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THE GENET ICS OF SEX DETERMINATION AND DIFFERENTIATION Jean-Louis Guenet Service de Genetique cellulaire du College de France et de l'Institut Pasteur 25, rue du Dr. Roux 75724 Paris Cedex 15

The primary sex (theABSTRACT type of the gonad) depends upon the presence (male) or absence (female) of a cell surface antigen called H-Y. If the omnipotent gonad differentiates toward the male type, it then produces a steroid hormone called testo­ sterone. If, in turn, testosterone is present in the growing embryo and if the appropriate receptors are present on a spe­ cific group of undifferentiated cells, then a male genital tract develops. If none of these conditions are fulfilled, a female phenotype results. Sex differentiation thus seems to be controlled by a very simple genetic system with a very limited number of genes. 14

Most of the vertebrate species reproduce sexually. This means that male individuals have to copulate with female partners to produce a new specimen of their own species (a

male ••. or a female Evolutionists!) . agree that confers immense benefits when considered as a source of individual variability because it furnishes material with which natural selection may operate. It necessitates however the differen­ tiation of very highly specialized tissues, like those of the gonads producing the gametes or those of the genital tracts. We will discuss the present status of our knowledge about sex differentiation with special reference to the mammals.

I.- IS SEX NECESSARY OR WHY DOES THE SEX EXIST Not all the ? vertebrate species have two sexes? some of them, among the fish or reptile classes, have only one sex. They reproduce parthenogenetically or gynogenetically. In true , eggs laid by the female spontaneously initiate development toward individuals. Thus, males are totally unnecessary. In a modified form called , copulation with males of other species is required. Alien sperms, however, function only as stimuli to initiate the development of unfertilized eggs. Aquarium fanciers should be familiar with the amazon molly (PoeeiZia formosa), a small fish of pleasing coloration and shape. As the name implies, there is no male amazon molly for they are the hybrid between sailfin and Mexican mollies that became an all female gynogenic species. Among whiptail lizzards (genus Cnemidophorus) inha­ biting arid regions of Arizona and New , all female species are also found along the boundaries between neighbo­ ring bisexual species. Sexual reproduction is thus not necessary. However when the are diploid (like most of the vertebrates), the bisexuality has selective advantages as a source of gene­ tic diversity. If, at a given locus, an of a given species 15 is a/a' (two allelic forms) and if the species reproduce par­ thenogenetically, all the offspring will be a/a'. On the contrary, if the species reproduce bisexually, then three genotypes (a/a, a/a' and a'/a') are expected. The fittest will be selected by evolution.

II.- WHY 2 SEXES INSTEAD OF 3, 4, OR MORE To make the probability of fertile? matings maxi­ mum,mathematicians have calculated that two sexes is optimal. It is also more simple to determine two sexes than three or more on a genetical basis.

III. - TWO SEXES IN THE SAME INDIVIDUAL OR IN TWO Another! way to dispense with bisexual life ? is, of course, to combine the male and the female organs into one body. Hermaphroditism is wide spread among fish. When an individual can simultaneously lay eggs and ejaculate sperms, it is called a synchronous hermaphrodite. Asynchronous her­ maphrodites are those which go through spontaneous sex rever­ sal with age. They can be protoandrous (males when young and females when old) or protogynous (the converse of 'the above). Among perch-like fish, synchronous hermaphroditism is the normal mode of reproduction as well as in many species of sea basses of the family Serranidae and the gonads of these fishes consist of a pair of ovotestis. Since both male and female regions of the gonad mature simultaneously to produce eggs and sperms prior to the spawning season, self-fertili­ zation is a possibility, except that their habit of spawning en masse permit cross-fertilization. Asynchronous hermaphroditism of the protogynous type is also seen in perch-like fish of the family Serranidae. Groupers and their allies, Epinephelus, Mycteroperca, exhibit a protogynous type of asynchronous hermaphroditism as well as the swamp eels of the order Synbranchiformes which are all females when young and change their sexes at about 2 to 3 years of age. There is a switch from ovary to testis which 16

takes from 3 to 5 months to complete. The protoandrous form is seen among progies and sea breams. The above examples should suffice to illustrate the point that there are ways other than being bisexual for di­ ploid organisms to generate enough genetic diversity among their progeny. In the mammalian species and in man, however, perma­ nent bisexuality is a rule. Thus it seems that sexual specia­ lization of the mammalian species has some selective advantage.

