Harnack and Strietzel, I956; Peiper and drumstick and lobe counts than the controls when Oehme, I956). The data so far suggest that, al- the average for all cells is considered, but that the though there may be considerable variation in indi- incidence of drumsticks is lower in cells of constant vidual cases, the average incidence of drumsticks lobe numbers. This suggests that in at least some in patients with three X chromosomes, both with conditions associated with chromosomal abnormali- and without a Y chromosome, is below normal; ties, the formation of drumsticks is inhibited to an while the admittedly limited data shown in the Table even greater extent than that of lobes (Mittwoch, indicate that in this condition also there may be an I964a). It is conceivable that hormonal influences, inhibition of nuclear lobing. as described by Caratzali et al. (1957) and Caratzali Jacobs et al. (I959) reported the presence of a (I963), may be responsible for the additional single cell with 2 drumsticks in a patient with XXX shortage of drumsticks. sex chromosomes. Both the existence and the The drumsticks in patients with 3 or more X rarity of double drumsticks has been confirmed by chromosomes present a special problem. As was Maclean (I962) who, surveying a large number of mentioned on p. S55 the Barr bodies in such cells in I2 patients with XXX sex chromosomes, patients present a very characteristic picture, since found an incidence of such cells of less than I in the maximum number of Barr bodies per cell is one 2,000 (Fig. I9). Two sessile nodules (Fig. 20) or less than the number of X chromosomes. In i drumstick and i sessile nodule occur somewhat

addition, the proportion of cells containing Barr more frequently than 2 fully filamented drumsticks copyright. bodies is also higher than in normal females (e.g. in the same cell. In an infant with 5 X chromosomes, Jacobs et al., 1959; Barr et al., 1959; Ferguson- described by Kesaree and Woolley (I963), there Smith et al., I960). By contrast, the incidence of were a number of cells with 2 drumsticks and some drumsticks in such patients appears to be below with 3, though no cells were found with 4 drum- normal. This is particularly pronounced if a sticks. The conclusion seems justified that the Y chromosome is also present (e.g. Barr et al., I959; theoretical maximum number of drumsticks, like Ferguson-Smith et al., I960; Maclean I962). that of Barr bodies, is one less than the number of Regarding female patients with XXX sex chromo- X chromosomes, but that owing to the low fre- http://jmg.bmj.com/ somes, although Johnston et al. (I96I) reported a quencies of drumsticks, particularly in chromo- \ on September 27, 2021 by guest. Protected ...:. ^:S.: :::...:.t 4.

:.:. ..;

:: "'' .. :*: .,:' ;v ..;. FIG. 19. Polymorphonuclear leucocyte with two drumsticks from a FIG. 20. Polymorphonuclear leucocyte with two sessile nodules from triple X female. (May-Grunwald-Giemsa, X 2,200.) (By courtesy a triple X patient. (May-Grunwald-Giemsa, X 2,200.) of the Editor of Cytogenetics.) .. <. < J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 66 Ursula Mittwoch

FIG. 2I, a, b. Polymorphonuclear leucocytes with large drumsticks from (a) an XXX female, (b) an XXXY male. Large drumsticks occur rarely in patients with multiple X chromosomes, but are frequent in patients with iso-chromosomes of the large arm ofthe X. somal abnormalities, the maximum number may be a head of normal size having a diameter of I.5 ,U rarely, if ever, reached. while the diameter of the Barr body is about i ,. Maclean (I962) also compared the size of drum- Davidson and Winn (I96I), who measured the sticks from patients with structurally abnormally ratios of the areas of drumsticks and Barr bodies X chromosomes with that of drumsticks from to their respective nuclei from enlarged prints, normal females. The 'drumstick/nucleus' ratio was found that the drumstick occupied a larger relative as area than the Barr in relation to the rest of the estimated D x ioo body

nucleus. Although the authors concluded that thiscopyright. N-D ' difference might be explained by difference in the where D = area of drumstick and N = area of shape and thickness of the nuclei in polymorpho- nucleus, in 3 patients with iso-chromosomes of the nuclear and epithelial cells, the alternative, i.e. that long arm of the X, i patient with a deletion of the drumsticks and Barr bodies are intrinsically different short arm of the X, and 2 normal female subjects. structures, remained for some time a possibility These studies indicated that the drumsticks of (Klinger, I957; Ashley, I962). Ashley (1957) iso-X chromosomes were larger, and those from thought that he could detect Barr bodies and drum- deleted X chromosomes were smaller, than those of sticks in the same cells. It should be remembered, http://jmg.bmj.com/ normal females. The results of Bamford, Cassin, however, that peripherally placed chromatin masses and Mitchell (I963) are in agreement with this. occur not infrequently in the leucocytes of both Although large drumsticks are primarily associ- sexes. ated with iso-chromosomes of the long arm of the In the majority of instances the results of tests on X, they may also be found, though much more Barr bodies and drumsticks in the same individuals rarely, in patients with multiple X chromosomes are in agreement, but a number of apparent dis- (Fig. 21). In general, the detection of large drum-

crepancies have been described. For instance, on September 27, 2021 by guest. Protected sticks will present little difficulty to an experienced Kosenow (I957) found that among IS patients with observer, since their intense staining and regular gonadal dysgenesis, i i had neither Barr bodies nor outline distinguishes them from other parts of the drumsticks, 3 had both Barr bodies and drumsticks, nucleus. On the other hand, it is sometimes not while i had Barr bodies in the buccal mucosa but possible to distinguish small drumsticks from other no drumsticks in the blood. The opposite situation, non-specific nuclear appendages. i.e. patients who lacked Barr bodies but in whom drumsticks were found, was reported by Ashley and The Relation of Drumsticks to Barr Bodies Jones (I958). Other apparent discrepancies between The significance of drumsticks and their relation findings in the buccal mucosa and in the blood were to Barr bodies remained in doubt for some time for described by Overzier (I958), Izakovic (I960), a number of reasons. To begin with the mor- Greenblatt, Manautou, Tigerman, Rogers, and phological appearances of the 2 structures in stained Sheffield (I957), Greenblatt, Aydar, and Gibson preparations are different. The drumstick is larger, (I962), and Turner and Zanartu (I962). J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from

Sex Chromatin 67 While a few of such results may be due to faulty drumsticks. It is considered by most haematolo- techniques, the results of chromosome analysis have gists that the lobulation of the nucleus gives a fairly shown that a considerable proportion of patients good indication of the age of the cell, the youngest with abnormalities of the sex chromosomes are, in cells having a nucleus shaped like a letter C, while fact, mosaic, with different sex chromosome com- old cells have 4 or 5 lobes (Whitby and Britton, plements in different cells (see Ford, I963). In the I963). As it appears that chromosomal abnormali- extensive data by Lindsten (I963), over one-third ties may interfere with the normal development of of the patients with Turner's syndrome were mo- these nuclei, the term 'maturity' may be preferable saics with XO cells and others containing a second to 'age'. X chromosome. In some cases the different karyo- Many problems of drumstick formation remain types are intermingled while in others different to be solved, together with related problems of the tissues are characterized by different chromosome physiology and pathology of the leucocytes. Simi- constitutions. Jacobs et al. (I960) described a larly, problems remain regarding details of the for- patient with Turner's syndrome, whose bone mation of Barr bodies, and further studies on the marrow cells appeared to be exclusively XO, while effect of hormones and other external agents on the the skin was predominantly XXX, but contained formation of drumsticks and Barr bodies are also XO cells. The buccal mucosa contained cells required. with single and double Barr bodies, but no drum- Nevertheless, work of recent years has made it sticks were present in the leucocytes. Thus the sex clear that the original suggestion by Davidson and chromatin findings are exactly as in the patient Smith (I954) that the drumstick is equivalent to described by Overzier (I958) before the advent of the Barr body was valid. In present-day terms, it chromosomal analysis. The results of chromosome may be concluded that both the Barr body and the studies have shown that many of the so-called drumstick of mammals are true sex chromatin, discrepancies between Barr body and drumstick representing heteropycnotic X chromosomes that findings provide, in fact, confirmation that both are present in addition to the first one. structures represent the sex chromatin in the cells in which they occur. copyright. Other apparent differences between drumsticks Sex Chromatin in Mammals Other than Man and Barr bodies have also been largely explained. A sex difference is found in the interphase nuclei As pointed out by Sohval (I963), some of the so- of many mammals, and the domestic cat was the called discrepancies between the 2 techniques are first species in which this was described. merely reflections of differences in the frequencies Moore and Barr (I953) undertook a systematic with which Barr bodies and drumsticks occur. investigation of the nerve cell nuclei of eutherian While Barr bodies may be seen in more than % mammals, with the following results. A sex differ- go http://jmg.bmj.com/ of suitable cells (Klinger, I957), drumsticks occur ence was found in the dog (Canis familiaris), mink only rarely in as much as I0 % of neutrophils, and (Mustela vison Schreber), marten (Martes americana their average incidence in normal females is about Turton), ferret (Mustela furo L.) raccoon (Procyon 3 %. Moreover, in patients with Klinefelter's lotor L.), and skunk (Mephitus mephitus Schreber), syndrome the incidence of Barr bodies appears to all of which belong to the order Carnivora; and in be no different from that of normal females, but the the goat (Capra hircus L.) and the deer (Odocoileus frequency of drumsticks is abnormally low (see virginianus Boddaert), both of which are members p. 64). It should be pointed out that, if only a few of the Artiodactyia. Cells from five regions of the on September 27, 2021 by guest. Protected drumsticks are to be expected within a given number nervous system were examined (pyramidal cells, of cells, none at all may be found in certain samples Purkinje cells spinal cord, dorsal root ganglion cells, merely by chance. As was described above (p. 64), and stellate ganglion cells). More than 8o % of such evidence has now been adduced that both the inci- cells from female animals contained a sex chromatin dence of drumsticks and the degree of lobing is mass, most of them adjacent to the nucleolus; likely to be below normal in a variety of chromo- similar masses were observed in up to 13 % of male somal abnormalities, and that, of the two, the cells, but usually the frequency was less than I0 %. reduction in the frequency of drumsticks is the more In the following animals a sex difference could severe. not be observed: rat (Rattus norvegicus Exleben), It is becoming clear that the drumstick is an mouse (Mus musculus L.), hamster (Cricetus auratus appendage of a morphologically highly specialized Waterhouse), and groundhog (Marmota monax L.), cell, and that the factors that cause the nucleus to belonging to the order Rodentia; and the rabbit divide up into lobes also affect the extrusion of (Oryctologus cuniculus L.), a member of the Lago- J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from

68 Ursula Mittwoch morpha. In these animals the cells of both sexes none have been found in rodents in which both tended to contain coarse chromatin clumps, usually sexes have non-specific appendages. Drumsticks adjacent to the nucleolus, which obscured any sex are present in the rabbit. chromatin that might have been present. The fact The reason why sex chromatin is clearly visible that sex chromatin is present in members of the in some animals and not in others, or only in certain carnivores and absent, or at least difficult to detect, cells of some animals, is not yet known. The in rodents and rabbits, has been largely confirmed by question is of fundamental importance, however, 1 ter workers (see Barr, I963, for review). In the for it touches on the significance ofthe sex chromatin rat (rodent), Klinger (I957) was able to demonstrate body itself. The problem that must be answered is sex chromatin only in spinal cord nerve cells; whether the difficulty of demonstrating sex chro- Shantaveerappa and Bourne (I962) found sex chro- matin in certain animals, e.g. rodents, is merely a matin in pia-arachnoid membrane and perineural technical one due to the 'coarseness' or clumping of epithelium of female rats. Klinger was also unable the chromatin, or whether the presence or absence to show sex chromatin in the rabbit, but Hulliger, of such clumps reflects a fundamental difference in Klinger, and Allgower (I963) found sex chromatin the organization ofthe DNA in different cells. This to be clearly visible in rabbit cells cultured in vitro. question will be further discussed later (p. 7I). Melander (I962) found sex chromatin in the rabbit from the fifth day of embryonic life onwards. Hay (I960) was unable to demonstrate sex chromatin in Sex Chromatin in Animals with Female the armadillo (Edentata) and the prairie dog and the Heterogamety mouse, both of them rodents. However, in another Following the discovery of a heteropycnotic body rodent, the porcupine, sex chromatin was present. in cells of female larvae of the spruce budworm, Prince, Graham, and Barr (i955) found a well- Choristoneura (Archips) fumiferana by Smith (I944, marked sex difference in the rhesus monkey, 1945a, b), Frizzi (1948) found a similar sex differ- Macacus rhesus. Hamerton, Fraccaro, de Carli, ence in the silk worm (Bombyx mori L.). Cells from Nuzzo, Klinger, Hulliger, Taylor, and Lang (I96I) the storage cells of the silk glands of larvae, which and Hamerton, Klinger, Mutton, and Lang (I963) were known to be female from the histology of thecopyright. found sex chromatin in the buccal mucosa of the gonads, contained a heteropycnotic body that was female gorilla and other apes belonging to the never found in the cells from males. The position Hominoidea. of the heteropycnotic body was variable, occurring In the hoofed animals (Artiodactyla) sex chro- either in the centre of the cells or at the poles of matin can be distinguished in neurones, but in elongated cells. Occasionally 2 bodies were seen in other cells the chromatin tends to be too coarse for the same cell. This body provided a simple means larvae from the second instar the distinction to be made (see Barr, I963). A for sexing onwards, http://jmg.bmj.com/ particularly interesting application of the nuclear when other means of sexing are very laborious. sexing method is the demonstration ofsex chromatin The silkworm is an animal of economic import- in the neurones of the bovine freemartin by Moore, ance and, though in common with other Lepidop- Graham, and Barr (I957). A freemartin is a sterile tera its chromosomes are not favourable for cyto- heifer, which is always born as a twin to a normal logical studies, a good deal is known about its male. The external genitalia are predominantly genetics (Tanaka, I953). At least 5 genes are known female, while the gonads resemble testes. The that show sex-linked inheritance (Tanaka, 1953), findings of sex chromatin confirm the interpretation and there can be no doubt that the female is the on September 27, 2021 by guest. Protected (Tandler and Keller, I9II; Lillie, I9I6) that the heterogametic sex. Frizzi (1948) postulates that the freemartin is a genetical female that has become heteropycnotic body represents the condensed masculinized as a result ofmale hormones, following Y chromosome. the fusion of chorions and vascular anastomosis in Another class of animals, in which the female is early embryonic life. the heterogametic sex, are the birds. Kosin and Drumsticks too are present in certain animals but Ishizaki (I959) found sex chromatin in interphase not in others. The subject has been recently cells of female chickens (Gallus domesticus). Cells reviewed by Davidson and Smith (I963). It is of smooth muscle of the duodenum as well as der- interesting that, in the various groups of animals, mal and epidermal cells at the base of growing there appears to be a close correlation between the feathers were particularly favourable for the demon- presence or absence of drumsticks with that of Barr stration of the sex chromatin body. This occurred bodies. Thus drumsticks have been demonstrated with an incidence varying between I5 and 56 % in in primates, carnivores, and hoofed animals, but cells from female birds, while a similar body was J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 69 seen in 0 4-5 4 % of male cells. The sex chromatin pattern of both X chromosomes in the male, as well body was situated at the periphery of the nucleus as the single X chromosome in the female, was not and its appearance resembled that of the Barr body different from that of the autosomes. However, in in mammals. the female, one of the larger micro-chromosomes, Kosin and Ishizaki conclude from their findings which appeared to be identical with the Y chromo- that the sex chromatin is an incompletely sex- some described by Frederic, exhibited a very heavy limited trait which is not necessarily related to the and comparative late uptake of thymidine. X chromosome. Although the data on sex chromatin in animals The finding of sex chromatin in the fowl was with female heterogamety available so far are scanty confirmed by Ohno, Kaplan, and Kinosita (I960) compared with those obtained in mammals, the and by Moore and Hay (I96I), while the presence of problems that are raised are of the greatest impor- sex chromatin in female chick embryos was also tance for a general understanding of the subject. reported by Ishizaki and Kosin (I960). Schmid's (I962) finding that the Y chromosome of On the other hand, neither Ashley and Theiss the hen shows late DNA replication is in agreement (i959) nor Miles and Storey (I962) were able to with the observation (Schmid, I963) that the Y demonstrate sex chromatin in the fowl. Ashley chromosome in man is similarly late labelling. On and Theiss examined sections of various tissues the other hand, the evidence available so far suggests from the fowl, duck, parrot, and parakeet. They that the situation with regard to the X chromosome were unable to establish a sex difference, since cells is very different from that found in mammals, as from both males and females contained many there appears to be no sex chromatin in the inter- chromatin crosses. Miles and Storey who exam- phase nuclei of cocks, while the DNA-labelling ined cells from the fowl, both grown in vitro as well pattern of both X chromosomes in the male and the as sections from various tissues, also found chromo- single X chromosome is like that of autosomes. centres present in both sexes. Since any comprehensive theory on the significance In the fowl at least i i sex-linked genes are known ofsexchromatin and heteropycnosis in mammalswill proving beyond doubt that the female is the hetero- have to take account of the findings in animals with

