Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.     

Development of the testes in female domestic fowls submitted to an experimental sex reversal during embryonic life

The sex differentiation of the female chick embryo can be totally inverted toward the male sex by an early extraembryonic testis grafting. This sex reversal remains permanent, as shown by three adult fowls described in this paper. They possess two testes associated with normally differentiated male excretory ducts and their Müllerian ducts have regressed. The development of male sex characteristics such as external features, behavior and complete spermatogenesis is evidence that these cocks have endocrine and exocrine capabilities similar to those of normal cocks. Although these cocks were able to mate with female fowls, they were sterile. A mechanism is discussed by which grafted testes induce such modifications in females. Hypotheses considering the heterogametic sex (female in birds) as exerting a dominant influence on the phenotypic sexual differentiation can be discarded in light of our results because a homogametic testis provokes the definitive sex inversion of a female.     

Charting the course of ovarian development in vertebrates

The decision of the embryonic gonad to differentiate as either a testis or an ovary is a critical step in vertebrate development. The molecular basis of this decision has been the focus of much study, particularly over the past decade. Here we contrast the knowledge of early gonadal development and the switch to testis differentiation with the lack of molecular understanding of ovarian development at early stages. We review current knowledge regarding mechanisms of ovarian morphogenesis and propose a model for the hierarchical control of development of the fetal ovary, incorporating the few genes already known to be important and several signals or factors that are hypothesised to exist in the early ovary.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Crossed graft of embryonic testis between quail and chick embryos: contribution to the study of the mechanism of female bird embryo masculinization]

Quail, or chick embryonic testes grafted respectively in the extraembryonic coelom of chick or quail embryos induce both a Mullerian duct regression and a masculinization of the female host gonads up to the differentiation of two testes, in some cases. Such a result confirms the fact evidenced previously in other bird species (chick and duck) that the testis-inducer is interspecific. Quail cells are not observed in histological sections of embryonic gonads of testis-grafted chicks. This allows to discard a possible influence of cells migrating from the graft to the host. The grafted testes act then through substance/s secreted in the blood stream. Present and previous experimental data strongly suggest that the same substance, i.e. the so-called anti-Mullerian hormone, is responsible for both MD regression and gonadal sex reversal.     

Testicular graft on the chick embryo

A testis from an 18-day-old chick embryo was transplanted into the extra-coelomic cavity of 3-4-day-old hosts. The embryos surviving at 17 days were sacrificed and their genital system was examined. Testis grafting produced inhibition of testicular development. Development of the female gonads was also inhibited. A more or less complete modification of sex was associated with this inhibition. The left ovary lost its cortex, but its medulla remained mostly ovarian in structure. The right gonad frequently acquired a typical testicular structure. These results confirm the possibility of obtaining sex reversal in the female chick embryo by testis grafting.     

Sex-specific localization of laminin alpha 5 chain in the differentiating rat testis and ovary

The localization of laminin (Ln) alpha 5, beta 1 and beta 2 chains in the differentiating rat testis and ovary was studied by immunolabeling light and electron microscopy. The initial formation of the male and female gonadal blastemas included an emergence of Ln alpha 5 and beta 1 chains, but not of Ln beta 2 chain. The sexual differentiation of the embryonic male gonadal cords included rapid sex-specific disappearance of the incipient Ln alpha 5 chain. The rete testis cords, in contrast, remained positive for Ln alpha 5 chain. In the postnatal testis, the Ln alpha 5 chain reappeared in Ln beta 1 chain-positive cord basement membranes, which also became positive for Ln beta 2 chain. The differentiating myoid cells also gradually became positive for both Ln alpha 5 and Ln beta 1 chains. In the ovary Ln alpha 5 chain persisted in BMs of the cords throughout the fetal phase. Small and newly formed follicles in the early postnatal rat ovary were also positive for Ln alpha 5 chain, whereas growing and large follicles were negative. During the early postnatal phase, Ln beta 1-chain positive follicular BMs became also positive for the Ln beta 2 chain. Basement membranes of testicular and ovarian surface epithelia contained the Ln alpha 5 chain throughout the study. The blood vessels of the male and female gonad showed differentiation-dependent variation in their reactivity for the Ln alpha 5 and beta 2 chains. The present results show that the Ln alpha 5 chain is an early molecular marker for sexual differentiation, which therefore may be regulated by the testis-determining factors. The results also show that in the early postnatal rat ovary, the follicular basement membranes are heterogeneous in their Ln content, which may offer a means to distinguish different follicular populations from each other and to identify the different stages of follicular growth.     

Sex differentiation and pubertal development of gonads in the viviparous mosquitofish, Gambusia affinis

The ontogenetic development of gonads from embryo to adult was observed histologically in the viviparous teleost, Gambusia affinis. Primordial germ cells (PGCs) appeared in the subendodermal space of the embryo 14 days before birth, and then transferred to the dorsal mesentery to form paired genital ridges 12 days before birth. The PGCs proliferated in the genital ridge, forming gonadal primordia 10 days before birth. All gonadal primordia differentiated to the ovary containing oocytes 2 days before birth, but then redifferentiated to the ovary and testis just after birth. This indicates that the mosquitofish is a juvenile hermaphroditic species. The characteristics of gonadal sex differentiation just after birth were enlargement of the oocytes in females, and invasion of somatic cells from the hilar region to an inner portion of the gonad in males. The paired ovary fused at the basal area 5 days after birth, then on the ventral and dorsal portions, developing into a single ovary 10 days after birth. During this time a single ovarian cavity was formed on the dorsal portion of the ovary. The paired testes fused only at the basal area and became a single testis having two main lobes 10 days after birth. The oocytes gradually developed and began vitellogenesis 100 days after birth, but did not reach maturation until 110 days after birth. Spermatogenic cells formed cysts at 20 days, began meiosis at 70 days, and matured to form sperm balls 90 days after birth. The male fish sexually matured earlier than the female.     

