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THESis on Sex determination and differentation of the subject REPRODUCTION BIOLOGY
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Umesh Rai and Brototi Roy Department of Zoology, University of Delhi Delhi- The concept of sex determination and differentiation has been baffling mankind since time immemorial. While Aristotle hypothesized in 335 B.C. that sex was determined by the heat generated during conception, Andreas Vesalius in 1543 A.D illustrated that women were but males, their genitalia turned inside out. In numerous cultures since then, women have been considered as the “default state of men”. However, in the 17th^ century gradually it was discovered that the females produced eggs that transmitted parental traits. Later, in the twentieth century Geddes and Thomson came forward with the hypothesis that constitution, age, nutrition and environment of parents decide the sex determination of the offspring. This environmental view of sex determination was again challenged by the discovery of sex chromosomes by McClung in 1902. Following this, more evidences gradually clinched to suggest the chromosomal concept of sex determination. Numerous experimental studies in vertebrates have now established beyond doubt that sex is determined either by chromosomal factors, environmental influences, or interplay of both, depending on the species/groups.
Environmental Sex Determination (ESD ): In some cold blooded vertebrates (fishes, reptiles), sex is determined not at the time of fertilization by sex chromosomes, rather, it is determined by the environment in which the early embryonic development takes place.
In various freshwater fishes as well as marine species from temperate or tropical habitats, it has been clearly shown that sex determination and consequent differentiation is influenced by the environmental factors like pH, salinity, social interaction and importantly, temperature (Baroiller & D’Cotta, 2001). In most thermosensitive species (some Atherinids,
Poecilids, Cichlids, a Siluriform), high temperature increases male to female sex ratios and vice-versa. Exceptionally, in European sea bass, Dicentrarchus labrax and channel catfish, Ictalurus punctatus high temperature induces female sex differentiation. Density and pH also plays a sex determining role in fishes as evidenced in paradise fish Macropodus opercularis, where the number of females is directly proportional to the density and in all Apistogramma species (cichlids) the proportion of males is higher at an acidic pH (4.5) than at neutral pH (6.5). The complete sex reversal can also be accomplished by exposing gonochoristic fish to exogenous sex steroids during gonadal differentiation. In addition to gonochorism, several types of hermaphroditism are seen in fishes. Social interaction such as disappearance of a male or female from a mixed group causes sex inversion in these species: from male to female (protoandrous, sea anemone fish, Amphiprion clarkii ), from female to male (protogynous, Red sea fish, Anthias squamipinnis ) and a few can change sex either way or multiple times (serial sex change, gobiid fish, Trima okinawae (Baroiller et al., 1999)
In amphibians, although sex chromosomes are invariably shown responsible for sex determination with either male heterogamety (XY/XX) or female heterogamety (ZW/ZZ), the environmental factor and hormones can override the genotypic mechanism of sex determination and induce sex reversal. The rearing of larvae of salamanders (ZZ/ZW) at high temperatures (30-32 oC) produces opposite effects in two different species. ZZ genotypic males of Algerian ribbed newt, Pleurodeles poireti become phenotypic females, whereas ZW genotypic females of Spanish ribbed newt, Pleurodeles waltl become phenotypic males. In another newt Triturus cristatus (XX/XY), more males than females, including some XX neomales are produced at higher temperature, and vice-versa at lower temperature. Also, in wood frog, Rana sylvatica , sex reversal of all females occurs when larvae are reared at high temperatures (32±2 oC). However, all the above said studies have been conducted at extreme temperature which is outside of the range normally experienced by these species, and
(TSD) is invariably seen in all tuatara and crocodiles, most tortoises, turtles and terrapins, and in some lizards (Pieau C, 1996). Generally, among most chelonians, incubation of eggs below transition range temperature (TRT) induces masculinization, above TRT leads to feminization, and at TRT both males and females or even intersex individuals are produced. On the contrary, in alligators, lizards and tuatara males are produced at high temperature and females at low temperature. However, exceptions to these trends are seen in crocodiles and a few turtles like snapping turtle, mud turtle, alligator snapping turtle etc., where eggs incubated below or above TRT develop into females and males are produced at TRT. In all TSD species there is a well defined thermosensitive period (TSP) when temperature affects sex determination and after this the embryos become refractory to temperature changes.
