Book - Sex and internal secretions (1961) 1

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Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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SECTION A Biologic Basis of Sex

Genetic and Cytologic Foundations for Sex

John W. Gowen, Ph.D.

Department of Genetics, Iowa State University, Ames, Iowa

I. Basic Literature

In the first edition of Sex and Internal Secretions published in 1932, Bridges discussed the closely prescribed problem of the genetics of sex, particularly as it was related to one species, Drosophila melanogaster. The treatment was sharply focused on the advances made, chiefly through his own researches, in understanding the functions of the genetic and cytologic factors operating during embryologic development which ultimately establish the sex types. The emphasis was on gene action through interchromosomal balances as they may aff"cct sex expression. The second edition of 1939 brought this material up-to-date and, at the same time, offered a much broader treatment by the inclusion of accumulated evidence on how differentiation for sex comes about in other forms. There is no substitute for a careful reading of these two presentations as background material for a present day understanding of sex determination. The current presentation makes no attempt, except in the barest outline, to repeat this early material other than in those aspects which bear on the advances that have been made since the printing of the second edition.

Immediately following the publication of the first edition of Sex and Internal Secretions there was a resurgence of interest in the problems considered in that book. The resulting research led to notable advances in available knowledge. Seven years later the second edition was published. A second wave of accomplished research appeared. In the interim of the past 21 years extensive advances have been recorded, particularly in understanding the mechanisms of sex differentiation in plants as well as in many animals. Among others, the data on Melandrium, Asparagus, Rumex, and Spinacia have been of first importance. Further information on Drosophila, Habrobracon, and Xiphophorus has notably broadened our viewpoints. Beginnings have been made to a better understanding of the conditions for sex separation in bacteria, protozoa, bees, birds, goats, mice, and man. To these specific contributions may be added the basic advances in understanding the methods by which genes are transmitted from one generation to the next, accomplish their actions in development, and by which chromosomes reorganize and reconstruct gene groups.

The period has also been one of excellent monographic treatments on different phases of the subject. Wilson's The Cell in Development and Inheritance (1928) and earlier, Morgan's Heredity in Sex (1914), Schrader's The Sex Chromosomes (1928) and Goldschmidt's Lymantria (1934) retain their pre-eminence. To this list have now been added the publication of extensive tabulations of chromosomes of cultivated plants by Darlington and Janaki Ammal (1945), of animal chromosomes by Makino (1951 ), and the yearly index to plant chromosomes beginning with 1956, compiled by a world-wide editorial group and published by the University of North Carolina Press, Chapel Hill, North Carolina. Those volumes give access to the basic chromosomal constitutions and their sex relations for far more species than were heretofore available. Inheritance information has been made more accessible and at the same time in more detail. The tabulations of mutants observed in particular species as in D. melanogaster (Morgan, Bridges, and Sturtevant, 1925; Bridges and Brehme, 1944) have been followered by those of corn, mouse, domestic fowl, rat, rabbit, guinea pig, Habrobracon, and many other animal forms, together with similar tabulations for cereals and a number of other plant species. Books of particular interest include those of Hartmann. Die Sexualitdt (1956), White, Animal Cytology and Evolution (1954), Goldschmidt, Theoretical Genetics (1955,1, Hartmann and Bauer, Allgemeine Biologic { 1953) , and Tanaka, Genetics of Silkworms (1952), and special reviews in various volumes of Fortschritte der Zoologie (1 to 12) (Wiese, 1960). Basic normal development of Drosophila has been presented in convenient book form in papers by Cooper, Sonnenblick, Poulson, Bodenstein, Ferris, Miller and Spencer, Biology of Drosophila (Demerec Ed., 1950) . Special papers having a direct bearing on the subject matter include particularly those of Pipkin (19401960) in analyzing the various chromosomes of Drosophila for sex loci, of Tanaka (1953) and Yokoyama (1959) for their studies and reviews of the genetics of silkworms, and of Westergaard (1958) on sex determination in dioecious flowering plants.

II. Mechanistic Interpretations of Sex

The records of search for basic mechanisms involved in the determination of sex have been foremost in the writings of man from the beginning of historical record. These ideas have included all forms of mechanisms both intrinsic and extrinsic to the organism. The most constructive advance, although not realized at the time, came through the recognition of cell structure, chromosome maturation and the discrete behavior of the inheritance. Differences in clu-omosome behavior were noted but not alwaj^s related to facts of broader significance. The period was one of expanding observations and development of ideas as they applied to species in general and also to the further complications which arose with more extended study. These complications included differences not only in one chromosome pair but in several. The significance of a balance between these chromosome types as well as with the environment was grasped by Goldschmidt and particularly by Bridges where his more favorable material brought out sharper contrasts in types and in the chromosome behavior. Ideas related to chromosome balance as they may affect developmental processes were developed. Goldschmidt emphasized that this balance could include factors in the cytoplasm as well as in the chromatin material. Bridges' observations on the other hand, pointed most strongly to the chromatin elements where changes in chromosome numbers were often accompanied by sharp differentiation of new sexual types.

Concepts of sex determination broadened. They came to include all the chromosomes in the haploid set, a genome, as a whole. The important concept of single chromosome difference being all important has been replaced with that of a balance between the chromosomes. It has sometimes been emphasized that it is this balance which is the most significant element in sex determination. Present day research seems to be pointing rather to even finer structures than the chromosomes in that the main search is on for the particular genes within the inheritance complex which contribute the characteristics of sex. Bridges' work has sometimes been asserted as committed to a particular type of balance in the chromosome. However, his writings 1 1932) make it clear that this balance is, in his judgment, basically due to the genes actually contained within the chromosomes rather than to the chromosomes themselves. This view is further emphasized in the second edition of Sex and Internal Secretions. "Sex determination in numerous forms with visible distinctions between the chromosome groups of the two sexes was at first interpreted on a 'quantitative' basis, as due to graded amounts of 'sex-chromatin.' But because even more species were encountered in which no visible chromosome difference was detected, the formula


tion was changed to include also 'qualitative differences in sex-chromatin.' Now, sex is being reinterpreted in terms of genes, with investigation by breeding tests penetrating to detail far beyond the reach of cytological investigation. The chromosomal differences are now treated as rough guides, and chromosomal determination is l)cing resolved into genie determination

Hence the difference in sex must be put on the same basis as that of any other organ differentiation in plant or animal, for example, the difference between legs and wings in birds— both modifications of ancestral limbs."

In contrast to what some have interpreted. Bridges was not committed to a specific chromosome balance, as for instance the X chromosomes to the A chromosomes in Drosophila, for all species. Rather he looked on that particular balance as but one mode of gene association in chromosomes that could bring about differences in gene action on development which would lead to sexual differentiation in its various forms. In that sense his ideas prepared him for, and were in conformity with, the types of sex differentiation which later became established in such forms as Melandrium and in the silkworm. In his concepts of sex determination and in his active interest in making genetics more specific, there can be little doubt that his theory would include those of the present and would also be welcome as refining and more sharply defining the significance of cytologic and genetic elements important to sex differentiation.

A. Concept of Sex Determination

As frequently happens, observations are made often before their significance is more than dimly understood. Background information may be insufficient for the facts to become clear. This was true when a lone chromosome was discovered as a part of the maturation complex in sperm formation of Pyrrhocoris. The association of the presence of this element with the idea of its being the arbitrator between male and female development in certain species was not made until 10 years later. Henking (1891) observed in Pyrrhocoris 11 paired chromosome elements and one unpaired element which after spermatocyte divisions led to sperm of two numerically different classes, one carrying 11 chromosomes plus the accessory chromosome and the other carrying only the 11 chromosomes. His observations were confirmed by several other investigators and in principle for other insect species over a decade following. The first suggestion that this chromosome behavior was related to sex diiTerentiation came from McClung's (1902) observations on the "accessory chromosome" of Xiphidiiim fasciatum. It is of some interest to examine the steps l)y which these conclusions were reached.

"The function exercised by the accessory chromosome is that it is the bearer of those qualities which pertain to the male organisms, primary among which is the faculty of producing sex cells that have the form of spermatozoa. I have been led to this belief by the favorable response which the element makes to the theoretical requirements conceivably inherent in any structure which might function as a sex determinant.

"These requirements, I should consider, are that: (a) The element should be chromosomic in character and subject to the laws governing the action of such structures, (b) Since it is to determine whether the germ cells are to grow into the passive, yolk-laden ova or into the minute motile spermatozoa, it should be present in all the forming cells until they are definitely established in the cycle of their development, (c) As the sexes exist normally in about (■(lual projoortions, it should be present in half the mature germ cells of the sex that bears it. (d» Such disposition of the element in the two forms of germ cells, paternal and maternal, should be made as to admit of the readiest response to the demands of enA'ironment i-egarding the ])i'oportion of the sexes, (el It should show variations in structure in accordance with the vai-iations of sex potentiality observable in different species, (f) In parthenogenesis its function would be assumed by the elements of a certain polar body. It is conceivable, in this regard, that another form of polar body miglit function as the non-determinant bearing germ cell." The important fact established by this reasoning was that a chromosome could be the visual differentiator between the fertilized eggs developing specific adult sexual differences. It w^as of little consequence perhaps that for sex itself, in this species, the chromosome arrangement was misinterpreted. The validity of the rules was probed through examinations of the cells of many species by many different observers.

B. Sex as Associated with Visible Chromosomal Differences

Variations in the chromosome complexes contributing to sexual differentiation were soon found. Probably the most frequent type observed in animals and plants was that in which a single accessory chromosome of the male was accompanied by, and paired with, another chromosome either of the same or of a different morphologic type. This other chromosome, as distinguished from the accessory chromosome now generally called the X, was designated the Y chromosome. For different species the Y ranged in size from complete absence, to much smaller, to equal to, to larger than the X. Besides the X and Y chromosomes, the autosomal or A chromosomes, the homomorpluc pairs, complete the species chromosome complement. In this terminology

Sperm (Y + A) + egg (X + A) = XY + 2A cells


of


lie determinino; tvpe


Sperm (X + A) + egg (X + A ) = 2X + 2A cells

of female detei'mining type

Variations in numbers and sizes of chromosomal pairs making up the autosomal sets of different species are familiar cytogenetic facts. These variations may extend from one pair to many chromosome pairs depending on the species. Similar variations may occur in chromosomes of eitluM- the X or Y types. The X is commonly a single chromosome but may be a compound of as many as 8 chromosomes in Aiicaris inciirva (doodricli. 1916). The Y mav be lacking entirely in some species or may be reduplicated in others. Males which form two types of sperm are often called heterozygous for sex, although the term digametic may be better, whereas the females are homogametic.

In other species the females show the chromosomal differences whereas the males are uniformly homogametic. These types are principally known in the Lepidoptera, more primitive Trichoptera, Amphibia, some species of Pisces, and Aves. The single accessory chromosome is present in the females whereas the males have two. It may l)e unpaired or be with another chromosome of different morphology and size. The sex types may be symbolized by designating the accessory chromosome by Z and its mate W as a means of separating possible differences between them and the X and Y chromosomes. The significance of these differences is not clearly established with the consequence that some investigators prefer to substitute the XY designation for the females and the XX condition for the males of these species. The zygotic formulae are:

Sperm ZA + egg ZA = 2Z + 2A male Sperm ZA + egg WA = ZW + 2A female

A third major chromosomal arrangement accompanying sex differentiation came to light as a result of Dzierzon's 1845 discovery of parthenogenesis in bees. Chromosomal and genetic studies have shown both Apis and Habrobracon of the Hymenoptera to be haploid, N, for each germinal cell in the male complex and diploid, 2N, in the female. N may stand for any number of chromosomes, as 16 for Apis or 10 for Habrobracon. The same chromosomal pattern characterizes, so far as known, the other genera of Hymenoptera. Haploidy vs. diploidy is viewed as the most obvious feature predicting differentiation toward the given sex type even though, as in Habrobracon, there is evidence for a particular chromosome of the N set carrying a locus for sex differentiating genes. The zygotic sex formulations are:

No sperm + egg N = N male Sperm N + egg N = 2X female

In development, as in some other species of widely diverse origins, chromosome polyploidy may take place causing the soma cells to differ from the germ cells in their chromosome coniponents.

The common phenotype for plants and lower animals has differentiated sex organs which are combined in the same individual. Plant species seldom depend on any but hermaphroditic types for their reproduction. Lewis' (1942) tabulation for British flora had but 8 per cent of all species depend on other than this form of reproduction. Those that had perfected dioecious systems were not all alike in the system adopted, although species with XY + 2A males and XX + 2A females were in high frequency. Dioecious reproduction was rarely the system common to a whole genus. Recent and multiple origins of dioecious types are indicated by the irregular distribution of species with bisexual reproduction within the different genera and families. Methods for preventing inbreeding have taken other channels as selfsterility genes reminiscent of fertility or sex alleles found in the older bacteria and protozoa. Animals of the lower phyla are those which are most frequently hermaphroditic. Within hermaphroditic species chromosomal distinctions are ordinarily absent. The higher forms with sex and chromosome differences, on the other hand, may show reversion to the hermaphroditic condition from the dioecious or bisexual states.

Other less common cytogenetic controls of sex development have become recognized and better understood. Discussion of their gene and chromosomal arrangements will be considered when these cases arise. The al)ove types will be sufficient to furnish a basis for interpreting the newer data.

C. Changing Methods Of Cytogenetics

The earlier studies of sex determination depended on the natural arrangements of chromosomes found in different species and on occasional chromosomal rearrangements occurring as relatively rare aberrant types. Further development of genetics has increased the tools for these studies. Mutant genes have been shown to control chromosome pairing in segregation (Gowen and Gowen, 1922; Gowen, 1928; Beadle, 1930).


Different drugs such as chloralhydrate and particularly colchicine and various forms of radiant ^energy have made it possible to create new types by doubling the chromosomes, by chromosome rearrangement and by changing the chromosome number. Better genetic tester stocks of known composition have been organized, which together with a more exact understanding of the inheritance structure of the species have made for more critical studies. Techniques, which have improved chromosome differentiation and structural analysis through better methods, better dyes, tracers to mark chromosome behavior (Taylor, 1957), and the use of hypotonic solutions in the study of cells (Hsu, 1952), have removed doubts that were created by the earlier technical difficulties. Instrumentation has improved for the measurement, physically and chemically, of cell components.

D. Chromosomal Association with Sex

The first genetic linkage groups were associated with the sex chromosomes and were soon shown to follow the patterns of the different chromosome sets observed within the different species. In man, hemophilia inheritance was observed to follow that which was presumed for the X chromosome. Barring in birds and wing-color pattern in Lepidoptera on the other hand were found to follow the chromosomal patterns of their species where the male was ZZ and the female Z or ZW for the sex chromosomes. The utility of these methods was further probed by Bridges' observations that when chromosome behavior in Drosophila resulted in sperm or eggs carrying uncxjx'cted chroiiiosomc combinations there followed ('(nmlly unexpected phcnotypes in the progeny. The cliaractei'istics of these unexpected progeny, in turn, I'ollowt'd those expected if genes for them were carried m the sex chromosomes. The use of these linkage groups as tracers, both as they naturally occur and as they may be reorganized through the treatment effects of such agents as radiant energy, open the possibility ol assigning sex effects to, not only chromosomes, but also to particular i-laces within the chromosomes.

Both polyploids and aneuploids, as occurring naturally and as marked by tracer genes, give insight into sex differentiation and its dependence on chromosome inheritance behavior. True polyploids in a species are formed as a consequence of multiplying the entire genome. The possible types may have a single set of chromosomes and genes in their nuclei (haploid ) , 2 sets (diploid) , 3 (triploid), 4 (tetraploid), and so on, representing the genomes 1. 2. 3. 4. to whatever level is compatible with life. Multiplying the genomes within the nucleus often increases cell size but seldom gives the organism overtly different sex characteristics.i Aneuploids, on the other hand, give quite different results, the result being dependent upon the particular chromosome that may be multiplied. Particular trisomies in maize, wheat, and spinach, for example, are distinguishable by marked differences in phcnotypic appearance that is not attributable to cell size but rather to abrupt deviations in particular characteristics. The genes in the particular trisomic set are unbalanced against those of the rest of the diploid sets within the organism. Their phenotypes express these differences.

E. Balance of Male and Female Determining Elements in Sex Determination

The foundations for the basic theory that sex determination rests on quantitative relationships of two genes or sets of genes localized in separate chromosomes rests largely on the work of Morgan, Bridges and Sturtevant, 1910, with Drosophila and the breeding work of Goldschmidt, 1911, with Lymantria. The work of Goldschmidt soon gave extensive descriptions of diploid intersexuality in L]imantria dispar. The details of this work scarcely net'd review because they have \)vvn repealed several times and have been smnmarized recently in Goldschnu(h's Theoretical Genetics (1955). Fioui (hildschmidt's viewpoint, the basic point was that definite conditions between the sexes, that is, interscxuality, could be pro.lnced at will l)y proper genetic combina, tions (crosses of subspecies of L. dispar)

• The notable exception of the Hynienoptera will I )c discussed later.


without any change in the mechanism of the sex chromosomes, and this in a typical quantitative series from the female through all intergrades to the male, and from the male through all intergrades to the female, with sex reversal in both directions at the end point. The consequence was: (1) the old assumption that each sex contains the potentiality of the other sex was proved to be the result of the presence of both kinds of genetic sex determiners in either sex; (2j the existence of a quantitative relation, later termed 'balance' (though it is actually an imbalance), between the two types of sex determiners decides sexuality, that is femaleness, maleness, or any grade of intersexuality; (3) one of the two types of sex determiners (male ones in female heterogamety, female ones in male heterogamety) is located within the X-chromosomes, the other one, outside of them; (4) as a consequence of this, the same determiners of one sex are faced by either one or two portions of those of the other sex in the X-chromosomes; (5) the balance system works so that two doses in the X-chromosomes are epistatic to the determiners outside the X, but one dose is hypostatic; (6j intermediate dosage (or potency) conditions in favor of one or the other of the two sets of determiners result, according to their amount, in females, males, intersexes, or sex-reversal individuals in either direction; (7) the action of these determiners in the two sexes can be understood in terms of the kinetics of the reactions controlled by the sex determiners, namely, by the attainment of a threshold of final determination by one or the other chain of reaction in early development; while in intersexuality the primary determination, owing to the 1X-2X mechanism, is overtaken sooner or later — meaning in higher or lower intersexuality — by the opposite one, so that sexual determination finishes with the other sex after this turning point. The last point is, of course, a problem of genie action."

Bridges developed his idea of "genie balance" as a consequence of his observations on chromosomal nondisjunction, particularly as it illustrated the loss or gain of a fourth chromosome in modifving nonsexual characters. The similarities and contrasts of this view from that of Goldschmidt are indicated by the following quotation (Bridges, 1932) : "From the cytological relations seen in the normal sexes, in the intersexes, and in the supersexes, it is plain that these forms are based upon a quantitative relation between qualitatively different agents — the chromosomes. However, the chromosomes presumably act only by virtue of the fact that each is a definite collection of genes which are themselves specifically and qualitatively different from one another. There are two slightly different ways of formulating this relation, one of which, followed by Goldschmidt, places primary emphasis upon the quantitative aspect of individual genes. The other view, followed by the Drosophila workers, emphasizes the cooperation of all genes which are themselves qualitatively different from one another and which act together in a quantitative relation or ratio. Goldschmidt developed his idea through work with the sex relations in Lymantria and has sought to extend it to ordinary characters. The other formulation, known as 'genie balance,' was developed from the ordinary genetic relations found in characters. Both are crystallizations of fundamental ideas with which the earlier literature was fairly saturated and no great claim to distinctive originality should be ventured for either or denied for one only. Both are physiological as well as genetic — that is, they are formulations of the action of genes, not merely statements of the genie constitutions of individuals nor merely studies of the way genes act. The physiological side has been emphasized by Goldschmidt and the genetic side by Drosophila workers. But Goldschmidt's tendency to represent the view of genie balance as without, or even as in opposition to, such physiological formulation is groundless — as groundless as would be the reciprocal contention that Goldschmidt's theory is only one of 'phenogenetics.'

"A common element in the foundation of both formulations is that if a gene is represented more than once in a genotype the phenotypic effect is expected to be different, though roughly in the same direction as before and roughly proportional to the quantitative change in the genie constitution."

Since the sexually different types observed by Bridges were accompanied by whole chromosomal differences, he could point to losses or gains of autosomes, with an internal preponderance of genes tending to develop male organs, as balanced by genes of the X chromosomes tending in the direction of producing female organs or of suppressing alternative male organs. The net effect of the X chromosome favoring femaleness, and of the set of autosomes favoring maleness, terminates in the development of male or female, according to the ratio of these determiners in the whole genotype. The effect of the X chromosome goes on the basis of whole numbers 1, 2, 3, 4, as does the similar variation in the sets of autosomes.