IV.- GENETIC SEX DETERMINATION AND THE ONE-TO-ONE SEX RATIO. The fact that in some fish, which are synchronous hermaphrodites, the gonad simultaneously function as both testis and ovary, and the fact that even in man, ovotestes are seen in pathological conditions, reveal that the embryonic gonad of vertebrates has a bipotential to develop into a tes­ tis as well as an ovary. It follows then that gonochorism (being bisexual) must owe its existence to a specific regula­ tory mechanism which makes testicular and ovarian developments mutually exclusive of each other. Sometimes, sexual determination depends upon envi­ ronmental influences as in the very strange case of the snap­ ping turtle of the Mississipi River (Chelydra) where it is determined by the temperature of incubation of the eggs. In most instances, however, sex determination is controlled by a genetic mechanism. We know that in most species of vertebrates there are homogametic sexes and hetero­ gametic sexes when the chromosomal complements are considered. In the mammals, the female is XX (this means that all the chromosomes can be paired) but the male is XY or heterogametic as one of the chromosomes (the Y) is unique and cannot be paired. The situation is reversed in the bird species where the female is (WZ) and the male (ZZ) . From the analysis of rare pathological conditions, we also know that the develop­ ment of a testis depends upon the presence or absence of a Y chromosome but not (like it is the case in the Drosophiia) from the ratio one X vs two XX. It has been found that the 17 presence of a single Y is sufficient for testes to differen­ t'iate even in individuals who have as many as four X in the karyotype (XXXX I Y constitution). The role that the Y chromosome plays in sexual differentiation of the mammals is however limited to this ini­ tial act of enticing an indifferent embryonic gonad to diffe­ rentiate toward a testicular direction. The differentiation of the genital tract itself is controlled by an hormonal in­ fluence and depends upon the presence or absence of testoste­ rone ; a male steroid hormone manufactured in the testes. We know from a series of experiments performed by Alfred Jost in Paris on rabbit and rat embryos that regardless of their sex chromosome constitutions, whether they be XY or XX, mammalian embryos have an inherent tendency to develop as females. Thus, if the testis is removed from the XX embryo early enough, it will develop all of the female characteristics while the male phenotype can easily be developed, out of an XX genotype, by continuously exposing it to testosterone. The female sex thus appears as the primordial sex, the manifestation of which requires no manipulation. The male, on another hand, is a female modified by an hormonal influence.

V.- MALE AND FEMALE OR THE TWO SIDES OF THE SAME COIN. A mammalian body is composed of a considerable num­ ber of cells which are grouped in different tissues with a sharply defined function. Both the male and the female however have heart, brain, blood cells, etc. and of course these cells must not be "inducible" by the testosterone as they have a basic physiological function totally unrelated with the sexuality. Inducible cells are restricted to the so-called Wolfian and Mullerion embryonal systems. If a gonad is of the male type and produces a normal amount of testoste­ rone, the Wolfian system is induced and, in addition to the testis, epididymis, vasa deferens, and seminal vesicles deve­ lop as well as a penis with a prostatic gland, and an erectile tissue. If the gonad is of the ovarian type, the Wolfian sys- 18

tern degenerate and a female genital tract develops from the Mullerian system. The cell types which can respond to testosterone are categorized as target cells of testosterone. On these cells, receptor sites are present for the small hydrophobic testosterone molecules to bind with. Thus maleness and femaleness can be reduced to two alternative states : the testosterone bound state and the unbound state. Two sides of a same coin.

VI.- THE GENETIC REGULATORY SYSTEM OF SEX DETERMINATION. Testosterone is a small hydrophobic molecule. By itself, it is incapable of recognizing a specific set of genes on the DNA double strands, but we know that there is a more complex molecule which is a protein called the Nuclear­ cytosol-androgen-receptor protein which plays the role of an intermediary between the cell surface (where it gets in con­ tact with the testosterone molecule) and the DNA of the cell (where it binds with a specific dormant set of genes to switch it on). Such a protein, which is under the primary influence of an inducer (the testosterone) controls the acti­ vity of a number of genes which in turn code for specific proteins. It is known as a regulatory protein. A gene however can only be identified and localized through its eventual mutation toward an inactive form. For the gene which codes for the regulatory protein Nuclear­ cytosol-androgen-receptor, we fortunately do have found such a mutation. It is known by the physicians as testicular feminization syndrom. It occurs in the human beings and has been discovered by Mary F. Lyon also in the mouse. Affected individuals despite a XY constitution have testes for gonad but a female type genital tract with a vagina uterus and well developed mammary glands. This syndrom results of an X-linked mutation called Tfm (testicular feminization) . 19

VII.- THE DIFFERENTIATION OF THE TESTIS IN THE EMBRYO DEPENDS OF A CELL SURFACE ANTIGEN. The correlation between the presence of a Y chromo­ some in the karyotype and the development of the undifferen­ tiated gonad into a functional testes is a rule. This rule however suffers some rare exceptions ; two of them are reci­ procal and complementary. The first one refers to the Sxr (Sex reversal) muta­ tion, an autosomal dominant change, which makes the XX geno­ type capable of development into phenotypic males. The second refers to the Myopus schisticolor (the wood lemming) species where half of the XY genotypes are asso­ ciated with a normal female phenotype. These two exceptions prove that something else must be more tightly correlated with the differentiation of the male sex. We know now that it is a cell surface antigen (a male specific antigen called H-Y) which induces the transformation of undifferentiated gonad into a testes. XX-Sxr/+ animals are H-Y positive like the normal XY males, and XY-female wood lemmings are H-Y negative like the normal XX females. The correlation is absolute.