Chapter 7 - Sexual reproduction, which greatly enhances...

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Unformatted text preview: Sexual reproduction, which greatly enhances genetic variation within species, requires mechanisms that result in sexual differentiation. A wide variety of genetic mechanisms have evolved in organisms leading to sexual dimorphism. Most often, specific genes, usually on a single chromosome, cause maleness or femaleness during development. In humans, the presence of extra X or Y chromosomes beyond the diploid number may be tolerated, but often lead to syndromes demonstrating distinctive phenotypes. - While segregation of sex-determining chromosomes should theoretically lead to a one-to-one sex ratio of males to females, in humans, this ratio greatly favors males at conception. In mammals, females contain two X chromosomes compared to one in males, but the extra genetic information in females is compensated for by random inactivation of one of the X chromosomes early in development. - In some reptilian species, temperature during incubation of eggs determines the sex of offspring NEIAEIS suave) 166 Chapter 7 n the biological world. a wide range of reproductive modes and life cycles are recognized. Organisms exist that display no evidence of sexual reproduction. Other species alternate between short periods of sexual reproduction and prolonged pe- riods of asexual reproduction. In most diploid eukaryotes. how- ever, sexual reproduction is the only natural mechanism resulting in new members of an existing species. Orderly transmission of genetic units from parents to offspring. and tints any phenotypic variability, relies on the processes of segregation and indepen— dent assorrment occuring during meiosis. Meiosis produces hap- loid gametes so that. following fertilization. the resulting offspring maintain the diploid number of chromosomes characteristic of their species. Hence, meiosis ensures genetic constancy within members of the same species. These events, which are involved in the perpetuation of all sexually reproducing organisms. depend ultimately on an effi- cient union of gametes during fertilization. In turn. successful mating between organisms. the basis for fertilization. depends on some form of sexual differentiation in organisms. Even though it is not overtly evident. this differentiation occurs for or— ganisms as low on the evolutionary scale as bacteria and single— celled eukaryotic algae. In evolutionarily higher forms of life. the differentiation of the sexes is more evident as phenotypic di- morphism in the males and females of each species. The shield and spear U‘. the ancient symbol of iron and Mars, and the mir- ror Q, the symbol of copper and Venus. represent the maleness and femaleness acquired by individuals. Dissimilar. or hetercmorphic chromosomes, such as the X—Y pair, often characterize one sex or the other, resulting in their label as sex chromosomes. Nevertheless. it is genes, rather than chromosomes, that ultimately serve as the underlying basis of sex determination. As we will see. some of these genes are present on sex chromosomes. but others are autosomal. Ex— tensive investigation has revealed a wide variation in sex chro- mosome systems. even in closely related organisms. suggesting that mechanisms controlling sex determination have undergone. rapid evolution in many instances. In this chapter. we will first review several representative modes of sexual differentiation by examining the life cycles of three model organisms often studied in genetics: the green alga Chimnydomonns; the maize plant. Zen. nmys; and the nee matode (roundworm). Coenorltubditis el'cgnns {most often re— ferred to as C. elegrmr). These will serve to contrast the different roles that sexual differentiation plays in the lives of cli- verse organisms. Then. we will delve more deeply into what is known about the genetic basis for the determination of sexual differences= with a particular emphasis on two other organisms: our own species. representative of mammals; and Drosopliila. subject of pioneering sex-determining studies. Sexual Differentiation and Life Cycles In multicellular organisms. it is important to distinguish be— tween primary sexual differentiation. which involves only the gonads where gametes are produced. and secondary Sex Determination and Sex Chromosomes sexual differentiation. which involves the Overall appear ance of the. organism. including clear differences in such organs as mammary glands and external genitalia. In plants and animals, the terms unisexual. dioecious. and gonochoric are equivalent: they all refer to an individual containing only male or only female reproductive organs. Conversely. the terms bisexual. monoccious. and hermaphroditic refer to individuals containing both male and female reproductive organs. a common occurrence in both the plant and animal kingdoms. These organisms can produce fertile gametes of both sexes. The term intersex is usually reserved for indi— viduals of intermediate sexual differentiation. who are most often sterile. Chlamydomonas The life cycle of the green alga Chlamydommmr. shown in Figure 7—1. is representative of organisms exhibiting only in— frequent periods of sexual reproduction. Such organisms spend most of their life cycle in the haploid phase. asexually producing daughter cells by mitotic divisions. However. under unfavorable nutrient conditions. such as nitrogen de— pletion. certain daughter cells function as gametes. Follow- ing fertilization. a diploid zygote. which can withstand the unfavorable environment. is formed. When conditions be- come more suitable, meiosis ensues and haploid vegetative cells are again produced. In such species. there is little vis- ible difference betWeen the haploid vegetative cells that re— produce asexually and the haploid gametes that are involved in sexual reproduction. The two gametes that fuse together during mating are not usually morphologically distinguish- able. Such gametes are called isogametes. and species pro- ducing them are said to be isogamous. In 1954. Ruth Sager and Sam Granik demonstrated that ga» metes in Chfomydomonas could be subdivided into two mating types. Working with clones derived from single hap- loid cells. they showed that cells from a given clone would mate with cells from some. but not all other clones. When they tested mating abilities of large numbers of clones. all could be placed into one of two mating categories. either mt+ or mf cells. “Plus” cells would only mate with “minus” cells. and vice versa. as represented in Figure 7—2. Follow: ing fertilization and meiosis. the four haploid cells {zoospores) produced were found to consist of two plus types and two minus types. Further experimentation established that there is a chemi- cal difference between plus and minus cells. When extracts were prepared from cloned Clilcnnydomorms cells (or their flagella) and then added to cells of the opposite mating type. clumping or agglutination occurred. No such agglutination occurred if the extract were added to cells of the mating type from which it was derived. These observations suggest that despite the morphological similarities between isogametes. a chemical differentiation has occurred between them. Therefore. in this alga. a primitive means of sex differenti- ation exists even though there is no morphological indicaw tion that such differentiation has occurred. FlGURE 7——1 The life cycle of Chlamydomonas. Unfavorable conditions stimulate the formation of isogarnetes of opposite mating type that may fuse in fertilization. The resulting zygote undergoes meiosis, producing two haploid cells of each mating type. The photograph shows vegetative cells of this green alga. Vegetative colony Vegetative colony 0 FIGURE 7—2 llustration of mating types during fertilization in Chlamydomonas. Mating will occur only when plus (+) and minus i—) cells are together. Zea mays As we first discussed in Chapter 2 (see Figure 2—16}, life cy- cles of many plants alternate between the haploid gametophyte stage and the diploid sporophyte stage. The processes of \? —-» ’L" Isogamete (n) 7.1 Sexual Differentiation and Life Cycles 167 Meiotic products (n) f Meiosis Mitosis Mitosis @e,® o‘fii‘eo Q) www Vegetative colony "7” cells (n) of ”+" cells (in) Nitrogen Nitrogen depletion depletion Fusion (fertilization) Pairing "+” lsogarnete (n) meiosis and fertilization link. the two phases during the life cycle. The relative amount of time spent in the two phases varies between the major plant groups. In some nonseed plants, such as mosses, the haploid gametophyte phase and the mor- phological structures representing this stage predominate. The reverse is true in seed plants. Maize (Zea mays). more popularly called corn, exemplifies a monoecious seed plant, where the sporophyte phase and the morphological structures representing this stage predominate dun'ng the life cycle. Both male and female structures are pre- sent on the adult plant. Thus. sex determination must occur dif- ferently in different tissues of the same organism, as illustrated in the life cycle of this familiar plant (Figure 723). The stamens, or tassels, produce diploid microspore mother cells. each of which undergoes meiosis and gives rise to four hapw loid microspores. Each haploid microspore in turn develops into a mature male microgametophyte—the pollen grain— which contains two sperm nuclei with identical genotypes. Comparable female diploid cells, known as megaspore mother cells, exist in the pistil of the sporophyte. Following meiosis, only one of the four haploid megaspores survives. it usually divides mitotically three times, producing a total of eight genetically identical haploid nuclei enclosed in the embryo sac. Two of these nuclei unite near the center of the embryo sac, becoming the endosperm nuclei. At the end of the micropyle, the sac where the sperm enters, three nuclei remain: the oocyte nucleus and two synergids. The other three antipodal nuclei are clustered at the opposite end of the embryo sac. 168 Chapter 7 Pollination occurs when pollen grains make contact with the silks [or stigma) of the pistil and develop extensive pollen tubes that grow toward the embryo sac. When contact is made at the micropyle= the two sperm nuclei enter the embryo sac. One sperm nucleus unites with the haploid oocyte nucleus and the other sperm nucleus unites with two endosperm nuclei. This process, known as doublefertilization, results in the diploid zy— gote nucleus and the triploid endosperm nucleus. respectively. Each ear of corn may contain as many as IOOO of these struc- tures, each of which develops into a single kernel. Each kernel, if allowed to germinate, gives rise to a new plant, the sporophyte. The mechanism of sex determination and differentiation in a monoecious plant such as Zea mays, where the tissues forming Sex Determination and Sex Chromosomes both male and female gametes are of the same genetic constitu- tion. was difficult to comprehend at first. However._ the discovery of a large number of mutant genes that disrupt normal tassel and pistil formation supports the concept that normal products of these genes play an important role in sex determination by afiecting the differentiation of male or female tissue in several ways. For example, mutant genes that cause sex reversal provide valuable information, When homozygous, all mutations clas- sified as tassel seed (is) interfere with tassel production and in- duce the formation of female structures. Thus, it is possible for a single gene to cause a normally 'monoecious plant to become functionally only female. 0n the other hand. the recessive [nu- tations sitkless (sit) and barren stalk (ba) interfere with the de- velopment of the pistil, resulting in plants with only functional male reproductive organs. Data gathered from studies of these and other mutants suggest that the products of many wild-type alleles of these genes in- teract in controlling sex determination. During development. certain cells are “determined” to become male or female struc- tures. Following sexual differentiation into either male or fe- male structures. male or female gametes are produced. How Do WE Know? How do we know that specific genes in maize play a role. in sexual differentiation? Caenorhabditis elegans The nematode worm Caenm-lmbdlris elegant [C elegans. for short; Figure 7—4(a)] has become a popular organism in genetic studies, particularly during the investigation of the genetic con- trol of development. lts usefulness is based on the fact that the her- maphroditic adult consists of exactly 959 cells. and the precise lineage of each cell can be traced back to specific embryonic orii gins. Among many interesting mutant phenotypes that have been studied, behavioral modifications have also been a favorite topic of inquiry. Microspore mother _ There are two sexual phenotypes i?