TABLE LI

Chromosomal numbers and kinds for the different

recognized sex types of Drosophila


Type


Superfemale . . . Triploid meta female

Female*

Female

Female

Female

Female

Female

Female

P'emale

Female

Female

Intersex*

Intersex

Intersex

Male

Male

Male

Male

Male*

Supermale


Chromosomes


X


Y


A


3



2


4



3


4



4


3



3


3


1


3


3


2


3


2



2


2


1


2


2


2 + vS


2


2


2 + yL


2


2


2


2


1



1


3



4


2



3


2


1


3




2



1


2



2


2



3


2




4




3


X/A Balance


1.5

L3

LO

LO

LO

LO

LO

1.0

LO

LO

LO

LO .75 . ()7 .()7 .50 .50 .50 .50 .50 .33


These forms are cited by Bridges from his own observations, from L. V. Morgan (1925) and from Sturtevant (unpublished). As yet but limited studies of these forms, which must be rare, have been published. Fourth chromosomes generally, but not always, equal mimber of the other individual chromosome gn)U])s.


Goldschmidt, since he was dealing with males and females of the diploid type, took a corresponding view for the Z chromosomes of his moths with this difference. Since the Drosophila chromosome pattern is XY + 2A for the males as contrasted to 2X + 2A for the females and the pattern for Lymantria is ZZ -|- 2A for the males and ZW + 2A for the females, it was necessary to use a relation which was reciprocal to that of Drosophila; the male determining element or elements were assigned to the Z chromosomes. Lymantria has a rather large number of different races found in different geographic locations. Within any one of these races this formulation apparently sufficed. However, from crosses between races it was soon observed that the progeny showed ranges in sexuality all the way from phenotypic males to phenotypic females although these females were actually genetic males. To Goldschmidt, this variation indicated different potencies of the male-determining element. Similar differences were attributed to the female element which he had first assigned to the cytoplasm but for which he later favored a W chromosome location.

In applying this postulate of discrete chromosome contributions to sex according to their number, Bridges made the further assumption that female-producing genes l)redominate in the X and are scattered through it in more or less random fashion as are the genes affecting so-called somatic characteristics as wing shape or bristle pattern. The quantitative relations for the different chromosomal types, together with their descriptions, are indicated in Table 1.1.

In the formulation of Table 1.1, the X and Y chromosomes are counted separately, whereas a set of A chromosomes (autosomes), is allowed a value of but one even though comjiosed of a 2nd, a 3rd, and a 4th chromosome for the haploid genome. The dii)loid set of autosomes is given a weight of two, and so on. From these data Bridges observed that the presence or absence of a Y chromosome did not affect the sex types. The X chromosomes and autosomes were, however, important. He held that their importance stood as the ratio of thcii' prcsuiiK'd iM'oducts to each other. The ratio is 1 for the perfect female and 0.5 for the perfect male. Between these values intersexual conditions develop. Beyond the value 1, development is overbalanced byexcess female genes resulting in the superfemale. Values less than 0.5 create a deficiency in the female elements or excess of male elements and a supermale results. Schrader and Sturtevant (1923) proposed another system. Instead of the ratio of X clu-omosomes to autosomes, they suggested that a straight difference between the products of the female determining elements of the sex chromosomes and the male effects of the autosomes causes the sex changes. ]3ridges criticized this system on the basis of the fact that progressive polyploidy did not change the sex type or ratio between the X and autosomes, whereas the numerical difference between them would be progressively increased. While keeping the X/A ratio as descriptive of the ultimate effects of the genes in these chromosomes, he modified their proposed weight from 1 : 1 for X:A to 1 for the X and 0.80 for the A.

All formulations for explaining sexual differences are beset with a lack of an unbiased quantitative scale by which these differences can be measured. The estimates of the changes in sexuality are left to the insight of the observer. It seems reasonable to suppose that the quantitative relations between the male and female sex determining elements should have intermediate values when the specimens under observation show a mixture of organs of either sex. This agrees with Bridges' considerations of this problem. It is not so clear, however, that the so-called supersexes^ really are what the names may connote to many readers. The superfemales, with their three X chromosomes and two sets of autosomes, are quite inviable; small in size, wings reduced and irregularly cut on the margins, ovaries developed to only the early pupal stage, and reproductive tracts much reduced in size.^ The supermales with one X

- Recently termed metafemales bj' Stern (1959b).

'^ Further development of the ovaries is able to take place in normal XX + 2A hosts. Larval XXX + 2A ovaries transplanted into fes/fes hosts IM-oduce eggs in the recipient host which on fertilization are capable of developing into adult imagoes. These imagoes show that there is a low per


chromosomc and three sets of autosomes are described as resembling males but are sterile. The wings arc somewhat spread and bristles less in size. They are late emerging and poorly viable. Neither type can be referred to as superior to normal female or normal male in anatomic develoi)ment or physiologic functioning. A new type, recently described by Frost (1960j, emphasizes this difficulty. Females, called triploid metafemales, Table 1.1 had 4X chromosomes and 3 second, 3 third, and 2 fourth autosomes. Viability was greater than superfemales but still low. Fertility was about 10 per cent. Progeny per female about 10. The flies were like triploids in bristles, eye and wing cells large, sex combs absent. They showed characteristics of superfemales in rough eyes, narrow wings without inner margins, and smaller body build. The distribution of these different Drosophila sex types (Table 1.1) showed that optimal development comes when the X/A values are 1.0 and 0.5. Any deviation away from these values tends to make the sex system less rather than more efficient.

In normal Drosophila sex differences are probably expressed in every cell making up their bodies. These differences are made visible to us only under special conditions. In adult organ differentiation, the sexual differences are manifest through such things as the body size of the males being about three-fourths that of the females, differences in coloration of the tergites, the appearance of sex combs, the development of the gonads into ovaries and testes, and the formation of a secondary reproductive system composed of several glands and ducts. The origin of these last elements is of particular interest. The ovaries can be distinguished from the testes as early as the second instar through their size and position within the fat body. They are located about two-thirds the larval length back from the mouth parts. The sex combs, on the other hand, take their origin from imaginal discs which are located in the head region, possibly one-third back from the mouth parts. The secondary reproduc centage of crossing over and high nondisjunction rate in the XXX + 2A ovaries and a high mortality rate in the offspring (Beadle and Ephrussi, 1937).


tive system for both the males and females takes its origin from a disc at the posterior extremity of the larvae. The testes or ovaries are only brought together with the secondary reproductive tracts during late development in the pupae, whereas the sex combs retain their separate development. The striking action of chromosome balance in sex determination is that all of these organs and others are jointly affected and simultaneously develop directly into either the male or female sexual types. Of these characteristics, the sex combs are particularly trustworthy indices of maleness. In the male, these combs are heavily sclerotized and pigmented. They have 9 to 13 rather blunt teeth on their margins. The normal females lack these sex combs entirely. The intersexes have well developed combs, whereas the triploid females and superfemales lack them. This all-or-none situation resembles that observed in the rest of the block of sexually differentiating characteristics found in normal males or females, save that in the intersexes development may result in the organs of either sex appearing in the primary or secondary reproductive systems. The all-or-none character of the sex combs may, however, be bridged in that the appearance of certain mutations leads to the production of these combs in all of the different sexual types. The combs differ in size, in thickness and length of the teeth, and their number. The sex combs meet the conditions of a quantitative character which may be counted or measured in unbiased units. Thus in the case of sex combs, it is possible to obtain quantitative information on the effects of clu'omosomal changes on the expression of this form of sexuality.

TABLE L2

Mean sex conih teeth for flies having (lijj'erent X and


A chromosome


numbers and heteroziiqons for



the Hr gene



Sex Type


Chromosome


Mean Sex


Genotype


Comb Teeth


Males



X + 2A


11.4


Intersex



XX + 3A


9.1


Female



XX + 2A


().9


Superfemale ....



XXX + 2A


5.0


Triploid female.



XXX + 3A


4.8


Two gene mutations in D. melanogaster have aided in this search. The first is the dominant gene, Hr, which causes the male reproductive tract to be added to that of the female when this gene is heterozygous (Gowen, 1942 j . The extensive effects of this gene on the whole sex determining system have been described by Gowen (1942, 1947) and Fung and Gowen (1957a). The second gene is that of Sturtevant's (1945) transformer, tra, which operates on the female phenotype to convert it to that which corresponds to the male in having sex combs, full male reproductive system including testes, while still leaving the female characteristic body size. These genes are located in the third chromosome of D. melanogaster. Crosses between them show allelomorphic effects. Combinations of these genes in conjunction with different chromosomal arrangements make possible a series of different sex types which are distinguishable from ordinary males and females (Gowen and Fung, 1957). As some of these types bear sex combs, a quantitative character is furnished in the variation of the sex comb teeth which may be used as an impersonal measure of departure of these types from ordinary males or females. The groups having particular interest are those carrying the Hr gene in heterozygous condition with the X and A chromosomes having various numbers. The mean numbers of sex comb teeth for these various groups are shown in Table 1.2.

Analysis of these data for the contributions made by the sex chromosomes and autosomes to the numbers of teeth found on the sex combs of these different genotypes shows the following relation.

Sex combs == 12.82 - 3.42 X + 0.89 A

From this equation it is seen that the X chromosome has four times as much effect on lowering the number of teeth on the sex combs as a set of autosomes. The direction of effect is, as would be expected, increasing the number of X chromosomes tends toward making the individual more female-like in that the sex combs become smaller and less l)ronounced. Increasing the number of autosome sets on the other hand tends to push the indiviihial toward the male type with larger sex combs. Some 95 per cent of the variation in sex comb teeth has been accounted for by this equation.

The above equation results when the effect of the sex chromosomes and autosomes is considered as operating on a simple additive basis. It is interesting to consider these effects on the basis of the ratio of sex chromosomes to autosomes as utilized by Bridges. As is customary, the male genotype is given a weight of 0.50, the intersex 0.67, the female 1.00, the superfemale 1.50, and the triploid female 1.00. With these values the data on the sex combs are fitted by the equation

Sex combs = 13.40 - 6.38 X/A

The fit of this equation to these data shows control of less of the variation in the sex comb teeth. Only 76 per cent of the variation is accounted for by these methods whereas 95 per cent is accounted for when the effects of the X and A chromosomes are considered as additive.

If it is agreed that the condition of the sex combs is a good unbiased measure of the degree of sexuality of the Drosophila, it follows that it would be more probable that the genes in the X chromosomes operate additively with those of the autosomes.

III. Sex Genes in Drosophila

A. MUTANT TYPES

Bridges' concept of sex determination turned on the action of sex genes located more or less fortuitously throughout the inheritance complex of the species. In Drosophila it happens that the major female determining genes seem to be located in the X chromosome and the male determining genes in the autosomes, whereas the Y chromosome seems essentially empty of sex genes. In support of this concept limited data are cited on specific genes affecting the reproductive system or its secondarily differentiated elements and two cases where genes affected the primary reproductive system as a whole. During the interim between 1938 and the present, the numbers of these genes and the breadth of their known effects have been notably increased. Again the genes as a whole affect every phase of sexuality, morphology, fertility,


and physiology. Single genes may occasionally alter both sexes or may frequently affect only male or only female phenotypes. Single genes may appear to influence two or more distinct characteristics observable in the developing flies, although this multiple phenotypic expression may go back to a gene action which is controlling a single event in development. Genes affecting the structural development of either male or female organs frequently are accompanied by sterility of various degrees. A very large category of genes is known only through its effects on sterility of either or both sexes. Experience has shown that when properly analyzed anatomic changes are probably basic to the sterility. In this sense genes for sterility should be considered genes for sex characters. Berg (1937) furnished data on the relative frequency of sterility mutations in the X chromosome as against those in the autosomes. 12.3 per cent mutations in which the males were sterile were found in the X chromosome against 4.5 per cent found in the second chromosome. These results show that the X chromosome has many gene loci occupied by genes capable of mutating to sterility genes which affect males. Sterility is also common for the females but requires more testing. The loci for these genes are widely distributed both within and among the chromosomes.

Genes affecting sex morphology are found in all Drosophila chromosomes. Of 17 which have been recently studied; 6 were in the 1st chromosome, 5 in the 2nd, 5 in the 3rd, and 1 in the 4th. Insofar as can be determined these genes are no different than those affecting other morphologic traits. They may be dominant, they may be recessive, and a limited number of them may show partial dominance. They affect a variety of sex characteristics and do not always involve sterility of one or the other sex. The loci occupied by these sex genes may have several alleles, some of which may lead to sterility, others not. Most affect characters like size and development of the ovaries, the characteristics of the eggs, duct development, spermathecae, ventral receptacles, parovaria, paragonia, sex combs, position of genitalia, and so on.


Altlioiigh Drosophila is the leader in furnishing types for analyses of the inheritance basis for sexuality, it is well known that similar conditions exist in other forms of life.

B. MAJOR SEX GENES

Major sex genes affecting the dichotomy of the sexes have appeared among the observed mutational types. Sturtevant (1920a, 1921 j in Drosophila simulans isolated a gene in its second chromosome which when homozygous could convert diploid females into intersexuals and render XY males sterile. Phenotypically these intersexuals were female-like in that they lacked sex combs and had 7 dorsal abdominal tergites, ovipositor of abnormal form, 2 spermathecae, and lacked the penis. They were male-like in having first genital tergite although abnormal in form, lateral anal plates, claspers, black pigmented tip to the abdomen. The gonads were rudimentary. The gene was recessive and, as expected, showed no effect on D. simulans X D. melanogaster hybrids.

In 1934 a new intersexual type was observed by Lebedeff in D. virilis. Intensive study of this type showed that it depended on a fairly complex inheritance. A 3rd chromosome recessive gene ix™ at 101.5 converted the XX females into sterile males, showing only one noticeable female characteristic, the presence of a rudimentary 5th stcrnite. Sterility could be accounted for, even though the internal genitalia were completely male, by the small size of the testes and the degeneration of the germ cells largely at the spermatocyte stage. Observable effects of this gene appeared only in females, XX + 2A. Genetic modifiers were isolated which acted on the ix"yix'" complex. Other gcnotyi)es were derived through recombinations of these genes. The resulting phenotypes showed a greater variation of the male-female mosaics. One of these types was sterile but fully hermaphroditic having a complete set of external and internal genitalia of both male and female. Genetically, this type was homozygous for the ix'" gene but in addition luid both a previously found dominant scmisu})pressor and also a second semisuppressor. Another line having the ix'" gene homozy


gous had a full set of male organs but there were rudimentary ovaries attached to the testes. This line showed the dominant semisuppressor as the restraining element on the developmental pattern of the ix'" gene. Another type was separable in that it was still more female-like, yet had male external genitalia and rudimentary testes. This type was the result of the homozygous ix'" genes operating in conjunction with 3 different semisuppressors. Extensive embryologic studies were interpreted as indicating that gonads, ducts, and genitalia had started as in females with the XX constitution. Shortly thereafter male organs appeared as new outgrowths from the same imaginal disc. The development of the two sets of organs was then simultaneous but still depended on the gene pools present in the particular strain.

Another intersexual type appeared in D. pseudoobscura as a mutation, presumed due to a single dominant gene (Dobzhansky and Spassky, 1941). In a series of cultures having "sex ratio," two cultures gave 234 females, 7 males, and 266 intersexes. The females' progeny transmitted only the normal condition to their Fs progeny. The males were sterile presumably because they lacked a Y chromosome. The intersexes were also sterile so that further study of the genetic condition became impossible. The evidence, however, is interpreted as showing that the intersexes were transformed females which had inherited a dominant gene governing this condition. The intersexes were characterized by two sets of more or less complete genital ducts and external genitalia but only one pair of gonads. One set of ducts and genitalia was almost always more female-like and the other more male-like. Sex combs were present, the distal comb had 2 to 4, and the proximal 4 to 6 teeth, compared with the normal male number of 4 to 7 on the distal and 6 to 9 on the proximal comb. Body develo{)ment was more like that of the female. Cytologic examination showed two of the intersexes had (wo ()\-aries each. One of these two had a rudimentary testis. The chroinosoiiu' complement was 2X + 2A.

A dominant gene which causes intersexiiality in diploid females of D. virilis was estal")lished by Briles. Stone (1942) located this gene in the second chromosome. Price ( 1949j placed the gene within the second chromosome near the locus of "brick" by inducing crossing over in males by exposing them to x-rays. Newby (1942) extensively studied the embryologic sequence in development of the organs of these intersexual types. The Ix^ gene did not affect the males, XY 4- 2A, but did change the females XX + 2A into intersexes when it was in the heterozygous condition. The intersexuals had 9 tergites; the first 6 were like those in the normal female and the last 3 were small and irregularly formed. There were 6 sternites, the first 5 being normal, but the 6th malformed. Anal valves were lateral as in the male but a third small valve was also present at the ventral side of the anus. The plates forming the claspers were of irregular pattern and found ventral to the anus. The vaginal plates were often extruded into a genital knob and were below the claspers. The knob occasionally became heavily pigmented. The internal organs ranged from nearly female through those which were of hermaphroditic type containing representative organs of both sexes to individuals almost wholly male. Newby concluded that intersexuality expresses itself as a response to the developmental pressures of both sexes, not as development in the one direction followed by a change.

Gowen in 1940 established a stock carrying the dominant gene Hr which had apl)eared as a mutant in one of his cultures of D. melanogaster. This gene affected diploid females of XX + 2A type but not the males of XY -I- 2A constitution. In the presence of the Hr gene the diploid phenotype of the females changed into a sterile type with male secondary reproductive system associated with the female counterpart. The first 6 segments were complete with 6 spiracles. The 7th was small with spiracle. The 8th was small but without spiracle. Sternite forming rudimentary ovipositor was usually protruded. Ninth and 10th segments resembled those of males with large tergal plates. Claspers were abnormal and had a pair of small plates flanking the anus majoi-ly in vertical position. Organs formed, although sometimes modified or missing, included: sex combs of 6.9 long slender teeth, gonads distinguishable from those of the ordinary male or female in the 3rd and possibly the 2nd instar, genital ducts male and/or female, male accessory gland, penis deformed, sperm pump, vas deferens, spermatheca, ventral receptacle often displaced, and occasionally parovaria. The primary gonads were often abnormal ovaries but in rarer instances bore a crude resemblance to testicular tissue. The yellow of the testes was frequently present as material clinging to the ovary.

Superfemales with one dose of the Hr gene had sex combs and developed parts of both the male and female external and internal reproductive systems. Sex combs had an average of 5 long and slender teeth. Abdominal segment 8 developed as in the female, and formed the vaginal plate. The latter was abnormal in shape, ordinarily becoming a sclerotic protuberance. Segments 9 and 10 developed more as in the male but were incomplete and abnormal. The genital arch did not develop but the inner lobe of tergite 9 showed irregular and abnormal growth as for the claspers. Segment 10 developed, as in the males, into longitudinal plates flanking the anus. The internal genitalia were underdeveloped but consisted of mixtures of male and female organs. Gonads were rudimentary but generally consisted of a pair of ovaries with small traces of yellow pigmentation.

The triploid fly with one dose of the Hr gene was largely female with developed ducts, ovaries and eggs, but was sterile. The male characteristics were small sex combs and dark abdominal plates. In superfemales, sex combs were present and teeth were intermediate between those of the diploid female and triploid female. The gene showed a dosage effect in triploids which was less than that observed in diploids and was in relation to the relative balance of the gene with its normal alleles, 1:2 for the triploid and 1 : 1 for the diploid. The developmental effects of Hr as well as the pigment producing potentialities of testes, ovaries and hermaphroditic gonads have been discussed by Fung and Gowen (1957a, b).

Hr has been shown to be allelomorphic to a recessive gene, tra, described by Sturtevant (1945) and known to be located in the 3rd chromosome. The location of this allele, tra, is at 44 to 45 or between the genes scarlet and clipped. When homozygous the gene transformed diploid females into sterile males. Heterozygotes showed no detectable differences from normal females of XX constitution. Males XY homozygous for tra or heterozygous for it were indistinguishable from normal males. The homozygotes XX, tra/tra were female in body size, but otherwise were nearly male in appearance. They had fully developed sex combs, male colored abdomens, normal male abdominal tergites, anal plates, external genitalia, genital ducts, sperm pumps, paragonia, and showed the usual rotation of the genital and anal segments through 360 degrees. They mated with females readily and normally. The testes, however, although normal in color, elongated, curved, and attached to the ducts were of small size. Testis size was never that found in normal brothers. The addition of a single Y or two Y's did not alter fertility.

The triploid females 3X + 3A homozygous for tra, had large bodies, ommatidia and wing cells. They resembled the diploid homozygous tra individuals in having male external genitalia, well developed ejaculatory ducts, sperm pumps, and accessory glands, testes elongated but narrower than those of normal males, and sex combs averaging about 9.6 teeth. They mated with females but were completely sterile.

Triploids with one or two doses of tra were like wild type triploids in having no sex combs and being female throughout. Intersexes having one or two doses of tra were similar to intersexes having only wild type genes in the locus.