gametic sex (Hutt, I949). Chromosome analysis in female heterogamety, further studies of these copyright. birds has always proved difficult owing to the phenomena in birds and Lepidoptera will clearly presence of large numbers of so-called micro- be of the greatest importance. chromosomes, some of which are at the limit of the resolving power of the light microscope in size. In addition, there are a number of pairs of chromo- Some Practical Applications of Sex somes of ordinary size, the macro-chromosome. Chromatin Determinations According to Ohno (I96I), the domestic fowl has The results of sex chromatin studies provided the 8 pairs of macrochromosomes, of which the fifth first indications of discrepancies between the http://jmg.bmj.com/ largest is unpaired in the female and paired in the genetical and phenotypic sex in some errors of male and therefore represents the X chromosome. human sexual development. Similarly, in present- Many authors, including Ohno, use the termin- day medical genetics, the presence or absence of ology ZZ and WZ to represent the X and Y chromo- Barr bodies and drumsticks will usually give the somes in animals with female heterogamety. first information regarding the numbers of X However, the XY terminology in female hetero- chromosomes present in patients in whom abnor- of the X chromosome may be suspected. gamety is simpler and is in accordance with the malities on September 27, 2021 by guest. Protected usage of Winge (1932); it has been adopted here. In general, the sex chromatin test will be most Regarding the presence of a Y chromosome in useful in detecting the classical XO type of Turner's female chickens, Ohno (I96I) did not arrive at a syndrome and the XXY type of Klinefelter's syn- definite conclusion, but Frederic (I96I) provided drome, as well as karyotypes with multiple X evidence that a Y chromosome is present in the chromosomes, but the test will not usually detect form of one of the largest microchromosomes. chromosomal mosaics, unless the X chromosome The total number of micro-chromosomes in the constitution is strikingly different in different fowl is thought to be about 6o (Ohno, I96I; Fred- tissues, as in the patient of Jacobs et al. (I960). For eric, I96I; Stenius, Christian, and Ohno, I963). instance, the detection of a single Barr body in a Tht problem of sex chromatin in the fowl was proportion of buccal mucosa cells in a male patient further tackled by Schmid (I962), who treated will suggest the presence of an XXY karyotype, but chicken cells grown in tissue culture with radio- it will not give any information regarding further active thymidine. He found that the labelling cell lines that might have XO, XY, or XX sex J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 70 Ursula Mittwoch chromosome constitutions. Also, in rare instances, Bergemann (I96I), comprising 3,800 males and the karyotype might be XXYY (Muldal and Ockey, 3,600 females, the incidence of Klinefelter's syn- I960). drome at birth may be estimated at about 2 per In patients with Turner's syndrome, who are i,ooo, that of the triple X condition at about i per XO/XX mosaics, sex chromatin is usually present i,ooo and that of Turner's syndrome without sex (de la Chapelle, I962), and such patients will not chromatin about 4 per I0,000. As was pointed out generally be detected as a result of sex chromatin above, this approach is likely to miss those cases of testing. A low incidence of sex chromatin bodies Turner's syndrome that are mosaics or have iso- may raise the suspicion of a mosaicism of this type, chromosomes. In the data by Lindsten (I963), sex but, particularly in buccal mucosa, a low incidence chromatin was present in the buccal mucosa of 20 of Barr bodies may occur for other reasons. out of 57 patients with Turner's syndrome, and in Patients with iso-chromosomes of the long arm the data by de la Chapelle (I962) there were I0 such of the X chromosome (XI) tend to have a high patients out of 23. As a first approximation it may proportion of the large Barr bodies (Jacobs et al., be assumed that the incidence of Turner's syndrome I960; de la Chapelle, I962; Lindsten, I963). The is about a third to a quarter higher than would be drumsticks are also larger and much more frequent estimated from sex chromatin studies. While the than normal (Maclean, I962; Bamford, Cassin, and results of these three surveys are in good mutual Mitchell, I963; de la Chapelle, I962); since they agreement a sex chromatin survey on newborn occur with a frequency of I0 per ioo neutrophils, babies in India (2,058 males and I,832 females) large drumsticks occurring with lesser frequencies failed to show any abnormalities (Subray and may suggest the presence of mosaicism of the type Prabhaker, I962). XXi/XXo with a fair degree of accuracy. Smaller Sex chromatin surveys on male patients in mental than normal Barr bodies are associated with deleted deficiency hospitals have been carried out by X chromosomes (Jacobs et al., I960, I96I; Lindsten, Mosier, Scott, and Cotter (I960), Barr, Shaver, I963). Carr, and Plunkett (I960), Ferguson-Smith et al. Although the drumsticks in such cases may also (I960), Sanderson and Stewart (I96I), Maclean,

be smaller than normal, small drumsticks are diffi- Mitchell, Harnden, Williams, Jacobs, Buckton,copyright. cult to distinguish from similar looking appendages Baikie, Court Brown, McBride, Strong, Close, and of the neutrophils that are not sex specific. Jones (I962), Hamerton, Jagiello, and Kirman (I962) From a genetical point of view, the most impor- and Davies (I963). Out of a total of 7,305 patients tant application of sex chromatin testing has been in examined, 67 were found to have sex chromatin, establishing the incidence of abnormalities of the giving an incidence of just under i %. This inci- sex chromosomes as a result of screening large dence is several times as high as that found at birth. populations. Preparations made from the oral The incidence of Klinefelter's syndrome is also mucosa are the most suitable for this purpose and increased among boys attending schools for the http://jmg.bmj.com/ have been used almost exclusively. The survey by educationally subnormal (see Israelsohn and Taylor, Shapiro and Ridler (I960) is exceptional in being I96I). based on leucocyte morphology, while Ridler, Surveys on female patients were carried out by Shapiro, and McKibben (I963) have utilized both Fraser, Campbell, MacGillivray, Boyd, and Lennox buccal mucosa and leucocytes. (I960), Sanderson and Stewart (I96I), Johnston Sex chromatin surveys have been carried out on et al. (I96I), Maclean et al. (I962), Hamerton et al.

series of newborn infants as well as on populations (I962), Davies (I963), and Ridler et al. (I963). The on September 27, 2021 by guest. Protected of the mentally retarded. The possibility of making total number of patients' surveys was 4,575, and sex chromatin surveys as the basis of post-mortem among these 9 patients with duplicated Barr bodies material has recently been suggested by Edwards were found, giving an incidence ofabout 4 per I,000. and Cameron (I964). In these patients an XXX sex chromosome consti- The largest survey on the sex chromatin of new- tution was either verified or may be assumed, and borns was carried out by Maclean, Harnden, Court it is clear that this condition also has a higher in- Brown, Bond, and Mantle (I964) who examined cidence among the mentally retarded than is found 10,725 liveborn males, born consecutively, and at birth. Among this group of patients only one had io,ooo females. Among the male infants, 21 were no Barr bodies (Maclean et al., I962), and another found to contain single Barr bodies, and among the patient with an iso-chromosome of the long arm of females, I2 had double Barr bodies, and in 4 no the X was identified by Hamerton et al. (I962). Barr bodies could be found. On the basis of this Judging from these data, the incidence of Turner's and 2 earlier surveys by Moore (i959) and by syndrome among the mentally retarded is about 4 J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 71 per io,ooo, and there is, therefore, no evidence that There can be no doubt that dosage compensation this is any higher than among the newborn. occurs also in mammals. For instance in man, the Thus, in spite of certain limitations of the buccal gene controlling the production of glucose-6- smear technique for sex chromatin investigations, phosphate dehydrogenase in erythrocytes is located it has provided results of the greatest interest to on the X chromosome, yet the enzyme is present in human cytogenetics. It is clear that without this equal amounts in males and females (Marks, I958) technique estimates of the frequencies ofthe various as well as in patients with abnormal numbers of abnormalities of the X-chromosome could not have X chromosomes (Grumbach, Marks, and Mori- approached the accuracy of those that are now shima, I962; Harris, Hopkinson, Spencer, Court available. Brown, and Mantle, I963). In the mouse, animals with an XO karyotype are viable and fertile females (Welshons and Russell, I959). Some Theoretical Considerations In the mouse, Lyon (I963) has presented evidence Concerning Sex Chromatin that in females heterozygous for 2 pairs of genes, The possibility that the sex chromatin body might with one gene borne on the X chromosome and the represent the second X chromosome which, being other on a piece of autosome translocated to the genetically superfluous, is present in a condensed other X chromosome, only i of the genes is acting and inactivated form, was suggested by Stewart in any patch of cells. It is postulated that the X (I960). Russell (I96I) on the basis of data relating chromosome bearing the translocation acts an integ- to the effect ofrearrangements ofthe X chromosome ral whole. Ohno and Cattanach (I962) have com- in the mouse formulated the hypothesis that in bined genetical studies of this type with cytological mammals any X chromosome in addition to the first observations. They used female mice having one assumes the properties of heterochromatin. How- normal X chromosome, Xn, and one bearing a ever, the inactive X hypothesis in its wider form translocation Xt, which made it i8 % longer than was advanced and developed by Lyon (I96I, I962, normal. The translocated chromosome contained I963). the wild type allele of the gene albinism which, in

The Lyon hypothesis consists of 3 parts. First, this combination, produces patches of wild-type copyright. it postulates that the genes on the sex chromatin colour alternating with patches of white. The forming X chromosome are inactive; secondly, authors found that in the prophase of mitosis in skin that the decision as to which of a pair of X chromo- cells, one chromosome was more condensed than the somes is to be inactivated is made early in embryonic rest. Moreover, it appeared that the condensed life and that, once the decision is made, the des- chromosome in cells from the white patches was cendants of each X chromosome will be like the larger than the condensed chromosome from the parent chromosome; and thirdly, that the original wild type patches, suggesting that the genes on the inactivation in each embryonic cell occurs at ran- condensed chromosome do not affect the phenotype. http://jmg.bmj.com/ dom, so that in some cells a paternal and in others a Further work along these lines would be most maternal X chromosome will be inactivated. There- desirable. At present, it is one of the ironies of this fore, as the embryo grows, patches of cells with subject, that although the theory of X-inactivation inactive X chromosomes of paternal origin will was originally formulated on the basis of genetic alternate with inactive X chromosomes of maternal data on the mouse, this species has proved so recal- origin. The theory was originally developed to citrant to the cytological demonstration of sex

explain the fact that female mice that are heterozy- chromatin. It may well be that the difficulties in this on September 27, 2021 by guest. Protected gous for sex-linked genes are piebald (Lyon, I96I). respect are merely technical, due to the presence of Since the effect of the theory would be that animals non-specific chromocentres. Nevertheless, the of both sexes have only a single dose of sex-linked significance of these chromocentres requires eluci- genes, it is thought to provide the basis for dosage dation. compensation in mammals (Lyon, I963). In man, work on 2 genes borne on the X chromo- The term 'dosage compensation' was introduced some is of particular relevance to the Lyon hypo- by Muller (I932) to describe the fact that in Dro- thesis. Women who are heterozygous for the gene sophila the effect of genes borne on the X chromo- causing glucose-6-phosphate dehydrogenase de- some is about the same in males where they occur ficiency tend to have an enzyme level that is inter- in single dose, and in females where they are present mediate between that of normal and deficient in double dose. Muller's explanation was that the subjects, and Beutler, Yeh, and Fairbanks (I962) genes on the X chromosome were balanced in such and Beutler and Baluda (I964), have pre- a way that the effects of dosage were cancelled. sented evidence that the erythrocytes of such J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 72 Ursula Mittwoch women are composed of 2 classes, with normal chromatin is formed in the embryo (p. 54) and and very low enzyme levels respectively. This therefore all X chromosomes might be presumed to finding was confirmed by Davidson, Nitowsky, be active. The possibility has also been raised that and Childs (I963) on cells grown in tissue culture. the X chromosome may have other effects on These authors succeeded in growing clones from embryonic development in addition to the mono- single cells and found that such clones from hetero- genic ones with which the Lyon hypothesis is zygous women either had a normal enzyme level specifically concerned (McKusick, I962). This, in or were deficient. turn, is connected with the bigger problem of the However, in women who are heterozygous for the role of the X chromosome in sexual differentiation sex-linked blood groups antigen Xg (Mann, Cahan, (Stern, I963). Gelb, Fisher, Hamper, Tippett, Sanger, and Race, The past io years or so have seen a tremendous i962), Gorman, di Re, Treacy, and Cahan (I963) increase in our knowledge of what we might call were unable to separate 2 classes of cells and Reed, formal human cytogenetics. Chromosomal abnor- Simpson, and Chown (I963), using a fluorescent malities have been shown to be the cause of a variety antibody technique, found that all the red cells from of human malformations; and the discovery of the such a woman contained the antigen. In a recent sex chromatin, and the development of suitable editorial (Lancet, I963) the following reasons for techniques to demonstrate it, have added a valuable this discordant finding were suggested: the X test to routine pathology. At the same time, we are chromosome may not be inactivated over its entire still far removed from any real understanding as to length (evidence that inactivating is not total has the physical part played by the chromosomes in the been obtained by Russell (I963) for the mouse); development of the cells and the organism which the antigen may be diffusible; or (unlikely in this we know they control. We do not know the function case, but very likely in some other instances) one of the chromosomes in normal development or in race of cells may have a selective advantage over the the pathogenesis of abnormalities. The Lyon other. It is clear that far more work is required on hypothesis has focused attention on some of the the behaviour of sex-linked genes in man and, problems involving the X chromosome. To solve

indeed, on the behaviour of autosomal genes also, these problems we shall require much more know-copyright. to enable one to make a full evaluation of the Lyon ledge of the relation between cytogenetics and the hypothesis in terms of gene action on the human emergence of the phenotype in man and other X chromosome. animals. On a more strictly cytological level, there are also Stern (I960) wrote: 'Dosage compensation forms many nuclear aspects that need to be resolved. For i chapter in the history of genetic systems. Its instance, the maximum number of Barr bodies is analysis is not yet a closed chapter.' Indeed, it has not present in every nucleus (p. 54). The reason only just begun and current work on sex chromatin for this is not yet known, and until it is we cannot will undoubtedly advance its development. http://jmg.bmj.com/ say what effect such nuclei have with respect to the action of their X chromosomes. REFERENCES A finding that is not strictly in accordance with Ashley, D. J. B. (1957). Occurrence of sex chromatin in the cells the Lyon hypothesis of random inactivation of the of the blood and bone marrow of man. Nature (Lond.), 179, 969. (1962). Human Intersex. Livingstone, Edinburgh and London. paternal or maternal X chromosome is that in - and Jones, C. H. (I958). Discrepancies in the diagnosis of patients with iso-chromosomes of the long arm of genetic sex by leucocyte morphology. Lancet, I, 240. the -, and Theiss, E. A. (I959). Nuclear sex in species showing male this chromosome appears to be invariably on September 27, 2021 by guest. Protected the X, homogamety. Anat. Rec., 135, 115. late labelling one (Muldal et al., I963; Giannelli Atkin, N. B. (I960). Sex chromatin and chromosomal variation in I963). However, in the present state it seems hardly human tumours. Acta Un. int. Cancr., I6, 41. -, and Klinger, H. P. (I962). The superfemale mole. Lancet, 2, fair to test the hypothesis in such an extreme 727. situation as that provided by the presence of an iso- Atkins, L., Book, J. A., Gustavson, K. H., Hansson, O., and Hjelm, M. (I963). A case of XXXXY sex chromosome anomaly with chromosome. On the other hand, there is as yet no autoradiographic studies. Cytogenetics, 2, 208. cytological evidence that in the case of normal X Austin, C. R., and Amoroso, E. C. (i957). Sex chromatin in early X chromosome cat embryos. Exp. Cell Res., 13, 419. chromosomes a Barr body forming Bamford, S. B., Cassin, C. M., and Mitchell, B. S. (I963). Sex invariably gives rise to another Barr body following chromatin determinations in selected cases of developmental sex mitotic division. abnormalities with an assessment of results. Acta cytol. (Philad.), 7, I5I. At present, we do not know the reason for the Barr, M. L. (I95i). The morphology of neuroglial nuclei in the cat, observed abnormalities in patients with abnormal according to sex. Exp. Cell. Res., 2, 288. - (1955). The sex chromatin and its application to errors in sex numbers of X chromosomes. It is conceivable that development. In Modern Trends in Obstetrics and Gynaecology, such abnormalities are laid down before the sex 2nd series, ed. K. Bowes, p. 117. Butterworth, London. J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from

