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.     

New insights on the mechanism of testis differentiation from the morphogenesis of experimentally induced testes in genetically female chick embryos

Embryonic testes grafted in the extraembryonic coelom of 3-day-old genetically female chick embryos may induce total and definitive reversal of gonadal sex differentiation. In this experimental condition, the left gonad becomes a testis instead of an ovary. This makes it possible to compare testicular and ovarian morphogenesis in animals having the same genetic sex and to discount what is due to differences in the genetic determination between male and female. The morphogenesis of such testes is marked by a disappearance of the cortical germinal epithelium. The medullary sex cords keep a narrow lumen instead of becoming large lacunae. The germ cells remain few in the sex cords and do not become meiotic. Furthermore, interstitial cell development is known to be very slow. As a consequence the gross size of the gonad is much smaller than that of an ovary. All these morphogenetic phenomena are unlike those observed during normal ovarian differentiation and evidence an inhibiting influence of the grafted testes. Since inhibition and masculinization are concomitant, inhibition appears to be the mechanism responsible for gonadal sex reversal. The extraembryonic situation of the grafted testes and their relation with the embryo only via the blood stream demonstrates the role of a secreted substance or substances still to be exactly identified. Previous data suggest that this could be the anti-Müllerian-hormone (AMH). Furthermore, previous and present results show that testis differentiation can be actively induced in a bird. This does not agree with the hypothesis that the gonads of the homogametic sex, i.e., the testes in birds, do not need any inducer in order to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reproductive cycle of yellowfin bream, Acanthopagms australis (Günther), with particular reference to protandrous sex inversion

Yellowfin bream, Acanthopagrus australis , of all age classes were collected from Moreton Bay, Australia. The species possessed typical sparid ovotestes in which the testis and ovary occur in separate zones. During the spawning period (June-August) juveniles, functional males and functional females could be distinguished by the macroscopic appearance of the gonad. The sex ratio of males to females decreases with age, indicating protandrous sex inversion.
Histological and structural study of the ovotestis showed all fish have previtellogenic cells in the ovarian zone but only juvenile and male fish have developing spermatogenic cells in the testis. Most juveniles become functional males by the age of two years but a small proportion of juveniles develop directly into functional females (primary females). Protandrous sex inversion commences after the spawning period when male fish appear with spermatozoa and no other spermatogenic cells in the testis. During the period November-January male fish with no spermatogenic cells are common and a reduction in size of the testis occurs so that by March-April the ovotestis becomes structurally and histologically similar to the female ovotestis. Some fish remain functional males during their whole life-history (primary males). In functional females vitellogenic cells are present in the ovary only during the spawning period and the testis remains very small in size.     

Abnormalities of the gonads of carp

Cyprinus curpio is usually dioecious, with well–developed paired gonads (ovaries or testes) of which the two parts are approximately equal in weight. However, a few instances of abnormal development have been observed. In one male fish, the right testis developed normally to maturity, while the left testis was vestigial. In a female fish, only the right component of the ovary was present. Some monoecious, synchronous hermaphrodite specimens have also been observed, and self fertilization demonstrated experimentally. It has been shown (Kossmann, 1971) that the progeny of such synchronous hermaphrodites may also have this characteristic. These studies demonstrate that even such a highly evolved teleost as the Carp may retain the primitive characteristic of hermaphroditism.     

The reproductive biology of blackspot sea bream Pagellus bogaraveo in captivity. I. gonadal development, maturation and hermaphroditism

Gonadal development, maturation, spawning and hermaphroditism were investigated in captive blackspot sea bream, Pagellus bogaraveo Brünnich, 1768, during the second, third and fourth years of life. The gonads of 224 fish were examined macroscopically and histologically. Undifferentiated gonads were found in fish smaller than 22.0 cm. Adult fish showed four gonadal differentiations: ovotestes with functional testis and quiescent ovary (Mf), ovotestes with functional ovary and regressed testis (mF), ovotestes with both ovary and testis at a resting stage (mf), and ovaries with no male tissue (F). The overall incidence of gonochoric females F was 41%. Functional males Mf were more frequent in age classes 1+ and 2+, whereas functional females mF predominated in the 3+ age class, above 25.0 cm TL. Histological examination revealed testicular degeneration and atrophy in functional females mF. On the basis of both histological data as well as size and age frequency distribution, it is suggested that the reproductive strategy of P. bogaraveo in captivity is characterized by protandrous hermaphroditism, with a high incidence of female gonochorism. Spawning occurred in March–April, at a size of 28.0 cm and age 3 in males and at 29.5 cm and age 4 in females. The gonosomatic index (GSI) remained constantly low (≤ 0.05) throughout the second and third years of life. A significant increase in GSI was noted in both males and females at the accomplishment of the fourth year of life, coinciding with the spawning season. The results are compared with information available on wild P. bogaraveo and discussed with a view to a possible exploitation of this species in aquaculture, through a reliable control of reproduction.     

Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Ovotestis development in B6-XY^POS mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY^POS ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.     

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.     

Lateral hermaphroditism in a C3H mouse.

A spontaneous case of true lateral hermaphroditism was observed in one of approximately 1000 necropsies of 12-wk-old female C3Hf-Wg mice (a substrain of C3H/He). Both the right ovary and abdominal left testis were functional as evidenced by the presence of oocytes in graffian follicles and spermatocytes maturing on sertoli cells. Both gonads communicated, the ovary via an oviduct and normal right uterine horn and the testis via an epididymus and vas deferens, with a vagina which ended in a blind pouch and was filled with squamous debris.     