Sturtevant obtained one superfemale which was homozygous for tra. It had male genitalia and sex combs with only about half the normal number of teeth. This individual argues for a greater balance toward the female side of sexual development than either the diploid or triploid females previously discussed. The evidence is, however, contradictory to that furnished by the Hr gene as indicated earlier.

A combination of two or more genes, Beaded and various Minutes, having well known phcnotypic effects, has sometimes


produced phenotypes which have been interpreted as peculiar, low grade types of intersexuality in males (Goldschmidt, 1948, 1949 and 1951). The data showed that the Beaded cytoplasm favors the low grade intersexual male whereas the Minute cytoplasm favors the reduced male with the hetcrozygote being intermediate. Just how far these types may be related to the other types strongly affected by specific genes is a matter of question, having at least other interpretations (Sturtevant, 1949).

In 1950, Milani trapped an inseminated female of D. subobscura w^iich segregated intersexual progenies. Spurway and Haldane (1954) studied these intersexual types. A recessive guiding development toward these intersexes was located on the 5th chromosome of subobscura. When present it caused the XX homozygous females, ix/ix + 2A, to have sex combs on both the first and second tarsal joints. The numbers of teeth making up the sex combs w^ere reduced as also were the sizes of the teeth. The illustration in Spurway and Haldane's (1954) paper indicates that the number of teeth was 7 on the first tarsal joint and 5 on the second joint, whereas the sex combs of the males had 11 teeth on the first joint and 9 on the second. A series of changes were observed in the genital plates which graded from those resembling true females to those approaching the male type.

C. OTHER CHROMOSOME GROUP ASSOCIATIONS IN DROSOPHILA AMERICANA

I), aniericana has 4 chromosomes in the female genome and 5 chromosomes in the male genome. As compared with D. virilis the X chromosome is fused with the 4th chromosome and the 2nd chromosome is fused with the 3rd, the 5th and 6th chromosomes are free in the female, whereas in the male genome the Y chromosome, 4th, 5th and 6th chromosomes are free and the 2nd and 3rd fused. Stalker (1942) has shown that the three female genomes are balanced and lead to triploid females as they do in D. melanogaster. D. aniericana triploids differ from their diploid sisters in having bigger ommatidia, larger wing cells and somewhat larger bodies. When these triploids are bred to diploid males they give rise to 6 chromosomally different types of offspring: diploid males, diploid females, triploid females, intersexes, females carrying a Y and a male limited 4th chromosome hut otherwise diploid, and tetraploid females. All intersexes cytologically show a Y and a 4th chromosome present. Intersexes without the Y are presumed also w^ithout male limited 4th chromosomes and would be expected to be inviable or very weak. No supermales or superfemales, that w^ould correspond with those found in D. melanogaster, were observed so are presumed to l)e inviable due to unbalance for the 4th chromosomes. Among 948 progeny of triploid females X diploid males there were 9 individuals that were phenotypically abnormal females. They had slightly spread, ventrally curved wings with slightly enlarged wing cells. In 8 of the 9 the first section of the costal vein was shortened so that no junction was made with the first vein at the distal costal break. Heads were large with rough eyes, thoraxes shortened, legs fre(luently malformed, and abdomens small with unusually wide 7th sternites. Genitalia were apparently normal with well developed ovaries. This type carries three doses of any genes contained in the 4th chromosome to two doses of the genes in the other chromosomes. Its phenotype represents a trisomic condition.

The sex characteristics of the flies observed in D. americana seem to follow the same patterns as those of D. melanogaster as judged by the numbers of the X chromosomes and autosomes. A Y chromosome in the intersex was not observed to affect sex expression. The intersexes could be grouped into six classes ranging from extreme male type to the most female type. The male type showed largely male organs, courted females, and had motile but nonfunctional sperm. The most female type had nearly normal ovipositor plates, well developed uterus, ventral receptacle, spermathecae and oviducts. At least one gonad showed egg strings, although a small patch of orange-red tissue was present at the tip. Two types of chromosomes were observed in the nuclei of these extreme female type intersexes; those like the chromosomes in any normal diploid cells and some which were so swollen as to be almost unrecognizable. Such swollen chromosomes were not found in the other classes of intersexes or in diploid or triploid individuals. They are suggestive of some noted by Metz (1959) in Sciara. Most of the intersexes were of the male type, 45 per cent, with decreasing numbers for each of the other five classes until those in the most female class constituted only about 4 per cent of the total.

D. LOCATION OF SEX-DETERMINING GENES

The problems of isolating and determining the modes of action of the factors normally operating in sex determination have received extensive study since they were reviewed by Bridges in 1939: Patterson, Stone and Bedichek (19371, Patterson (1938), Burdette (1940), Pipkin (19401942, 1947, 1959), Poulson (1940), Stone (1942), Dobzhansky and Holz (1943), Crow (1946), and Goldschmidt (1955). From his work on Lymantria dispar, Goldschmidt concluded that sexual differentiation was controlled by a major male factor, M in the Z chromosome and a factor F directing development toward the female and at first assumed to be in the cytoplasm but later considered to be in the W chromosome. The heterogametic female of this species would then be FM and the male be MM. In considering this problem, Goldschmidt attempted to distinguish between the sex determiners responsible for the F/M balance and modifiers affecting special developmental processes (Goldschmidt, 1955). At the other extreme Bridges' study of triploids led him to consider that sex in Drosophila was determined by the interaction of a number of female tendency genes found largely in the X chromosome and of genes having male bias located largely in the autosomes. These numerous genes were considered as being distributed throughout the whole inheritance complex. Search for the more exact locations of these genes within the different chromosomes of Drosophila has largely taken the form of determining the variation in sex types as induced by the addition or deletion of various pieces of the different chromosomes to either the normal male, normal female, or triploid complexes.



The sections of the chromosomes added were derived from previous translocations generally to the 4th chromosome and were of varying lengths determined through cytologic and genetic study. Summaries of these comparisons are found particularly in the papers of Pipkin (1940, 1947, 1959). In their search for a major female sex factor in the X chromosome, Patterson, Stone and Bedichek (1937), Patterson (1938), Pipkin (1940), and Crow (1946) finally were unable to show that any single female sex determiner, located in the sex chromosome, was of primary importance to sex. Evidence for multiplicity of genes with a bias toward female determination was found by Dobzhansky and Schultz (1934) and by Pipkin (1940). They were able to transform diploid intersexes into weakly functioning hypotriploid females by the addition of long fragments of the X chromosome to the 2X + 3A intersex complement. Short sections of the X chromosome in some cases shifted the sex type in the female direction. Pipkin found that additions of short X chromosome sections, to the 2X -|- 3A chromosome sets, although covering in succession the entire X chromosome, were insufficient to make the flies other than of the intersexual type. Longer and longer fragments from either the left or right end of the X chromosome caused a qualitatively progressive shift toward femaleness. Weakly functional hypotriploid females resulted when either right- or left-hand sections of two translocations with t-lz (17) and Iz-v (W13) breaks were present in the 2X + 3A chromosome complement. These duplication intersexes possessed 1 or 2 sex comb teeth when reared at 22 °C. and up to 5 well developed teeth when reared at 18°C. These facts give support to the multiple sex gene theory of Bridges or at least that quantitative differences in sex potencies exist within the X chromosome. This conclusion was further strengthened by the hypointersexes lacking a short portion of one of their two X chromosomes although possessing three of each autosome, inasmuch as these types were shifted strongly in the male direction. These studies of the X cliromosonie show that several parts of this chi-omosomc are concerned witli female dif


ferentiation and that the effects are irregularly additive.

Similar search of the autosome II and III for genes of male potency showed that small shifts in the male direction were found in hyperintersexes for several short regions of chromosome III but for none of chromosome II (Pipkin, 1959, 1960). Tl^ree slightly different right-hand end regions of chromosome III produced the largest shifts in the male direction in hyperintersexes, but no increase in number of sex comb teeth. These changes were comparable with those produced in the female direction by the addition of very short sections of the X-chromosome to the 2X + 3A intersex complement. On the other hand, none of the seven different hypointersexes lacking a short section of the 3rd chromosome from the 2X + 3A complement showed a shift in the female direction. This is rather surprising as hypointersexes for two short regions in the X chromosome were shown by Pipkin (1940) to shift the sex type in the male direction as was to be expected. From these results Pipkin (1959) derives the conclusion that 3rd chromosome aneuploids as well as those of the 2nd chromosome and X chromosome support the deduction that dosage changes of portions of the X chromosome are more powerful than dosage changes of portions of either of the large autosomes in affecting sex balance. This view receives further support through changes of size and number of sex comb teeth as observed in the chromosomal types carrying the gene Hr and reviewed earlier.

Influence of the Y chromosome on sexual differentiation has generally been ruled out as XO nondisjunctional flies are male although they are sterile (Bridges. 1922). Similarly Dobzhansky and Schultz (1934) ruled out the Y chromosome as an effective influence on sex types of triploid intersexes of D. melanogaster since the mean sex type of Y -(- 2X 4- 3A intersexes did not differ significantly from the sex tyyies of siblings 2X + 3A. "

The steps taken in the studies of chromosome IV are of interest. Dobzhansky and Bridges in 1928 concluded that the 4th chromosomes play no part in sex determination in D. melanogaster. The evidence was of two kinds. Triploid females were outcrossed separately to males of two diploid stocks. The triploid daughters from these crosses were again crossed to males like their fathers. This repeated outcrossing to the different stocks resulted in a shift in the grade of the intersexes in both cases, in one case to a very high proportion of extreme male-like intersexes and in the other to nearly as high a proportion of extreme female-type intersexes. These results were interpreted as showing that the grade of development of the sexual characters was dependent on genetic modifiers. The second experimental test consisted of subjecting a triploid stock to selection toward a line which produced a high proportion of extreme male type intersexes and to another line which would have a high proportion of extreme female type intersexes, each being much higher than the original stock. The l)rocedure established a line with a high proportion of extreme male type and another line which was not so extreme in its proportions but was definitely higher in female type intersexes than the original stock. Again these results w'ere interpreted as indicating the selection of modifying genes of unknown positions within the inheritance complexes.

This evidence had been preceded by Bridges' (1921) discussion in which he wrote "the fourth-chromosome seems to have a disproportionately large share of the total male-producing genes; for there are indications that triplo-fourth intersexes are predominately of the 'male-type', while the dijjlo-fourth intersexes are mainly 'femaletype'." In 1932, Bridges concluded for Drosophila intersexes that, in spite of the favorable genetic checks, in repeated and varied tests, it has been impossible to state with any assurance whether the 4th chromosome is or is not a large factor in the variability encountered.

In our own work a stock of attached X triploids has for many years consistently produced only male type intersexes. This is in contrast to what we frequently see within other lines of triploids as made up utilizing the cIIIG gene (Gowen and Gowen, 1922). Lines established from these triploids ordinarily have three intersexual types: male,


intermediate, and female. These lines, however, may be subjected to selection in both directions. In our experience, male intersex lines are established rapidly and remain relatively permanent. On the other hand, female intersex lines take many more generations and are less stable. These lines have been extensively examined for their 4th chromosome constitutions (Fung and Gowen, 1960). The male intersex lines seldom show more than two 4th chromosomes. On the other hand, the female intersex lines rarely show two 4th chromosomes but generally have more than three, the number sometimes going as high as four. More tests are needed but the evidence would seem to indicate that the fourth chromosome does have sex genes. These genes, contrary to the first notion of Bridges, are more frequently of the female determining type than of the male determining type. This would make the 4th chromosome like the X in that it carries an excess of female influencing genes and is not like the rest of the autosomes which have an excess of male determining genes. These observations are of particular interest in view of Krivshenko's (1959) paper. In this investigation on D. busckii, cytologic and genetic evidence was presented for the homology of a short euchromatic element of the X and Y chromosome with each other and also with the 4th chromosome or microchromosome of D. melanogaster. This conclusion is based on (1) observed somatic pairing of the X and Y of D. busckii by their proximal ends in ganglion cells and the conjugation of the short euchromatic elements of these chromosomes at their centromeric regions in the salivary gland cells; (2) the presence in the short Y chromosomal element of normal allelomorphs to four different mutant genes of the short X chromosomal element; (3) the presence in the short element of the D. busckii X chromosome of chromosome IV mutants: Cubitus interruptus. Cell and shaven of D. melanogaster. These considerations furnish proof for the homology of this X chromosomal element with the 4th chromosome of Drosophila.

These observations of Krivshenko support our findings that the 4th chromosome of D. melanogaster has an excess of female determining functions. It would further show that autosomes may behave differently with regard to their sex-determining properties according to the chance distribution of sex genes which happen to fall within them as they do in Rumex (Yamamoto, 1938j. The finding that the chromosome IV has a bias toward female tendencies further strengthens Bridges' multiple sex gene theory and weakens the theory of an all-or-none action of the whole X chro


IV. Sex under Special Conditions

A. Species Hybridity

Hybrid progeny coming from species crosses are apt to represent but a very few of the possible genotypes of the total number that conceivably could come from the gene pool. The hybrid phenotypes may display three kinds of characteristics. The common set is that derived from genes in either or both parents through ordinary meiotic segregations and dominance. The second set shows intermediate development of the characters found in the two parent species. The third set of characters that complete the animal is new to those observed in either parent species. These new characters may be the loss of a few dorsocentral and scutellar bristles, broken or missing cross veins, or abnormal bands in the abdomen as in D. simulans x D. melanogaster hybrids (Sturtevant, 1920b), extra antigenic substances as found in dove hybrids (Irwin and Cole, 1936), or more numerous characteristics as in the mule. Frequent among these new characteristics is sterility. The sterility may extend to either or both sexes and affect the secondary sex ratios. As Sturtevant (1920b) points out, crosses between the domestic cow and male bison give male offspring with humps derived from the bison which are so large as to prevent their being born alive. The female hybrids lack these humps and are conse(iuently born normally. The abnormal sex ratio observed at time of birth is due to causes external to the hybrid itself and attributable to the structure of the mothers.

A comparable case was found during the study of female sterility in interspecies hybrids of Drosophila pseudoobscura in which Mampell (1941) showed that in the hybrids of certain strains, the females produced no or few offspring because of interspecies lethal genes connected with a maternal effect. Comparable cases as well as those dependent on other mechanisms are known for other groups. The progeny may also be altered to give new sex types, generally intersexes. These intersexes often replace either the male or the female sex group. However, despite their apparent relation, the changes in the sex ratio and the appearance of intersexes can have different causes. D. simvlans X D. melanogaster hybrids emphasize that there may be no relation between the peculiar hybrid sex ratios and the intersexes since extreme differences in sex ratio occur but no intersexual types.

Species, however, may have natural differences in the sex potencies of their X chromosomes and/or their autosomes. In crosses between D. repleta and D. neorepleta involving a sex-linked recessive white-eyed mutant type of D. repleta Sturtevant (1946) obtained about 15 per cent fertile matings in 500 mass cultures, a total of 532 females to 635 males. All progeny as expected were wild type in character. The males, however, had long narrow testes and were totally sterile, a condition later shown to be due to a gene in the X chromosome located near the white locus. Females suggested intersexuality in having three anal plates instead of the usual two. Mating of Fi hybrid females to white D. repleta males gave 9 per cent fertility, the 179 offspring being distributed as 70 wild type females, 9 white females, 42 wild type males, and 58 white males, although the expectation for the classes was equality. Evidence indicates that some of the 9 white females were intersexes as were possibly some of the white males. The wild-type males again had the long narrow testes and sterility of the Fi male progeny. Wild type females were moderately fertile. By continued backcrossing to 1). repleta males having white or whitesinged, a female line was picked up which continued to have the unusual sex ratios but had more fertility. It was presumed that the D. neorepleta gene responsible for the unusual ratios was originally associated with MHother gene that decreased fertility in females largely of D. repleta constitution and that the foundation female for the more fertile line came as a result of a crossover between an infertility gene and that responsible for the unusual sex ratios. Continued back crosses of females of this line to white D. repleta males have been made. Out of 33 fertile cultures, 16 gave approximately equal ratios of wild-type and white females, wild-type and white males; and 17 gave 472 wild-type females, 5 white females, 63 white intersexes, 482 wild-type males, and 339 white males. The white females presumably represented crossovers between the loci of white and the critical gene in the X derived from D. neorepleta. The intersexes were of extreme type with gonads very small (rudimentary ovaries in those cases where they were found at all). External genitalia were missing or of abnormal male type. Other somatic characteristics included weakness which prevents emergence and accounts for the loss of about 88 per cent of the flies expected in that class. The intersexual condition was suggested as being caused by an autosomal dominant gene derived from D. neorepleta which so conditions the eggs before meiosis that two D. repleta X chromosomes result in the development of intersexes rather than females. The action of this gene occurs before meiosis and may in fact be absent from the intersexes themselves. This was confirmed by crosses of white brothers of the intersexes to pure D. repleta females when the offspring were normal for both sexes; but when these Fi daughters were mated to D. repleta males only intersexes and males resulted. This last cross further showed that although this gene was derived from D. neorepleta in D. neorepleta cytoplasm, the D. neorepleta cytoplasm was not necessary for the intersexes to result. The case also has an important bearing on the location of the sex-determining factors, for in this cross the characteristics were only secondarily governed by the cytoplasm through earlier determination by genes of the mothers' nuclei.


Significant parallels are found between the autosomal gene of D. neorepleta and the third chromosome Ne gene of D. melanogaster (Gowen and Nelson, 1942) described in the section on high male sex ratio. The D. neorepleta gene caused the cytoplasms of the eggs laid by mothers carrying it to become more male potent. The female potencies of two X chromosome D. repleta zygotes were unable to balance these male elements. Many died late in development. Those able to emerge became intersexes. The Ne gene also sensitizes the cytoplasms of all eggs of mothers carrying it causing any 2X + 2A, 3X + 3A, 3X + 2A or 2X -h 3A intersexes of female type to die in the eggs at 10 to 15 hours whereas males XY + 2A and male-type intersexes live.

Other mechanisms for causing sex and sex ratio changes are known, i.e., Cole and Hollander (1950), but few are as well worked out as that of D. repleta x D. neorepleta. New mechanisms will certainly be found for the opportunities for genetic analysis of sex in hybrids are many.


B. Mosaics For Sex

Recent genetic work has emphasized the fact that individual D. melanogaster may be composed of cells of more than one genie or chromosome constitution. The main type of sex mosaic is the gynandromorph composed of cells of female constitution on one side, XX + 2A, and male, X + 2A on the other, the loss of the X chromosome coming at an early cleavage (Morgan and Bridges, 1919; L. V. Morgan, 1929; Bridges, 1939). The mosaic areas are large since the cells of each type may be in nearly equal numbers.

At the other extreme Stern (1936) has shown that phenotypic mosaics may develop as a consequence of the somatic chromosome pairs crossing over at late stages in embryologic development. Special genes, Minutes, materially increase the frequencies of these crossovers. The proportion of the body occupied by the cross-over type cells is small because crossing over takes place so late in development.


Recently another agent in the form of a ring chromosome has been discovered which greatly increases the production of sex mosaics. Some ring chromosomes are relatively stable whereas others are quite unstable, the instability depending to some extent on aging of the eggs and environmental factors (Hannah, 1955). The instability is manifest by frequent gynandromorphs, XO males, and dominant lethals among the rod and ring zygotes. It has been suggested that the instability is due to heterochromatic elements. Hinton (1959) has observed the chromosome behavior of these types in Feulgen mounts of whole eggs that were in cleavages 3 to 8. He found strikingly abnormal chromosome behavior in these cleaving nuclei. For some cell divisions chromosome reproduction was interpreted as being through chromatid-type breakage fusion bridge cycles. As a result of this behavior mosaics are formed which are intermediate between those of the half gynandromorphs and those which occur much later because of somatic crossing over. In terms of volume of cells included, the abnormal types may include only a few cells of the total organism, a fair proportion of the cells, or a full half of the whole body. These unstable ring chromosome mosaics may be a part of the secondary reproductive system or for that matter any other region of the body. When the mosaic cells are incorporated in the region of sex organ differentiation male or female type organs or parts of organs may develop as governed by the cell nuclei being X, XX or some fraction thereof.


Gynandromorplis appear sporadically and rarely in many species but in some instances genes which activate mechanisms for their formation are known. In the presence of recessive homozygous claret in the eggs of D. siniulans, gynandromorphs constitute a noticeable percentage of the emerging adults. The gene nearly always operates on the X received from the mother causing it to be eliminated from the cell. The resulting gynandromorphs are similar to those of D. melanogaster. The fact that the claret gene should affect the X and a particular X chromosome is suggestive of the manner in which given chromosomes arc eliminated in Sciara. Other types of sex mosaics will be found in the descriptions of other species, i)articularly in the Hymenoptci'a.