Temperature influences sex determination by modulating the activity of steroidogenic enzymes and thereby affecting the hormonal environment of the embryo inducing either masculinization or feminization. In freshwater turtle, Eyms orbicularis, the aromatase activity responsible for aromatization of testosterone into estradiol remains low at male producing temperature (25 oC) and high at feminizing temperature (30 oC). Moreover, exogenous sex steroids can override the effect of temperature on sex determination in TSD species. Earlier, it was thought that the hormone induced sex reversal is unilateral towards the female sex, since administration of estradiol or testosterone induces embryos to develop as females even if eggs are incubated at male sensitive temperature. However, in case of red-eared slider turtles, Trachemys scripta , instead of testosterone, administration of dihydrotestosterone (DHT, a non aromatizable androgen) during TSP altered the unilateral pattern of sex reversal and induces masculinization. Identical result is observed when aromatase inhibitor (AI) is applied to eggs incubated at female sensitive temperature. Although sex is genetically determined in the Indian garden lizard, Calotes versicolor , exogenous androgen induces sex reversal. DHT is found to be effective only between stages of initialization of genital ridge
and formation of sex cords (Ganesh et al., 1995). Previous experiences of unilateral sex reversal towards females after administration of testosterone to eggs incubated even at male sensitive temperature might be due to the aromatization of testosterone into estradiol, the female sex steroid.
Chromosomal Sex Determination (CSD): Before the karyotyping of human chromosomes, it was considered that sex is determined by the number of X chromosomes present in an individual. It was observed that in drosophila, males had single X chromosome (XY or XO) and the presence of 2 or more X chromosomes (XX, XXX, or XXY) always conferred female phenotype. It was thus considered that the Y chromosome was a null chromosome. However, in 1966, a landmark observation was made by Jacobs and Ross. They described two sisters who had female external genitalia but 46 XY karyotype in which the Y chromosome consisted only of its long arm. It seemed therefore, that the testicular determining region of the Y chromosome normally resides in its short arm. This was confirmed by high resolution banding studies of an XX male which revealed that some material from the short arm of Y chromosome had been translocated to one of his X chromosomes. With advanced techniques, it became apparent that sex determining gene on short arm of Y chromosome is responsible for the development of testis in mammals. Also, testicular development is seen impaired in several clinical syndromes resulting from autosomal deletions or mutations. Now autosomal genes such as SOX 9, WNT 4, SF 1, DMRT 1 etc are shown to play a crucial role in the downstream events of sex differentiation initiated by SRY (Fig 1).
SRY (Sex determining region on Y chromosome): SRY gene is located near the tip of the short arm of Y chromosome that encodes a transcription factor of 204 amino acids. The central 79 amino acids encode the HMG (high mobility group) box. In the entire SRY protein, only HMG domain shows sequence conservation. HMG box functions as DNA-
other transcription factors activate the expression of Amh gene (anti Müllerian hormone gene). The binding of SOX 9 HMG box bends the DNA which bring SF1 and GATA 4 in close proximity to each other and along with WT1 and HSP 70 form a tightly associated protein complex that activates transcription of the Amh gene. The complete absence of AMH transcripts is seen in XY mice mutant for HMG box (DNA binding domain) of Sox 9 gene suggesting that Sox 9 is required for AMH expression.
Although experimental evidences in non mammalian vertebrates show that Sox 9 has a conserved role in sex determination, it is important to mention here that its expression in alligator and chicken begins well after pre Sertoli cell differentiation and AMH expression. It seems that Sox 9 , in non mammalian vertebrates may be involved in Sertoli cell organization, rather than early testicular determination (Pask & Graves, 1999).
SF1 (Steroidogenic factor 1): SF1 gene transcribes a protein, otherwise known as Ad4BP, belonging to the orphan nuclear receptor family. Initially, SF1 was described to regulate the production of cytochrome P-450 steroid hydroxylase enzymes that are necessary for synthesis of steroids, and thus, are expressed in many steroidogenic tissues, including adrenal gland, ovary and Leydig cells of the testis. Subsequently, SF1 transcripts were detected in the mouse urogenital ridge even at the stage of the indifferent gonad (9-12 dpc) and mutation in Sf1 gene was shown to cause complete dysgenesis of gonad in both sexes. This suggests its role in early formation of the indifferent gonad and, thereby, Sf1 is placed upstream of Sry in sex determination pathway. However, Sf1 also plays an important role in the downstream testicular differentiation pathway initiated by Sry. Sf1 activates testicular differentiation by influencing both, Leydig and Sertoli cells. SF1 in Leydig cells regulates steroid biosynthesis and in Sertoli cells it binds to Amh promoter region and activates the expression of AMH in collaboration with other transcriptional factors. The importance of SF1 , located on
chromosome 9, for testis development and AMH regulation in humans is demonstrated by XY patient heterozygous for SF1 where the individual has malformed fibrous gonads and retains fully developed Müllerian duct structures (Achermann et al., 1999).