Transmission et expression des caractères ‘ovaire’ et ‘testicule’ dans des clones de Némertes allophéniques produits par multiplication végétative de chimères bipartites mâle/femelle et femelle/mâle chez Lineus sanguineus

Summary

Two stable strains of bilaterally allophenic nemertines were obtained 15 years ago by vegetative multiplication of two bipartite chimeras constructed from symmetrical worm halves of opposite sexes.

From year 1 to 3 of the organismal cloning, the ‘testis’ and ‘ovary’ characteristics differentiated at the onset of the sexual development in respectively male and female sides of allophenic nemertines. Subsequently, unilateral sex reversal by substitution of ovaries for testes regularly occurred as a secondary process.

The 4th year, transient ovotestes were the only type of gonad showing features of masculinity in clones. ‘Pure’ testes did not differentiate.

From year 5 to 9, the ‘ovary' characteristic alone expressed in the two sides of the allophenic worms’ body. Such a transmission of the primary feminization phenotype by organismal cloning showed that the genetic determinant of the testicular differentiation either had been eliminated or was permanently repressed.

From year 10 up to date, the differentiation of the single ‘ovary’ characteristic was again the usual pattern of the gonadogenesis. However, some worms from a sub-clone differentiated a few transient testes in year 10. Although this male-gonad-phenotype reminiscence was discreet, it showed that the testicular determinant had not been eliminated.

The present data support the hypothesis that allophenic worms derived from vegetative multiplication of male-female chimeras retain genetically male and female cells but that interactions between cells of two genetic sexes eventually result in complete repression of the testicular determinant.     

Influence of embryonic and adult testis on the differentiation of embryonic ovary in the mouse.

The development of 11 1/2-13 1/2-day embryonic mouse ovaries subjected to the influence of adult and embryonic testes was investigated. The environment of adult testis caused severe restriction of ovarian growth, but did not produce any effects which might be considered as masculinization. The presence of embryonic testis was distinctly unfavourable to the embryonic ovary, resulting in restriction of the growth of the latter and degeneration of oocytes. Reversal of the course of differentiation of genetically female germ cells has never been observed.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321–337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25–33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ “oogonia” were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm. © 1995 wiley-Liss, Inc.     

PITX2 controls asymmetric gonadal development in both sexes of the chick and can rescue the degeneration of the right ovary

The gonads arise on the ventromedial surface of each mesonephros. In most birds, female gonadal development is unusual in that only the left ovary becomes functional, whereas that on the right degenerates during embryogenesis. Males develop a pair of equally functional testes. We show that the chick gonads already have distinct morphological and molecular left-right (L-R) characteristics in both sexes at indifferent (genital ridge) stages and that these persist, becoming more elaborate during sex determination and differentiation, but have no consequences for testis differentiation. We find that these L-R differences depend on the L-R asymmetry pathway that controls the situs of organs such as the heart and gut. Moreover, a key determinant of this, Pitx2, is expressed asymmetrically, such that it is found only in the left gonad in both sexes from the start of their development. Misexpression of Pitx2 on the right side before and during gonadogenesis is sufficient to transform the right gonad into a left-like gonad. In ZW embryos, this transformation rescues the degenerative fate of the right ovary, allowing for the differentiation of left-like cortex containing meiotic germ cells. There is therefore a mechanism in females that actively promotes the underlying L-R asymmetry initiated by Pitx2 and the degeneration of the right gonad, and a mechanism in males that allows it to be ignored or overridden.     

Testes of XX ↔ XY chimeric mice develop from fetal ovotestes

The majority of XX ? XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12–14 postcoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX ? XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

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.     

Behavioral and morphological asymmetries in chukar Alectoris chukar copulation

Birds often exhibit greater reproductive tract development on the left side than right side. Behavioral evidence from the three species for which data has been published indicates that these species copulate more frequently on the left side of females than on the right side. Missing from the literature are studies that compare asymmetry in copulation behavior to asymmetry in reproductive tract morphology of the same individuals of both sexes within a single species. To better understand the potential for cryptic sexual selection to influence avian copulation, we measured asymmetries in chukar Alectoris chukar copulation using 24 male and 29 female chukar brought into captivity from the wild. Chukar copulated (n=37) more from the left side (n=30) of females than the right side (n=7). The left testis of males was consistently greater in size, mass and volume than the right testis. The left ovary and oviduct of females was consistently functional with no observable development of the right ovary or oviduct. Left-side bias in direction of copulation, larger left testes, and functional left vaginal openings may act in concert to deliver spermatozoa to the oviduct, promoting fertilization.     

Why boys will be boys and girls will be girls: Human sex development and its defects

Among the most defining events of an individual's life, is the development of a human embryo into male or a female. The phenotypic sex of an individual depends on the type of gonad that develops in the embryo, a process which itself is determined by the genetic setting of the individual. The development of the gonads is different from any other organ, as they possess the potential to differentiate into two functionally distinct organs, testes, or ovaries. Sex development can be divided into two distinctive processes, “sex determination,” which is the commitment of the undifferentiated gonad into either a testis or an ovary, a process that is genetically programmed in a critically timed manner and “sex differentiation,” which takes place through hormones produced by the gonads, once the developmental sex determination decision has been made. Disruption of any of the genes involved in either the testicular or ovarian development pathway could lead to disorders of sex development. In this review, we provide an insight into the factors important for sex determination, their antagonistic actions and whenever possible, references on the “prismatic” clinical cases are given. Birth Defects Research (Part C) 108:365–379, 2016. © 2016 Wiley Periodicals, Inc.     

A study of meiosis in chimeric mouse fetal gonads

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.     

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.