C. Parthenogenesis in Drosophila

Parthenogenesis is of interest as it changes the sex ratios in families and brings to light new sex types and novel methods for their development (Stalker, 1954), A survey of 28 species of Drosophila showed a low rate of parthenogenesis in 23 species. Adult progeny were obtained for only 3 species. For D. 'parthenogenetica the original rate was 8 in 10,000 whereas that for D. polymorpha was 1 in 19,000. These rates could be increased by selection of higher rate parents: 151 and 70 per 10,000 unfertilized eggs of the first and second species respectively.


D. parthenogenetica diploid virgins produced diploid and triploid daughters as well as rare XO sterile diploid males. Triploid virgins produced diploid and triploid females and large numbers, 40 per cent, of sterile XO diploid males. Diploid virgins heterozygous for sex-linked recessive garnet produced homozygous and heterozygous diploid females as well as +/+/g and + /'g/g triploid females. No homozygous wild-type or homozygous garnet triploid females or garnet mosaics were found. Diploid females crossed to fertile diploid males produced few if any polyploid progeny or jjrimary X chromosome exceptional types. Of the unfertilized eggs from diploid virgins which started development, 80 per cent died in late embryonic or early larval stages. The parthenogenesis in diploid females depended on two normal meiotic divisions followed by fusion of two of the derived haploid nuclei to form diploid progeny, or the fusion of three such nuclei to form triploitl progeny. In the triploid virgins similar fusions of the maturation nuclei may produce diploid and triploid females but the large number of diploid XO sterile males were picsunicd to be the result of cleavage without prior nuclear fusion. Such cleavages without fusion in eggs of dijiloid virgins would lead to the production of haploid embryos. They were presumed i-esponsible for the large early larval and embryonic <h'atlis. These observations have been confirmed by the study of S|)rackling (1960) involving some 2200 eggs at various stages of cleavage. Evidence from XXY diploid virgins indicated that biiuiclcar fusion in unfertilized eggs involved two terminal haploid nuclei or two central nuclei. The fact that tetraploids were not observed as progeny of ti'iploid vii'gins was considered indicative of relative inviability of this type. Successful parthenogenesis was under partial control of the inheritance as 80 generations of selection increased the rate about 20-fold. Similarly outcrossing to bisexually reproducing males and reselection resulted in both pronounced increases in and survival of the parthenogenetic types.


Carson, Wheeler and Heed (1957) and Murdy and Carson (1959) have established a strain of Drosophila mangabeirai with only thelytokous reproduction. Males have been captured in nature but are rare. Fecundity of the virgin females is low but the egg hatch is 60 per cent and 80 per cent survive to adult stage. The progeny of the virgins are always diploid. In meiotic spindle formation D. mangabeirai differs from other Drosophila species in that its orientation increases the probability for fusion of two haploid nuclei into structurally heterozygous diploid females. The study of Feulgen whole mounts of freshly laid eggs indicated automictic behavior with two meiotic divisions followed by a fusion of two of the four haploid meiotic products. The absence of adult structural homozygotes in wild populations is probabl}^ explained by death during early development and by possible fusion of second division meiotic products derived from different secondary oocytes. Stalker (1956a) postulated such selective fusion in order to account for the heterozygous condition in females of Lonchoptera dubia, an automictic, parthenogenetic fly in which males are rare or unknown.

A

case in which jihenotypically rudimentary females, supposed homozygous for r/r 

give rare and unexpected type progeny, has suggested that polar body fusion may also take place in D. melanogaster (Goldschmiclt, 1957). The rudimentary mothers producing the peculiar type are interpreted as formed by a most unusual series of events: a fertilization nucleus derived from the fusion of an r containing egg fertilized by an r containing sperm and a polar copulation nucleus derived from the fusion of a polar body containing an r genome and one having wild type. Both cell types become incorporated into the ovary. The progeny which come from these supposed rudimentary mothers are presumed to be derived from the maturation into eggs of the cells derived from the heterozygous i)olar copulation nuclei. If these progeny-producing rudimentary mothers arise in the presumed manner they give the basis for a parthenogenetic mode of reproduction and the sex types which have been described in other Drosophila species by Stalker and Carson. There are similar, as well as other forms of parthenogenesis which affect sex (Smith, 1955) or assist in maintaining trijiloid conditions (Smith-White, 1955). A number of these types have been reviewed by Suomalainen (1950, 1954). The reader may be referred to this material for other cases and chromosome behaviors.


D. Sex Influence of the Y Chromosome

The first function discovered for the Y chromosome in D. melanogaster was that it was necessary to male fertility (Bridges, 1916). Two and possibly more Y chromosome-borne, genetic factors were involved (Stern, 1929). Gamete maturation when these factors were lacking ceased just short of the sperm's becoming motile (Shen, 1932). The motility conferred on the sperm by the presence of the Y chromosome factors was fixed for the testes at an early stage of development as transplantation experiments, sterile testes to fertile larvae and fertile testes to sterile larvae, showed motility to be a property determined by early localized somatic influences on the developing gametes or predetermined in the diploid phase (Stern and Hadorn, 1938). This Y chromosome function was sex limited, because females without a Y were the normal fertile females and those with an extra Y also were fertile.


Neuhaus (1939) followed by Cooper (1952, 1959) and Brosseau (1960) further analyzed the Y chromosome for fertility loci. The latter showed at least two fertility loci on the short arm and five on the long arm of the Y chromosome. Data are compatible with a linear order of the genes. An additional fertility factor common to the X and Y was suggested. The Y chromosome fertility factors consequently fall in line with Bridges' concept of multiple gene loci distributed in a more or less random manner which may affect sex.


The Y chromosome has other attributes which help to explain its significance to secondary if not primary sex characteristics. As with many other species it has loci for genes which are also found in the X chromosome, i.e., bobbed, as well as a limited number of bands in the salivary gland chromosome. Nucleolus organizers and at least two specialized pairing organelles similar to those in the X chromosome are found within the Y chromosome (Cooper, 1952, 1959). One of the most significant properties is the effect of extra Y chromosomes on the variegation observed for various gene phenotypes either in the normal chromosome pattern or in that accompanying translocation. In variegation one Y chromosome as extra to the normal complex is sufficient to eliminate or more rarely to much reduce the variegated expression (Gowen and Gay, 1933). When the Y chromosomes are 2 above the normal complement, the phenotypes gain two new features (Cooper, 1956). Both males and females become variegated in the expression of their eye characteristics. The males become sterile. These effects are unexpected for they are counter to any previous trends in the Y effects on these characteristics. They have reversed the direction of the effects as established by the two previous chromosome types. The variegation of the XX2Y + 2A females and X3Y + 2A males resembles that of the XX + 2A females and XY + 2A males but is more extreme, whereas the XXY + 2A and X2Y + 2A are largely nonvariegated. The fertility relations are equally aberrant: the X3Y + 2A males have the same type sterility through loss of sperm motility as that of the XO + 2A males. Bundles of sperm are formed but they do not become motile. Full-sized Y chromosomes are not required to bring about these effects, because females having a whole Y plus a piece of a second, or males with 2Y plus a piece of a third, will show the effects.


The fractional Y chromosomes furnish opportunities to test for the partial independence of the variegation and sterility effects. The two Y hyperploid males differ in their degree of fertility according to the fraction of the Y chromosome which may be present whereas the effects on variegation may be constant among groups. This is in accord with the sterility being in part independent of the factors causing the variegations. Similarly, the variegations may be shown to be partially free of the action of some elements that are not themselves members of the two sets of factors influencing fertility in the normal male.


Other phenotypic irregularities appear; eye facets may be roughened, legs shortened, and wing membranes become abnormal. On the negative side two extra Y chromosomes in females homozygous for the transformer gene, tra, do not increase in maleness or function.


The variegations of the so-called V-type position effects with translocations are suppressed, as in the normal type described, by one extra Y but are nonetheless variegated when two extra Y chromosomes are present. That these effects are caused by there being two supernumerary Y's is indicated by the fact that XX2Y females are fertile and X3Y males sometimes lose a Y chromosome in their germinal tracts and become fertile. A number of mechanisms have been suggested to account for these results but most have proven unsatisfactory. A balance interpretation for the X, Y and autosomes like that for sex, as suggested by Cooper (1956) is compatible with the somatic cell variegations for euchromatic loci transferred to the heterochromatic regions and the sterility-fertility relations expressed by the different chromosomal types.


The variegation effects of the Y chromosome take on further significance. Baker and Spofford (1959) have shown that 15 different fragments of the Y chromosome when studied for their contributions to variegation differ in their effects with the differences often not related to the size of the fragment, thus indicating that the Y chromosome has linearly differentiated factors capable of modifying the variegated phenotype.


Other indications of genetic activity of the Y chromosome were given by Aronson (1959) in her study of the segregation observed in 3rd chromosome translocations. A deficiency for the region of the 3rd chromosome centromere is lethal when homozygous. Both males and females are fully \'iable when this deficiency is heterozygous. XO or haplo IV deficient males die. However, in the presence of a Y chromosome the deficient haplo IV males become viable. The lability of this last class indicates that the Y chromosome is genetically active, can compensate for the autosomal deficiency, and thus alter the progeny sex ratio.


In D. virilis the situation is somewhat different from that observed by Cooper in 1956 in D. melanogaster. Baker (1956) has shown that, in a translocation, males having two Y chromosomes plus a Y marked with a 5th chromosome peach are fertile. These results seem to indicate a species difference in the effect of the extra Y on fertility or the Y with the inserted peach locus is not a complete Y and in consequence the true composition of these males is X + 2Y plus a fragment of the Y. The X chromosomal associations in the multichromosomal types are shown to be by trivalents or by tetravalents. The segregation data indicate that the pattern of disjunction of trivalents is a function of the particular Y chromosome involved. In X2Y males with normal Y's or with one normal and one marked Y, the Y's disjoin almost twice as frequently as they do from trivalents with two identical Y's. Tetravalent segregation is almost entirely two by two, with no preference for any of the three types of disjunction.


An odd situation was reported by Tokunaga (1958) in substrains of Aphiochaeta vanthina Speiser. When a male of the substrain was crossed to individuals bearing 3rd chromosome genes of the original strains, the mutant genes for brown, and so on, behaved as if they were partially sexlinked in the following generations. On the other hand, when a female carrying the partially sex-linked genes on the X and Y chromosomes. Abrupt or Occhi chiari, was crossed to the male of the substrain the characters segregated as though they were autosomal. As a working hypothesis it was suggested that in this species the Y chromosome had the major male determining factors. The "special" male arose as a translocation of these factors to the third chromosome with the consequent change in linkage relations. Data on the role of the X chromosome in sex determination in this species have not yet been obtained but if they support the interpretation they indicate real differences between this species and that of Drosophila in the location of the sex genes. The results are reminiscent of those obtained by Winge in Lebistes as well as those in Melandrium and other forms in which the Y or W chromosomes may contain strong sex genes for either sex. They would further support the thesis that sex genes may be distributed to almost any loci within the inheritance complex.


E. Maternal Influences on Sex Ratio

Aside from chance and specific genetic factors, sex ratio is subject to effects from agents intrinsic in the cells of the mothers (Buzzati-Traverso, 1941; Magni, 1952). Strains of Drosophila bifasciata (Magni, 1952, 1953, 1957), D. prosaltans (Cavalcanti and Falcao, 1954), D. willistoni and D. paulistorum Spassky, 1956 have been isolated which were nearly all of the female sex even though the mothers were outbred to other strains having normal ratio bisexual progeny. Study of these strains by the above workers and Malogolowkin (1958) IVIalogolowkin and Poulson (1957), and Malogolowkin, Poulson and Wright (1959), as well as by Carson (1956) have shown inheritance strictly through the mother regardless of the genetic nature of the males to which they were bred. The unbalanced ratio was retained even when the original chromosomes had been replaced by homologous genomes from lines giving normal male and female progenies. Transmission of this unbalanced sex ratio was through the cytoplasm. Eggs fertilized by Y-bearing sperm died early in the course of development. Malogolowkin, Poulson and Wright (19591 have shown that the high female ratio and embryonic male deaths may be transferred from affected females to those which do not normally show the condition through injection of ooplasm from infected females. Ooplasm of this same type is, w^hen injected into males, suflficient to cause death to occur within 3 days. In the females a latent period of 10 to 14 days after ooplasm injection was apparently necessary to establish egg sensitization. Once established the condition could be transmitted through the female line for several generations. The ooplasm injections were not as efficient in establishing these lines as females found naturally infected. Unisexual broods may fail to appear, in some instances fail to transmit the condition or produce intermediate progeny ratios, as well as in some instances to skip a generation. The infectious agent was found to vary in different species. Magni (1953, 1954) for D. bifasciata found that temperature above normal tended to remove the cytoplasmic agent making it ineffective. This result has a parallel to the action of temperature on certain viruses, as for instance that involved in one of the peach tree diseases. On the other hand, Malogolowkin (1958) ior D . willistoni found no temperature effect. Malogolowkin further found that the cytoplasmic factor was not independent of chromosomal genes since some wildtype mutant strains induced reversions to normal sex ratio. Recently Poulson has established the complete correlation of the high female progeny characteristic with the presence of a Trepomena in the fly's lymph. As with mouse typhoid variations, lethality was genotype dependent.


These cases have interest from more than the sex ratio viewpoint. Cytoplasmically inherited susceptibility of some strains of D. 7nelanogaster to poisoning by carbon dioxide as studied by L'Heritier (1951, 1955) depended on the presence of some cytoplasmic entity which passed through the egg cytoplasm and also, but less efficiently, by means of the male sex cells to the progeny. The carbon dioxide susceptibility was transmitted through injections of hemolymph or transplantation of organs of susceptible strains. The transmissible substance had a further property of heat susceptibility. The COo susceptibility differed from that of "sex ratio" in being partially male transmitted. It agrees with sex ratio" D. bifasciata in being heat susceptible but differs from D. willistoni "sex ratio" in this respect.


D. bifasciata may behave differently than D. willistoni in that Rasmussen (1957) and Moriwaki and Kitagawa (1957) both conducted transplantation experiments with negative results. However, it is jiossible that these experiments may be affected by a different incubation period for the injected material as contrasted with that for D. willistoni. A range of possibilities evidently exist for extrinsic effects in sex ratio.

Carson (1956) found a female producing strain of D. borealis Patterson, which carried on for a period of 8 generations, produced 1327 females with no males. The strain showed no chromosomal abnormalities. It had 3 inversions. The females would not produce young unless mated to males from other strains having biparental inheritance. This requirement, together with gene evidence, showing that the females were of biparental origin, is against the female progenies being derived by thelytokous reproduction.

F. Male-Influenced Type of Female Sex Ratio

Sturtevant in 1925 described exj^eriments with a stock of D. affinis in which a great deficiency of sons was obtained from certain males, regardless of the source of females to which such males were mated. The few males obtained from such matings were normal in behavior but some of the sons of females from such anomalous cultures again gave very few sons (]Morgan, Bridges and Sturtevant, 1925).


Gershenson (1928) in a sampling of 19 females caught in nature found two that were heterozygous for a factor causing strong deviations toward females (96 per cent to 4 per cent males) whereas the normal D. pseudoobscura ratio was nearly 1 to 1. The factor was localized in the X chromosome and was transmitted like an ordinary sex-linked gene. Its effect was sex limited as it was not manifest in either heterozygous or homozygous females. It had no effect on the development of zygotes already formed but strongly influenced the mechanism of sex determination through the almost total removal of the spermatozoa with the Y chromosome from the fertilization process. Egg counts showed the divergent ratio toward females was not caused by death of the male zygotes inasmuch as there was no greater mortality from such cultures than from controls giving 1 to 1 sex ratios.

Sturtevant and Dobzhansky (1936) showed that an identical or nearly identical phenotypc to that obscM'ved in Drosophila obscura was present in D. pseudoobscura. The D. pseudoobscura carrying the factor were scattered over rather wide geographical areas. Comparable types also were found in two other species D. athabasca and D. azteca. This genie "sex ratio"' sr lies in the right limb of the X of races A and B of D. pseudoobscura. Like the other cases analyzed, males carrying sr have mostly daughters and few sons regardless of the genotypes of their mates. Structurally the female sex ratio came through modification of the development of the sex cells of the male to give a majority of X-bearing sperm. Cytologic study showed that in "sex ratio" males the X underwent equational division at each meiotic division, whereas the autosomes behaved normally. The Y chromosome lagged on the first spindle, remained much condensed, and its spindle attachment end was not attenuated. The Y chromosome was not included in either telophase group of the first meiotic division but was left behind. It was sometimes noted in one of the daughter cells where it formed a small nucleus. Among 64 spermatocytes examined, all had an X chromosome and none a Y chromosome in their main nuclei. The micronuclei containing Y played no further part in the division, became smaller and exceedingly contracted. The final fate of the micronuclci was uncertain, but there was no indication that any spermatids died or were abnormal.

G. High Male Sex Ratio of Genetic Origin

In 1920, Thompson described a recessive mutant in D. melanogaster which killed all homozygous females but changed the male phenotypes only slightly, the wings standing erect above the back. The locus of the mutant was 38 in the X chromosome. This mutant was a leader for a class in which the genes affect only one sex but are innocuous to the other.

Bobbed-lethal, a sex-linked gene found i)y Bridges, is a gene of this class but one in which the mechanism of protection to the other sex is known. It kills homozygous females but does not kill the males because of the wild type allele which the males have in their Y chromosomes. The presence of this bobbed-lethal in a population consequently leads to male ratios higher than wild type.


A recessive gene in chromosome II, discovered by Redfield (1926), caused the early death of the majority of female zygotes and led to a sex ratio of about 1 female to 5.5 males. The effect was transmitted througli both males and females. The missing females may have died largely in the egg stage although disproportionate losses also occurred in the larvae and pupae. The maternal effect was attributed to an influence exerted by the chromosome constitution of the mother on the eggs before they left the mother's body. Because of this maternal lethal, the families from these mothers have the normal number of sons but few or no daughters.


A culture was observed by Gowen and Nelson (1942) which yielded only male progeny, 136 in all. Some of the male progeny were able to transmit the male-producing characteristics to half of their daughters without regard to the characteristics of the mates to which they were bred. The inheritance was without phenotypic effect on the males. When present in the heterozygous condition in females, the female's own phenotype gave no indication of the gene's presence. The inheritance was sex limited in that it affected only the eggs laid by the mothers carrying it. In these eggs it acted as a dominant lethal for the XX zygotes. The gene was found located in the 3rd chromosome between the marker genes for hairy and for Dicheate at approximately 31. The X eggs carrying the gene, Ne, died in the egg stage at between 10 and 15 hours under 25°C. temperature. The gene was as effective in triploids as in diploids. One dose of this gene in triploids caused them to have only male progeny and male type intersexes. The presence of this gene caused the elimination of any embryos with chromosomal capacities for initiating and developing the primary or secondary female sexual systems. Since the gene itself was heterozygous in the female the meiotic divisions of the eggs would cause the gene to pass into the polar bodies as often as to remain in the fertilization nucleus yet the lethal effects are found in all eggs. The antagonism was between the egg cytoplasm and the XX fusion products. Supernumerary sperm of the Y type were not sufficient to overcome the lethal effects. An XY fertilization nucleus was necessary for survival.


This case has parallel features with that observed by Sturtevant (1956) for a 3rd chromosome gene that destroys individuals bearing the first chromosome recessive gene for prune. The gene responsible was found to be a dominant, prune killer, K-pn, which was located in the end of the 3rd chromosome at 104.5. Prune killer was without phenotypic effect so far as could be observed except that it acted as a killer for flies containing any known allele of prune. It was equally effective in either males or females, hemizygous or homozygous, for prune. The larvae died in the 2nd instar but no gross abnormalities were detected in the dying larvae. This case has interest for the Ne gene in that the killer gene occupies a locus in the 3rd chromosome although removed by about 70 units from Ne, and acts on a specific genotype, the prune genotype, whereas Ne acts on the specific XX genotype. The known base for action of the prune killer is genetically much narrower than that for the female killer, Ne, in that prune killer acts on an allele within a specific locus in the X chromosome, and Ne acts on a type coming as a product of the action of two whole sex chromosomes. It is, of course, conceivable that when ultimately traced each action may be dependent upon specific changes of particular chemical syntheses. The action of these genes is also of interest from another viewpoint.


In mice there is a phenotype caused by the homozygous condition of a recessive gene (Hollander and Gowen, 1959) which acts on its own specific dominant allelic type in its progeny so as to cause an increased number of deaths between birth and two weeks of age, as well as causing the long bones to break and the joints to show large swellings. The lethal nature of this interaction is not a product of the mother's milk nor does it show humoral effects such as those observed with erythroblastosis in the human. The interallelic interaction is sex limited in that it is confined to the mother and is without effect when the male has the same genotype.


H. Female-Male Sex Ratio Interactions

A case in D. affinis involving the interaction of a "female sex ratio" factor and an autosomal "male sex ratio" factor has been studied by Novitski (1947). Starting from stock which had a genetic constitution for the "sex ratio" X chromosome which ordinarily causes males carrying it to produce only daughters, he was able to establish a recessive gene in chromosome B in whose presence only male offspring resulted. The genetic constitution of the male was alone important. High sterility accompanied the "male sex ratio" males breeding performance. The "male sex ratio" parents yielded 95 fertile cultures having an average of 25 individuals per culture. Females appeared in 10 of these cultures with an average of 3 per culture for those producing females. The total sex ratio was 77 males per female. The males were morphologically normal. They carried an X chromosome of their mothers. Cytologic observation of spermatogonial metaphases of 3 progeny showed that the Fi males may or may not have carried a Y of their fathers. This agreed with the tendency for sterility in these "male sex ratio" males. The ventral receptacles of the females from 4 such sterile cultures when examined proved devoid of sperm. The occasional female offspring of the "male sex ratio" males had one X chromosome from each parent. Females mated to "male sex ratio" males show large numbers of sperm in the ventral receptacles (7 out of 8 cases) , although such females had usually produced no or very few offspring. The sperm, if capable of fertilization, must have had a lethal effect on the zygotes. The recessive nature of the factor in chromosome B indicated that its potential lethal effect originated during spermatogenesis rather than at the time of fertilization. This lethal period corresponds to that when the "female sex ratio" factor in the X chromosome is active.


The research on sex ratio in Drosophila reviewed shows that through the interplay of the sex chromosome-located and autosomal-located factors all types of sex ratios from only females in the family to only males in the family may be generated.

V. Sex Determination in Other Insects

Sciara, a fungus gnat, offers sex-determining mechanisms quite different from any yet offered in Drosophila. Sciara is unique, yet in its uniqueness, it illustrates basic facts that were rediscovered in other species only through the study of abnormal forms some of which were created as induced developmental, chromosomal, or hormonal abnormalities. The complexities responsible for the remarkable facts were analyzed by Metz and his collaborators. The following brief review is based upon Metz's (1938) summarization of the chromosome behavior problem to which the reader is referred for further information. Of Sciara species studied 12 out of 14 have their basic chromosome groups composed of three types of chromosomes: autosomes, sex chromosomes, and "limited" chromosomes. Two species, S. ocellaris Comstock and S. reynoldsi Metz, lack the "limited" chromosomes. Two sets of autosomes and three sex chromosomes are found in the zygotes of all Sciara species at the completion of fertilization. The behavior of these chromosomal types will be considered in sequence.


The autosomes behave normally in somatic mitosis and in oogenesis. There is cytologic and genetic evidence for synapsis, crossing over, random segregation, and regular distribution of chromosomes and genes in the female.


In spermatogenesis the story is quite different. The first maturation division is unipolar. Genetic evidence shows a complete, selective segregation of the maternally derived autosomes and sex chromosomes from those of paternal origin. The paternal homologues move away from the pole, are extruded, and degenerate. The second spermatocyte division is likewise unequal and one of the products, a bud, degenerates. Thus a spermatogonial cell passing through meiosis gives rise to only one sperm. At the second spermatocyte division all of the chromosomes (maternal homologues) except the sex chromosome undergo an equational division but both halves of the sex chromosome enter into the same nucleus. This nucleus becomes the sperm nucleus. The chromosomes at the opposite pole form a bud and degenerate. Fertilization of the Sciara egg is normally monospermic not polyspermic.


The sex chromosomes XXX of the fertilization nucleus are derived, two from the sperm and one from the oocyte. Their destiny depends on whether the egg they are in develops into a male or a female imago. In male early development, those nuclei which are to become male soma lose the two sex chromosomes XX, contributed by the father's sperm, to give X + 2A soma. These chromosomes fail to complete mitosis at the 7th or 8th cleavage division and are left to degenerate in the general cytoplasm when there are no true cells in the soma region and no membranes surround the nuclei. Those embryos which are to become female soma at the same cleavage cycle eliminate but one paternal X chromosome. On a chromosome basis the soma cells respectively become X, plus two sets of autosomes, give rise to males on differentiation, or become XX + 2A and develop female organs (Du Bois, 1932). The germ line nuclei for each sex, on the other hand, remain unrestricted in their development. They retain their XXX constitutions until the first day of larval life, or about 6 hours before the formation of the left and right gonads (Berry, 1941), when they eliminate a single paternally derived X. The X which is rejected or makes its own exit is always one of two sister chromosomes contributed by the father. The process of loss is strikingly different from that noted in the soma-building nuclei. Both cell walls and nuclear membranes are present. The path of the X chromosome is through the nuclear membrane into the cytoplasm where degeneration eventually takes place. The loss occurs at a time when there is no mitotic activity and the chromosomes are separated from the cytoplasm by an intact nuclear membrane. Some Sciara species are characterized by females which produce only unisexual families — practically all females or practically all males. Genetically, the sex of progeny is accounted for by sex chromosomes, X' and X, which are so designated because of their physiologic properties. Mothers having only female progeny are characterized by always having the X' chromosome in all their cells, X'X + 2A, whereas mothers whose progeny are all males are XX + 2A (Moses and Metz, 1928). The all female and all male broods are observed in about equal numbers. The male has no influence on sex ratio. Normally the progeny in all female broods will be X'X + 2A or XX + 2A in soma and germ lines, whereas the all male progeny broods will be only X + 2A in the soma and XX + 2A in the germ line.


Studies of Grouse (1943, 1960a, b) show nondisjunction may occur and eggs entirely lacking sex chromosomes or having double the normal number may be produced. When the nondisjunctional eggs lacking X chromosomes are from X'X females and are fertilized by sperm contributing two sister X chromosomes, one X (as expected of X'X mothers, not two as expected of XX cells) is eliminated at the 7th or 8th cleavage to give cells which develop into "exceptional" males with XO + 2A soma and XX + 2A germ line, all X chromosomes being of paternal origin. The casting out of the single X chromosome is consequently controlled by the characteristics of the egg cytoplasm derived from the X'X mother rather than by the kind of chromosomes present in the nuclei at the 7th or 8th cleavage stage of the eml)ryo.


Similarly when nondisjunctional eggs having both XX chromosomes of the XX mothers are fertilized by the normal spermbearing XX chromosomes, the zygotes are XXXX. At the 7th or 8th cleavage both paternal X chromosomes are eliminated and the resulting soma develops into an exceptional" female, XX + 2A, often with 3 X's in her germ line. These observations confirm the earlier interpretations that the X'X or XX constitution of the mother conditions her eggs respectively to cast out 1 or 2 parental X chromosomes at the 7th or 8th cleavage according to the cytoplasmic constitution of the egg. Somatic sex development is visually determined only after this stage when the chromosomes of the somatic cells take on the familiar aspect of either XX + 2A for the females or X + 2A for the males.


Although at present there are few examples in other species, this cytoplasmic conditioning of early embryologic development by the mother's genotype may be a common rather than a rare occurrence of development. The expression of the events to which this control may lead may be quite different in different cases. The spiral of snail shells was an early recognized case (Sturtcvant, 1923) . In Drosophila, a gene acting through the mother's genome allows only male embryos to survive (Gowen and Nelson, 1942), suggesting that this gene acts through her cytoplasm in a manner comparable to that of the X' and X chromosomes of Sciara. In either case the genotype would not be considered a primary sex factor, profound as the effect on sex may ultimately be.


Sciara also has strains or species where the progeny of single matings are of both sexes, bisexual, yet the chromosomal elimination mechanisms remain as described above save for the fact that they now operate within broods rather than between broods. Sex ratios for the families within these bisexual strains are extremely variable, 1:0 or 0:1 ratios not being infrequent and 1 : 1 ratios being the exception. An instance of a bisexual strain arising from a unisexual line has been analyzed by Reynolds (1938). The females were found to be 3X in their germ line and produced 2X eggs which developed into daughters and X eggs which became sons. Loss of the extra X from this line resulted in the line's return to unisexual reproduction.


In general, bisexuality as contrasted with unisexuality seems to be caused by differences in the X'X-XX mechanisms, either specific for the mechanism, or induced by modifying genes which cause different embryos within the same progeny to cast out sometimes one or sometimes two X chromosomes from all their somatic nuclei. This casting out occurs uniformly for all nuclei of a given embryo as is shown by the fact that sex mosaics or gynandromorphs are as seldom observed in bisexual as unisexual strains.


Some species of Sciara have large chromosomes which are in essence "limited" to the germ line since they are lost from the somatic nuclei at the 5th or 6th cleavage division. Other species lack these chromosomes, yet agree with the rest in the behavior of their remaining chromosomes and in sex determination. So far as is now known the "limited" chromosomes seem empty of inheritance factors influencing either sex or unisexual vs. bisexual broods or for that matter other characteristics (their persistence seems to make this latter unlikely I. Their presence does lead to a question of the efficacv of desoxvribonucleic acid (DNA) as the all inclusive agent in inheritance for they are clearly DNA-positive.


These cytogenetic observations clear up most of the events leading to sex differentiation and the delayed time in embryologic development when it takes place, but, as INIetz points out, other puzzling questions are raised. Observed chromosome extrusions from cell nuclei are rarities today even though discovered for Ascaris 60 years ago. Why should this mechanism be so well defined in Sciara? Why should the mode of elimination be so accurately timed and yet be different in method and time for the soma and germ cell lines? Two of the three X chromosomes in the fertilized egg are sisters from the father and should be identical. What is special in the 7th or 8th cleavage that causes elimination in the soma plasm but not in the germ plasm? Why should the casting out of one of these same paternal X's in the germ plasm of both sexes be reserved for a much later stage in cleavage and by a different procedure?


The observations of Grouse (1943, 1960a), obtained from translocations and species crosses, are critical in showing that the chromosome first moA'ing in its entirety to the first differentiating pole of the second spermatocyte division is the X and is the one taking part in the later postfertilization sex chromosome elimination of the 7th or 8th cleavage. The X heterochromatic end of the chromosome and not the centromere region seems to be responsible for the unique behavior of this chromosome (Grouse, 1960b). Induced nondisjunctional eggs of X'X (female-producing) mothers having as a consequence no sex chromosomes, when fertilized by the normal sperm bringing in XX chromosomes, develop into embryos eliminating one of these X's at the 7th or 8th cleavage, as expected of the eggs of X'X mothers, and become "exceptional" males with X + 2A soma and XX paternal germ line. Imagoes of these embryos become functional but partially sterile males transmitting the paternal X, a reversal of the normal condition. Similarly the nondisjunctional eggs retaining both X's, XX from male-producing mothers, and having 4 X's on fertilization eliminate the two paternal X's, as occurs normally in XXX eml)ryos, to become "exceptional" females with 2X or sometimes in species hybrids 3X soma.


Phenotypic sex in Sciara on this evidence depends on the adjustment of the X chromosome numbers and their contained genes with this adjustment regularly taking place, not at fertilization as in most forms like Drosophila, but at the 7th or 8th cell cleavage of embryologic development. The X' vs. X chromosomes of the mother predispose her haploid egg cytoplasm to time specific chromosome elimination. The germ line cells on the other hand may regularly have other chromosome numbers than the soma or exceptionally tolerate other numbers as extra X's (Grouse, 1943), or extra sets (Metz, 1959).


Triploids of 3X and 3A soma have not been found in pure Sciara species. Salivary gland cells of a triploid hybrid S. ocellaris x aS. reynoldsi have two sets of S. ocellaris and one of S. reynoldsi chromosomes (Metz, 1959). Ghromosome banding in 2X -I- 3A, 3X + 3A larval glands showed the »S. ocellaris homologues were heterozygous. The 3X + 3A type chromosomes were of typical female appearance. The 2X:3A-type had X chromosomes of typical male type, very thick, pale and diffuse. The mosaic salivaries were a mixture of the two types of cells. These results suggest that the 3X:3A would be a typical female. The intersexes 2X -I- 3A conceivably have a range in male and female organ development depending on how the S. ocellaris and S. reynoldsi chromosomes act. If the chromosomes are equivalent, the gene expression would be expected to be that of a 2X to 3A or a phenotypic intersex. If they act separately the S. ocellaris genes would be in balance for female determination, whereas the S. reynoldsi autosomal genes would have no X S. reynoldsi genes to balance them and presumably would be overwhelmingly male in effect. The phenotypic outcome would be in doubt.


The maternally governed cleavage and chromosomal sequence occurring before the 8th embryonic cell division, although related to the subsequent events leading to the particular sex, is unique to Sciara and its relatives. Phenotypic sex expression apparently starts with the somatic nuclei having either X + 2A or XX + 2A as their chromosomal constitutions. Sciara is like Drosophila and many other genera in its basic sex-determining mechanism. There is a sexindifferent period when the maternal genotype may influence development through cytoplasmic substances. This is followed by active differentiation when the sex phenotypes may be influenced by various agencies but when passed becomes fixed for phenotypic sex. The indifferent period may represent a fairly large number of early cell generations as in amphibia or fish. Somatic and germ cells are labile but are normally susceptible to control only by forces developed within the organism as present knowledge indicates for Sciara. In Sciara, only the somatic cells conform in chromosome number to their expected phenotypes. The germ cells show not only a regulated instability of their chromosome number but the number is different from that of the supporting somatic cells. The fact that sex mosaics in Sciara may develop both an ovary and testis in the same animal (Metz, 1938; Grouse, 1960a, b) shows localized phenotypic sex determination occurring fairly late in development rather than the generalized determination that would occur if sex were established at fertilization.


The germ line chromosomal differentiation from that of the somatic tissue has parallels in a number of fairly widely dispersed species. The significance of these types to soma line determination and to the reversibility or irreversibility of differential gene activation in differentiated cells subsequent to embryogenesis has been considered by Beermann (1956). Although these types are yet in the fringe areas of our knowledge surrounding the general paths taken in the evolution of sex determining mechanisms, they promise to have more generality as clarification of gene action becomes more exact.


B. Apis and Habrobracon

Hymenopteran interi:)retations of sex determination have largely turned on studies of the honey bee. Apis mellijera Linnaeus and Habrohracon juglandis Ashmead. Dzierzon early established that the drones of honey bees come from unfertilized eggs, whereas the females, queens, and workers come from fertilized eggs so that sex differentiation is marked by an N set of chromosomes for the male and 2N for the female. This mechanism was satisfactory until it was realized that on a balanced theory the doubling of a set of chromosomes, if truly identical, should not change the gene balance and consequently not the sex. Further doubt was cast on the N-2N hypothesis for sex determination by the discovery of diploid males by Whiting and "Whiting (1925). These difficulties were resolved by Whiting (1933a, b) with the suggestion that the two genomes in the female were not truly alike but actually contained a sex locus linked with the gene for fused which was occupied by different sex alleles as Xa and Xb . In this A'iew the heterozygous condition would lead to the production of females, whereas in the haploid or homozygous condition either of these genes would react to produce males. Difficulties with this hypothesis became evident in that the ratios of males to females in certain crosses were significantly divergent from those which were expected. Evidence was collected and trials made to interpret these difficulties (Whiting, 1943a, b) by assuming that on fertilization by Xa-bearing sperm the polar body spindle would turn so as to cast out the Xa chromosome and retain the complementary Xb in the majority of cases instead of in a random half. Snell (1935) offered the hypothesis that there could be several loci for sex genes, such that the X locus might be a master locus, but when this locus was in homozygous condition the sex would then be controlled by genes in other loci in the genome. As pointed out by Bridges (1939) this hypothesis would satisfy that of sex gene balances postulated in Drosophila and of change in emphasis on loci for sex genes as observed by Winge (1934) in Lebistes. However, the work of Bostian (1939) called both hypotheses into question. Consideration of these further results led to the multiple allele hypothesis of complementary sex determination for Habrobracon (Whiting, 1943a) taking a similar form to that of the sterility relations observed for a single locus in the white clover of INIelilotus. Under this system the sex locus would be occupied by multii)le alleles, any one of which or any combination of identical alleles would be male-producing, but any combination of two different alleles would l»e female determining. By having a sufficient number of these genes the jiroduction of diploid males would be curtailed to a point at which their various frequencies could be explained. In support of this liyi)othesis, multiple alleles to the number of at least 9 were found to exist for this single sex locus.


In principle at least, the honey bee could have the Habrobracon scheme of sex determination. Rothenbuhler (1958) has recently collected the researches which test this possibility. Tests of the multiple allele hypothesis as applied to the honey bee were made by Mackensen (1951, 1955) who interpreted evidence for inviable progeny produced by mating of closely related individuals as proof that this species as well as Habrobracon juglandis follows the multiple allelic system. The discovery of male tissue of bii)arental origin in mosaic bees from related i)arents was considered as further evidence for the multiple allelic theory of sex determination (Rothenbuhler, 1957).


Most recent cytologic evidence supports the concept that there are 16 chromosomes in the gonadal cells of the male and 32 in those of the female (Sanderson and Hall, 1948, 1951; Ris and Kerr, 1952; Hachinohe and Onishi, 1952; Wolf, 1960». Hachinohe and Onishi (1952) found 16 chromosomes as characteristic of the meiosis in the drone. Wolf observed a nucleus in both bud and spermatocyte of the only maturation (equational) division.


The greatest progress has been made in understanding the mechanisms of sex mosaicism in the Hymenoptera species. These mosaics, although ordinarily of rather rare occurrence, have a direct bearing on sex determination and development. In Apis, iiolyspermy furnishes the customary basis for their formation. One sperm fertilizes the haploid egg nucleus and another sperm, which has entered the egg instead of degenerating as it ordinarily does, enters into mitotic cleavage and eventually forms islands of haploid cells of paternal origin among the diploid cells derived from the fertilized egg (Rothenbuhler, Gowen and Park, 1952). Evidence showing that genetic influences affect the sperm nucleus toward stimulating its independent cleavage is found to exist in Apis material (Rothenbuhler, 1955, 1958). Tliousands of gynandromorphs have been observed in Apis, all but a small number of which have been produced in this manner. This method of initiating sex mosaics also exists for Habrobracon (Whiting, 1943b) but is rather rare. In Habrobracon the frequent mode has a different origin. The gynandromorph is formed from the cleavage products of the normal fertilization of the egg nucleus combined with those of a remaining nuclear product of oogonial meiosis. Under these conditions the female tissue is 2N of biparental origin and the male tissue is N of maternal origin (Whiting, 1935, 1943b). This type is less frequent in Apis but one specimen has been described by Mackensen (1951).


A number of other ways in which sex mosaics may occur are occasionally expressed in these species. Three different kinds of male tissue have been observed in individual honey bee mosaics produced by doubly mated queens — haploid male tissue from one father, haploid male tissue from the other genetically different father, and diploid male tissue of maternal-paternal origin. In other cases, the diploid, biparental tissue was female and associated with two kinds of male tissue (Rothenbuhler, 1957, 1958) . Cases where the haploid portions of the sex mosaics are of two different origins, one paternal and the other maternal, while the female portion is representative of the fertilized egg, are known in Habrobracon (Whiting, 1943b). Similarly, Taber (1955) observed females which were mosaics for two genetically different tissues and which he accounted for as the result of binucleate eggs fertilized by two sperm. Mosaic drones of yet another type were observed by Tucker (1958) as progeny of unmated queens. They were interpreted as the cleavage products from two of the separate nuclei formed in meiosis. These cases represent a number of the possible types that arise through meiotic or cleavage disfunctions under particular environmental or hereditary conditions.


Tucker (1958) studied the method by which impaternate workers were formed from the eggs of unmated queens. For this purpose he used genetic markers, red, chartreuse, ivory, and cordovan. Observations Avore nuult' i)ii 237 workers from hotcrozygous mothers. For the chartreuse loeus. 12 to 20 per cent were homozygous, for x\w i\ory locus 1.8 per cent, and (he cordovan locus on lesser numbers per t-ent. An egg which is destined to become an automictic worker, a gynandromorph with somatic male tissue or a mosaic male, is retained within the queen for an unusually long time during which meiosis is suspended in anaphase 1. Normal reorientation of first division spindle is i)ossibly inhibited by this aging so that after the egg is laid meiosis II occurs with two second division spindles on sejiarate axes as Goldschmidt conjectured for rarely fertile rudimentary Drosophila (19")7i. Two polar l)odies and two egg prontich^i are formed. The polar bodies take no fiu'ther part in develoinnent. In most of the unusual eggs the two egg pronuclei unite to form a diploid cleavage nucleus which develops into a female. Rarely the two egg pronuclei develop separately as two haploid cleavage nuclei to form a mosaic male. Two unlike haploid cleavage nuclei, one descending from each of the two secondary oocytes after at least one cleavage di\ision. unite to form a dijiloid cleavage nucleus which develops together with the remaintler of the haploid cleavage nuclei to produce a gynandromorph with mosaic male tissues. The male and female tissues within these unusual gynandromor[ihs or female types were identical with normal drone or normal female tissues so were probably haploid and diploid resiiectively. Genetic segregation observed within the mothers of automictic workers allows the estimation of the distance between the locus of the gene and its centromere. "With random recombination and "central union" Tucker estimates this distance for the chartreuse locus to centromere as 28.8 units and for the ivory to its centromere 3.6 units. Four lines of bees of diverse origin all showed a low percentage of automictic or gynandromorphic types produced from queens in each line. Various chance environmental conditions apparently influence the rate of production of these types. However, there were some females in two of the lines with higher frequencies inciicating that innate factors may have significant eft'ects on their frequencies. Observations on Drosophila species — I), porthenogeuetica (Stalker, 1956b), I). ))ia)njabeirai (INIurdy and Carson, 1959) and D. nielanogaster (Goldschmidt, 1957) — strongly suppt)i-t this A-iew.