“ . CE” (m'cmgamemphyte) in these worms: males, which have pm“ St‘gmfl MEIOSIS 3 of 4 ' -. only testes, and hermaphrodites that .- degenerate contain both testes and ovaries. Dur- . Megiipore 4.. ing larval development of hennaph- . . mo . _. l ceiler rodites. testes form that produce SPOROPHYTE STAGE Me as ores Megagametophyte sperm, which is then stored. Ovaries mph-m g P are also produced, but oogenesis Endosperm figgféfif’frm does not occur until the adult stage is Embryo . = Antipodal nude-l =} Spelrm reached several days later. The de- nuc el Endosperm nuclei veloped eggs are fertilized by the DOUBLE Synergids Tube stored sperm during the process of MATURATION FERTILIZATION 8%:ng ' POLLINATION nucleus selgfertjlizationl - r. r Pollen Tube Diploid zygote Embryo sac (megagametophyte) FIGURE 7—3 The life cycle of maize (Zea mays). The diploid sporophyte bears stamens and pistils that give rise to haploid microspores and megaspores, which develop into the pollen grain and the embryo sac that ultimately house the sperm and oocyte, respectively. Following fertilization, the embryo develops within the kernel and is nourished by the endosperm. Germination of the kernel gives rise to a new sporophyte (the mature corn plant), and the cycle repeats itself. 7.2 X and Y Chromosomes Were First Linked to Sex Determination Early in the 20th Century 169 (b) Hrmaphrodite Male (> 99%) (< 1%) Cross-fertilization I Hermaphrodite Male (50%) (50%) FIGURE 7—4 (a) F‘hotomicrograph of a hermaphroditic nematode, C. elegans; (b) The outcomes of self-fertilization in a hermaphrodite and a mating of a hermaphrodite and a male worm. The outcome of this process is quite interesting [Figure 7—4tbil. The vast majority of organisms that result, like the parental worm, are hermaphrodites; less than ] percent of the offspring are males. As adults, males can mate with hetmaphrodites, producing about hall" male and half hermaphrodite offspring. The genetic signal that determines maleness in contrast to here maphroditic deveIOpment is provided by genes located on both the X chromosome and autosomes. C. elegans lacks a Y chromo some altogether. Hermaphrodites have two X chmmOsomes, while males have only one X chromosome. It is believed that the ratio of X chromosomes to the number of sets of autosomes LiIe timately determines the sex of these worms. A ratio ol‘ 1.0 (two X. chromosomes and two copies of each autosome) results in her maphrodites and a ratio oi'0.5 results in males. The absence of a heteromorphic Y chromosome is not uncommon in organisms. / Problem 7.22 on page 185 asks you to devise an exper- imental approach to elucidate the original findings re- garding sex determination in a marine worm. Hint: An obvious approach would be to attempt to iso- late and devise experiments with the unknown factor affecting sex determination. An alternative approach, more in tune with genetic analysis, could involve the study of mutations that alter the normal outcomes. X and Y Chromosomes Were First Linked to Sex Determination Early in the 20th Century How sex is determined has long intrigued geneticists. In 1891. H. Henking identified a nuclear structure in the sperm of certain insects, which he labeled the X—boa‘y. Several years later, Clarance McCIung showed that some grasshopper sperm contain an unusual genetic structure, which he called a hetemciirmiiosome, but the rest lack this structure. I-le mis- takenly associated its presence with the production of male progeny. In [906. Edmund B. Wilson clarified the findings of Henking and McClung when he demonstrated that female somatic cells in the insect Pi'oreiwr contain 14 chromo- somes. including 2 X chromosomes. During oogenesis, an even reduction occurs. producing gametes with 7 chromo- somes. including one X. Male somatic cells. on the other hand, contain only 13 chromosomes. including a single X chromosome. During spermatogenesis, gametes are pro- duced containing either 6 chromosomes, without an X, or 7 chromosomes, one of which is an X. Fertilization by X-bearing sperm results in female offspring. and fertiliza~ tion by X-deficient sperm results in male offspring [Figure 7—5taJ]. The presence or absence of the X chromosome in male ga~ metes provides an efficient mechanism for sex determination in this species and produces a 1:1 sex ratio in the resulting off- spring. The mechanism, now called the XX [X0 or Prater-tor mode of sex determination, depends on the random distribu- tion of the X chromosome into half of the male gametes dur- ing segregation. Wilson also experimented with the hemipteran insect Lygueus rtrrictrs. in Which both sexes have 14 chromosomes. Twelve of these are autosomes. In addition, the females have 2 X chro- mosomes. while the males have only a single X and a smaller heteroeltrornOsome labeled the Y chromosome. Females in this species produce only gametes of the (6A + X) constitu- tion, but males produce two types of gametes in equal propor- tions: (6A + X) and {6A + Y}. Therefore, following random fertilization. equal numbers of male and female progeny will be produced with distinct chromosome complements. This mode of sex determination is called the Lygaeus or XX/XY type [Figure 7—5(b)_]. In Pi‘orennr and Lygaeus insects. males produce unlike gt 7 metes. As a result, they are described as the heterogametic sex, and in effect, their gametes ultimately determine the sex of the progeny in those species. In such cases, the female. who has like sex chromosomes, is the homogametic sex. pro ducing uniform gametes with regard to chromosome num— bers and types. The male is not always the heterogametic sex. In other orv ganisms, the female produces unlike gametes. exhibiting ei- ther the Pivreiior (XX/X0) or Lygnens (XX/KY) mode of sex determination. Examples include moths and butterflies, most birds. some fish. reptiles, amphibians. and at least one species of plants (FI‘GgCJfffl orientalis). To immediately distinguish sit— uations in which the female is the heterogametic sex. some 170 Chapter 7 (a) Protenor mode x lllili A tosomes XS PCutosomes X XX Female (12A + 2X) X0 Male (12A + X) iillll iill alfifiafl% l ‘ Autosomes X Male (12A + X) Female (12A + 2X) 1 :1 sex ratio Autosomes Y XY Male (12A + X + Y) Gamete formation illiii lilil i lilll- WM Gamete formation (1)) Lygaeus mode liliii’ Autosomes X5 XX Female (12A + 2X) >< Gamete formation :i ii l: liil lit Autosomes Y Autosomes Xs Male (12A + X + Y) Female (12A + 2X) 1 :1 sex ratio i l i i FiGURE 7~5 (a) The Protenor mode of sex determination where the heterogametic sex (the male in this example) is X0 and produces gametes with or without the X chromosome; (b) The Lygaeus mode of sex determination, where the heterogametic sex (again. the male in this example) is XY and produces gametes with either an X or a Y chromosome. In both cases, the chromosome composition of the offspring determines its sex. Sex Determination and Sex Chromosomes workers use the notation ZZ/ZW. where ZW is the heterogal mous female. instead of the XX/‘XY notation. The situation with fowl (chickens) illustrates the difficulty in establishing which sex is heterogametic and whether: the Protenor or Lygneus mode is operable. While genetic evidence supported the hypothesis that the female is the heterogametic sex. the cytological identification of the sex chromosome was not accomplished until 1961. because of the large number of chromosomes [78) characteristic of chickens. When the sex chromosomes were finally identified, the female was shown to contain an unlike chromosome pair. including a heteromorphic chromosome (the W chromosome). Thus. in fowl. the female is indeed heterogamet’ic and is characterized by the Lygneus type of sex determination. The Y Chromosome Determines Maleness in Humans The first attempt to understand sex determination in our own species occurred almost 100 years ago and involved the exam- ination of chromosomes present in dividing cells. Efforts were made to accurately determine the diploid chromosome num- ber of humans, but because of the relatively large number of chromosomes, this proved to be quite difficult. In 1912. H. von Winiwarter counted 47 chromosomes in a spermatogonial metaphase preparation. It was believed that the sex—determining mechanism in humans was based on the presence of an extra chromosome in females. who were thought to have 48 chro- mosomes. However. in the 19203. Theophilus Painter observed between 45 and 48 chromosomes in cells of testicular tissue and also discovered the small Y chromosome, which is now known to occur only in males. In his original paper, Painter fae vored 46 as the diploid number in humans. but he later con- cluded incorrectly that 48 was the chromosome number in both males and females. For 30 years. this number was accepted. Then. in 1956. Joe Hin Tjio and Albert Levan discovered a better way to prepare chromosomes. This improved technique led to a strikingly clear demonstration of metaphase stages showing that 46 was in- deed the human diploid number. Later that same year. C. E. Ford and John L. Hameiton. also working with testicular tissue. confirmed this finding. The familiar karyotype of humans (Figure 7—6) is based on Tjio and Levan’s technique. Within the normal 23 pairs of human chromosomes. one pair was shown to vary in configuration in males and females. These two chromosomes were designated the X and Y sex chromo- somes. The human female has two X chromosomes, and the human male has one X and one Y chromosome. We. might believe that this observation is sufficient to conclude that theY chromosome determines maleness. However. several other interpretations are possible. TheY could play no role in sex determination: the presence of two X chromosomes could cause femaleness; or maleness could result from the lack of a second X chromosome. The evidence that clarified which explanation was correct awaited the study of variations in the human sex chromosome composition. As such investigations revealed. the Y chromosome does indeed determine maleness in humans. 7.3 The Y Chromosome Determines Maleness in Humans 171 A a: v y“f~.¢= a” .c-"vm-“l ‘ I "W. I "ta/f- -“ amt-V’s». 6*? (“an Q “f; [0 dJ ;. Ln C‘J‘T‘s ; Q : Ills" “NJ fir“.- ‘ E NJ“ «as if“? is ire his mg M 6 7 s 9 [0 ll 12 i”. .33- if I”? is if? .13. 14~ '15 l6 I7 13 '3'" 9% n r i _. I9 20 2| 22 X X a n ‘ g V 3‘ _ It i... t )iiétt it '3‘; tr rt it .43... a 4t FIGURE 7—6 The traditional human karyotypes derived from a normal female and a normal male. Each contains 22 pairs of autosomes and two sex chromosomes. The female (a) contains two X chromosomes, while the male (b) contains one X and one Y chromosome (see arrows). Klinefelter and Turner Syndromes About 1940, scientists identified two human abnormalities char- acterized by aberrant sexual development, Klinefelter syn- drome and Turner syndromef“ Individuals with Klinefelter syndrome have genitalia and internal ducts that are usttally male, but their testes are rudimentary and fail to produce sperm. They are generally tall and have long arms and legs and large hands and feet. Although some masculine development does occur. feminine sexual development is not entirely suppressed. Slight enlarge- ment of the breasts (gynecomastia) is common, and the hips are often rounded. This ambiguous sexual development. re- ferred to as intersexuality. may lead to abnormal social devel- opment. Intelligence is often below the normal range. In Turner syndrome, the affected individual has female ex~ ternal genitalia and internal ducts. but the ovaries are rudi— mentary. Other characteristic abnormalities include short stature (usually under 5 feet). skin flaps on the hack of the neck, and underdeveloped breasts. A broad, shieltllike chest is sometimes noted. Intelligence is often normal. In 1959, the karyotypes ofindividuals with these syndromes were determined to be abnormal with respect to the sex chro- mosomes. Individuals with Kline‘l’elter syndrome have more than one X chromosome. Most often they have an XXY com~ plement in addition to 44 autosomes [Figure 7—7(a)J. People with this karyotype are designated 47.XXY. Individuals with Turner syndrome are most often monosomic and have only 45 chromosomes, includingjust a single X chromosome. They are designated 45,X [Figure 7—7(b)]. Note the convention used in *Although the possessive form of the names of most syndromes (eponyms) is sometimes used te.g.. Klinefelter’s), the current prefer— ence is to use the nonpossessivc form, which we have adopted for all human syndromes. designating the above chromosome compositions. The num- ber indicates the total number of chromosomes present, and the information after the comma designates the relevant (levi- ation from the normal diploid content. Both conditions result from nondisiunction. the failure of the X chromosomes to seg— regate properly during meiosis. (See Figures 2~l7.) These Klinefelter and Turner karyotypes and their corre— sponding sexual phenotypes allow us to conclude that the Y chromosome determines maleness in humans. In its absence. the sex of the individual is female. even if only a single X chromosome is present. The presence of the Y chromosome in the individual with Klincfelter syndrome is sufficient to determine maleness. even though male development is not complete. Similarly, in the absence of a Y chromosome. as in the case of individuals with Turner syndrome. no mas- culinization occurs. Klinefelter syndrome occurs in about 2 of every 1000 tnale births. The karyotypcs 48.XXXY. 48,XXYY, 49,XXXXY, and 49,XXXYY are similar phenotypically to 47.XXY. but mani- festations are often more severe in individuals with a greater number of X chromosomes. Turner syndrome can also result from karyotypes other than 45K. including individuals called mosaics whose somatic cells display two different genetic cell lines, each exhibiting a dif- ferent karyotype. Such cell lines result from a mitotic cn'or dur— ing early development. the most common chromosome combinations being 45,X /46.XY and 45.x /46.XX. Thus, an embryo that began life with a normal karyotype can give rise to an individual whose cells show a mixture ofkaryotypes and who exhibits this syndrome. Turner syndrome is observed in about I in 2000 female births. a frequency much lower than that for Klinefelter syndrome. One explanation for this difference is the observation that a substantial majority of 45.x fetuses die in utero and are aborted spontaneously. Thus. a similar frequency of the two syndromes may occur at conception. 172 Chapter 7 (a) r 2 3 "i if; it i} if lt 6 ‘ll 12 )ittg ii ’35 H 13 16 17 ‘3 Kr] fer Sex chromosomes it t4 H t: 21 Sex Determination and Sex Chromosomes ‘ s. .1 :3 i; r: lerUlh ninfl 6 7 B 9 10 ll l2 13 i4 15 16 r? I is - - t l t e i t }X 19 20 21 22 Sex chromosomes FIGURE 7—7 The karyotypes and phenotypic depictions of individuals with (a) Kline-falter syndrome [47,XXY) and [b] Turner syndrome (45,)0. How Do We Know? What key information cancerning the sex chromosome composition of Klinefelter and Turner syndrome individuals proves that X chromosomes play no role in human sex de- termination, while showirrg that theY chromosome causes maleness and its absence causes femaleness in humans? 47,)(XX Syndrome The presence of three X chromosomes along with a normal set of autosomes (47,XXX) results in female differentiation. This syndrome, which is estimated to occur in about i of IZOD female births, is highly variable in expression. Frequently, 47,XXX women are perfectly normal. In other cases, underdeveloped secondary sex characteristics, sterility, and mental retardation may occur. In rare instances, 48,XXXX and 49,XXXXX kary- otypes have been reported. The syndromes associated with these karyotypes are similar to but more pronounced than the 47.XXX. Thus, in many cases. the presence of additional X chromosomes appears to disrupt the delicate balance of genetic information essential to normal female development. 47,XYY Condition Another human condition involving the sex chromosomes, 47,XYY, has also been intensively investigated. Studies of this condition. where the only deviation from diploidy is the pres- ence of an additional Y chromosome in an otherwise normal male karyotype, have led to an interesting controversy. In 1965, Patricia Jacobs discovered 9 of 3 l 5 males in a Scot- tish maximum security prison to have the 47,XYY karyotype. These males were significantly above average in height and had been incarcerated as a result ofantisocial (nonviolent) criminal acts. Of the nine males studied, Seven were of subnormal in- telligence. and all suffered personality disorders. Several other studies produced similar findings. The possible correlation be— tween this chromosome composition and criminal behavior piqued considerable interest and extensive investigations of the phenotype and frequency of the 47,XYY condition in both crim- inal and noncriminal populations ensued. Above-average height (usually over 6 feet} and subnormal intelligence have been gen- erally substantiated, and the frequency of males displaying this karyotype is indeed higher in penal and mental institutions com~ pared with unincarcerated males. (See Table 7.1.) A particu- lariy relevant question involves the characteristics displayed by XYY males who are not incarcerated. The only nearly constant association is that such individuals are over 6 feet tall! A study addressing this issue was initiated to identify 47.XYY individuals at birth and to follow their behavioral patterns during preadult and adult development. By [994. the two investigators. Stanley Walzer and Park Gerald, had identified about 20 XYY newborns in 15.000 births at Boston Hospital for Women. How- ever, they soon came under great pressure to abandon their re- search. Those opposed to the study argued that the investigation could not be justified and might cause great harm to those indi- viduals who displayed this karyotype. The opponents argued that (I) no association between the additionalY chromosome and ab- normal behavior had been previously established in the popula- tion at large. and (2) “labeling” these individuals in the study might create a self-fulfilling prophecy. That is. as a result of par- ticipation in the study, parents, relatives. and friends might treat individuals identified as 47,XYY differently ultimately produc- ing the expected antisocial behavior. Despite the support of a gov- ernment funding agency and the faculty at Harvard Medical School. Waiver and Gerald abandoned the investigation in 1995. Since Waller and Gerald’s work. it has become apparent that many XYY males are present in the population who do not cite hibit antisocial behavior and who lead normal lives. Therefore. 7.3 The Y Chromosome Determines Maleness in Humans Setting Restriction Newborns No height restriction Control population Mental—penal FREQUENCY or XYY INDIVIDUALS IN VARIOUS SETTINGS 173 Number Number Frequency Studied XYY XYY 28,366 29 0.10% 4,239 82 1.93 Penal No height restriction 5.805 26 0.44 Mental No height restriction 2,562 8 0.31 Mental—penal Height restriction 1,048 48 4.61 Penal Height restriction 1,683 31 1.84 Mental Height restriction 649 9 1.38 Source: Compiled from data presented in Hook, 1973, Tables 1—8. Copyright 1973 by the American Association for the Advancement of Science. we must conciude that there is no consistent correlation be.- twcen the extraY chromosome and the predisposition of males to behavioral problems. Sexual Differentiation in Humans Once researchers had established that. in humans. it is the Y chromosome that houses genetic information necessary for maleness. they made efforts to pinpoint a specific gene or genes capable of providing the “signal” responsible for sex determi- nation. Before we deive into this topic. it is useful to consider how sexual differentiation occurs in order to better compre— hend how humans deveiop into sexually dimorphic males and females. During early development, every human embryo un— dergoes a period when it is potentially hermaphroditic. By the fifth week ofgestation. gonadal primordia (the tissue that will form the gonad) arise as a pair of ridges associated with each embryonic kidney. Primordial germ cells migrate to these ridges. where an outer cortex and inner medulla form. The cortex is capable of developing into an ovary, while the inner medulla may develop into a testis. In addition. two sets of an differentiated male (Wolff—lair] and female [Mullerian) ducts exist in each embryo. If the cells of the genital ridge have the XY constitution. de- velopment of the medullary region into a testis is initiated around the seventh week. However. in the absence of the Y chromosome. no male development occurs. and the cortex of the genital ridge subsequently forms ovarian tissue. Parallel development of the appropriate male or female duct system then occurs, and the other duct system degenerates. A sub- stantial amount of evidence indicates that in males, once testes differentiation is initiated, the embryonic testicular tissue se- cretes two hormones that are essential for continued male sex- ual differentiation. In the absence of male development. as the 12th week of fetal development approaches. the oogonia within the ovaries begin meiosis and primary oocytes can be detected. By the 25th week of gestation. all oocytes become arrested in meiosis and remain dormant until puberty is reached some 10 to 15 years later. In males. on the other hand. primary spermatocytes are not prow duced until puberty is reached. The Y Chromosome and Male Development The human Y chromosome. unlike the X, has long been thought to be mostly blank ge-ncticaliy. It is now known that this is not true. even though the Y chromosome contains far fewer genes than does the X. Current analysis has revealed numerous genes and regions with potential genetic function. some with and some without homologous counterparts on the X chromosome. For example. present on both ends of theY chromosome are the so-called pseudoautosomal regions (PARS) that share ho- mology with regions on the X chromosome and which synapse and recombine with it during meiosis. The presence of such a pairing region is critical to segregation of the X and Y chro— mosomes during male game-togenesis. The remainder of the chromosome. about 95 percent of it. does not synapse or re- combine with the X chromosome. As a result, it was originally referred to as the nonrecrmtbining region qf'rhe YtNRY). More recently. researchers have designated this region as the male- specific region of the Y (MSY). As you will see. some portions of the MSY share homology with genes on the X chromosome. and some do not. The human Y chromosome is diagrammed in Figure 7—8. The MSY is divided about equally between eucliromaric regions containing functional genes and heremchmmarr‘c regions lack- in g genes. Within euchromatin. adjacent to the PAR of the short arm of the Y chromosome, is a critical gene that controls male sexual development. called the. sex-determining region 1" (SRY). In humans. the absence of a Y chromosome almost al- ways leads to female development, thus this gene is absent from the X chromosome. Ski/encodes a gene product that somehow triggers the undifferentiated gonadal tissue of the embryo to form testes. This product is called the testis-determining fac- tor (TDF). SRY (or a closely related version) is present in all mammals thus far examined, which is indicative of its essential function throughout this diverse group of animals. Our ability to identify the presence or absence of DNA se- quences in rare individuals whose expected sex chromosome composition does not correspond to their sexual phenotype has provided evidence that SR Y is the gene responsible for male sex determination. For example, there are human males who have two X and no Y chromosomes. Often, attached to one of their X 174 Chapter 7 Euchrornatin ’v Centrornere —- Euchromatin 7-" MSY Heterochromatin PAR —. Human Y Chromosome FIGURE 7—8 The various regions of the human Y chromosome. chromosomes is the region of the Y that contains SRY. There are also females who have. one X and one Y chromosome. TheirY is almost always missing the SRY gene. TheSe observations argue strongly in favor of the role of SR? in providing the primary sig- nal for male development. Further support of this conclusion involves an experiment using transgenic mice. These animals are produced from fertilized eggs injected with foreign DNA that is subse quently incorporated into the genetic composition of the de— veloping embryo. In normal mice, a chromosome region designated Sr); has been identified that is comparable to SRY in humans. When mouse DNA containing Sry is injected into normal XX mouse eggs, most of the offspring develop into males. The question of how the product of this gene triggers the embryonic gonadal tissue to develop into testes rather than ovaries is under intensive investigation. In humans, other au— tosornai genes are believed to be part of a cascade of genetic expression initiated by SRY. Examples include the SOXQ gene and WT] (on chromosome 11), originally identified as an oncogene associated with Wilms tumor, which affects the kidney and gonads. Another, SF], is involved in the regula— tion of enzymes affecting steroid metabolism. In mice, this gene is initially active in both the male and female bisexual genital ridge. persisting until the point in development when testis formation is apparent. At that time, its expression per- sists in males, but is extinguished in females. The link be tween these various genes and sex determination brings us c105er to a complete understanding of how males and females arise in humans. Sex Determination and Sex Chromosomes Key: PAR: Pseudoautosomal region SRY: Sex-determining region of the Y MSY: Male-specific region of the Y Some recent. findings by David Page and his many colleagues have now provided a reasonably complete picture of the MSY region of the human Y chromOsome. This work, completed in 2003, is based on information gained through the Human Genome Project, where the DNA of all chromosomes has now been sequenced. Page has spearheaded the detailed study of theY chromosome for the past several decades. The MSY consists of about 23 million base pairs ('23 Mb) and can be divided into three regions. The first region is the X—transpused region. It contains about 15 percent of the MSY and was originally derived from the X chromosome during human evolution (about 3 to 4 million years ago). The X—transposed region is 99 percent identical to region Xq21 of the modern human X chromosome. Two genes, both with X-chromOsome homologs. are present in this region. The second area is designated the X—degenemtiue region. Containing about 20 percent of the MSY. this region contains DNA sequences that are even more distantly related to those present on the X chromosome. The X-degenerative region contains 27 single-copy genes, including numerous pseudagertes, whose sequences have degenerated sufficiently during evolution to render them nonfunctional. As with the genes present in the X- transposed region, all share some homology with counterparts on the X chromosome. These 2’? genetic units include [4 that are capable of being transcribed, and each is present as a sin- gle copy. One of these is the SRY gene, discussed above. Other X-degenerative genes that encode protein products are ere pressed ubiquitously in all tissues in the body, but SRY is ex- pressed only in the testes. The third area, the ampliconic region, contains about 30 percent of the MSY, including most of the genes closely as- sociated with testes development. These genes lack counter— parts on the X chromosome and their expression is limited to the testes. There are 60 transcription units divided among 9 gene families in this region, most represented by multiple. copies. Members of each family have nearly identical {>98 percent) DNA sequences. Each repeat unit is an amplicon and is contained within seven segments scattered across the euchromatic regions present on both the short and long arms of the Y chromosome. Genes in the ampliconic region ene code proteins specific to the development and function of the testes, and the products of many of these genes are di- rectly related to fertility in males. It is currently believed that a great deal of male sterility in our population can be linked to mutations in these genes. This recent work has provided a comprehensive picture of the genetic information present on this unique chromosome. This information clearly refutes the so-called “wasteland” the- ory, prevalent only 20 years ago, that depicted the human Y chromosome as almost devoid of genetic information other than a gene or two that caused maleness. The knowledge we have gained provides the basis for a much clearer picture of how maleness is determined. Additionally, this information provides important clues as to the origin of the Y chromosome during human evolution. 7.5 Dosage Compensation Prevents Excessive Expression of X-Linked Genes in Humans and Other Mammals 175 I. ‘ Problem 7.28 on page 185 concerns itself with sox9, a gene on an autosome, that when mutated appears to in- hibit normal human male development. Hint: Some genes are activated and produce their nor- mal product as a result of expression of products of other genes found on different chromosomes—4n this case, perhaps one that is on the Y chromosome. The Ratio of thales to Females in Humans Is Not 1.0 The presence of heteromorphic sex chromosomes in one sex of a species but not the other provides a potential mechanism for producing equal proportions of male and female offspring. This potential is premised on the segregation of the X and Y (or Z and W) chromosomes during meiosis, such that half of the gametes of the heterogametic sex receive one of the chromosomes and half receive the other one. As we learned in the previous section, in humans. small psettdoautosomal regions of pairing homology do exist at both ends of the X and theY chromosomes. Provided that both types of gametes are equally successful in fertilization and that the two sexes are equally viable during development. a one—to-one ratio of male and female offspring results. Given the potential for the production of equal numbers of both sexes. the actual proportion of male to female olfsprin g has been investigated and is referred to as the sex ratio. We can assess it in two ways. The primary sex ratio reflects the pro- portion of males to females conceived in a population. The secondary sex ratio reflects the proportion of each sex that is born. The secondary sex ratio is much easier to determine, btlt has the disadvantage of not accounting for any disproportion- ate embryonic or fetal mortality. When the secondary sex ratio in the human population was determined in 1969 by using worldwide census data. it was found not to equal 1.0. For example, in the Caucasian popula- tion in the United States, the secondary ratio was a little less than 1.06, indicating that about 106 males were born for each 100 females. (In 1995, this ratio dropped to slightly less than i .05.) in the AfricanuAmetican population in the United States, the ratio was 1.025. In other countries the excess of male births is even greater than reflected in these values. For example, in Korea, the secondary sex ratio was 1 .15. Despite these ratios, it is possible that the primotfir sex ratio is 1.0. and that it is altered between conception and birth. For the secondary ratio to exceed 1.0, then, prenatal female moo tality would have to be greater than prenatal male mortality. However, this hypothesis has been examined and shown to be false. In fact. just the opposite occurs. In at Carnegie Institute study. reported in 1948. the sex of approximately 6000 cm— bryos and fetuses recovered from miscarriages and abortions was determined. and fetal mortality was actually higher in males. On the basis of the data derived from that study. the pri— mary sex ratio in U.S. Caucasians was estimated to be 1.079. It is now believed that this figure is much higher—between 1.20 and 1.60, suggesting that many more males than females are conceived in the human population. It is not clear why such a radical departure from the expected primary sex ratio of | .0 occurs. To come up with a suitable ex- planation, we must examine the assumptions upon which the theoretical ratio is based: 1. Because of segregation. males produce equal numbers of X- and Y—bearing sperm. 2. Each type of sperm has equivalent viability and motility in the female reproductive tract. 3. The egg surface is equally receptive to both X- and Y- bearing sperm. While no direct experimental evidence contradicts any of these assumptions. the htnnan Y chromosome is smaller than the X chromosome and therefore of less mass. Thus, it has been speculated that Y-bearing sperm are more motile than waearing sperm. If this is true. then the probability of a fertilization event leading to a male zygote is increased. providing one possible explanation for the observed primary ratio. How Do WE Know? What key experimental observations demonstrate that the primary sex ratio in humans strongly favors males at conception? Dosage Compensation Prevents Excessive Expression of X-Linked Genes in Humans and Other Mammals The presence of two X chromosomes in normal human fe— males and only one X in normal human males is unique com- pared with the equal numbers of autosomes present in the cells of both sexes. On theoretical grounds alone, it is possi- ble to speculate that this disparity should create a “genetic dosage“ problem between males and females for all X-linked genes. Recall that in Chapter 4 we discussed the topic of X— Iinkage, the inheritance of traits under the control of genes located on one of the sex chromosomes. There, we saw that during meiosis. sex chromosomes, like autosomes, are subject to the laws of segregation and independent assortment during their distribution into gametes. Because females have two copies of the X chromosome and males only one. there is the potential for females to produce twice as much of each gene product for all aninde genes. The additional X chromo- somes in both males and females exhibiting the various syn— dromes discussed earlier in this chapter should compound this dosage problem even more. In this section, we will de- scribe certain research findings regarding X-linked gene ex» pression that demonstrate a genetic mechanism allowing for dosage compensation. 176 Chapter 7 Barr Bodies Murray L. Barr and Ewart G. Bertram’s experiments with fe- male cats. as well as Keith Moore and Barr’s subsequent study with humans, demonstrate a genetic mechanism in mammals that compensates for X chromosome dosage disparities. Barr and Bertram observed a darkly staining body in interphase nerve cells of female cats that was absent in similar cells of males. in humans, this body can be easily demonstrated in fe- male cells derived from the buccal mucosa (cheek cells) or in fibroblasts (undifferentiated connective tissue cells), but not in similar male cells (Figure 7—9). This highly condensed struc— ture, about 1 am in diameter, lies against the nuclear envelope of interphase cells. It stains positively in the Feulgen reaction, a cytochemjcal test for DNA. Current experimental evidence demonstrates that this body. called asex chromatin body or simply a Barr body1 is an inac» tivated X chromosome. Susumo Ohno was the first to suggest that the Barr body arises from one of the two X chromosomes. This hypothesis is attractive because it provides a mechanism for dosage compensation. If one of the two X chromosomes is inac- tive in the cells of females, the dosage of genetic information that can be expressed in males and females is equivalent. Convincing but indirect evidence for this hypothesis comes from the study of the sex chromosome syndromes described earlier in this chapter. Regardless of how many X chromosomes exist. all but one of FIGURE 7—9 Photomicrographs comparing cheek epithelial cell nuclei from a male that fails to reveal Barr bodies (bottom) with a female that demonstrates Barr bodies (indicated by an arrow in the top image). This structure, also called a sex chromatin body, represents an inactivated X chromosome. Sex Determination and Sex Chromosomes them appear to be inactivated and can be seen as Barr bodies. For example. no Barr body is seen in Turner 45,X females; one is seen in Klinefelter 47,XXY males: two in 47.XXX females; three in 48,XXXX females; and so on (Figure 7—10). Therefore, the number of Barr bodies follows an N i 1 rule where N is the total number of X chromosomes present. Although this mechanism of inactivation of all but one X chromosome increases our understanding of dosage compen- sation, it further complicates our perception of other matters. Because one of the two X chromosomes is inactivated in nor- mal human females. why then is the Turner 45.x individual not entirely normal? Why aren’t females with the triple-X and tetra-X karyotypes (47,XXX and 48,)(XXX) completely un- affected by the additional X chromosome? Further, in Kline- felter syndrome (47,XXY), X chromosome inactivation effectively renders such individuals 46.XY. So why aren’t these males unaffected by the extra X Chromosome in their nuclei? One possible explanation is that chromoesome inactivation does not normally occur in the very early stages of develop- ment of those cells destined to form gonadal tissues. Another possible explanation is that not all of each X chromosome fortn- ing a Barr body is inactivated. If either hypothesis is correct. overexpression of certain X-linked genes might occur at criti— cal times during development despite apparent inactivation of additional X chromosomes. The Lyon Hypothesis In mammalian females, one X chromosome is of maternal origin, and the other is of paternal origin. Which one is in- activated? Is the inactivation random? is the same chromo- some inactive in all somatic cells? In 1961. Mary Lyon and Liane Russell independently proposed a hypothesis that an» swers these questions. They postulated that the inactivation of X chromosomes occurs randomly in somatic cells at a point early in embryonic development. Further, once inacti- Nucleus ,C to lasm // y P Barr body 7 46, x v 45, x 46, [XX (“l—1:0) 47m Y(iv—i=1) 47am _ _ 48,l3fiitlix _ 4anxviN “2) 49,?![1EXYW—I'3) FIGURE 740 Barr body occurrence in various human karyotypes, where all X chromosomes except one (N — 1) are inactivated. 7.5 Dosage Compensation Prevents Excessive Expression of X-Linked Genes in Humans and Other Mammals 177 vation has occurred. all progeny cells have the same X chro- mosome inactivated. This explanation. which has come to be called the Lyon hy- pothesis. was initially based on observations of female mice heterozygous for X-linked coat color genes. The pigmentation of these heterozygous females was mottled. with large patches expressing the color allele on one X and other patches CXA pressing the allele on the other X. Indeed. such a phenotypic pattern would result if different X chromosomes were inactive in adjacent patches of cells. Similar mosaic patterns occur in the black and yellow-orange patches of female tortoiseshell and calico cats (Figure 7—11}. Such X-linked coat color patterns do not occur in male cats because all their cells contain the single maternal X chromosome and are therefore hemizygous for only one X-linked coat color allele. The most direct evidence in support of the Lyon hypothesis comes from studies of gene expression in clones of human li- broblast cells. Individual cells may be isolated following biopsy and cultured in suite. If each culture is derived from a single cell. it is referred to as a clone. The synthesis of the enzyme glucose- 6-phosphate dehydrogenase (GfiPD) is controlled by an X-linked gene. Numerous mutant alleles of this gene have been detected. and their gene products can be differentiated from the wild-type en, zyme by their migration pattem in an electrophoretic lield. Fibroblasts have been taken from females heterozygous for different allelic forms of GtSPD and studied. The Lyon hy— pothesis predicts that if inactivation of an X chromosome oce curs randomly early in development and is permanent in all progeny cells. such a female should show two types of clones. each showing only one electrophoretic form of GoPD. in ap— proximately equal proportions. In [963. Ronald Davidson and colleagues performed an ex- periment involving 1% clones from a single heterozygous female. Seven showed only one form of the enzyme, and 7 showed only the other form. What was most important was that none of the [4 showed both forms of the enzyme. Studies of (36PD mutants thus provide strong support for the random permanent inactivate tion of either the maternal or paternal X chromosome. How Do WE Know? What experimental evidence supports our belief that X chromosomal inactivation of either the paternal or maternal member is random in mammalian females? The Lyon hypothesis is generally accepted as valid: in fact. the inactivation of an X chromosome into a Barr body is sometimes referred to as lyonization. One extension of the hypothesis is that mammalian females are mosaics for all heterozygous X-linked alleles—some areas of the hotly exu press only the maternally derived alleles. and others express only the paternally derived alleles. Two especially interest- ing examples involve red-green color-blindness and anhidrotic ectodermal dysplasia. both X-Iinked recessive disorders. In the former case, hemizygous males are fully color-blind in all retinal cells. However. heterozygous fe- males display mosaic retinas with patches ol’defective color perception and surrounding areas with normal color per- ception. Males hemizygous for anhiclrotic ectodermal dys- plasia show absence of teeth. sparse hair growth. and lack of sweat glands. The skin of females heterozygous for this dis— order reveals random patterns of tissue with and without sweat glands (Figure 7—12). In both examples. random in— activation of one or the other X chromosome early in the development of heterozygous females has led to these oc— currenees. Mgr/5W ‘ Problem 7.32 on page 186 is concerned with Carbon Copy, the first cloned cat, who was derived from a so- matic nucleus of a calico cat. Hint: The donor nucleus was from a differentiated ovar- ian cell of an adult female cat, which itself had inacti- vated one of its X chromosomes. FIGURE 7—11 (a) A calico cat, where the random distribution of orange and black patches illustrates the Lyon hypothesis. The white patches are due to another gene; (b) A tortoiseshell cat, which lacks the white patches characterizing calicos. 178 Chapter 7 FIGURE 7—12 Depiction of the absence of sweat glands (shaded regions) in a female heterozygous for the X-linked condition anhidrotic ectodermal dysplasia. The locations vary from female to female, based on the random pattern of X chromosome inactivation during early development, resulting in unique mosaic distributions of sweat glands in heterozygotes. The Mechanism of Inactivation The least understood aspect of the Lyon hypothesis is the. mech— anism of chromosome inactivation in mammals. How are al~ most all genes of an entire chromosome inactivated? Recent investigations are beginning to clarify this issue. A single region of the mammalian X chromosome, called the X-inactivation center (Xic)*, is the major control unit. Genetic expression of this region, located on the proximal end of the p arm, occurs only on the X chromosome that is inactivated. The constant as sociation of expression of Xic and X chromosome inactivation supports the conclusion that this region is an important genetic component in the inactivation process. The Xic is about 1 Mb {106 base pairs) in length and is known to contain several putative regulatory units and four genes. One of these, X-inactive specific transcript (Xist)*, is now be- lieved to represent the critical locus within the X to. Several in- terestng observations have been made regarding the RNA that is transcribed from it, with much of the underlying work hav- ing been done by using the mouse Xisr gene. First, the RNA product is quite large and lacks what is called an extended open reading frame (ORE). An ORF includes the information nec- essary for translation of the RNA product into a protein. Thus, the RNA is not translated, but instead serves a structural role in the nucleus, presumably in the mechanism of chromosome On the human X chromosome, the inactivation center and the key gene are designated as XIC and XIST, respectively. Sex Determination and Sex Chromosomes inactivation. This finding has led to the belief that the RNA products of Xisr spread over and coat the X chromosome hear- ing the gene that produced it, creating some sort of molecular “cage” that entraps it, leading to its inactivation. Inactivation is therefore said to be ctr-acting. Second. transcription of Xisr occurs initially at low levels on all X chromosomes. As the inactivation process begins, how— ever, transcription continues and is enhanced only on the X c'hromosonre(s) that becomes inactivated. In 1996, a research group led by Graeme Penny provided con- vincing evidence that transcription of Xist is the critical event in chromosome inactivation. These researchers were able to intro- duce a targeted deletion [7 kb) into this gene that destroyed its ac- tivity. As a result, the chromosome bearing the mutation lost its ability to become inactivated. Several interesting questions re: main unanswered. First, in cells with more than two chromo— somes, what sort of “counting” mechanism exists that designates all but one X chromosome to be inactivated? Second, what "blocks" the Xic of the active chromosome, preventing tran- scription of Xisr? Third, how is inactivation of the same X chro- mosome or chromosomes maintained in progeny cells, as the Lyon hypothesis calls for? The inactivation signal must be stable as cells proceed through mitosis. Whatever the answers to these questions, we have taken an exciting step toward understanding how dosage compensation is accomplished in manunals. The Ratio of X Chromosomes to Sets of Autosomes Determines Sex in Drosophila Because males and females in Dmsophiln melrmognstcr {and other Drosophila species) have the same general sex chromo- some composition as humans (males are KY and females are XX), we might assume that the Y chromosome also causes maleness in these flies. However, the elegant work of Calvin Bridges in l9 [6 showed this not to be true. He studied flies with quite varied chromosome compositions, leading him to the conclusion that theY chromosome is not involved in son de— termination in this organism. Instead, Bridges proposed that both the X chromosomes and autosornes together play a criti- cal roIe. in sex determination. Recall that in the nematode C. et- egans, which lacks aY chromosome. the sex chromosomes and autosomes are also critical to sex determination. Bridges’ work can be divided into two phases: ( l ) A study of offspring resulting from nondisjunction of the X chromosomes during meiosis in females and (2') subsequent work with proge eny of females containing three copies of each chromosome. called triploid (371) females. As we have seen previously in this chapter and earlier (see Figure 2—17), nondisjuncti on is the fail- ure of paired chromosomes to segregate or separate during the anaphase stage of the first or second meiotic divisions. The re- sult is the production of two types of abnormal gametes, one of which contains an extra chromosome (n + 1) and the other of which lacks a chromosome (11 — 1). Fertilization of such gametes with a haploid gamete produces (2n + l ) or (2n 7 l) zygotes. As in humans, it nondisjunction involves the X chromosome, in addition to the normal complement of 7.6 The Ratio of X Chromosomes to Sets of Autosomes Determines Sex in Drosophila 179 autosomes. both an XXY and an X0 sex chromosome compo- sition may result. (The “O” signifies that there is neither a sec- ond X nor a Y chromosome present.) Contrary to what was later discovered in humans, Bridges found that the XXY flies were normal females. and the X0 flies were sterile males. The presence of the Y chromosome in the XXY i‘lies did not cause maleness. and its absence in the X0 flies did not produce fe- maleness. From these data. he concluded that the Y chromou some in Drosophila lacks male-determining factors, but since the X0 males were sterile. it does contain genetic information essential to male fertility. Bridges was able to clarify the mode of sex determination in Dmsopliila by studying the progeny of triploid females (3n). which have three copies each of the haploid complement of Chromosomes. Drosophilrt has a haploid number of 4. thereby displaying three pairs of autosomes in addition to its pair of sex chromosomes. Triploid females apparently originate from rare diploid eggs fertilized by normal haploid sperm. Triploid females have heavy-set bodies. coarse bristles, and coarse eyes. and they may be fertile. Because of the odd number of each chromosome (3). during meiosis, a wide range of chromosome complements is distributed into gametes that give rise to off- FIGURE 7—13 Chromosome compositions, the '95" *3 ratios of X chromosomes to sets of autosomes. ' and the resultant sexual morphology in Drosophila melanogaster. The normal diploid male chromosome composition is shown as a reference on the left (XY/ZA}. Normal diploid male Chromosome compositlon spring with a variety of abnormal chromosome constitutions. A correlation among the sexual morphology, chromosome com- position, and Bridges” interpretation is shown in Figure 7—13. Bridges realized that the critical factor in determining sex is the ratio of X chromosomes to the number of haploid sets of au- tosomes (A) present. Normal (2X:2A] and triploid (3X:3A) fe- males each have a ratio equal to l .0, and both are fertile. As the ratio exceeds unity (3X:2A, or 1.5. for example). what was orig- inally called a superfemale is produced. Because this type of fe- male is rather weak. infertile. and has lowered viability. it is now more appropriately called a metafemale. Normal (XY:2A) and sterile (X0:2A) males each have a ratio of l :2. or 0.5. When the ratio decreases to l:3. or 0.33. as in the case of an XYz3A male. infertile metamalcs result. Other llies recovered by Bridges in these studies contained an X:A ratio intermediate between 0.5 and 1.0. These flies were generally larger. and they exhibited a variety of morphological abnor- malities and rudimentary bisexual gonads and genitalia. They were invariably sterile and expressed both male and female morphology. thus being designated as intersexes. Bridges” results indicate that in Diosophilu. factors that cause a fly to develop into a male are not localized on the sex 7. list _ _ chromosomes to autosome sets Sexual morphology Chromosome formulation Metafemale Intersex 2 sets of autosomes + X Y lntersex Metamale 180 Chapter 7 chromosomes, but are instead found on the autosomes. Some female-determining factors. however. are localized on the X chromosomes. Thus, with respect to primary sex determina— tion, male gametes containing one of each autosome plus a Y chromosome result in male offspring. not because of the pres- ence of the Y, btit because of the lack of a second X chromo— some. This mode of sex determination is explained by the genie balance theory. Bridges proposed that a threshold for maleness is reached when the X:A ratio is 1:2 (leA), but that the pres- encc of an additional X (,XX22A) alters the balance and results in female differentiation. Numerous mutant genes have been identified that are involved in sex determination in Drosophila. The recessive autosomal gene Imnsfomter (Ira). discovered over 50 years ago by Alfred H. Sturtevant, clearly demonstrated that a single autosomal gene could have a profound impact on sex determination. Fe- males homozygous l'or rm are transl'orn'icd into sterile males, but homozygous males are unaffected. More recently, another gene, Sex-lethal (SM), has been shown to play a critical role. serving as a “master switch” in sex de— termination. Activation of the X-linked le gene, which relies on a ratio of X chromosomes to sets of autosomes that equals 1.0, is essential to female development. In the absence of acti— vation, resulting, for example, from an X:A ratio of 0.5. male development occurs. It is interesting to note that mutations that inactivate the Sr! gene, as originally studied in 1960 by Her- mann J. Muller. kill female embryos. but have no effect on male embryos, consistent with the role of the gene. as described. While it is not yet exactly clear how this ratio influences the 5x] locus. we do have some insights into the question. The Ex] locus is part ofa hierarchy of gene expression and exerts con— trol over still other genes. including rra (discussed in the pre- vious paragraph) and (st (doublesar), as well as others. Only in females is the wi|d~type allele of rm activated by the prod— uct ofo/ which, in turn. influences the expression of dsx. De— pending on how the initial RNA transcript of dsx is processed (spliced), the resultant dsx protein activates either male— or female—specific genes required for sexual differentiation. Each step in this regulatory cascade requires a form of processing called RNA splicing, in which portions of the RNA are re— moved and the remaining fragments “spliced” back together prior to translation into a protein. In the case of the Sr! gene. its transcript may be spliced in several different ways, a phe- nomenon called alternative splicing. Two different RNA tran- scripts are produced in females and males. ln potential females, the transcript is active and initiates a cascade of regulatory gene expression. ultimately leading to female differentiation. In po- tential males, the transcript is inactive. leading to a different pattern of gene activity, whereby male differentiation occurs. We will return to this topic in Chapter 17 when alternative splic— ing is again addressed as one of the mechanisms involved in the regulation ofgenetic expression in eukaryotes. Dosage Compensation in Drosophila Since Drosophila females contain two copies of X-linked genes whereas males contain only one copy. a dosage problem ex— ists. as it does in mammals such as humans and mice. However, Sex Determination and Sex Chromosomes the mechanism of dosage compensation in Drosophila differs considerably from that in mammals, since X chromosome in- activation is not observed. Instead, male X-Iinked genes are transcribed at twice the level of the comparable genes in fe» males. Interestingly, if groups of X-linked genes are moved (translocated) to autosomes. dosage compensation also affects them even when they are no longer part of the X chromosome. As in manmtals. considerable gains have been made recently in understanding the process of dosage compensation in Drosophila. At least four autosomal genes are known to be in— volved, under the same master—switch gene. le, that induces fe- male differentiation during sex determination. Mutations in any of these genes severely reduce the increased expression of X- linked genes in males. causing lethality. Evidence supporting a mechanism of increased genetic ac— tivity in males is now available. The well-accepted model pro- poses that one of the autosomal genes. mlc (mu/class), encodes a protein that binds to numerous sites along the X chromo- some, causing enhancement of genetic expression. The prod- ucts ofthe other three autosomal genes also participate in and are required for mle binding. This model predicts that the master-switch le gene plays an important role during dosage compensation. in XY flies, Sr] is inactive; therefore, the autosomal genes are activated. causing enhanced X chromosome activity. On the other hand, Sr] is ac— tive in XX females and functions to inactivate one or more of the 1nale~specific autosomal genes. perhaps mle. By dampen— ing the activity of these autosomal genes. it ensures that they will not serve to double gene expression of X-linked genes in females. which would further compound the dosage problem. Tom Cline has proposed that, before the aforementioned dosage compensation mechanism is activated. 5x] acts as a sen- sor for the expression of several other X-linked genes. In a sense, Sr] counts X chromosomes. When it registers the close of their expression to be high, for example. as the result of two X chro— mosomes. the St! gene product is modified. and it dampens the expression of the autosomal genes. Although this model may yet be modified or refined, it is useful in guiding future research. Clearly, an entirely different mechanism of dosage compen- sation exists in Drosophila (and probably many related organ— isms) than that in mammals. The development of elaborate mechanisms to equalize the expression of X-linked genes demonstrates the critical nature of gene expression. A delicate balance of gene products is necessary to maintain normal de- velopment of both males and females. Drosophila Mosaics Our knowledge of sex determination and X~linkage in Drosophila (Chapter 4) helps us to understand the unusual ap- pearance of a unique fruit fly. shown in Figure 7—14. This fly was recovered from a stock where all other females were het- erozygous for the X-linked genes white eye (\t’) and miniature wing (in). It is a bilateral gynandromorph. which means that one—half of its body (the left half) has developed as a male and the other half (the right half) as a female. We can account for the presence of both sexes in a single fly in the following way. lf a female zygote (heterozygous for tit/rite 7.7 Temperature Variation Controis Sex Determination in Reptiles FIGURE 7—14 A bilateral gynandromorph of Drosophila meianogaster formed following the loss of one X chromosome in one of the two celis during the first mitotic division. The left side of the fly, composed of male cells containing a single X, expresses the mutant white-eye and miniature-wing alleles. The right side is composed of female cells containing two X chromosomes heterozygous for the two recessive alleles. eye and miniature wing) were to lose one of its X chromosomes during the first mitotic division. the two cells would be of the XX and X0 constitution. respectively. Thus. one cell would be female and the other would be male. Each of these cells is re- sponsible for producing all progeny cells that make up either the right half or the left half of the body during embryogenesis. In the case of the bilateral gynandromorph. the original cell of X0 constitution apparently produced only identical prog- eny cells and gave rise to the left half of the fly, which. be- cause of its chromosomal constitution, was male. Since the male half demonstrated the white. miniature phenotype. the X chromosome bearing the It”. at; alleles was lost, while the w, m-bearing homolog was retained. All cells on the right side of the body were derived from the original XX cell. lead— ing to female development. These cells. which remained Case I 181 heterozygous for both mutant genes, expressed the wild—type eye—wing phenotypes. Depending on the orientation of the spindle during the first mitotic division. gynandromorphs can be produced where the "line" demarcating male versus female development occurs along or across any axis of the fly’s body. Temperature Variation Controls Sex Determination in Reptiles We conclude this chapter by discussing several cases involving reptiles where the environment. specifically temperature. has a profound influence on sex determination. In contrast to chromosomal or genotypic sex determination (CSD or GSD). which describes all examples thus far presented, the cases that We will now discuss are categorized as temperature- dependent sex determination (TSD). As we shall see. the in- vestigations leading to this information may well come cIOser to revealing the true nature of the primary basis of sex deter— mination titan any finding previousiy discussed. in many species of reptiles, sex is predetermined at concep- tion by sex chromosome composition, as is the case in many or- ganisms already considered in this chapter. For example. in many snakes, including vipers. a ZZ/ZW mode is in effect. where the female is the heterogamous sex. However. in boas and pythons. it is impossible to distinguish one sex chromo- some from the other in either sex. in lizards. both the XX/XY and ZZ/ZW systems are found. depending on the species. In other reptilian species. including all crocodiles. most turtles. and some lizards. sex determination is achieved according to the incubation temperature of eggs during a critical period of em bryonic development. Since 1980. it has become clear that TSD is quite widespread among reptiles. Interestingly. there are three distinct patterns of TSD. as il- lustrated in Figure 7715. In the first two. low temperatures yield 100 percent females, while high temperatures yield 100 per— cent males (Case I}. orjust the opposite occurs (Case II}. In the third pattern [Case III). low and high temperatures yield 100 percent females. while intermediate temperatures yield 100; Percent femaie on c:- l Percent female FT rp MT FT Case ll Case Ill 100‘ 2 M E .2 .4 50 C 8 b 0. fl 0 i l , J Tp MT FT Tp MT Tp FT Temperature —-— Temperature —- Temperature —- FIGURE 7—15 Three different patterns of temperature-dependent sex determination (TSD) in reptiles, as described in the text. The relative pivotal temperature Tp is crucial to sex determination during a critical point during embryonic development (FT : femaleudetermining temperature; MT : male»determining temperature). 