Sex Chromatin 73

(I963). The sex chromatin. In Intersexuality, ed. Claus -, and Winn, S. (I96I). The relationship between the sex nodule Overzier, p. 48. Academic Press, London and New York. and the sex chromosomes. In Proceedings of the Conference on London ed. W. M. -, and Bertram, E. G. (1949). A morphological distinction Human Chromosomal Abnormalities, 1952, between neurones of the male and female, and the behaviour of Davidson, and D. R. Smith, p. 28. Staples Press, London. the nucleolar satellite during accelerated nucleoprotein synthesis. Davies, T. S. (I963). Buccal smear surveys for sex chromatin. Brit. Nature (Lond.), I63, 676. med. Jr., I, I541. -, H. A. (I950). The morphology Bertram, L. F., and Lindsay, DeMars, R. (I962). Sex chromatin mass in living, cultivated human of the nerve cell nucleus, according to sex. Anat. Rec., 107, 283. cells. Science, I38, 980. -, and Carr, D. H. (I960). Sex chromatin, sex chromosomes and Edwards, J. H., and Cameron, A. H. (I964). Postmortem nuclear sex anomalies. Canad. med. Ass. J., 83, 979. sexing. Lancet, I, 328. , Pozsonyi, J., Wilson, R. A., Dunn, H. G., Jacobson, M. A. W., and Handmaker, S. D. T. S., and Miller, J. R. (I962). The XXXXY sex chromosome Ferguson-Smith, A., Johnston, abnormality. ibid., 87, 89I. (I960). Primary amentia and micro-orchidism associated with an XXXY sex-chromosome constitution. ibid., 2, I84. -, Shaver, E. L., Carr, D. H., and Plunkett, E. R. (1959). An and unusual sex chromatin pattern in three mentally deficient subjects. Ferrier, P., Ferrier, S., Stalder, G., Biihler, E., Bamatter, F., associated with diploid- J. ment. Defic. Res., 3, 78. Klein, D. (I964). Congenital asymmetry and satellites. ibid., i, 80. -, -, and (I960). The chromatin-positive Kline- triploid mosaicism large felter syndrome among patients in mental deficiency hospitals. Ford, C. E. (I963). The cytogenetics of human intersexuality. In ibid., 4, 89. Intersexuality, ed. C. Overzier, p. 86. Academic Press, London Barton, D. E., David, F. N., and Merrington, M. (I963). Numerical and New York. analysis of chromosome patterns. Ann. hum. Genet., 26, 349. Fraccaro, M., Ikkos, D., Lindsten, J., Luft, R., and Kaijser, K. Bergemann, E. (I96I). Geschlechtschromatinbestimmungen am (ig6oa). A new type of chromosomal abnormality in gonadal Neugeborenen. Schweiz. med. Wschr., 91, 292. dysgenesis. Lancet, 2, 1I44. Bertram, E. G., Bertram, L. F., and Lindsey, H. A. (1950). The -, Kaijser, K., and Lindsten, J. (ig6ob). A child with 49 nucleolus-associated chromatin in nerve cells of cat and man. chromosomes. ibid., 2, 899. Anat. Rec., Io6, 299. -, Klinger, H. P., and Schutt, W. (I962). A male with XXXXY Beutler, E., and Baluda, M. C. (i964). The separation of glucose-6- sex chromosomes. Cytogenetics, I, 52. phosphate-dehydrogenase-deficient erythrocytes from the blood -, and Lindsten, J. (1959). Observations on the socalled of heterozygotes for glucose-6-phosphate-dehydrogenase defi- chromatin' in human somatic cells cultivated in vitro. Exp. ciency. Lancet, I, I89. Res., 17, 536. -, Yeh, M., and Fairbanks, V. F. (5962). The normal human -, and (I960). A child with 49 chromosomes. Lancet, 2, female as a mosaic of X-chromosome activity. Studies using the I303. gene for G-6-Pd deficiency as a marker. Proc. nat. Acad. Sci. Fraser, J. H., Campbell, J., MacGillivray, R. C., Boyd, E., (Wash.), 48, 9. Lennox, B. (I960). The XXX syndrome; frequency among Bishop, A., and Bishop, 0. N. (i963). Analysis of tritium-labelled mental defectives and fertility. ibid., 2, 626. human chromosomes and sex chromatin. Nature (Lond.), 199,930. Frederic, J. (196I). Contribution a l'etude du caryotype chez Book, J. A., and Santesson, B. (i96i). Nuclear sex in triploid XXY poulet. Arch. Biol. (Liege), 72, I85. human cells. Lancet, 2, 318. Frizzi, G. (1948). L'eteropicnosi come indice di riconoscimento dei copyright. R. A. (1956). Ric. Bradbury, J. T., Bunge, R. G., and Boccabella, sessi in 'Bombyx mori L.'. sci., I8, I19. Chromatin test in Klinefelter's syndrome. J. clin. Endocr., I6, 689. und der Geitler, L. (I937). Die Analyse des Kernbaus Kernsteilung Briggs, D. K. (1958). The individuality of nuclear chromatin with Gerris lacustris der Wasserlaufer Gerris lateralis und (Hempitera, to neutrophil leukocytes. Z. particular reference polymorphonuclear und die Somadifferenzieurung. Zellforsch., 26, Blood, I3, 986. Heteroptera) 641. -, and Kupperman, H. S. (1956). Sex differentiation by leuko- besonderer (1938). tYber den Bau des Ruhekerns mit Ber;uck- cyte morphology. clin. Endocr., I6, I I 63. Biol. Zbl., sichtigung der Heteropteren und Dipteren. 58, I52. Caratzali, A. (I963). Sex appendages, ARNETH formula, and the Somakerne (1939). Die Entstehung der polyploiden menstrual cycle. Cytogenetics, 2, 15. ohne Kernteilung. Heteropteren durch Chromosomenteilung -, A. Considerations sur le determinisme and Phleps, (1958). Chromosoma (Berl.), I, I. http://jmg.bmj.com/ des corpuscules sexuels des leucocytes. C.R. Soc. Biol. (Paris), of German, J. L., III (i962a). Synthesis deoxyribonucleic I52, 1114. during interphase. Lancet, I, 744. cor- , and Turpin, R. (1957). Variations quantitatives des Trans. (i962b). DNA synthesis in human chromosomes. puscles sexuels des granulocytes neutrophiles au cours du cycle Acad. Sci., 24, 395. menstruel. Bull. Acad. Mid. (Paris), 141, 496. Giannelli, F. (I963). The pattern of X-chromosome deoxyribo- Carpentier, P. J., Stolte, L. A. M., and Visschers, G. P. (1956). nucleic acid synthesis in two women with abnormal Determination of genetic sex by the vaginal smear. clin. Endocr., some complements. Lancet, I, 863. I6, s55. L. G., and Rowley, J. Carr, D. H. (i963). Chromosome studies in abortuses and stillborn Gilbert, C. W., Muldal, S., Laitha, (I962). Time-sequence of human chromosome duplication. infants. Lancet, 2, 603. (Lond.), I95, 869. -, R. An XXXX sex Barr, M. L., and Plunkett, E. (i96i). Determination of early Glenister, T. W. (I956). on September 27, 2021 by guest. Protected chromosome complex in two mentally defective females. Canad. ibid., 177, II35. med. Ass. J3., 84, 13I. embryos. de Castro, N. M. (i963). Frequency variations of "drumsticks' of Gorman, J. G., di Re, J., Treacy, A. M., and Cahan, (I963). peripheral blood neutrophiles in the rabbit in different alimentary application of -Xga antiserum to the question of in female J. Lab. Med., 6I, 642. conditions. Acta anat. (Basel). 52, 341. heterozygotes. clin. de la Chapelle, A. (i962). Cytogenetical and clinical observations in Graham, M. A. (0954a). Detection of the sex of embryos female gonadal dysgenesis. Acta endocr. (Kbh.), Suppl. 65. nuclear morphology in the embryonic membrane. (Lond.), Davidson, R. G., Nitowsky, H. M., and Childs, B. (i963). Demon- 173, 31I0. nuclei of the cat early stration of two populations of cells in the human female hetero- (1954b). Sex chromatin in cell from Anat. 119, 469. zygous for glucose-6-phosphate dehydrogenase variants. Proc. embryo to maturity. Rec., nat. Acad. Sci. (Wash.), 50, 48i. -, and Barr, M. L. (1952). A sex difference morphology nuclei in somatic cells of the cat. ibid., 112, 709. -, and Smith, D. R. (1954). A morphological sex difference in of metabolic the polymorphonuclear neutrophil leucocytes. Brit. med. J7., 2, 6. Greenblatt, R. B., Aydar, C. K. and Gibson, H. H. (I962). -, and (I958). The neutrophil sex nodules in Klinefelter's crepancies in the evaluation of sex chromatin in oral syndrome. In Symposium on Nuclear Sex, ed. D. R. Smith, and peripheral blood specimens. Acta cytol. (Philad.), 6, 103. W. M. Davidson, p. 93. Heinemann, London. , Manautou, J. M., Tigerman, N. R., Rogers, L., Jr., -, and (i963). The nuclear sex of leucocytes. In Inter- Sheffield, F. H. (I957). A simplified staining technique sexuality, ed. C. Overzier, p. 72. Academic Press, London and study of chromosomal sex in oral mucosal and peripheral New York. smears. Amer. J. Obstet. Gynec., 74, 629. J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 74 Ursula Mittwoch