WT1 (Wilms’ tumor 1) : The WT1 gene first came into focus in patients with Wilms’s tumor where mutation of this gene led to embryonic kidney tumor. Later, mutation in this gene was seen associated with the disruption of bipotential gonadal development. The presence of WT transcripts in developing gonads substantiates its role in early bipotential gonadogenesis.
WT1 encodes variant transcripts by alternative splicing, alternative translation start sites, and RNA editing. These variants give rise to different zinc finger DNA-binding protein isoforms that fall under two categories (designated as +KTS and – KTS) depending on the presence or absence of three amino acids (KTS, lysine-threonine-serine) between two zinc fingers. Experimental evidences in mice show that the -KTS isoform is responsible for development of undifferentiated gonad. WT1 +KTS isoform plays an important role in testicular differentiation possibly by regulating Sry expression, since potential WT1 binding sites are present upstream of both mouse and human SRY. Further more, mutation in WT gene causes persistence of Müllerian duct in males. In recent years, WT1-KTS isoform is shown to physically interact with SF1 and synergistically up regulate AMH expression.
GATA4/FOG2: GATA4, a member of the GATA family of transcription factors, contain a zinc finger DNA-binding domain that binds to the consensus sequence WGATAR in the 5’- flanking region of target genes. In mice, it is detected as early as embryonic day 11.5 in the somatic cells of primitive gonads of both sexes and therefore, GATA4 in conjunction with other transcription factors play role in development of bipotential gonad (Viger et al., 1998). The GATA4 expression remains maintained in Sertoli cells throughout embryonic development, whereas it is down regulated shortly after differentiation of ovary on embryonic
downstream genes, e.g. Am h and other steroidogenic genes. Thus, it is considered as ovary– determining gene.
WNT 4 : WNT 4 is another important ovary determining gene. Like DAX 1, its expression is turned off with the differentiation of testis. WNT 4 knock out XX mice shows masculinization as ovarian differentiation ceases and its cells express testis-specific markers, including Amh and testosterone producing enzymes (Vainio et al., 1999). This suggests that WNT 4 is obligatory gene for female sex differentiation.
In marsupials too, the control of testis determination is vested in the Y chromosome, though it is the smallest of any mammals (Graves and Shetty, 2001). However, the number of X chromosome plays a critical role in other aspects of sex differentiation (Table.1). Single X chromosome in XO animals leads to the development of empty scrotum (without testis), whereas scrotum fails to develop in XXY animals having testis. In fact, the presence of two X chromosomes leads to the development of pouch and mammary glands in lieu of the scrotum (Manolakou et al., 2006). Thus, marsupials are different from other mammals with regard to the accessory sex organ differentiation since the formation of scrotum, pouch and mammary glands in marsupials are dependent on genes present on X chromosomes rather than on gonadal hormones as in eutherian (Pask and Benfree, 2001).
Table 1: X-linked secondary sexual differentiation in marsupials
Single X chromosome (scrotum) Double X chromosome (pouch and mammary glands)
Y chromosome Absence of Y chromosome (testis) (no testis) (testis) (^) XY XO (males with scrotum and testis) (males with empty scrotum) XXY XX (males with testis, pouch and (females with pouch and mammary gland) mammary glands)
However, the monotremes are unusual because they have multiple set of sex chromosomes rather than the single pair usually found in marsupials and eutherians. Recent report demonstrates the presence of 10 sex chromosomes in platypus arranged as X1Y1X2Y2X3Y3X4Y4X5Y5 (Rens et al., 2004). Interestingly, genes present on X chromosome situated at one end of the ‘sex chromosome chain’ are orthologous with those on the human-X chromosome, while genes on chromosome situated at the other end of the chain are homologous with those on the bird-Z chromosome. Hence, the platypus provides an important link between the chromosomal sex determination in mammals and birds.