182 Chapter 7 various proportions of males. The third pattern is seen in vari- ous species of crocodiles, turtles, and lizards, although other members of these groups are known to exhibit the other pat— terns. Two observations are noteworthy. First, under certain temperatures in all three patterns, both male and female off— spring result. Secondly. the pivotal temperature (P) is fairly narrow, usually iess than SEC. and sometimes only 1°C. The central question raised by these observations is what metabolic or physiological parameters that lead to the differ- entiation of one sex or the other are being affected by temper ature? The answer is thought to involve steroids (mainly estrogens] and the enzymes involved in their synthesis. Stud- ies have clearly demonstrated that the effects of temperature on estrogens. androgens. and inhibitors of the enzymes con trolling their synthesis are involved in the sexual differentia— tion of ovaries and testes. One enzyme in particular. aromatase, converts androgens (male hormones such as testosterone) to estrogens (female hormones such as estradiol). The activity of this enzyme is correlated with the pathway followed during CHAPTER SUMMARY Sex Determination and Sex Chromosomes gonadal differentiation activity and is high in developing ovaries and low in developing testes. Researchers in this field, includ— ing Claude Pieau and colleagues, have proposed that a ther- mosensitive factor mediates the transcription of the reptilian aromatase gene which leads to temperature-dependent sex de- termination. Several other genes are likely to be involved in this mediation. The involvement of sex steroids in gonadal differentiation has also been documented in birds. fishes. and amphibians. Thus, sex-determining mechanisms involving estrogens seem to be characteristic of nonmammalian vertebrates. The reg- ulation of such a system, while temperature dependent in many reptiles, appears to be controlled by sex chromosomes [XXfXY or ZZ/ZW) in many of these other organisms. A final intriguing thought on this matter is that the product of SRY, a key component in mammalian sex determination, has been shown to bind in vitro to a regulatory portion of the aro- matase gene. whereby it could act as a repressor of ovarian development. i. In sexually reproducing organisms. meiosis. which both creates genetic variability and ensures genetic constancy, depends on fertilization. Fertilization ultimately relies on some form of sex- ual differentiation. which is achieved by a variety of sex— determining mechanisms. 2. The genetic basis of sexual differentiation is usuaily related to different chromosome compositions in the two sexes. The heterogzunetic sex either lacks one chromosome or contains a unique heteromorphic chromosome. usually referred to as the Y chromosome. 3. In humans, the study of individuals with altered sex chromosome compositions has established that theY chromosome is responsible for male differentiation. The absence of theY leads to female dif— ferentiation. Similar studies in Dmsophiln have excluded the Y in such a role. instead demonstrating that a balance between the num- ber of X chromosomes and sets of autosomes is the critical factor. 4. The primary sex ratio in humans substantially favors males at conception. During embryonic and fetal deveiopment, male mor- tality is higher than that of females. The secondary sex ratio at birth still favors males by a small margin. 5. Dosage compensation mechanisms limit the expression of X- linked genes in females, who have two X chromosomes, as compared with males. who have only one X. In mammals. compensation is achieved by the inactivation of either the ma- ternal or paternal X early in development. This process results in the formation of Barr bodies in female somatic cells. In Di‘ornphiim compensation is achieved by stimulation of genes located on the single X chromosome to double their genetic expression. 6. The Lyon hypothesis states that. early in development, inactiva- tion is random between the maternal and paternal X chromor somes. All subsequent progeny cells inactivate the same X as their progenitor cell. Mammalian females thus develop as ge— netic mosaics with respect to their expression of heterozygous X-iinked alleles. 7. in many reptiies. the incubation temperature at a critical time during cmbryogenesis is often responsible for sex determination. Temperature influences the activity of enzymes involved in the metabolism of steroids related to sexual differentiation. Genetics, Technology, and Society 183 GENETICS, TECHNOLOGY, AND SOCIETY A Question of Gender: Sex Selection in Humans L The desire to choose a baby’s gender is as pervasive as human nature itself. Throughout time, people have resorted to varied and sometimes bizarre methods to achieve the preferred gender of their offspring. In me- dieval Europe, prospective parents would place a hammer under the bed to help them conceive a boy, or a pair of scissors to con ceive a girl. Equally effective were practices based on the ancient belief that semen from the right testicle created male offspring and that from the left testicle created females. Men in ancient Greece would lie on their right side during intercourse in order to conceive a boy. Up until the 18th century, European men would tie off (or remove) their left testicle to increase the chances of getting a male heir. In some cultures, efforts to control the sex of offspring has a darker side—female in— fantlcide. In ancient Greece, the murder of female infants was so common that the male:female ratio in some areas approached 4: 1. Some societies, even to present times, continue to practice female infanticide. In some parts of rural India, hundreds of fam- ilies are reported to admitting to this prac- tice, even as late as the 19905. In t997, the World Health Organization reported popu- lation data showing that about 50 million women were "missing" in China, likely due to institutionalized neglect of female chil- dren, The practice of female infanticide arises from poverty and age-old traditions. in these cultures, sons work and provide in» come and security, whereas daughters not only contribute no income but require large dowries when they marry. Under these con- ditions, it is easy to see why females are heid in low esteem. In recent times, sex—specific abortion has replaced much of the traditional female in- fanticide. Amniocentesis and ultrasound techniques have become lucrative busi- nesses that provide prenatal sex determi— nation. Studies in India estimate that hundreds of thousands of fetuses are aborted each year because they are female. As a result of sex—selective abortion, the female: male ratio in India was 927: 1000 in 1991. in some Northern states, the ratio is as low as 600: 1000. Although sex de- termination and selective abortion of fe- male fetuses was outlawed in India and China in the mid—1990s, the practice is thought to continue. in Western industrial countries, advances in genetics and reproductive technology offer parents ways to select their children ‘5 gender prior to implantation. Following in vitro fer- tilization. embryos can be biopsied and as sessed for gender. Only sex«selected embryos are then implanted. The simplest and least in- vasive method for sex selection is preconcep- tion gender selection (PGS), which involves separating ><— and Y—Chi’Ofl’lOSDme bearing spermatozoa. The only effective PGS method devised so far involves sorting the sperm based on their DNA content. Due to the dif- ferent size of the X and Y chromosomes, X- bearing sperm contain 2.8—3.0 percent more DNA than Y—bearing sperm. Sperm samples are treated with a fluorescent DNA stain, then passed single file through a laser beam in a FluorescenceActrvated Cell Sorter (FAC 5) ma- chine. The machine separates the sperm into two fractions based on the intensity of their DNA-fluorescence. Using this method, human sperm can be separated into X- and Y— chromosome fractions, with enrichments of about 85 percent and 75 percent, respectively. The sorted sperm are then used for standard intrauterine insemination. The Genetics and IVF Institute (Fairfax, Virginia) is presently using this PGS technique in an FDA-approved clin- ical trial, As of January 2002, 419 human pregnancies have resulted from the method. The company reports an approximately 80 to 90 percent success rate in producing the de sired gender. The emerging PGS methods raise a num- ber of legal and ethical issues. Some people feel that prospective parents have the legal right to use sex-selection techniques as part of their fundamental procreative liberty. Others believe that this liberty does not extend to custom designing a child to the parents' spec~ ifications. Proponents state that the benefits far outweigh any dangers to offspring or so— ciety, The medical uses of P65 are a clear case for benefit. People at risk for transmitting X- linked diseases such as hemophilia or Duchenne muscular dystrophy can now en- hance their chance of conceiving a female child who will not express the disease. As there are more than 500 known X—Iinked dis- eases and they are expressed in about 1 in 1000 live births, PGS could greatly reduce sufu fering for many families. The greatest number of people undertake PGS for nonmedical reasons—Le, to "bal- ance" theirfamilies. It is possible that the abil— ity to intentionally select the desired sex of an offspring may reduce overpopulation and eco- nomic burdens for families who wouid re- peatedly reproduce to get the desired gender. In some cases, PGS may reduce the number of abortions of female fetuses. It is also possible that PGS may increase the happiness of both parents and children, as the child would be more “wanted.” On the other hand, some argue that PGS serves neither the individual nor the common good. It is argued that 965 is inherently sexist, based on the concept of superiority of one sex over another, and leads to an increase in linking a child's worth to gen— der. Some fear that large—scale $365 will rein— force sex discrimination and lead to sex-ratio imbalances. Others feel that sexism and dis- crimination are not caused by sex ratios and would be better addressed through education and economic equality measures for men and women. The experience so far In Western countries suggest that sex-ratio imbalances would not resultfrom PGS. More than half of couples in the United States who use PGS re- quest female offspring. l-Iowever, the conse- quences of widespread P65 in some Asian countries may be more problematic. Both India and China already have sex-ratio imbalances, which contribute to some socially undesirable side effects, such as millions of adult men being unable to marry. Some critics of P65 argue that this tech- nology may contribute to social and economic inequality, if it will be available only to those who can afford it. Other critics fear that social approval of P65 will open the door to ac- cepting other genetic manipulations of chil- dren for characteristics such as skin or eye color. It is difficult to predict the full effects that PGS will bring to the world. But the gendereselection genie is now out of the bot~ tie and will be unwilling to step back in, References Sills, ES, Kirman, I., Thatcher, 5.5. III, and Palermo, GD. 1998. Sex-selection of human spermatozoa: Evolution of current techniques and applications. Arch. Gynecol. Obstet. 261 :109—1 1 5. Robertson, JA 2001. Preconception Gender SE— Iection. Am. J. Bioethics 122—9. Web Sites Microsort technique, Genetics 8: IVF Institute. Fair- fax, Virginia. ottori'vwwmmr‘crosorthet Female Infanticide, Gendercide Watch. h tips/Mum gendercide.org/casejnfanticide. h rmi 184 Chapter 7 1. in Drvsgplrila, the X chromosomes may become attached to one another (XX) such that they always segregate together. Some flies contain both an attached X chromosome and a Y chromosome. What sex would such a fly be? Explain why this is so. Solution: The fly would be a female. The ratio of X chromo- somes to sets of autosomes would be 1.0, leading to normal female development. TheY chromosome has no influence on sex deter— mination in Drosopliilo. Given this answer. predict the sex of the offspring that would occur in a cross between this fly and a normal one of the opposite sex. Solution: All flies would have two sets of autosomes, but each would have one of the following sex chromosome compositions: tndfitixa a metafemale with 3 X's (called a trisomic) (2) Y —> a female like her mother {3) XY 6 a normal male (4} YY —> no development occurs Sex Determination and Sex Chromosomes If the offspring just mentioned were allowed to interbreed. what would be the outcome? Solution: A stock would be created that would generate the at, tached X females generation after generation. 