Morishima, Grumbach, M. M., Marks, P. A., and (1962). -, and Schwarzacher, H. G. (1g60). The sex chromatin and Erythrocyte glucose-6-phosphate dehydrogenase activity heterochromatic bodies in human diploid and polyploid nuclei. X-chromosome polysomy. Lancet, 1330. biophys. biochem. Cytol., 8, 345.

A. Sex chromatin and the and Morishima, (1962). -, and (I962). XY/XXY and sex chromatin positive cell mosomes: on the origin of sex chromatin from a single X distribution in a 60 mm human fetus. Cytogenetics, i, 266. some. Acta cytol. (Philad.), 6, 46. Kosenow, W. (I957). Abweichende Ergebnisse bei der Geschlecht- -, and Taylor, J. H. (x963). Human sex chromosome sbestimmung an Leukocyten- und Mundepithel-Kernen. Klin. abnormalities in relation to DNA replication and heterochromati- Wschr., 35, 75- nization. Proc. nat. Acad. Sci. (Wash.), 49, 58x. -, and Scupin, R. (1956). Die Bestimmung des Geschlechts mit Guard, H. R. (I959). A new technic for differential staining Hilfe einer Kernanhangsformel der Leukocyten. Acta haemat. sex chromatin, and the determination of its incidence (Basel), 15, 349. vaginal epithelial cells. Amer. clin. Path., 32, 145. Kosin, I. L., and Ishizaki, H. (I959). Incidence of sex chromatin in Gutherz, S. (1907). Zur Kenntnis der Heterochromosomen. Gallus domesticus. Science, 130, 43. mikr. Anat., 69, 491. Lancet (I963). [Editorial!] Lyonisation of the X chromosome. Hamerton, J. L., Fraccaro, M., de Carli, L., Nuzzo, 2, 769. Lennox, B. Sex in tumours. ed. H. P., Hulliger, L., Taylor, A., and Lang, E. M. (x96x). (I963). chromatin In Intersexuality, p. York. chromosomes of the gorilla. Nature (Lond.), I92, C. Overzier, 462. Academic Press, London and New Jagiello, G. M., and Kirman, B. H. (1962). Sex-chromosome Lillie, F. R. (I9I6). The theory of the free-martin. Science, 43,6II. abnormalities in population of mentally defective children. Brit. Lindsay, H. A., and Barr, M. L. (I955). Further observations on

med. J., I, 220. the behaviour of nuclear structures during depletion and restora- -, Klinger, H. P., Mutton, D. E., and Lang, E. (x963). tion of Nissl material. Anat. (Lond.), 89, 47. somatic chromosomes of the Hominoidea. Cytogenetics, Lindsten, J. (I963). The Nature and Origin of X Chromosome Aberra- von Harnack, G. A., and Strietzel, H. N. (X956). Die Altersabhan- tions in Turner's Syndrome. Almqvist and Wiksell, Stockholm. gigkeit der geschlechtsbedingten Leukocytenmerkmale. Klin. Fraccaro, M., Ikkos, D., Kaijser, K., Klinger, H. P., and Luft, Wschr., 34, 401. R. (I963). Presumptive iso-chromosomes for the long arm of the Harnden, D. G. (I96I). Nuclear sex in triploid XXY X in man. Analysis of five families. Ann. hum. Genet., 26, 383. Lancet, 2, 488. London, D. R., Kemp, N. H., Ellis, J. R., and Mittwoch, U. (I964). Harris, H., Hopkinson, D. A., Spencer, N., Court Turner's syndrome with secondary amenorrhoea and sex chromo- and Mantle, D. (i963). Red cell glucose-6-phosphate some mosaicism. Acta endoct (Kbh.), 46, 34I. genase activity in individuals with abnormal numbers Luers, T. (1956). Vergleichende Untersuchungen uber morpholo- chromosomes. Ann. hum. Genet., 27, 59. gische Geschlechtsunterschiede der neutrophilen Leukozyten- Hay, J. C. Further observations on the (ig60). sex kerne bei Mensch und Kaninchen. Blut, 2, 81. mammalian cells. Anat. Rec., 136, 315. and Petzel, C. (I958). Zellkernmorphologische Geschlechts- Heitz, E. Das Heterochromatin der Moose. (1928). Jb. diagnose bei Pelger-Anomalie der Blutkorperchen. ibid., 4,I85. Bot., 69, 762. Lyon, M. F. (1961). Gene action in the X-chromosome of the Hulliger, L.,Klinger, H. P., and Allgower, M. (x963). mouse (Mus musculus L.). Nature (Lond.), 190, 372. as a marker in some rabbit cells. Experientia (Basel), matin 19, (i962). Sex chromatin and gene action in the mammalian 240. copyright. X-chromosome. Amer. J. hum. Genet., 14, 135. Hunter, W. F., and Lennox, B. (1954). The (i963). Attempts to test the inactive-X theory of dosage com- Lancet, 2, 633. pensation in mammals. Genet. Res., 4, 93. Hutt, F. B. (I949). Genetics of the Fowl. McGraw-Hill, York, McKusick, V. A. On the X chromosome of man. Quart. Toronto, London. (1962). Rev. Biol., 37, 69. Ishizaki, H., and Kosin, I. L. Sex chromatin (I960). Maclean, N. (i962). The drumsticks of polymorphonuclear leuco- embryos. Exp. Cell Res., 2I, 197. cytesin sex-chromosome abnormalities. Lancet,I, 1154. Israelsohn, W. J., and Taylor, A. I. (I96I). Chromatin Harnden, D. G., Court Brown, W. M., Bond, J., and Mantle, presumed Klinefelter's syndrome. Survey of boys D. J. (1964). Abnormalities of sex chromosome constitution in schools for educationally subnormal children. Brit. I, newborn babies. ibid.,I, 286.