The problem of sex determination in Hal)rol)i'acon presently stands as a function of multii)le alleles in one locus, the heterozygotes being female and the azygotes and homozygotes male. The occasionally diploid males are regularly produced from fertilized eggs in two allele crosses after inbreeding. These diploid males are of low viability and are nearly sterile. Their few daughters are triploids, their sperm being dijiloid. Apis probably follows the same scheme, as a few cases of mosaics with diploid male tissue are known and close inbreeding results in a sufficient number of deaths in the egg to account for diploid males which might be formed. ^lormoniella (Whiting, 1958), however, shows that this scheme for sex determination does not hold for all Hymenoptera. In this form diploid males may occur through some form of mutation. They may then develop from unfertilized eggs laid by triploid females. In contrast to Habrobracon the dijiloid males are highly viable and fertile. Their sperm are diploid and their numerous daughters triploid. Virgin triploid females produce 6 kinds of males, 3 haploid and 3 dij^loid. Similarly ]\Ielittobia has still a different and as yet unexplained form of sex determination. Haploid eggs develop into males. After mating many eggs are laid which develop into nearly 97+ per cent females. The method of reproduction is close inbreeding but no dijiloid males or "bad" eggs are formed. The prol)lem of the sex determining mechanism remains open (Schmieder and Whitiuii, 1947).


The silkworm, Bombyr mori, differs from Drosophila, Lymantria and the species thus far discussed in having a single region in the ^^' chromosome (Hasimoto, 1933; Tazima, 1941, 1952) occupied by a factor or factors of high female potency. The strong female potency has thus far been connnon to all races. The chromosome patterns of the sexes are like those of Abraxas and Lymantria: males ZZ + 2A and females ZwV 2A. The diploid chromosome number is 56 in both sexes. Extensive, well executed studies have revealed no W chruniosome loci for genes expressed as morphologic traits. From radiation-treated material it has been possible to pick up a translocation of chromosome II to the W chromosome as well as a cross-over from chromosome Z. This chromosome together with tests of hypoploids and hyperploids have materially aided in understanding how the normal chromosome complexes determine sex. The sex types resulting from different chromosome arrangements have been summarized by Yokoyama 1 1959) and are presented in Table 1.3.


Whenever the W chromosome was absent a male resulted. Extra Z or A chromosomes did not influence the result. Similarly with a W chromosome in the fertilized egg a female developed. Again extra Z or A chromosomes did not influence the result.


A full Z chromosome was essential to survival. Hypoploids deficient for different amounts of the Z chromosome in the presence of a normal W chromosome all died without regard to the portion deleted. Hyperploids for the Z chromosome, on the other hand, when accompanied with a W chromosome all lived and showed no abnormal sexual cliaracteristics. Parthenogenesis led to the pioduction of both sexes, although the males were more numerous than the females. Diploidy was necessary for the eml)ryo to go beyond the blastoderm stage. Triploid and tetraploid cells were often found. High temperature treatments led to merogony (Hasimoto, 1929, 1934). The exceptional males were homozygous for a sexlinked recessive gene and were explained by assuming that the egg nuclei were inactivated by the high temperature and the exceptional males developed from the union of two sperm nuclei. This conclusion was supported by cytologically observed l)olyspermy (Kawaguchi, 1928) and by cytologic observation of the union of two sperm nuclei by Sato ( 1942 ) . Binucleate eggs were also believed to occur, which when fertilized by different sperm may each construct half of the future body. This type of mosaicism was influenced by heredity iGoldschmidt and Katsuki, i927, 1928, 1931 ). Polar body fertilization was also believed to occur, one side of the embryo originating from the ordinary fertilized egg nucleus and the other side from the union of


TABLE 1.:^ Sex in Bombyx iitori (Summarized by T. Yokoyama, 1959.)


Sex


Chromosome Types and Numbers



W


z


A


Male


W II.W.ZL

w w

WW WW


zz

zz

zzz

z

z

zz zzz zz zz


AA


Male

Male

Female

Female

Female

Female

Female

Female


AAA AAA

AA

AA

AAA

AAAA

AAA

AAAA


nuclei of two of the polar bodies. Similarly, dispermic merogony was noted following the formation of one part of the body from the fertilization nucleus, the other part from the union of two sperm nuclei, the result being a gynandromorph or mosaic.


VI. Sex Determination in Dioecious Plants

A. Melandrium T Lychnis

Over the last 20 years studies on several species of dioecious plants have made notable advances in unclerstanding the mechanisms by which sex is determined.

Melandrium album has been shown to have the same chromosome arrangement as Drosophila. The male has an X and Y plus 22 autosomes, whereas the female has XX plus 22 autosomes. Sex-linked inheritance is known for genes borne in the X chromosomes as well as for genes born in the Y chromosome. The X and Y chromosomes are larger than any of the autosomes with the Y chromosome about 1.6 times that of the X in the materials studied by Warmke ( 1946) . Separate male and female plants are characteristic of the species. By use of colchicine and other methods, Warmke and Blakeslee (1939), Warmke (1946), and Westergaard (1940) have made various l^olyploid types from which they could derive other new X, Y and A chromosome combinations from which information was obtained on the location of the sex determining elements. The Y chromosome carries the male determining elements, the X chromosome the female determining elements. The guiding force of the elements in the Y chromosome during development is sufficient to override the female tendencies of several X chromosomes. Data derived by each of these investigators are shown in Table 1.4.

From these data Warmke (1946) concluded that the balances between the X and the Y chromosomes essentially determined sex with the autosomes of relatively little importance. Where no Y chromosomes were present but the numbers of X chromosomes ranged from 1 to 5, only females were observed, even though the autosomes varied in number from two to four sets. When a Y chromosome was present the individual was of the male type unless the Y was balanced by at least 3 X chromosomes when an occasional hermaphroditic blossom was formed.

TABLE L4

Numhers of X, Y, chromosomes and A, autosome sets

and the sex of the various Melandrium plants

(Data from H. E. Warmke, 1946; and

M. Westergaard, 1953.)




Ratio



Chromosome


Warmlce



Westergaard


Constitution






X/A



4A 5X


Female


1.3



4A 4X


Female


1.0


Female


4A 3X


Female


0.8



4A 2X


Female


0.5



3A 3X


Female


1.0


Female


3A 2X


Female


0.7


Female


2A 3X


Female


1.5



2A 2X


Female


1.0


Female



Bisexual*


X/Y



4A 4X Y


4.0


Male


4A 3X Y


Malet


3.0


Male


4A 2X Y


Malet


2.0


Male


4A X Y


Male


1.0



3A 3X Y


Malet


3.0



3A 2X Y


Malet


2.0


Male


3A X Y


Male


1.0



2A 2X Y


Malet


2.0



2A X Y


Male


1.0


Male


4A 4X YY


Malet


2.0



4A 3X YY


Male


1.5



4A 2X YY


Male


1.0


Male


2A X YY


Male


0.5



  • Occasional staminate but never carpellate

blossom.

t Occasional licrina])hr(i(lit ic blossom.


When 4 X chromosomes were present together with a Y, the plants were hermaphroditic but occasionally had a male blossom. Two Y chromosomes almost doubled the male effect. Two Y chromosomes balanced 4 X chromosomes to give a majority of male plants. Only an occasional plant showed an hermaphroditic blossom. Autosomal sex effects, if present, were only observed when plants had 4 sets and 3 or 4 X chromosomes balanced by a Y chromosome. Warmke used the ratio of the numbers of X to Y chromosomes as a scale against which to measure clianges from complete male to hermaphroditic types. No mention is made of quantitative measures of the sex character changes with increasing X chromosome dosages. This is of interest since in many forms changes in chromosome balance are accompanied by changes of phenotype which are unrelated to sex. That such phenotypic changes do accompany changes in autosomal balance in Melandrium are proven, however, by further observations of Warmke in 4 trisomic types coming from crosses of triploids by diploids. Of 36 such trisomies analyzed, 5 or 6 of them were of different growth habits and morphologic types. These differences did not affect the sex patterns since all were females. Warmke and Blakeslee in 1940 observed an almost complete array of chromosome types from 25 to 48 in progeny derived from crosses of 3N x 3N, 4N X 3N, and 3N x 4N. Out of about 200 plants studied, only 4 were found to show indications of hermaphroditism. These types were 2XY and 3XY. As noted from the table, even the euploid plants would occasionally be expected to have an hermaphroditic blossom. Of the 200 plants, all with a Y (XY, 2XY, 3XY) were males and all plants without the Y (2X, 3X, 4X) were females. In an 8-year period up to 1946. Warmke was able to observe only one male trisomic. From these facts he concluded that the autosomes are unimportant in the sex determining mechanism utilized by this species. In their crosses they were unsuccessful in getting a 5XY plant, the point at which the female factor influence of the X chromosomes might be expected to nearly equal or slightly surpass that of the single Y. From the j^hysiologic side the obscrvation of Strassburger in 1900, as quoted by both Warmke and Westergaard, that the fungus Ustilago violacea when it infects Melandrium will cause diseased plants to produce mature blossoms with well developed stamens (filled with fungus spores) as well as fertile pistils, shows that these females have the potentialities of both male and female development. The case suggests that sex hormone-like substances may be produced by the fungus which acting on the developing Melandrium sex structures cause sex reversions. Should this be true, Melandrium cells would have a parallel with those of fish where sex hormones incor|)orated in the developing organism in sufficient quantities can cause the soma to develop a phenotype opposite to that expected of their chromosomal type. For other aspects see Burn's chapter and Young's chapter on hormones.

Westergaard's studies (1940) with European strains of Melandrium were in progress at the same time as those of Warmke and Blakeslee. In their broad aspects both sets of data are concordant in showing the l^rimary role of elements found within the Y chromosome in determining the male sex and of elements in the X chromosomes for the female sex. Examination of Table 1.4 shows that the strain used by Westergaard has a Y chromosome containing elements of greater male sex potentialities than the strain used by Warmke.

A similar difference appeared in the sex potencies of the autosomes of European strains. Instead of obtaining essentially only male and female plants in crosses involving aneuploid types, Westergaard obtained from 3N females (3A + 3X) x 3N males (3A + 2XY) 10 plants which were more or less hermaphroditic, 21 females, and 15 males. Studies of the offspring of these hermaphrodites through several generations showed that their sex expression required effects by both the X chromosomes and certain autosome combinations which under special conditions counterbalanced the female suppressor in the Y chromosome. Increasing the X chromosomes from 1 to 4 increased the hermaphrodites from to 100 per cent in the presence of a Y chromosome. However, in euploids these types would be all males. The significance of the autosomes is further shown by the fact that among 205 aneuploid 3XY plants, 72 were males and 133 were hermaphrodites.


As pointed out for Drosophila, quantitative studies on the effects of sex chromosomes and autosomes in Melandrium are handicapped by not having a suitable scale for the evaluation of the different sex types. The data presented by Westergaard and by Warmke make this difficulty become particularly evident. In the interest of quantizing the X, Y, and A chromosome on sex the author has assigned a value of 1 for the male type, 3 for the female type, and 2 when the types are said to be hermaphroditic. When the types are mixed, as for example, in the data of Warmke where he says a particular type is male with a few blossoms, the type is assigned a value of 1.05 or 1.10, depending on the numbers of these blossoms. His bisexual type which comes as a consequence of Y, 4X and 4A chromosome arrangement is given a value of 2, although possibly the value should be somewhat higher as it may well be that the fully bisexuals are further along in the scale toward female development than the hermaphroditic types. The data are treated on the additive scale both as between chromosomal types and within chromosomal type. This is apparently unfair if we examine the work of Westergaard in which it looks as if particular autosomes rather than autosomes in general make a contribution to sex determination. The results, when these methods are used, are as follows:


Westergaard in Tables 1 to 5 of his 1948 paper gives information on sex types with a determination of the numbers of their different kinds of chromosomes. Analysis of these data by least square methods shows that the sex type may be predicted from the equation

Sex type = - 1.37 Y + 0.10 X

+ 0.01 A + 2.34

This equation fits the data fairly well considering that the correlation between the variables and the sex type is 0.87. This analysis again shows that the Y chromosome has a strong effect toward maleness. The X chromosomes are next in importance with an effect of each X only about 1/13 that of the Y and in the direction of femaleness, the autosomes have one tenth the effect of the X chromosomes but they too have a composite effect toward femaleness. It is to be remembered that the Y chromosome variation is limited to 2 chromosomes whereas the X chromosomes may total 4, and the autosomes may range from 22 up to 42, so that the total effect of the autosomes is definitely more than their single effects. These data are for aneuploids. Examining Westergaard's data for 1953 for the euploids and assigning the value of 1.5 for the type observed when there was one Y chromosome, four X, and four sets of autosomes, we have the following equation:

Sex value = -1.29 Y + 0.10 X - 0.01

(autosome sets) + 2.53

In these data, as distinct from those above, the autosomes are treated as sets of autosomes since they are direct multiples of each other so the value of the individual autosome is but 1/11 that given in the equation.

This equation shows no pronounced difference from that when the aneuploids were utilized. The Y chromosomes have slightly less effect toward the male side. The X chromosomes have practically identical effects but there has been a shift in direction of the autosomal effects on sex, although the value is small. The constants are subject to fairly large variations arising through chance.


In Table 10 of Westergaard's 1948 paper he presented data on the chromosome constitution and sex in the aneuploids which carried a Y chromosome. These data are of particular interest as the plants are counted for the i)roi)ortions of those which are male to those which are hermaphroditic. The plants with a Y chromosome plus an X are all males. Those which have either one, two or three Y chi'omosomes balanced by two X chromosomes have 89 per cent males. The plants with three X chromosomes and one oi- two Y's have 36 p(T cent males and those which have one or two ^■ chroiiiosoincs and four X chromosomes have no males. The woik iiiv()l\-cd in getting these data is, of course, large indeed and is definitely handicapped by the diiiicuHies in obtaining certain types. Thus the XXYYY and the 4X + 2Y types depend only on one plant. There are eight observations but the fitting of the data for the X and Y constitutions eliminates three degrees of freedom from that number so that statistically the observations are few. The data do have the advantage that the sex differences can be measured on an independent quantitative scale. The equation coming from the results is:

Percentage of males = 13.2 Y - 36.4 X

+ 134.4

These results show that the Y chromosome increases the proportion of males and the X chromosome increases the proportion of intersexes. The data are not comparable with those analyzed earlier as these data are describing simply the ratio between the males and intersexes, instead of the relations betw'een the males, intersexes, and females. The equation fits the observations rather well, as indicated by the fact that the correlation between the X's and Y's and the percentages of males is 0.98, but there are large uncertainties.

As a contrast to these data we have those presented by Warmke (1946) in his Tables 2 and 3. These data give the numbers of X and Y chromosomes found within the plants but not the numbers of autosomes, the autosomes being considered as 2, 3, and 4 genomes. Analyzing these data in the same manner as those of Westergaard's Table 1, we find that the Sex type = -1.05 Y + 0.22 X -0.04 A

-f2.25

As indicated for Westergaard's data, the A effect is now in terms of the diploid type e(iualing 2, the trijiloid 3, and the tetral)loid 4.

The Y chromosome has a i)ronounccd effect toward maleness, the effect l)eing someW'hat less in Warmke's data than that of Westergaard's. The X chromosomes on the other hand, have nearly twice the female infhience in Warmke's data that they do in the phiiits grown by Westergaartl. A difference in sign exists for the effect of the A rhi'oinosnnie genom(\s as well as a difference in [\\v (|uantitati\'e effect. Th(^ values for both sets of data are small and toward the male side. As Westergaard points out, the strains used by these investigators are of different geographic origins. The evolutionary history of the two strains may have a bearing on the lesser Y and greater X effects on the sex of the American types. Chromosome changes seem to have occurred in the strains before the studies of Warmke and Westergaard and will be discussed.


The location of the sex determiners has been studied by both investigators utilizing techniques by which the Y chromosome becomes broken at different places. These breakages may occur naturally and at fairly high rates in individuals which are Y + 2X + 2A. These facts suggest that the breakage of the Y chromosome occurs in meiosis since the breakage comes in selfed individuals of highly inbred stocks where heterozygosity is not to be expected, in the Y chromosome which has no homologue thus does not synapse, and in the second meiotic

  • ls in mice and man.

    E. Sex in Man: Chromosomal Basis

    A surprise even to its discoverers, Tjio and Levan (1956), came with the observation that the somatic number of chromosomes in cultures of human tissue was 46 rather than the previously supposed 48. Search for the true number has been going on for more than half a century. In early investigations the numbers reported varied widely. Difficulties of proper fixation and spreading of the chromosomes of human cells accounted for most of this variation and the numerous erroneous interpretations. Among the observations that of de Winiwarter (1912) was of particular interest in showing the chromosome number as 46 autosomes plus one sex chromosome with the Y being absent. This number was also found later by de Winiwarter and Oguma (1926). Observations by Painter (1921, 1923) showed 46 chromosomes plus an X and a Y, a total of 48. This number was subsequently reported by a series of able investigators, Evans and Swezy (1929), Minouchi and Ohta (1934), Shiwago and Andres (1932), Andres and Navashin (1936), Roller (1937), Hsu (1952), Mittwoch (1952), and Darlington and Haque (1955). As Tjio and Levan indicated, the acceptance of 48 as the correct number, with X and Y as the sex chromosome arrangement, was so general that when Drs. Eva Hanson-Melander and S. Kullander had earlier found 46 chromosomes in the liver cells of the material they were studying they temporarily gave up the study. In the few years since 1956, the acceptance of 46 chromosomes as the normal complement of man has become nearly universal. There are 22 paired autosomes plus the X and Y sex chromosomes.


    The reasons which have warranted this change of viewpoint are no doubt many, but three improvements in technique are certainly significant. The first came as a consequence of simplifying the culture of human somatic cells. The second followed Hsu's (1952) recognition that pretreatment of these cells before fixation with hypotonic solutions tended to better spreads of the chromosomes on the division plates when subsefiuently stained by the squash techniciuo. Pretreatment of the cultures with colchicine made the studies more attractive by increasing the numbers of usable cells that were in the metaphase of cell division.


    Ford and Hamerton (1956) in an independent investigation, closely following that of Tjio and Levan, observed that the human cell complement contained 46 chromosomes. They, too, agreed with Painter and others that followed him that the male was XY and the female XX in composition. A flood of confirming evidence soon followed: Hsu, Pomerat and Moorhead (1957), Bender (1957), Syverton (1957), Ford, Jacobs and Lajtha (1958), Tjio and Puck (1958), Puck (1958), Chu and Giles (1959), and a number of others.


    In most instances the results of the different investigators were surprisingly consistent in showing that the individual cell chromosome counts nearly always totaled 46. This was no doubt due in part to the desirability of single layers of somatic cells for identifying and separating the different chromosomes into distinct units. Chu and Giles' results illustrate this consistency. For 34 normal human subjects, including 29 American whites and 4 American Negroes, and one of unknown race, and regardless of sex, age, or tissue, the diploid chromosome number of the somatic cells was overwhelmingly 46. In only five individuals were other numbers observed in isolated cells. Out of 620 counts, 611 had 46 chromosomes; two individuals, whose majority of cells showed 46, had 3 cells with 45 chromosomes; three other individuals, the majority of whose cells showed 46, had 6 cells with 47 chromosomes. Average cell plates counted per individual was nearly 20.


    The only recent observations at variance with these results were those of Kodani (1958) who studied spermatogonial and first meiotic metaphases in the testes from 15 Japanese and 8 whites. In these studies at least several good spermatogonial metaphases in which the chromosomes could be counted accurately, and secondly at least 15 spermatocyte metaphases in which the structure of individual chromosomes could be observed clearly, were made on each specimen. The numbers of cells studied in metaphase were generally above these numbers, one reaching 60 metaphases. Some variation was noted within individuals. Among individuals, numbers of 46, 47, and 48 were observed. Among 15 Japanese, 9 had 46, 1 had 47, and 5 had 48 chromosomes, whereas among the whites 7 had 46, and 1 had 48. Sixteen of the 23 individuals had 46 chromosomes. Karyotype analyses indicated that the numerical variation was caused by a small supernumerary chromosome. On the basis of these observations it would appear that individuals within races may vary in chromosome number and yet be of normal phenotype. However, in view of the extensive observations by others, it seems unlikely that the variation between individuals is as large as that indicated. It will require much further study to establish any other number than 46 as the normal karyotype of man. This is particularly true in view of the work of Makino and Sasaki (1959) and Alakino and Sasaki cited by Ford (1960), in which they studied the human cell cultures of 39 Japanese and found without exception 46 chromosomes, and the earlier work of Ford and Hamerton (1956) on spermatogonial material where they, too, found 46 chromosomes in that tissue. The best features of these human chromosome studies will come in the identification of the individual chromosomes making up the human group. The chromosome pairs may be ordered according to their lengths. The longest chromosome is about 8 times the length of the smallest. The chromosomes may be classified according to their centromere positions. The chromosomes are said by most observers to be fairly easily separated into 7 groups. Separation of the individual chromosome pairs from each other and designation of the pairs so that they can be identified by trained investigators in all good chromosome preparations is not possible according to some ciualified cytologists and admitted difficult by all students. However, standardized reporting in the rapidly growing advances in human cell studies should refine observations, reduce errors, and encourage better techniques. With this in mind, 17 investigators working in this field met in Denver in 1959 in what has come to be called the "Denver conference" (Editorial, 1960). From an examination of the available evidence on chromosome morphologies an idiogram was set up as a standard for the somatic chromosome complement of the normal human genome. A reproduction of this standard is presented in Figure 1.1, as kindly loaned by Dr. Theodore T. Puck for this purpose.



    Fig. 1.1. Id



    The autosomes were first ordered in relation to their size and such attributes as would help in their positive identification. Numbers were given to each chromosome as a means of permanent identification. Basically, identification is assisted by the ratio of the length of the long arm to that of the short arm; the centromeric index calculated from the ratio of the length of the shorter arm to the whole length of the chromosome ; and the presence or absence of satellites. Classification is assisted by dividing the chromosome pairs into seven groups. Groups 1-3. Large chromosomes with approximatel}^ median centromeres. The three chromosomes are readily distinguished from each other by size and centromere position. Group 4-6. Large chromosomes with submedian centromeres. The two chromosomes are difficult to distinguish, but chromosome 4 is slightly longer. Group 6-12. Medium sized chromosomes with submedian centromeres. The X chromosome resembles the longer chromosomes in this group, especially chromosome 6, from which it is difficult to distinguish. This large group is the one which presents major difficulty in identification of individual chromosomes. Group 13-15. INledium sized chromosomes with nearly terminal centromeres ("acrocentric" chromosomes). Chromosome 13 has a prominent satellite on the short arm. Chromosome 14 has a small satellite on the short arm. No satellite has been detected on chromosome 15. Group 16-18. Rather short chromosomes with approximately median (in chromosome 16) or sul>median centromeres. Group 19-20. Short chromosomes with approximately median centromeres. Group 21-22. Very short, acrocentric chromosomes. Chromosome 21 has a satellite on its short arm. The Y chromosome belongs to this group. Separations of the human chromosome pairs into the seven groups is not as difficult as designating the pairs within groups (Patau, 1960). The svstem is a notable advance in summarizing visually the current information in the hope that availability of such a standard will promote further refinements, lessen misclassification, and contribute to a better understanding of the problems by cytologists and other workers in the field.


    1. Xuclear Chromatin, Sex Chromatin

    Sexual dimorphism in nuclei of man (Barr, 1949-59) and certain other mammals may be detected by the observable presence of nuclear chromatin adherent to the inner surfaces of the nuclear membrane. The material is about 1 /x in diameter. It frequently can be resolved into two components of equal size. It has an affinity for basic dyes and is Feulgen and methyl green positive. Nuclear chromatin can be recognized in 60 to 80 per cent of the somatic nuclei of females and not more than 10 per cent of males. It is known to be identifiable in the females of man, monkey, cat, dog, mink, marten, ferret, raccoon, skunk, coyote, wolf, bear, fox, goat, deer, swine, cattle, and opossum, but is not easily usable for sex differentiation in rabbit and rodents because these forms have multiple large particles of chromatin in their nuclei. The tests can be made quickly and easily on skin biopsy material or oral smears. Extensive utilization of the presence or absence of nuclear chromatin in cell samples of man has been made for assigning the presumed genetic sex to individuals who are phenotypically deviates from normal sex types. (See also chapters by Hampson and Hampson, and by Money.) Numerous studies on normal individuals seem to support the test's high accuracy. However, in certain cases involving sexual modification, questions have arisen which are only now being resolved. In male pseudohermaphroditism, sex, determined by nuclear chromatin, is male, thus agreeing with the major aspects of the phenotype. For female pseudohermaphroditism, individuals with adrenal hyperplasia or those without adrenal hyperplasia give the female nuclear chromatin test. For cases listed as true hermaphrodites Grumbach and Barr (1958) list 6 of the male type and 19 of the female type. For the syndrome of gonadal dysgenesis they list 90 as male and 12 as female among the proved cases and 15 more as female among those that are suspected. In the syndrome of seminiferoustubule dysgenesis where there is tubular fibrosis, 9 are listed as male and 18 female. Where there is germinal aplasia, 15 are listed as male and 1 as female. The seeming difficulties in assigning a sex constitution to some of these types are now being dissipated through the study of the full chromosome complements which are responsible for these different disease conditions. As observations on different chromosome types have been extended, evidence has accumulated to show that the numbers of sex nuclear chromatins, for at least some of the nuclei making up the organism, often equals (n — 1) times the number of X chromosomes. The majority of male XY nuclei are chromatin negative as are most of the Turner XO type. Female nuclei XX have a single chromatin positive element as do the XXY and XXYY types. The XXX and XXXY have 14 and 40 per cent respectively with two Barr bodies in cases for which quantitative data are available. However, a child with 49 chromosomes, but whose cultured cell chromosomes appear as single heteropycnotic masses making identification of the individual chromosomes difficult, showed 50 per cent of the cell nuclei with three Barr elements (Fraccaro and Lindsten, 1960) . The chromosome constitution of these nuclei was interpreted as trisomic for 8, 11, and sex chromosomes. Sandberg, Crosswhite and Gordy (1960) report the case of a woman 21 years old having various somatic changes which does not fit this sequence. The chromosome number was 47 and the nuclei were considered trisomic for the sixth largest chromosome. Two chromatin positive bodies were ])rosent in the nuclei.


    2. Chrotnosome Complement and Phenotyppe in Man

    Experience of the past 50 years has emphasized that genes and trisomies or other types of aneuploid chromosome complexes may lead to the development of abnormal phenotypes expressing a variety of characteristics. Drosophila led the way in illustrating how the different gene or chromosome arrangements may affect sex expression. Investigations of human abnormal types, particularly those with altered sex differentiation, have reccntly .^liown that man follow.- other species in this regard. The Y carries highly potent male influencing factors. Gene differences often lead to characteristic phenotypes of unique form.

    3. Testicular Feminization

    The testicular feminization syndrome illustrates one of these types. As described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959), "In complete expression of this syndrome the external genitalia are female, pubic and axillary hair are absent or scanty, the habitus at puberty is typically female, and there is primary amenorrhoea. The testes can be found either within the abdomen, or in the inguinal canals, or in the labia majora, and as a rule the vagina is incompletely developed. An epididymis and vas deferens are commonly present on both sides, and there may be a rudimentary uterus and Fallopian tubes. The condition is familial and is transmitted through the maternal line." A sex-linked recessive, a sex-limited dominant, and chromosome irregularities of the affected persons have been postulated as mechanisms causing the apparent inheritance of this condition. Chromosome examinations of the cells of affected persons have shown 46 as the total number and X and Y as the sex complement. The karyotype analysis agrees with the Barr nuclear chromatin test in that the cells are chromatin-negative but both are at variance with the sex phenotypes in the sense that aside from suppressed testes the patients are so completely female. Genetically, Stewart (1959) has described two color-blind patients with the testicular feminization syndrome in the first five patients he reported. The limited data from these cases suggest that the genie basis for this condition is either independent or but loosely linked with color blindness. This evidence does not exclude sex-linkage but does make it less probable. The third hypothesis of autosomal inheritance may take one of several forms. A recessive gene which affects only the male phenotypes when in homozygous condition is apparently untenable because the matings from which these individuals come are of the outbreeding type and the ratios apparently do not differ from the one-to-one ratio expected of a heterozygous dominant instead of that required for an autosomal recessive. The hypothesis advanced by Witschi, Nelson and Segal (1957), that the presence of an autosomal gene in the mother converts all her male offspring into phenotypes of more or less female constitution, in a manner comparable to that of the Ne gene in Drosophila (Gowen and Nelson, 1942) which causes the elimination of all the female type zygotes, is also made unlikely by the ratios of normal to testicular feminization phenotypes observed in the progenies of these affected mothers. The evidence favors a simple autosomal dominant, acting only in the male zygotes and perhaps balanced by some genes of the X chromosome, which have sufficient influence on the developing male zygote to guide it toward an intermediate to nearly female phenotype. The observations of Puck, Robinson and Tjio ( 1960) indicate that the action of a gene for this condition may not be entirely absent in the female, because in heterozygous condition in an XX individual it seemed to delay menarche as much as 8 years. If this delay be diagnostic for the heterozygote, it will further assist in the genetic analysis of this problem. Evidence on this point should be a part of the genetic studies.


    Cases closely similar to those described by Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox (1959) are presented by Sternberg and Kloepfer (1960). The patients show no trace of masculinity. They are remarkably uniform in anatomic expression. Except for failure to menstruate due to lack of uteri they undergo normal female puberty. Cryptorchid testes, usually intra-abdominal, if removed precipitate menopause symptoms. Four unrelated cases were found in this one study with 7 additional cases traced through pedigree information. A total of 11 affected individuals was found in 6 sibships having 26 siblings of whom 5 were normal males. In each kindred the inheritance was compatible with that of a sex-linkecl recessive gene. A chromosomal study of a thyroid tissue culture from one case revealed 46 chromosomes with normal XY male configuration. The individuals observed were designated as simulant females."

    4. Superfemale

    The human superfemale has been recognized by Jacobs, Baikie, Court Brown, MacGregor, Maclean and Harnden (1959) in a girl of medium height and weight, breasts underdeveloped, genitalia infantile, vagina small, and uterocervical canal 6 cm. in length. Ovaries appeared postmenopausal with normal stroma, and as indicated by a biopsy specimen, deficient in follicle formation. Menstruation was thought to have begun at age 14, but was irregular, occurring every 3 to 4 months and lasting 3 days. The last spontaneous menstruation was at 19. Estrogen therapy caused some development of the breasts and external genitalia, vagina, and uterus with slight uterine bleeding. The patient's parents were above 40 years of age, mother 41, at time of her daughter's birth.


    Examination of sternal marrow cultures showed 47 chromosomes in over 80 per cent of the cells examined. The extra chromosome was the X, the chromosomal type being XXX plus 22 pairs of autosomes. Buccal smears showed 47 per cent of nuclei contained a single chromatin body and 14 per cent contained 2 chromatin bodies as expected of a multiple XX or XXX genotype. In comparison, 25 smears from 20 normal women had 36 to 51 per cent chromatin positive cells but none of these contained 2 chromatin bodies. Two chromatin bodies were seen in some cells of the ovarian stromal tissue. The patient showed a lack of vigor, mentally was subnormal, was underdeveloped rather than overly developed in the phenotypic sexual characteristics. Examination of the patient's mother showed her to be XX plus 22 pairs of autosomes, the normal 46 chromosomes.


    Other cases show that types with XXX plus 22 pairs of autosomes are of female l)henotype but may vary in fertility and development of the secondary sexual characteristics from nonfunctional to functional females bearing children ( Stewart and Sanderson, 1960; Eraser, Campbell, MacGillivray, Boyd and Lennox, 1960). The triplo X condition in man has a greater range of development and fertility than in Drosophila. In man ovaries may develop spontaneously. In Drosophila they require transplantation to a diploid female host where they may attach to the oviducts and release eggs for fertilization (Beadle and Ephrussi, 1937).


    These cases present confirmation of two facts already mentioned for Drosophila. They show that when the X chromosome has primarily sex determining genes, the organism generally becomes unbalanced when 3 of these X chromosomes are matched against two sets of autosomes. The resulting phenotypes are female but relatively undeveloped rather than overdeveloped. The second is that the connotations evoked by the prefix "super" are by no means applicable to this human type or to the Drosophila type.


    The characteristics of the patient also suggest that the autosomes may be carrying sex genes opposing those of female tendencies as observed in both Drosophila and Rumex genie imbalance.

    5. Klinefelter Syndrome

    In the Klinefelter syndrome there is male differentiation of the reproductive tracts with small firm descended testes. Meiotic or mitotic divisions are rare, sperm are ordinarily not found in the semen. The type is eunuchoid in appearance with gynecomastia, high-pitched voice, and sparse facial hair growth. Seminiferous tubules showing an increased number of interstitial cells are atrophic and hyalinized. Urinary excretion of pituitary gonadotrophins is generally increased, whereas the level of 17-ketosteroids may be decreased. The nuclear chromatin is typically female. Of the dozen or more cases studied (Jacobs and Strong, 1959; Ford, Jones, Miller, Mittwoch, Penrose, Ridler and Sha])iro, 1959; Bergman and Reitalu quoted by Ford, 1960), only one, having but 5 metaphase figures, had less than 47 chromosomes in the somatic cells and XXY sex chromosomes. That case was thought to have typical female chromosomes XX + 22 AA. Two other cases were of particular interest as indicating further chromosome aberration. Ford, Jones, Miller, Mittwoch, Penrose, Ridler and Shapiro (1959) studied one patient who displayed both the Klinefelter and Mongoloid syndromes. The chromosome number was 48, the sex chromosomes being XXY and the 48tli chromosoinc being small acrocentric.


    This individual had evidently developed from an egg carrying 2 chromosomal aberrations, one for the sex chromosomes and the second for one of the autosomes. The other case, Bergman and Reitalu as cited by Ford (1960), had 30 per cent of its cells with an additional acrocentric chromosome which had no close counterpart in the normal set.


    Data where the Klinefelter syndrome occurs in families showing color blindness (Polani, Bishop, Ferguson-Smith, Lennox, Stewart and Prader, 1958; Nowakowski, Lenz and Parada, 1959; and Stern, 1959a) further test the XXY relationship and give information on the possible position of the color blindness locus with reference to the kinetochore. Polani, Bishop, FergusonSmith, Lennox, Stewart and Prader (1958) tested 72 sex chromatin-positive Klinefelter patients for their color vision and found that none was affected by red-green color blindness. Nowakowski, Lenz and Parada ( 1959) tested 34 cases and detected 3 affected persons, 2 of whom were deuteranomalous and one protanopic. Stern (1959a) l^oints out that these cases and their ratios are compatible with the interpretation of the Klinefelter syndrome as XXY. One of the deuteranomalous cases had a deuteranomalous mother and a father with normal color vision. This case could have originated from a nondisjunctional egg carrying 2 maternal X chromosomes fertilized by a sperm carrying a Y chromosome. The other two cases had normal fathers with heterozygous mothers. There are several explanations by which the color-blind Klinefelter progenies could be obtained. The heterozygotes might manifest the color-blind condition. The second hypothesis, which is favored, is that of crossing over between the kinetochore and the color-blind locus at the first meiotic division to form eggs each carrying 2 X chromosomes, one homozygous for color blindness, and the other for normal vision. An equational nondisjunction would form eggs homozygous for color blindness which on fertilization by the Y chromosomes of the male would give the necessary XXY constitution for the color-blind male which is Klinefelter in phenotype. A third possibiHty is that these exceptions may arise without crossing over as the result of nondisjunction at the second meiotic division.


    If the hypothesis of crossing over is accepted, the color-blind locus separates freely from its kinetochore and would suggest that the position of the locus is at some distance from the kinetochore of the X chromosome.


    A disturbed balance between the X and the Y chromosomes alters the sexual type. A single Y chromosome, contributing factors important to male development, is able to alter the effects of two sets of female influencing X chromosomes. Yet two Y chromosomes in a complex of XXYY plus 44 autosomes seem to have little or no more influence than one Y (Muldal and Ockey, 1960). The locations of the sex-influencing genes in man are thus more like those of the plant Melandrium than of Drosophila in which the male-determining factors occur in the autosomes. The relative potencies of the male sex factors compared with those of the female, however, are much less than those in Melandrium.

    6. Turner Syndrome

    Turner's syndrome or ovarian agenesis further substantiates the female influence of the X chromosomes. The cases occur as the developmental expression of accidents in the meiotic or mitotic divisions of the chromosomes. These accidents lead to adults unbalanced for the female tendencies of the X chromosome. The gonads consist of connective tissue. The rest of the reproductive tract is female. Growth stimuli of puberty are lacking, resulting in greatly reduced female secondary sexual development. Patients are noticeably short and may be abnormal in bone growth. In its more extreme form, designated as Turner's syndrome, the individuals may show skin folds over the neck, congenital heart disease, and subnormal intellect, as well as other metabolic conditions. Earlier work (Barr, 1959; Ford, Jones, Polani, de Almeida and Briggs, 1959) shows that 80 per cent of the nuclear chromatin patterns are of the male type. Evidence from families having both this condition and color blindness suggested that at least some of the Turner cases would be found to have 45 chromosomes, the sex chromosome being a lone X (Polani, Lessof and Bishop, 1956). Work of Ford, Jones, Polani, de Almeida and Briggs, (1959) has confirmed this hypothesis and added the fact that some of these individuals are also mosaics of cells having 45 and 46 chromosomes. The 45 chromosome cells had but one X, whereas the 46 had two X's. This finding may explain the female-chromatin cell type observed in about 20 per cent of the cases having the Turner syndrome. Such mosaics of different chromosome cell types could also be significant in reducing the severity of the Turner syndrome and in increasing the range of symptoms which characterize this chromosome-caused disease as contrasted with those characterizing Turner's disease. Further cases observed in other investigations, Fraccaro, Kaijser and Lindsten (1959), Tjio, Puck and Robinson (1959), Harnden, and Jacobs and Stewart cited by Ford (1960) have all shown 45 chromosome cells and a single X chromosome. As with the XXX plus 44 autosome super females, the Turner type, X plus 44 autosomes, also shows a rather wide range in development from sterility with extensive detrimental secondary effects to nearly normal in all respects. Bahner, Schwarz, Harnden, Jacobs, Hienz and Walter (1960) report a case which gave birth to a normal boy. Other cases have been described (Hoffenberg, Jackson and jVIuller, 1957; Stewart, 19601 in which menstruation was established over a period of years. The XO type in man and Melandrium is morphologically female. In Drosophila on the other hand, the XO type is phenotypically nearly a perfect male. It is further to be noted that the X chromosome of Drosophila appears to have a less pronounced female bias than that of man when balanced against its associated autosomes, inasmuch as the XO + 2A type in Drosophila is male as contrasted with the XO + 2A type in man which is female. At the same time it seems that the autosomes in the human may be influential in that the female gonadal development is suppressed instead of going to completion as it does in the XX type.


    7. Hermaphrodites

    Hermaphroditic phenotypes in man, to the number of at least 74 (Overzier, 1955), have been observed and recorded since 1900. Types with a urogenital sinus predominated. The uteri were absent in some cases, even when complete external female genitalia were present. Ovotestes were found on the right side of the body in over half the cases; separate left ovaries or testes were about equally frequent ; in three cases separate testis and ovary were indicated. The left side of the body showed a different distribution of gonad types; about onefourth had ovotestes, another fourth ovaries, and one-twelfth testes. Unilateral distribution of gonad types was most frequent. The presence or absence of the prostate seemed to have significance because it is sometimes absent in purely female types. In recent literature similar cases have been called true hermaphrodites. This is an exaggeration in terms of long established practice in plants and animals where true hermaphroditism includes fully functioning gametes of each sex.


    Hungerford, Donnelly, Nowell and Beck (1959» have reported on a case of a Negro in which the culture cells had the chromosome complement of a normal female 46, with XX sex chromosomes. Unfortunately, the possibility that this case may be a chromosome mosaic was not tested by karyotype samples from several parts of the body.


    Harnden and Armstrong (1959) established separate skin cultures from both sides of the body of another hermaphroditic type. The majority of the cells were apparently of XX constitution with a total of 46 chromosomes. However, in one of the 4 cultures established, some 7 per cent of cells had an abnormal chromosome present, suggesting that the case might involve a reciprocal translocation between chromosomes 3 and 4 when the chromosomes were ordered according to size. All the other cell nuclei were normal. The fact that the majority of the cells in these two cases were XX and with 46 chromosomes seems to predicate against the view that either changes in chromosome number or structure of the fertilized egg are necessary for the initiation of hermaplu'odites.