2. The Xg cell—surface antigen is coded for by a gene that is lo— cated on the X chromosome. There is no equivalent gene on the Y chromosome. Two codominant alleles of this gene have been iden- tified: Xg] and Xg2. A woman of genotype Xg2y’Xg2 bears chil- dren with a man of genotype Xgl/Y. and they produce a son with Klinefelter syndrome of genotype Xgl,’Xg21’. Using proper genetic terminology, briefly explain how this individual was generated. in which parent and in which meiotic division did the mistake occur? Solution: Because the son with Klinefelter syndrome is XgI/Xg2l’. he must have received both the Xgl allele and the Y chromosome from his father. Therefore. nondisjunction must have occurred during meiosis I in the father. PROBLEMS AND DISCUSSION QUESTIONS 1. As related to sex determination, what is meant by (a) homomon phic and heteroinorphic chromosomes and lb] isogamous and heterogarnous organisms? 2. Contrast the life. cycle of a plant such as Zea ways with an ani— mal such as C. L’fé’gwls. 3. Discuss the role of sexual differentiation in the life cycles of C lilrtrnydomonas. Zea mays. and C. elegans. 4. Distinguish between the concepts of sexual differentiation and sex determination. Contrast the Pr‘utcnm' and ngaeus modes of sex determination. Describe the major difference between sex determination in Dm.rr.2plzila and in humans. 7. What specific observations (evidence) support the conclusions you have drawn about sex determination in Drosopfn'la and humans? 8. Describe how nondisjunction in human female gametes can give rise to Klinefelter and Turner syndrome offspring following ferA tilization by a normal male gamete. 9. An insect species is discovered in which the heterogametic sex is unknown. An X-linked recessive mutation for reduced wing (in) is discovered. Contrast the F] and F: generations from a cross between a female with reduced wings and a male with normal—sized wings when (a) the female is the heterogametic sex; ('b') the male is the heterogametic sex. 10. Based on your answers in Problem 9. is it possible to distinguish between the Premier and Lygneirs mode of sex determination based on the outcome of these crosses? 11. When cows have twin calves of unlike sex (fraternal twins}. the female twin is usually sterile and has masculinized reproductive organs. This calf is referred to as a freemanin. ln cows. twins may share a comnton placenta and thus fetal circulation. Predict why a freemartin develops. 9‘5" 12. An attachedix female fly. fifi’ (see the “Insights and Solutions” box ). expresses the recessive X-linked white eye phenotype. [t is crossed to a male fly that expresses the X—linked recessive minia- ture wing phenotype. Determine the outcome of this cross re- garding the sex. eye color. and wing size of the offspring. I3. Assume that rarely. the attached X chromosomes in female gun metes become unattached. Based on the parental phenotypes in Problem 12, what outcomes in the F1 generation would indicate that this has occurred during female meiosis? 14. It has been suggested that any male—determining genes contained on the Y chromosome in humans cannot be located in the lim- ited region that synapses with the X chromosome during meio- sis. What might be the outcome if such genes were located in this region? 15. What is a Barr body, and where is it found in a cei]? 16. Indicate the expected number of Barr bodies in interphase cells of the following individuals: Klinefelter syndrome: Turner syn— drome; and kai'yotypes 47,XYY. 47,XXX, and 48.30004. 17. Define the Lyon hypothesis. 18. Can the Lyon hypothesis be tested in a human female who is homozygous for one allele of the X-iinked capo gene? Why. or why not? 19. Predict the potential effect of the Lyon hypothesis on the retina of a human female heterozygous for the Xrlinked redngreen coior- blindness trait. 20. Cat breeders are aware that kittens expressing the X—linhed cal— ico coat pattern and tortoiseshell pattern are almost invariably females. Why? 21. What does the apparent need for dosage compensation mecha- nisms suggest about the expression of genetic information in nor- mal diploid individuals? 22. 23. 24. The marine echiurid worm Bonellt'cr virt'dilr is an extreme exam- ple ofthe environment's influence on sex determination. Undif- ferentiated larvae either remain frecsswimming and differentiate into females or they settle on the proboscis of an adult female and become males. 1f larvae that have been on a female proboscis for a short period are removed and placed in seawater. they de- velop as intersexes. If larvae are forced to develop in an aquar- ium where pieces of proboscises have been placed, they develop into males. Contrast this mode of sexual differentiation with that of mammals. Suggest further experimentation to elucidate the mechanism of sex determination in B. viria'ir. How do we know that the primary sex ratio in humans is as high as 1.40 to 1.00? Devise as many hypotheses as you can that might explain why so many more human male conceptions than human female coa~ ccptions occur. Extra-Spicy Problems The X-linked dominant mutation in the mouse. Thrift-nit:rfi'nnl nizmion (Uni). eliminates the normai response to the testicular hormone testosterone during sexual differentiation. An XY mouse hearing the Tfin allele on the X chromosome develops testes. but no further male differentiation occurs—the external genitalia of such an animal are female. From this information. what might you conclude about the role of the Tfin gene product and the X and Y Chromosomes in sex determination and sexual differenti- ation in mammals? Catt you devise an experiment. assuming you can "genetically engineer" the chromosomes of mice, to test and confirm your explanation? Campomelic dysplasia (CMDll is a congenital human syndrome, featuring malfomiation of bone and eartiiage. It is caused by an autosomal dominant mutation of a gene located on chromosome | 7'. Consider the following observations in sequence. and in each Case. draw whatever appropriate conclusions are warranted. ta) Of those with the syndrome who are karyotypically 46.XY. approximately 75 percent are sex reversed. exhibiting a wide range of female characteristics. (h) The nomnutant form of the gene. called SOXQ. is ex- pressed in the developing gonad of the XY male, but not the XX female. (cl The SOX9 gene shares 7] percent amino acid coding se- quence homology with the Y—linked SRY gene. (d) CMDI patients who exhibit a 46,XX karyotype develop as females. with no gonadal abnormalities. In the wasp. Brat-0n lichen»: a form of parthenogenesis t where unfertilized eggs initiate development) resulting in haploid or~ ganisms is not uncommon. All haploids are males. When off- spring arise from fertilization. females almost invariably result. P. W'. Whiting has shown that an X—linked gene with nine multi- ple alleies (Xu. Xb. etc.) controls sex determination. Any ho- mozygous or hemizygous condition results in males and any heterozygous condition results in females. If an Xanj, female mates with an X” male and lays 50 percent fertilized and 50 per- cent unfertilized eggs. what proportion of male and female off— spring will result? Shown in the right column are two graphs that plot the percent- age of males occurring against the atmospheric temperature dur~ ing the early development of fertilized eggs in (a) snapping turtles and to] most lizards. Interpret these data as they relate to the ef- fect of temperature on sex determination. 25. 26. 3|. Extra-Spicy Problems 185 In mice, the Sr}: gene [see Section 7.3) is located on the Y chro- mosome very close to one of the pseudoautosomal regions that pairs with the X chromosome during male meiosis. Given this in- formation. propose a model to explain the generation of unusual males who have two X chromosomes (with an Siy-containin g piece of theY chromosome attached to one X chromosome]. The genes encoding the red and green color-detecting proteins of the human eye are located next to one another on the X chro— mosome and probably arose during evolution from a common ancestral pigment gene. The two proteins demonstrate 76 per- cent homology in their amino acid sequences. A normal-visioned woman with one copy of each gene on each of her twu X nitro- mosomes has a red color—blind son who was shown to contain one copy of the green—detecting gene and no copies of the red- detecting gene. Devise an explanation at the chromosomal level (during meiosis) that explains these observations. (:1) Snapping turtles 100 50- % Males 20 30 40' (b) Most lizards 100 'e ' SO 9/0 Males .l 20 3O 40 Temp. (°C) CC (Carbon Copy]. the first cloned cat, was created from an ovarian cell taken from her genetic donor. Rainbow. The diploid nucieus from the cell was extracted and then injected into an enucleated egg. The resulting zygote was then allowed to develop in a petri dish and the cloned embryo was implanted in the uterus of a surrogate mother cat. who gave birth to CC. Rainbow is a calico cat. CC’s surrogate mother is a tabby. Geneticists were very interested in the outcome of cloning a calico cat, because they were not certain if the cat would have patches of ortmge and black, just orange, or just black. Taking into account the Lyon hypothesis. explain the basis of the uncertainty. 186 Chapter 7 Carbon Copy with her surrogate mother. SELECTED READINGS Amory. J.K., et a1. 2000. Klinefeltet"s syndrome. Lancet 356133 3—35. Avner. P._. and Heard, E. 2001. X-chromosome inactivation: Count— ing. choice. and initiation. Nature Reviews 2:59—67. Burgoyne. RS. 1998. The mammalian Y chromosome: A new per- spective. Bioessayr 20:363u66. Carrel. L., and Willard. H.F. E998. Counting on Xist. Nature Gener— it's19:211—12. Court-Brown. WM. 1968. Males with an XYY sex Chromosome coma piement. J. Med. Genet. 5341—59. Davidson. R.. Nitowski. 1-1.. and Chiids. B. 1963. Demonstration of two populations of cells in human females heterozygous for glucose-ovphosphate dehydrogenase variants. Pror: Nail. .‘it'fld. Sci. USA 501481785. Erickson, JD. 1976. The secondary sex ratio of the United States. l969e71: Association with race. parental ages. birth order. paternal education and legitimacy. Ann. Hum. Genet. (London) 40:205—12. Hodgkin, J. 1990. Sex determination compared in Dmmphiiri and Caenon’rabdiris. Nature 344:721—28. Hook, EB. 1973. Behavioral implications ofthe humans XYY geno— type. Science 179:139—50. Irish. BE. 1996. Regulation of sex determination in maize. BioErsays 18363—69. Jacobs. P.A.. et a1. 1974. A cytogenetic survey of [1.680 newborn in— fants. Ann. Hum. Genet. 37359—76. .legalian. K.. and Lahn. B.T‘. 2001. Why the Y is so weird. Sci. Am. (Feb) 28456431. Sex Determination and Sex Chromosomes 32. Let‘s assume hypothetically that Carbon Copy (see Problem 31 J is indeed a calico with black and orange patches, along with the patches of white characterizing a calico cat. Would you expect CC to appear identical to Rainbow? Explain why or why not. 33. When Carbon Copy was born (see Problem 31}. she had black patches and white patches, but completely lacked any orange patches. The knowledgeable students of genetics were not stir- prized at this outcome. Starting with the somatic ovarian ceil used as the source of the nucleus in the cloning process. explain how this outcome occurred. Koopman. P.. et at. 1991. Male development of chromosomally fe- male mice transgenic for Si'y. Nature 35121 17—] 1. Lahn. BI. and Page. DC. 1997. Functional coherence ofthe human Y chromosome. Science 278:675—80. Lucchesi. J. 1983. The relationship between gene dosage. gene ex- pression. and sex in Drrisopitiin. Dev. Genet. 33275782. Lyon, MP. 1972. Xichromosome inactivation and developmental pat- l'ems in mammals. Biol. Rev. 4711735. . I988. Xachromosome inactivation and the location and ex- pression of Kilinked genes. Am. J. Hum. Genet. 42:8—16. . I998. Xichromosome inactivation spreads itself: Effects in autosomes. Am. J. Hum. Genet. 63:17—19. McMillen. M.M. 1979. Differential mortality by sex in fetal and neonatal deaths. Science 204:89—91 . Penny. G.D.. et al. 1996. Requirement for Xist in X chromosome in- activation. Nature 379: 131—37. Pieau. C. [996. Temperature variation and sex determination in rep- tiles. BitiEssrivtr 1 8: 19—26. Westergaard. M. 1958. The mechanism of sex determination in dioe- cious flowering plants. Adi: Genet. 9:217—81. Whiting. PW. [939. Multiple alleles in sex determination in Hribr'ubmcon. J. Morpimingy 662323455. Witkin. H.A.. et a1. 1996. Criminality in XYY and XXY men. Science 193:547—55. ...
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