Birds have distinguished sex chromosomes, except ratitae (e.g.ostrich, emu) which have karyologically indistinguishable or slightly differentiated sex chromosomes (Schartl, 2004). The sex chromosomes have been designated as Z and W chromosome. Unlike mammals, males are homogametic (ZZ) while females are heterogametic (ZW). However, whether W chromosome carrying dominant female determining genes ( ASW : avian sex specific W-linked, and FET1 : female expressed transcript 1) or the Z chromosome carrying a dosage-dependent male determinant gene ( Dmrt1 ), or both features determine the sex is not yet clear. Nevertheless, many of the downstream sex determining genes ( Sox9 , Amh , Dmrt1 , Sf1 , Wt1 and Dax 1 ) are reported in birds though the order of their expression in developing gonad may be different from that reported in mammals. Chromosomal sex determination is prevalent also in amphibians, though environmental and hormonal factors are reported to cause sex reversal. Although in most of the fishes and reptiles sex is determined by environmental factors, downstream sex determinant genes are reported. However, their interaction, temporal expression and the initial trigger does not follow any taxonomic scheme.
Fig 3. Embryologic events in male sex differentiation depicted in temporal fashion. The line depicts the increase in fetal serum testosterone concentrations. The word activity refers indirectly to the action of AMH in causing Müllerian duct regression and androgens to induce male sex differentiation (Hughes, 2001)
The sexually indifferent bipotential gonad develops from the ventromedial surface of the mesonephros near the kidneys at around 4 weeks in the human fetus and 9.5 days post-coitum (dpc) in the mouse. The somatic cells of gonad are derived from the mesonephros and coelomic epithelium that covers the coelomic surface of the gonadal ridge. These cells proliferate in the gonadal primordia and form the sex cords. Several factors such as Lhx (LIM Homeobox gene1), Emx2 (homolog of empty spiracles homeobox gene 2), Pax 2 (paired box gene 2), WT1and SF1 are involved in cell proliferation and development of the
bipotential gonad. The gonad is subsequently colonized by primordial germ cells (PGC) that originate from epiblast-derived cells present in the yolk sac near the base of allantois and migrate through the hindgut to invade the indifferent gonad. Germ cell migration is under the influence of stem cell factor (SCF), mast cell growth factor (MGF), and extra cellular matrix proteins like fibronectin and laminin (Bendel-Stenzel et al., 1998). During migration the germ cells proliferate but do not differentiate. The primordial germ cells are distinguishable
from the other cell type because of their large size and large round nuclei. Histologically, they are identified by high alkaline phosphatase activity and glycogen. PGCs along with the somatic cells form the “gonadal ridge”. The formation of gonadal ridge is completed within 5 to 6 week of gestation in human embryos. No sexual difference can be observed in the gonads until the 6th^ week of embryonic life in humans and 11.5 dpc in mice.
Male Sex Differentiation In XY fetus of 6-7 weeks, the first sign of testis differentiation is seen with the aggregation of the pre Sertoli cell (derived from mesonephros) around the germ cell now called gonocytes, to form the testicular cords. These cords lose contact with the surface epithelium and become separated from it by a thick extra cellular matrix, the tunica albuginea. By the end of 9 week, the mesenchyme that separates the seminiferous cords gives rise to interstitial cells. Later these are differentiated into steroid secreting Leydig cells. Although differentiation of Leydig cells in the initial phase is independent of gonadotropin action, its proliferation and differentiation in the first and second trimesters of the fetal life depends on placental hCG, and thereafter, controlled by fetal pituitary LH (Josso et al., 2005).
The masculinization of the genital tract starts with the regression of Müllerian duct under the influence of anti Müllerian hormone (AMH) secreted from Sertoli cells. Shortly after the Müllerian duct regression, the portion of Wollfian duct adjacent to testis is differentiated into epididymis, the central portion becomes vas deferens, and the distal end of the duct near the urogenital sinus develops into seminal vesicle. The prostrate gland develops as a series of outgrowths from the urogenital sinus (Fig. 4). The virilization of the Wollfian duct is under the control of testosterone as the phenomenon is seen inhibited after the administration of anti-androgen or testosterone antibody at the critical period of sex differentiation (Elger et al ., 1970).
in the gubernaculums development in INSL3 mutant mice ( Nef & Parada ,1999). Mutation of this gene has been detected in cryptorchid patients. Moreover, androgens mediate the disappearance of the cranial suspensory ligament (van der Schoot & Elger, 1992) and are required for the inguinoscrotal phase of testicular descent.
Female Sex Differentiation:
In females, the primary sex cords undergo degeneration and a new set of sex cords is then produced by the epithelium. These sex cords reside in the periphery and hence, are called cortical sex cords. The primordial germ cells proliferate by mitosis and give rise to oogonia that enter into meiosis by 10th^ week in human fetus and form the oocyte. The cortical cords surround the oocytes and form granulosa layer. The formation of primordial follicle, oocyte surrounded by a single layer of flattened granulosa cells, commences around 16th^ week of gestation. This is the pool from where Graffian follicles are formed (primordial-primary- secondary-tertiary/Graffian) by week 23-24 in human fetus. Oocytes proceed to the diplotene stage and remain arrested till the time of puberty.
Female differentiation of the internal genital tract is characterized by the regression of the Wollfian duct that disappears at 90 days of human fetal development. Vestiges remain in the form of Gartner canals and Rosenmüller organs. The Müllerian duct differentiates into oviducts, uterus, cervix, and upper vagina. Although the role of estrogen in female fetal development was obscure earlier, in recent years it is seen indispensable for Müllerian duct differentiation using null mouse model. The secretion of estradiol in XX gonads starts at the same time when testosterone synthesis begins in XY gonads (George & Wilson, 1978). Nonetheless, the female sex differentiation in eutherian mammals takes place in a sea of factors from the placenta, maternal circulation and the fetal gonads. Therefore, one should be cautious while describing the role of any factor in female sex differentiation.
Anomalies of Sex Determination and Differentiation:
The anomalies at any step of sex determination/differentiation leads to disorders. The alterations might be chromosomal, gonadal or phenotypical. The opportunities for mishaps are considerably high in males than females since the male sex development is very active and highly complicated process. The chromosomal abnormalities occur due to numeric changes in the sex chromosomes. For example, only one X chromosome is present in females with Turner’s syndrome (XO). The bilateral streak gonads and incomplete development of sexual characteristics with primary amenorrhea at puberty is reported in these females. On the other hand, an extra X chromosome in males due to non-disjunction of X chromosomes during oogenesis leads to Klinefelter’s syndrome (XXY). Males with this syndrome have small testis, azoospermia resulting in infertility, low concentrations of testosterone, high levels of gonadotropins and poor virilization. Similarly, triple X (XXX) syndrome is reported in females.
In case of gonadal anomalies, genetically females (XX) generally have male external genitalia. This results either due to defects in sex determining genes on autosomes or linking of a testis determining gene, otherwise present on Y chromosome, with autosome. These females have either cryptorchid testis or atleast one ovo-testis. The gonadal dysgenesis is also true for 46 XY males with defects in Y chromosome or sex determining genes on autosomes.
Phenotypical anomalies arise due to imbalance in hormone milieu or its responding machinery during development. In this case, the chromosomal sex and the gonadal sex match up, but the ambiguity in external phenotype results in pseudo-hermaphroditism. Female (XX) pseudo-hermaphroditism occurs as a result of excess androgens during embryo development. The masculinization of external genitalia is prevalent, though these females have normal ovaries. In case of XY males, pseudo-hermaphroditism might arise due to androgen
to play a pivotal role. DAX1 represses the synergistic action of SF1 and WT1 on AMH expression and also downregulates the other steroidogenic genes. Like the importance of SRY for testis differentiation in males, WNT 4 is assigned as the ovary determining gene in females. Subsequent to formation of testis or ovary from the bipotential gonads, secondary sex determination commences in which hormones play a crucial role. The virilization of Wollfian duct into epididymis, vas deferens and seminal vesicle takes place under the influence of testosterone. However, DHT, and not the testosterone, is responsible for the masculinization of male external genitalia. Further, the testicular descent into the scrotal sacs is regulated by INSL3 and androgen. The knowledge on hormonal regulation of female sex differentiation is rudimentary, however, the indispensable role of estrogen is implicated in the stabilization and differentiation of Müllerian duct into female genital tract. Unlike eutherians, the sex differentiation in marsupials is independent of hormonal control.