    Ferguson-Smith (1960) describes two cases of gynandromorphic type in which the reproductive organs on the left side were female and on the right side were male. The recognizable organs were Fallopian tube, ovary with primordial follicles only, immature uterus in one case, none in the other, rudimentary prostate, small testis and epididymis, vas deferens, bifid scrotum, phallus, perineal urethra, pubic and axillary hair, breasts enlarging at 14 years. Testicular development with hyperplasia of Leydig cells, germinal aplasia, and hyalinization of the tubules was suggestive of the Klinefelter syndrome. Nuclear-chromatin was positive in both cases. Modal chromosome number was 46. The sex chromosomes were interpreted as XX. The 119 cell counts on one patient showed a rather wide range; 7 per cent had 44 chromosomes, 13 per cent had 45, 62 per cent had 46, and 18 per cent had 47 chromosomes. The extra chromosome within the cells containing 47 chromosomes was of medium size with submedian kinetochore as generally observed for chromosomes of group 3.


    It is surprising that the male differentiation in these four and other hermaphroditic cases (Table 1.5) is as complete as it is. Other observations show that the Y chromosome contains factors of strong male potency, yet in its absence the hermaphrodites develop an easily recognized male system. It is not complete but the degree of gonadal differentiation is as great as that observed in the XXY 4- 2A Klinefelter types. The bilateral sex differentiation in hermaphrodites would seem to require other conditions than those heretofore considered.


    Another case of hermaphroditism is that presented by Hirschhorn, Decker and Cooper (1960). The patient's j:)henotype was intersexual with phallus, hypospadias, vagina, uterus. Fallopian tubes, two slightly differentiated gonads in the position of ovaries. The child was 4 months old. Culture of bone marrow cells showed that the individual was a mosaic of two types. About 60 per cent of the cells had 45 chromosomes of XO karyotype, and 40 per cent had 46 chromosomes with a karyotA'^pe XY. The Y chromosome when present was larger than Y chromosomes of normal individuals. The change in size may be related to the association of the XO and XY cells and be similar etiologically to the case discussed by yivtz (1959) in Sciara triploids.


    There are mosaics in Drosophila formed from the loss by the female in some cells of one of lioi- X chromosomes, as for instance in ring chromosome types, which may display primary and secondary hermaphroditic development. For this to happen the altered nuclei apparently find their way into the region of the egg cytoplasm which is to differentiate into the reproductive tract. As seen in the adults, organ tissue of one chromosome type is cell for cell sharply differentiated from that of the other chromosome type with regard to sex. These observations indicate that for these mosaics the basic chromosome structure of the cell itself determines its development. In fact most mosaics of this species show this cell-restricted differentiation. Several problems arise when these well tested observations are considered in comparison with those now arising in the chromosome mosaics of the sex types in man. It would seem unlikely that the bone marrow cells or for that matter any somatic cells not a part of the reproductive tract would operate to modify the adult sex or a part thereof. Rather the developmental secjuence should start from cell differences within the early developing reproductive tract. Circulatory cells or substances would be of dubious direct significance from another viewpoint. All cells of the body would ultimately be about equally affected by any cells or elements circulating in the blood. With strong male elements and strong female elements the result expected would be a reduction in sex development of either sex instead of the sharply differentiated organ systems which are observed. This raises the question, are the chromosomally differentiated cell sex mosaics primary to or secondarily derived from the tissues of the ultimate hermaphrodites? Study of the cell structure of the sex organs themselves as well as much other information will be necessary to clear up this problem.


    There are, however, other types of controlled sex development, as by various genes, which lead to the presence of both male and female sexual systems. Genes for these phenotypes are relatively rare, but once found are transmitted as commonly expected. The inherited hermaphroditic cases in Drosophila are certainly relevant to the testicular feminization syndrome in man. Are they equally pertinent to the highly sporadic hermaphroditic forms just considered for man? If so, they indicate a genie basis for these types which would probably be beyond the range of the microscope to detect. The low frequency of true hermaphrodites in the human, together with lack of information on possible inheritance mitigates against the genie explanation ; although genie predisposition acting in conjunction with rare environmental events as occurs in our Balb/Gw mice (Hollander, Go wen and Stadler, 1956 ) could explain the rare hermaphrodites observed in that particular line of mice and a limited number of its descendants.

    8. XX XY + U Autosome Type

    The XXXY -I- 44 autosome type in the human has been studied by Ferguson-Smith, Johnson and Handmaker (1960) and Ferguson-Smith, Johnston and Weinberg (1960). The two cases described were characterized by primary amentia, microorchidism and by two sex chromatin bodies in intermitotic nuclei. The patients were similar in having disproportionately long legs; facial, axillary, and abdominal hair scant; pubic hair present; penes and scrota medium to well developed ; small testes and prostates; vasa deferentia and epididymides normally developed on both sides of the body and no abnormally developed Miillerian derivatives. Testes findings were like those in Klinefelter cases with chromatinpositive nuclei, small testes with nearly complete atrophy, and hyalinization of seminiferous tubules and islands of abnormal and pigmented Leydig cells in the hyalinized areas. The few seminiferous tubules present were lined with Sertoli cells but were without germinal cells. Nuclear chromatin was of female type. About twofifths of the nuclei had double and twofifths single sex chromatin. The modal chromosome count for bone marrow cells was 48, 75 per cent of the cells having this number. Chromosome counts spread from 45 to 49. This type, XXXY plus 44 autosomes, may be looked upon as a superfemale plus a Y or a Klinefelter plus an X chromosome. In either case the male potency of the genes in the Y chromosome is able to dominate the female tendencies of XXX to develop nearly complete male phenotypes.


    TABLE 1.5 Chromosome kinds and numbers for different recognized sex types in man


    External Type


    Male

    Female

    Female (rare hemophilic)

    Eunuchoid female Female


    Female Female Female


    Female Female


    Male . Male. Male.


    Male. Male.


    Male . . . Female. Female .


    Female . Female .


    Male. Male.


    Hermaphrodite


    Hermaphrodite. Intersex


    Numbers of Chromosomes


    chromosoil rearrange


    1 + X fragment

    1 2 2


    2 2 Interpreted a.s XX trisomic for Sand 11 or as XXXX 4X + Yl


    3X+ Yj 1 3 3


    2l /mosaic


    Missing or Extra Chromosomes


    ? Chr mosome 3

    orT(X;A)


    Small autosome

    ? Large chromosome or

    T(X;A)


    X or Y

    (X or Y) +21


    46,47,48 cliromosomes X, Y X or Y -fA set


    Sex Chromatm


    Negative Positive

    Negative Negative Positive


    Negative Positive Negative

    Negative


    Negative Negative


    Negative ±

    Negative Positive Positive


    Triple positi


    Negative Double posit Double posit

    Negative

    Double posit Negative


    Negative Negative


    Designating Term


    Normal male Normal female


    Pure gonadal dysgenesis CJonadal dysgenesis


    Testicular feiuinizati

    Turner

    Turner type female


    Tinner? gave birth to boy Turner


    Turner

    Klinefelter

    Klinefelter mongoloid


    Klinefelter Klinefelter


    Klinefelter


    Klinefelter Sujierfemale

    Superfemale gave birtli to children

    Testicular deficiency


    Precocious puberty


    Triploid


    Hermaphrodite or intersex dei)cnding on definition


    Investigators*


    2 3, 4, 5,


    5, 12, 13, 14, 40, 41

    41


    16, 17, 18, 40, 41

    19


    40

    23, 40

    24, 25

    41

    26


    29, 30, 31,. 32, 33 35, 39, 40

    34

    41


    Tjio and Levan, 1956.

    Nilsson, Bergman, Reitalu and Waldenstrom, 1959. Harnden and Stewart, 1959. Stewart, 1960b. Stewart, 1960a.

    Elliott, Sandler and Rabinowitz, 1959.

    Jacobs, Baikie, Court Brown, Forrest, Roy, Stewart and Lennox, 1959. Stewart, 1959

    Lubs, Vilar and Bergenstal, 1959. Sternberg and Kloepfer, 1960. Puck, Robinson and Tjio, 1960. Ford, Jones, Polani, de Almeida and Briggs, 1959. Fraccaro, Kaijser and Lindsten, 1959. Fraccaro, Kaijser and Lindsten, 1960a.


    15. Bahner, Schwarz, Harnden, Jacobs, Hienz and Walter, 1960.

    16. Jacobs and Strong, 1959.

    17. Bergman, Reitalu, Nowakowski and Lenz, 1960.

    18. Nelson, Ferrari and Bottura, 1960.

    19. Ford, Jones, Miller, Mittwoch, Penrose, Hidlor and Shapiro, 1959.

    20. Ford, Polani, Briggs and Bishor), 1959.

    21. Crooke and Hayward, 1960.

    22. Muldal and Ockey, 1960.

    23. Jacobs, Baikie, Court Broun, MacCJregor, Maclean and Harnden, 1959.

    24. Eraser, Campbell, .MacCiillivray, Boyd and Lennox, 1960.

    25. Stewart and Sanderson, 1960.

    26. Jacobs, Harnden, Court Brown, Cohl.stein, Close, MacGregor, Maclean and Strong, 1960.

    27. Ferguson-Smith, Johnston and Handiiiaker, 196(

    28. Book and Santesson, 1960.

    29. Harnden and Armstrong, 1959.

    30. Hungerford, Donnelly, Nowell and Beck, 1959.

    31. Ferguson-Smith, Johnston and Weinberg, 1960.

    32. deAssis, Epps, Bottura and Ferrari, 1960.

    33. Gordon, O'Gorman, Dewhurst and Blank, 1960

    34. Hirschhorn, Decker and Cooper, 1960.

    35. Sasaki and Makino, 1960.

    36. Bloise, Bottura, deAssis, and Ferrari, 1960,

    37. Fraccaro, Kaijser and Lindsten, 1960c.

    37a. Fraccaro and Lindsten, 1960.

    38. Fraccaro, Ikkos, Lindsten, Luft and Kaijser, 1960.

    39. Harnden, 1960.

    40. Ferguson-Smith and Johnston, 1960.

    41. Sandberg, Koepf, Crosswhite and Hauschka, 1960.

    42. Hayward, 1960.


    Both cases had severe mental defects but it should be remembered that they were sought in institutions for which this is a criterion of admittance. Their mental ability was distinctly less than that of Klinefelter XXY cases which have come under study. The pattern of the XXXY effects on the reproductive tract, however, was comparable with that observed in the XXY genotypes. The effects of one Y chromosome were balanced by either two or three X chromosomes to give nearly equal phenotypic effects.

    9. XXY + 66 Autosome Type

    XXY + 66 autosome type was established by Book and Santesson (1960) for an infant boy having several somatic anomalies which may or may not be relevant to the sex type. Externally the genitalia were normal for a male of his age, penis and scrotum with testes present in the scrotum. Again the Y chromosome demonstrates its male potencies over two X's even in the presence of three sets of autosomes. The case is of particular significance since further development may indicate what male potencies an extra set of autosomes may possess.

    10. Summary of Types

    Other types of sex modifying chromosomal combinations and their contained genes have been observed particularly as mosaics or as chromosomal fragments added or substracted from the normal genomes. No doubt other types will be discovered during the mushroom growth of this period. Time can only test the soundness of the observations for the field of human chromosomal genetics and cytology is difficult at best requiring special aptitudes and experience. Mistakes, no doubt, will be made. The status of the subject is summarized in Table 1.5.

    11. Types Unrelated to Sex

    Other cases not related to sex or only secondarily so were scrutinized during the course of these studies. The information gained from them is valuable as it strengthens our respect for the mechanisms involved. The sex types which are dependent on loss or gain of the X and/or Y chromosomes belong to the larger category of monosomies or trisomies. Numbers of autosomal monosomic and trisomic syndromes have also been identified in the course of these investigations. Similarly, not all cases that have been studied have turned out to be associated with chromosomal changes. This in itself is important since it lends confidence in those that have, as well as redirects research effort toward the search for other causes than chromosomal misbehavior. The first trisomic in man was identified through the study of Mongolism. The condition affects a number of primary characteristics but not those of sex, for males and females occur in about equal numbers. The broad spectrum of these effects points to a loss of balance for an equally extensive group of genes in the two sexes. The common association of characteristics making up these Mongoloids, together with their sporadic appearance and their change in frequency with maternal age, all suggest the findings which Lejeune, Gautier and Turpin (1959a, b) and Lejeune, Turi)in and Gautier (1959a, b) were able to demonstrate so successfully. They established that the tissue culture cells of Mongoloid imbeciles had 47 chromosomes and that the extra chromosome was in the small acrocentric group. Lejeune, Gautier and Turpin (1959a, b) have now confirmed these observations on not less than nine cases. Jacobs, Baikie, Court Brown and Strong (1959), Book, Fraccaro and Lindsten (1959) and Fraccaro (cited by Ford, 1960) as well as later observers have substantiated the results on more than ten other cases. The well known maternal age effect, whereby women over 40 have a chance of having IMongoloid offspring 10 to 40 times as frequently as those of the younger ages, would seem to point to non-disjunction in oogenesis as the most important cause of this condition. Some women who have had previous JMongoloid progeny have an increased risk of having others. This is an important consideration in that genetic factors may materially assist in bringing about nondisjunction in man as they are known to do in Drosophila (Gowen and Gowen, 1922; Gowen, 1928). The products of the nondisjunctions approach those expected on random distribution of the chromosomes (Gowen, 1933) so that occasionally more than one type of chromosome disjunction will appear in a given individual. Such a case is that illustrated by Ford, Jones, Miller, Mittwoch, Penrose, R idler and Shapiro (1959) in which the nondisjunctional type included not only that for the chromosome important to Mongolism but also the sex chromosomes significant in determining the Klinefelter condition. This individual showed 48 chromosomes, 22 pairs of normal autosomes, 3 sex chromosomes XXY, and a small acrocentric chromosome matching a pair of chromosomes, the 21st, within the smallest chromosomes of the human idiogram. The analysis of Mongolism showed the way for the separation of the various human sex types through chromosome analyses.


    Chromosome translocations furnish another means of establishing an anomaly that may then continue on an hereditary basis as either the male or female may transmit the rearranged chromosomes. Polani, Briggs, Ford, Clarke and Berg (1960), Fraccaro, Kaijser and Lindsten (1960b), Penrose, Ellis and Delhanty (1960) and Carter, Hamerton, Polani, Gunalp and Weller (1960) have studied Mongoloid cases which they interpreted in this manner. In some cases the rearranged chromosomes have been transmitted for three generations. Several of the translocations were considered to include chromosomes 15 and 21.


    Another trisomic autosomal type was rc])ortcd by Patau, Smith, Therman, Inhorn and Wagner (1960). The patient was female and had 47 chromosomes. The extra chromosome was a medium-sized acrocentric autosome belonging to the D group. Despite extensive malformations affecting several organs the patient lived more than a year. Another female iiortraying the same syndrome has since been found, so other cases may be expected. Among the characteristics are mental retardation, minor motor seizures, deafness, apparent micro or anophthalmia, horizontal palmar creases, trigger thumbs, Polydactyly, cleft i)alate, and hemangiomata.


    The third trisomic type was also reported by Patau, Smith, Therman, Inhorn and Wagner (1960). Six individuals have been observed. The characters affected are mental retardation, hypertonicity (5 patients), small mandible, malformed ears, flexion of fingers, index finger overlaps third, big toe dorsiflexed (at least 4), hernia and/or diaphragm eventration, heart anomaly (at least 4), and renal anomaly (3). The sexes were two males and four females. The extra chromosome was in the E group and was diagnosed as number 18. Edwards, Harnden, Cameron, Crosse and Wolff (1960) have described a similar case but they consider the trisomic to be number 17. Ultimate comparisons of these types no doubt will decide if this is a 4th trisomic or if all the cases belong in the same group.


    The Sturge-Weber syndrome apparently is caused by another trisomic. Locomotor and mental abilities are retarded. Hayward and Bower (1960) interpret the 3 chromosomes responsible as the smallest autosomes, number 22, of the human group.


    Trisomic frequencies should be matched by equal numbers of monosomies. Turpin, Lejeune, Lafourcade and Gautier (1959) have reported polydysspondylism in a child with low intelligence, dwarfing, and multil)le malformations of spine and sella turcica. The somatic cell chromosome count was only 45 but one of the smallest acrocentric chromosomes appeared to have been translocated, the greatest part of this chromosome being observed on the short arm of one of the 3 longer acrocentric chromosomes. Th(> condition appears to be unique and not likely to be found in other unrelated famiVws. However, the phenotyj^ic effects were so severe that all members of the proband's family would seemingly be worthy of careful sur\'('y for their chromosome characteristics.

    The comi)lex pattern of multiple anomalies renders each syndrome distinct from the othei's. Chromosome losses or gains from the normal diploid would be expected to lead to the complex changes. Mongolism is influenced by age of the mother and probably to some extent by her inheritance. It is to be expected that the other trisomies may show parallel relations. Other trisomies may be expected although, as the chromosomes increase in size, a group of them will have less opportunity to survive because of loss of balance with the rest of the diploid set. Thus far most of these conditions affect the sex phenotypes. This is in accord with the results in Drosophila. Changes in the balance of the X chromosomes are less often lethal than the gain or loss of an autosome. Other animals show like effects. In plants, loss or gain of a chromosome, although generally detrimental, often causes less severe restrictions on life. Harmful effects are observed but do not cause early deaths. This may be because many aneuploids are within what are presumably polyploid plant species.

    Ford (1960) has collected the data on 13 different phenotypes that could come under suspicion of chromosomal etiology as examined by a number of workers. Careful cytologic examination of patients suffering from one or another of these diseases has shown that the idiograms were normal in both number and structure of the chromosomes. The disease conditions were: acrocephalosyndactyly, arachnodactyly

    (Marfan's syndrome), chondrodystrophy, Crouzon's disease, epiloia, gargoylism, Gaucher's disease, hypopituitary dwarfism, juvenile amaurotic idiocy, Laurence-MoonBiedl syndrome. Little's disease, osteogenesis imperfecta, phenylketonuria, and anencephalic types. To this list Sandberg, Koepf , Crosswhite and Hauschka (1960) have now been added neurofibromatosis, Lowe's syndrome, and pseudohypoparathyroidism.

    F. Sex Ratio in Man

    Sex ratio studies on human and other animal populations have always been large in volume. The period since 1938 is no exception. Geissler's (1889) data on family sex ratios, containing more than four million births, have been reviewed and questions raised by several later analysts. Edwards (1958) has reanalyzed the clata from this population and considered these points and reviewed the problems in the light of the following questions: (1) Does the sex ratio vary between families of the same size? (2) Do parents capable of producing only unisexual families exist? (3) Can the residual deviations in the data be satisfactorily explained? Probability analyses were based on Skellam's modified binomial distribution, a special case of the hypergeometrical. The following conclusions were drawn. The probability of a birth being male varies between families of the same size among a complete cross-section of this 19th century German population. There is no evidence for the existence of parents capable of producing only unisexual families. With the assumption that proportions of males vary within families, the apparent anomalies in the data appear to be explicable. These studies have a bearing on the variances observed in further work dealing with family differences such as that of Cohen and Glass ( 1959) on the relation of ABO blood groups to the sex ratio and that of Novitski and Kimball (1958) on birth order, parental age, and sex of offspring. Novitski and Kimball's data are of basic significance, for the interpretations are based on a large volume of material covering a one-year period in which improved statistical techniques were utilized in the data collection, in showing that within these data sex ratio variation showed relatively little dependence on age of mother, whereas it did show dependence on age of father, birth order, and interactions between them. These observations have direct bearing on the larger geographic differences observed in sex ratios as discussed by Russell (1936) and have recently been brought to the fore through the studies of Kang and Cho (1959a, b). If these data stand the tests for biases, they are of significance in showing Korea to have one of the highest secondary sex ratios of any region, 113.5 males to 100 females, as contrasted with the American ratio of about 106 males to 100 females. Of similar interest is the lower rate of twin births, 0.7 per cent in Korea vs. about 1 per cent in Caucasian populations and the fact that nearly twothirds of these tW'in births in Korean peoples are identical, whereas those in the Caucasian groups are only about half that number. The reasons for these differences must lie in the relations of the human X and Y chromosomes and autosomes and the balance of their contained genes. Little or nothing is known about how these factors operate in the given situations.


    Acknowledgment

    In formulating and organizing the material on which this paper is based I have been fortunate in the helpful discussions and analytical advice contributed so generously by Doctors H. L. Cai'son, K. W. Cooper, H. V. Grouse, C. W. Metz, S. B. Pipkin, W. C. Rothenbuhler, and H. D. Stalker, and others having primary research interests in this field. To them, and particularly to my research associates Doctors S. T. C. Fung and Janice Stadler, our secretary Gladys M. Dicke and to my wife, ]\Iarie S. Gowen, I tender grateful acknowledgment for their contributions to the manuscript. Direct reference is made to some 300 investigations in the body of the paper but the actual number, many of which could equally well have been incorporated, that furnished background to these advances is much larger, more than 1600. To this vast effort toward understanding the foundations for sex we are all indebted.

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