V. Gonadal dysgenesis in two sisters Izakovic, (I96o). Mitchell, J. M., Harnden, D. G., Williams, J., Jacobs, P. A., http://jmg.bmj.com/ nuclear sex pattern and female characteristics in Buckton, K. A., Baikie, A. G., Court Brown, W. M., McBride, nuclear leukocytes. clin. Endocr., 20, 1301. J. A., Strong, J. A., Close, H. G., and Jones, D. C. (1962). A

Jackson, W. P. U., Shapiro, B. G., Uys, C. J., and survey of sex-chromosome abnormalities among 4514 mental (I956). Primary male hypogonadism with female defectives. ibid.,I, 293. Lancet, 2, 857. Mann, J. D., Cahan, A., Gelb, A. G., Fisher, N., Hamper, J., Jacobs, P. A., Baikie, A. G., Court Brown, W. M., T.N., Tippett, P., Sanger, R., and Race, R. R. (1962). A sex-linked N., and D. G. Evidence Maclean, Harnden, (i959). blood group. ibid., i, 8. ence of the human 'super female'. ibid., 2, 423. Marberger, E., Boccabella, R. A., and Nelson, W. 0.(955). Oral Harnden, D. G., Buckton, K. E., Court Brown, smear as a method of chromosomal sex detection. Proc. Soc. exp.

M. J., McBride, J. A., MacGregor, T. N., and Biol. (N. Y.), 89, 488.

(I96I). Cytogenic studies in primary amenorrhoea. ibid.,,, 1183. Marks, P. A. (1958). Red cell glucose-6-phosphate and 6-phospho- on September 27, 2021 by guest. Protected , Court Brown, W. M., Goldstein, J., Close, gluconic dehydrogenases and nucleoside phosphorylase. Science,

MacGregor, T. N., Maclean, N., and Strong, J. (1960). 127, I338. Abnormalities involving the X chromosome in women. I, Melander, Y. (1962). Chromosomal behaviour during the origin of 1213. sex chromatin in the rabbit. Hereditas (Lund), 48, 645.

on the so-called James, J. (i96o). Observations Merrington, M., and Penrose, L. 5. (i964). Distances which involve Z. Zellforsch., 59, 597. satellited chromosomes in metaphase preparations. Ann. hum. Johnston, A. W., Ferguson-Smith, M. A., Handmaker, Genet., 27, 257. Jones, H. W., and Jones, G. (i966). The triple-X Miles, C. P. (1959). Sex chromatin in cultured human tissues. Clinical, pathological, and chromosomal studies in Nature (Lond.),I84, 477. retarded cases. Brit. med. 2, 1046. (-96o). Morphology and functional relations of sex chromatin Kesaree, N., and Woolley, P. V.(I963). A phenotypic in cultured amnion celi. Exp. Cell Res., 2, 324.

49 chromosomes, presumably XXXXX. Pediat., Peripheral sex Nature J. I099. ((969). position of chromatin. (Lond.), 9I1, 626. Klinger, H. P. (ig95). The sex chromatin in fetal meternal portions of the human placenta. Acta anat. (Easel), 371. and Storey, S. D. (1962). Nuclear chromocenters of cultured cells. Cell Res., 27, 377. (I958). The fine structure of the sex chromatin Exp. chicken ExP. Cell Res., 14, 207. Miller, 0. J., Breg, W. R., Mukherjee, B. B., Gamble, A. Van N., and Ludwig, K. (1957). A universal and Christakos, A. C.(I963a). Non-random distribution of chromatin body. Stain Technol., 32, 235. chromosomes in metaphase figures from cultured human leuco- J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from Sex Chromatin 75

cytes. II. The peripheral location of chromosomes 13, 17-18 and Overzier, C. (I958). Diskrepanz der Geschlechtsdiagnose. Klin. 21. Cytogenetics, 2, 152. Wschr., 36, 845. Academic Press, London and New -, Schmickel, R. D., and Tretter, W. (i96i). A family (I963). Intersexuality. with an XXXXY male, a leukaemic male, and two 2i-trisomic York. mongoloid females. Lancet, 2, 78. Park, W. W. (1957). The occurrence of sex chromatin in early human (Lond.), 369. , Mukherjee, B. B., Breg, W. R., and Gamble, A. Van N. and macaque embryos. J. Anat. 91, (I963b). Non-random distribution of chromosomes in metaphase Peiper, U., and Oehme, J. (I956). Die Abhangigkeit geschlechts- figures from cultured human leucocytes. I. The peripheral gebundener Leukocytenmerkmale bei Feten und Fruhgeboreren location of the Y-chromosome. Cytogenetics, 2, I. vor der Reife. Klin. Wschr., 34, I067. Minouchi, 0. (1928). Spermatogenesis of the albino rat (Mus Plunkett, E. R., and Barr, M. L. (I956). Testicular dysgenesis; norvegicus albus). jap. Zool., I, 235. affecting the seminiferons tubules principally with chromatin- Mittwoch, U. (I957). Some observations on the leucocytes in mon- positive nuclei. Lancet, 2, 853. I, golism. J. ment. Defic. Res., 26. Polani, P. E., Hunter, W. F., and Lennox, B. (I954). Chromosomal - (i96i). Nuclear sex and chromosome structure in mongolism sex in Tumer's syndrome with coarctation of the aorta. ibid., 2, and the Klinefelter syndrome. In Proceedings of the Conference on 120. M. Human Chromosomal Abnormalities, London 1959, ed. W. Prince, R. H., Graham, M. A., and Barr, M. L. (i955). Nuclear p. 97. Staples Press, London. Davidson, and D. R. Smith, morphology, according to sex, in Macacus rhesus. Anat. Rec., 122, nuclear size in cultured human fibro- (i963). Barr bodies and 153. 975. blasts. Nature (Lond.), 198, Reed, T. E., Simpson, N. E., and Chown, B. (1963). The Lyon - Frequency of drumsticks in normal women and in ((964a). hypothesis. Lancet, 2, 467. patients with chromosomal abnormalities. ibid., 201, 317. Reitalu, J. Observations on the so-called sex chromatin in (x964b). Barr bodies and their relation to nuclear size. Cyto- (I957). man. Acta Genet. med. (Roma), 6, 393. genetics, 3, 62. A. C., Shapiro, A., and McKibben, W. R. (I963). Sex -, Atkin, N. B., and Ellis, J. R. (1963). Barr bodies in triploid Ridler, M. in female subnormal patients. Brit. cells. ibid., 2, 323. chromatin abnormalities J. -, and Delhanty, J. D. A. (i96i). Nuclear sex in triploid XXY Psychiat., 109, 390. human cells. Lancet, 2, 552. Riis, P., Johnsen, S. G., and Mosbech, J. (1956). Nuclear sex in Klinefelter's syndrome. Lancet, I, 962. Moore, K. L. (x959). Sex reversal in newborn babies. ibid., I, 217. , and Barr, M. L. (1953). Morphology of the nerve cell nucleus Ross, A. (I962). The demonstration of sex chromatin in cultured in mammals, with special reference to the sex chromatin. comp. cells. med. Lab. Technol., I9, I12. Neurol., 98, 213. Rowley, J., Muldal, S., Gilbert, C. W., Lajtha, L. G., Lindsten, J., -, and (1954). Nuclear morphology, according to sex, in Fraccaro, M., and Kaijser, K. (1963). Synthesis of deoxyribo- human tissues. Acta anat. (Basel), 21, 197. nucleic acid on X-chromosomes of an XXXXY male. Nature -, and (1955). Smears from the oral mucosa in the detection (Lond.), 197, 251. of chromosomal sex. Lancet, 2, 57. Russell, L. B. (I96I). Genetics of mammalian sex chromosomes. --, Graham, M. A., and Barr, M. L. (i953). The detection of Science, 133, I795. chromosomal sex in hermaphrodites from a skin biopsy. Surg. (I963). Mammalian X-chromosome action: inactivation Obstet., 96, 641. ibid., 976. Gynec. limited in spread and in region of origin. 140, copyright. bovine , and (1957). The sex chromatin of the Sanderson, A. R., and Stewart, J. S. S. (I96I). Nuclear sexing with freemartin. J. exp. Zool., 135, IOI. aceto-orcein. Brit. med. J7., 2, io65. and Hay, J. C. (i96i). Sexual dimorphism in intermitotic Shantaveerappa, T. R., and Bourne, G. H. (I962). A perineural 139, 3I5. nuclei of birds. Anat. Rec., epithelium. J7. cell. Biol., 14, 343. DNA Moorhead, P. S., and Defendi, V. (i963). Asynchrony of Schmid, W. (I962). DNA replication patterns of the heterochromo- chromosomes of human diploid cells. 7. Cell. Biol., I6, synthesis in somes in Gallus domesticus. Cytogenetics, I, 344. 202. (I963). DNA replication patterns of human chromosomes. Morishima, A., Grumbach, M. M., and Taylor, J. H. (I962). ibid., 2, 175. origin Asynchronous duplication of human chromosomes and the Schneiderman, L. J., and Smith, C. A. B. (I962). Non-random of sex chromatin. Proc. nat. Acad. Sci. (Wash.), 48, 756. distribution of certain homologous pairs of normal human chromo- fre- http://jmg.bmj.com/ Mosier, H. D., Scott, L. W., and Cotter, L. H. (I960). The somes in metaphase. Nature (Lond.), 195, 1229. with mental quency of the positive sex-chromatin pattern in males Schrader, F. (1928). Die Geschlechtschromosomen. Borntraeger, deficiency. Pediatrics, 25, 291. Berlin. J., Rowley, J., Muldal, S., Gilbert, C. W., Lajtha, L. G., Lindsten, Schwarzacher, H. G. (I963). Sex chromatin in living human cells M. Tritiated thymidine incorporation in an and Fraccaro, (i963). in vitro. Cytogenetics, 2, 117. isochromosome for the long arm of the X chromosome in man. Serr, D. M., Ferguson-Smith, M. A., Lennox, B., and Paul, J. Lancet, I, 86i. (I958). Representation of the X-chromosome in intermitotic -, and Ockey, C. H. (i960). The 'double male': a new chromo- nuclei in man. Nature (Lond.), 182, 124. some constitution in Klinefelter's syndrome. ibid., 2, 492. A., and Ridler, M. A. C. (I960). The incidence of Kline- Muller, H. J. (1932). Further studies on the nature and causes of Shapiro, in a mentally deficiency hospital. ment. Defic. 6th Int. Cong. Genet., Ithaca, 193I, l, 213. felter's syndrome gene mutation. Proc. on September 27, 2021 by guest. Protected Res., 4, 48. 0. (i956). Sex differences in human nuclei with par- Nelson, W. Heni, F. (I963). Ein Extrachromosom ticular reference to the 'Klinefelter-syndrome', a gonadal agenesis Siebner, H., Klaus, D., and bei der Pelger-Huetschen Kernanomalie. Med. Welt. (Stuttg.), and other types of hermaphroditism. Acta endocr. (Kbh.), 23, 227. Ohno, S. (i96i). Sex chromosomes and microchromosomes of 877. Smith, D. W., Marden, P. M., McDonald, M. J., and Speckhard, M. Gallus domesticus. Chromosoma (Berl.), II, 484. Lower incidence of sex chromatin in buccal smears of -, and Cattanach, B. M. (i962). Cytological study of an X-auto- (I962). newborn females. Pediatrics, 30, 707. some translocation in Mus musculus. Cytogenetics, I, 129. Kaplan, W. D., and Kinosita, R. (i959). Formation of the sex Smith, S. G. (I944). The diagnosis of sex by means of heteropyc- chromatin by a single X-chromosome in liver cells of Rattus nosis. McGill. med. 13, 451. norvegicus. Exp. Cell Res., I8, 4I5. (I945a). Heteropycnosis as a means of diagnosing sex. Hered.,

, and (I960). On the sex chromatin of Gallus 36, I95. domesticus. ibid., I9, i8o. (I945b). The diagnosis of sex by means of heteropycnosis. , and Makino, S. (I96I). The single-X nature of sex chromatin Sci. Agric., 25, 566. in man. Lancet, I, 78. Sohval, A. R. (I963). Chromosomes and sex chromatin in normal, , , Kaplan, W. D., and Kinosita, R. (I96I). Female germ and anomalous sexual development. Physiol. Rev., 43, 306. in size of nuclear cells of man. Exp. Cell Res., 24, io6. -, and Casselman, W. G. B. (I96I). Alteration Lancet, 2, Orsi, E. V., and Ritter, H. B. (I958). A report of sex chromatin in sex-chromatin mass (Barr body) induced by antibiotics. human tumor tissue culture. Exp. Cell Res., 15, 244. I386. J Med Genet: first published as 10.1136/jmg.1.1.50 on 1 September 1964. Downloaded from 76 Ursula Mittwoch

-, Gabrilove, J. L., Gaines, J. A., and Soffer, L. J. (i956). growth phases of human cells in tissue culture. Exp. Cell Res., 28,

Observations on the sex chromatin in testicular disorder. J. Mt 588. Sinai Hosp., 23, 647. Tonomura, A., Toyofuko, Y., and Matsunaga, E. (1962). The Stenius, C., Christian, L. C., and Ohno, S. (x963). Comparative frequency of so-called drumsticks in the polymorphonuclear cytological study of Phasianus cokchicus, Meleagris gallopavo, and neutrophil leucocytes of Japanese females. Yap. hum. Genet., 7, Gallus domesticus. Chromosoma (Berl.), 13, 515. 60. Stern, C. (1960). Dosage compensation-development of a concept Turner, H. H., and Zanartu, J. (1962). Ovarian dysgenesis in identi- and new facts. Canad. J. Genet. Cytol., 2, 105. cal twins: discrepancy between nuclear chromatin pattern in (1963). The genetics of sex determination in man. Amer. J3. somatic cells and in blood cells. clin. Endocr., 22, 66o. Med., 34, 715. Turpin, R., and Bernyer, C. (1947). De l'influence de l'h6r6dite sur Stewart, J. S. S. (ig6o). Genetic mechanisms in human intersexes. la formule d'Arneth (cas particulier du mongolisme). Rev. Hemat., Lancet, I, 825. 2, 189. Subray, N., and Prabhaker, N. (i962). Sex chromatin anomalies in Welshons, W. J., and Russell, L. B. The Y-chromosome as newborn babies in India. Science, 136, I i6. (I959). Tanaka, Y. (1953). Genetics of the silkworm, Bombax mori. Advanc the bearer of male determining factors in the mouse. Proc. nat. Acad. Sci. (Wash.), 45, 560. Genet., 5, 239. Tandler, J., and Keller, K. (I9i i). Ueber das Verhalten des Chor- Whitby, L. E. H., and Britton, C. J. C. (X963). Disorders of the ions bei verschiedengeschlechtlicher Zwillingsgraviditat des Blood, gth ed. Churchill, London. Rindes und uber die Morphologie des Genitales der weiblichen Wiedemann, H. R. (1958). The result of haematological determina- Tiere, welche einer solchen Graviditat entstammen. Dtsch. tion of the genetic sex in disturbances of sexual development. In tierarztl. Wschr., I9, 148. Symposium on Nuclear Sex, ed. W. M. Davidson, and D. R. Smith, Taylor, A. I. (I963a). Nuclear sex of embryonic tumours. Brit. p. 102. Heinemann, London. med. ., I, 377. -, Romatowski, H., and Tolksdorf, M. (i956). Geschlechts- (I963b). Sex chromatin in the newborn. Lancer, 1, 912. bestimmung aus dem Blutbilde. Munch. med. Wschr., 98, IO90, Taylor, J. H. (i960). Asynchronous duplication of chromosomes in II08. Wilkins, L., Grumbach, M. M., and van Wyk, J. J. Chromo- cultured cells of Chinese hamster. J. biophys. biochem. Cytol., 7, (1954). 455. somal sex in 'ovarian agenesis'. J. clin. Endocr., 14, I270. -, Woods, P. S., and Hughes, W. L. (1957). The organization Wilson, E. B. (1925). The Cell in Development and Heredity, 3rd ed. and duplication of chromosomes as revealed by autoradiographic Macmillan, London and New York. studies using tritium-labeled thymidine. Proc. nat. Acad. Sci. Winge, 0. (1932). The nature of sex chromosomes. Proc. 6th Int. (Wash.), 43, 122. Congress Genetics, Ithaca, 1931, 1, 343. Therkelsen, A. J., and Petersen, G. B. (1962). Frequency of sex- Wintrobe, M. M. (196I). Clinical Haematology, sth ed. Kimpton, chromatin-positive cells in the logarithmic and postlogarithmic London. copyright. http://jmg.bmj.com/ on September 27, 2021 by guest. Protected