Germ cells, gonads and sex reversal in marsupials.

The formation of the testis or ovary is a critical step in development. Alterations in gonadal development during fetal or postnatal life can lead to intersexuality or infertility. Several model systems have been particularly useful in studying gonadal differentiation, the eutherian mammal and amphibia, fish, and birds. However, marsupials provide a unique opportunity to investigate gonadal development and the interactions of genes and hormones in gonadal differentiation and germ cell development in all mammals. On the one hand the genetic mechanisms appear to be identical to those in eutherian mammals, including the testis-determining SRY gene. On the other hand, marsupials retain in part the plasticity of the amphibian gonad to hormonal manipulation. It is possible to induce female to male and also male to female gonadal sex reversal in marsupials by hormonal manipulation, and oestradiol can induce male germ cells to enter meiosis at the time the oogonia do. In addition, in marsupials the development of the scrotum and mammary glands are independent of testicular androgens and instead are controlled by a gene or genes on the X-chromosome. Thus marsupials provide a number of opportunities for manipulating the sexual differentiation of the gonads that are not possible in eutherian mammals and so provide a unique perspective for understanding the common mechanisms controlling sexual development.     

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.     

Gonadal morphogenesis and sex differentiation in cultured Ussuri catfish Tachysurus ussuriensis

The objective of this study was to investigate the optimal developmental time to perform sex reversal in Ussuri catfish Tachysurus ussuriensis, to develop monosex breeding in aquaculture. Systematic observations of gonadal sex differentiation of P. ussiriensis were conducted. The genital ridge formed at 9 days post fertilization (dpf) and germ cells begin to proliferate at 17 dpf. The ovarian cavity began forming on 21 dpf and completed by 25 dpf while presumptive testis remained quiescent. The primary oocytes were at the chromatin nucleolus stage by 30 dpf, the peri‐nucleolus stage by 44 dpf and the cortical alveoli stage by 64 dpf. The germinal vesicle migrated towards the animal pole (polarization) at 120 dpf. In presumptive testis, germ cells entered into mitosis and blood vessels appeared in the proximal gonad on 30 dpf. The efferent duct anlage appeared on 36 dpf and formation of seminal lobules with spermatogonia and lobules interstitium occurred at 120 dpf. Therefore, gonadal sex differentiation occurred earlier in females than in males, with the histological differentiation preceding cytologic differentiation in T. ussuriensis. This indicates that undifferentiated gonads directly differentiate into ovary or testis between 17 and 21 dpf and artificial induction of sexual reversal by oral steroid administration must be conducted before 17 dpf.     

Lectin-binding carbohydrates in sexual differentiation of rat male and female gonads.

Development and sexual differentiation of the mammalian gonad involve changes in the type and distribution of different proteins and glycoproteins in and around the epithelial gonadal cords, the future seminiferous tubules in the testis, and follicles in the ovary. To study changes in cellular carbohydrate-containing compounds in the sex-specific morphogenesis of rat gonads, sections from embryonic, fetal and early postnatal gonads were labelled with seven different fluorescein isothiocyanate (FITC)-conjugated plant lectins of various carbohydrate-binding specificities. Double labelling of laminin with tetramethylrhodamine isothiocyanate (TRITC)-conjugated antibodies was used to outline the epithelial tissues. From the results we conclude that the abundance and similar distribution of carbohydrates in the early gonads of both sexes supports their sexually indifferent nature. Furthermore, the basement membranes of the differentiating gonadal cords in both sexes have common features, which differ, however, from those of the other developing urogenital organs. Changes in carbohydrate composition appear with the sexual differentiation of the gonads; the similarity of the changes in lectin binding to the gonadal cords of embryonic and fetal male, and to postnatal female, suggests similar mechanisms of cell-cell interactions in both sexes although activated at different developmental stages. These carbohydrate specificities at the tissue level should be taken into account together with cell-type specific changes, e.g. in the formation of the zona pellucida, when the phenomenon of polymorphic expression of different compounds is integrated into theories of epithelial differentiation.     

Lectin-binding carbohydrates in sexual differentiation of rat male and female gonads

Summary Development and sexual differentiation of the mammalian gonad involve changes in the type and distribution of different proteins and glycoproteins in and around the epithelial gonadal cords, the future seminiferous tubules in the testis, and follicles in the ovary. To study changes in cellular carbohydrate-containing compounds in the sex-specific morphogenesis of rat gonads, sections from embryonic, fetal and early postnatal gonads were labelled with seven different fluorescein isothiocyanate (FITC)-conjugated plant lectins of various carbohydrate-binding specificities. Double labelling of laminin with tetramethylrhodamine isothiocyanate (TRITC)-conjugated antibodies was used to outline the epithelial tissues. From the results we conclude that the abundance and similar distribution of carbohydrates in the early gonads of both sexes supports their sexually indifferent nature. Furthermore, the basement membranes of the differentiating gonadal cords in both sexes have common features, which differ, however, from those of the other developing urogenital organs. Changes in carbohydrate composition appear with the sexual differentiation of the gonads; the similarity of the changes in lectin binding to the gonadal cords of embryonic and fetal male, and to postnatal female, suggests similar mechanisms of cell-cell interactions in both sexes although activated at different developmental stages. These carbohydrate specificites at the tissue level should be taken into account together with cell-type specific changes, e.g. in the formation of the zona pellucida, when the phenomenon of polymorphic expression of different compounds is integrated into theories of epithelial differentiation.     

Gadd45g Is Essential for Primary Sex Determination,Male Fertility and Testis Development

In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g^−/− XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g^−/− XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Müllerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans.