Sexual Differentiation In Vertebrates Research Articles

Mini review: Asymmetric Müllerian duct development in the chicken embryo.

Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract. In humans, the Müllerian ducts differentiate into the Fallopian tubes, uterus and upper portion of the vagina. In birds and reptiles, the Müllerian ducts develop into homologous structures, the oviducts. The genetic and hormonal regulation of duct development is a model for understanding sexual differentiation. In males, the ducts typically undergo regression during embryonic life, under the influence of testis-derived Anti-Müllerian Hormone, AMH. In females, a lack of AMH during embryogenesis allows the ducts to differentiate into the female reproductive tract. In the chicken embryo, a long-standing model for development and sexual differentiation, Müllerian duct development in females in asymmetric. Only the left duct forms an oviduct, coincident with ovary formation only on the left side of the body. The right duct, together with the right gonad, becomes vestigial. The mechanism of this avian asymmetry has never been fully resolved, but is thought to involve local interplay between AMH and sex steroid hormones. This mini-review re-visits the topic, highlighting questions in the field and proposing a testable model for asymmetric duct development. We argue that current molecular and imaging techniques will shed new light on this curious asymmetry. Information on asymmetric duct development in the chicken model will inform our understanding of sexual differentiation in vertebrates more broadly.

It's a trap?! Escape from an ancient, ancestral sex chromosome system and implication of Foxl2 as the putative primary sex-determining gene in a lizard (Anguimorpha; Shinisauridae).

Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally to genetically influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades are not conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate that Shinisaurus has replaced the ancestral anguimorph ZW system on LG7 with a novel ZW system on LG3. The linkage group, LG3, corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this ~1 Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor, Foxl2, critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex-determining gene for Shinisaurus.

CYP19A1 (aromatase) dominates female gonadal differentiation in chicken (Gallus gallus) embryos sexual differentiation.

Cytochrome P450 Family 19 SubFamily A member 1 (CYP19A1) gene encodes an aromatase which regulates the sexual differentiation in vertebrates by initiating and maintaining 17β-Estradiol (E2) synthesis. Here, we described the spatiotemporal expression pattern of CYP19A1 and its functional role in the embryonic gonad development in amphoteric chickens (Gallus gallus). Results showed that CYP19A1 exhibited a sexually dimorphic expression pattern in female gonads early at embryonic day 5.5 (HH 28) and robustly expressed within the cytoplasm in ovarian medullas. Most importantly, we induced the gonadal sex reversal by ectopically delivering the aromatase inhibitor (AI) or estradiol (E2) into chicken embryos. To further explore the role of CYP19A1 in chicken embryonic sexual differentiation, we successfully developed an effective method to deliver lentiviral particles with CYP19A1 manipulation into chicken embryos via embryonic intravascular injection. The analysis of interference and overexpression of CYP19A1 provided solid evidences that CYP19A1 is both necessary and sufficient to initiate sex differentiation toward female in chicken embryos. Collectively, this work demonstrates that CYP19A1 is a crucial sex differentiation gene in the embryonic development, which provides a foundation for understanding the mechanism of sex determination and differentiation in chickens.

Sexual and somatic development of wood frog tadpoles along a thermal gradient.

All amphibian species are known to have genetic sex determination. However, a variety of environmental conditions can moderate sexual differentiation, in some cases leading to sex reversal and skewed sex ratios. While there has been a recent focus on chemically-induced sex reversal in amphibians, temperature can also influence sexual differentiation. Building upon a classic 1929 study by Emil Witschi, we assessed temperature-mediated sex reversal. Witschi found that the wood frog sex ratio is 100% male at a high temperature (32°C) compared to a 50:50 sex ratio at 20°C. This pattern is consistent with multiple models of environmentally mediated sexual differentiation in vertebrates. To better understand thermally mediated sex reversal, we raised wood frogs at temperature increments of ∼1°C between 19 and 34°C. Mirroring earlier findings, wood frog metamorph sex ratios are indistinguishable from 50:50 at the lowest temperature and entirely male at the highest temperatures. In between, sex ratios become increasingly male-dominated as temperatures increase, implying a steadily increasing tendency toward female-to-male sex reversal in warmer environments. There was no evidence of a threshold temperature effect on reversal patterns. We also show that, compared to males, females metamorphose larger and later in cooler conditions but earlier and smaller under warmer conditions. While the ecological relevance in this species is unknown, these results conform to the Charnov-Bull model of sex determination (in which female-to-male sex reversal can increase fitness to genetic females at higher temperatures), suggesting the system would reward further study.

Expression changes of mRNAs encoding kisspeptins and their receptors and gonadotropin-releasing hormones during early development and gonadal sex differentiation periods in the brain of chub mackerel (Scomber japonicus)

In recent years, brain kisspeptin system has been shown to be involved in diverse reproductive function, including sexual differentiation in vertebrates. Our previous reports demonstrated that the chub mackerel (Scomber japonicus) brain expresses two kisspeptin (kiss1, kiss2), two kisspeptin receptor (kissr1, kissr2) and three gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) genes. In the present study, using quantitative real-time PCR (qRT-PCR) assays, we analysed expression changes of these genes during early development (0–30dphs) and gonadal sex differentiation periods (37–60dphs). Absolute expression level of kiss–kissr–gnrh in the whole head was higher between 0 and 15dphs, in comparison to later developmental periods. Histological analyses revealed presence of sexually differentiated males and females with testicular and ovarian features at 37, 45, and 60dphs. In both males and females, kiss2, kissr1, and kissr2 levels were higher at 37dph, in comparison to 45 and 60dphs, with kiss1 showing no significant differences. Levels of all three gnrh mRNAs were higher at 45dph, in comparison to 60dph. Changes in the expression level of kiss–kissr–gnrh mRNAs in different brain regions of sexually differentiated males and females indicated differences in their regional distribution. These results suggest possible involvement of Kiss–KissR–GnRH systems during early development and gonadal sex differentiation in the chub mackerel.

Increased exposure to yolk testosterone has feminizing effects in chickens, Gallus gallus domesticus

Competing for food by altricial and semiprecocial bird nestlings is a behaviour well known for its sensitivity to maternal androgens during prenatal development. Whether a similar effect is present in precocial species that do not beg is less well known. We therefore increased yolk testosterone levels within the physiological range at the onset of incubation to study its effects on food competition behaviour in the domestic chicken, Gallus gallus domesticus. We found an increase in competitiveness in testosterone-treated male domestic chicks, raising their level to that of the females. This is in line with the decrease in circulating plasma levels of males in the direction of the levels in females, and the overall decrease in androgen receptor densities after prenatal treatment as found previously. Hormones are known to have long-lasting organizing effects on behaviour and to affect sexual differentiation in vertebrates. Although research into hormone-mediated maternal effects has been productive, only a few studies describe (the ambiguous) effects into adulthood. Therefore we followed our animals into adulthood and recorded androgen-dependent social behaviour and secondary sexual characteristics, body mass and circulating plasma testosterone levels and checked whether these variables were treatment dependent. Treatment had a near significant effect on comb colour (both brightness and chroma). Again treatment caused a shift towards a more female-like phenotype. This suggests that, in contrast to earlier suggestions, maternal androgens may interact with (but not disrupt) sexual differentiation of brain and behaviour and the development of secondary sexual characteristics.

Exposure of Xenopus laevis tadpoles to finasteride, an inhibitor of 5-α reductase activity, impairs spermatogenesis and alters hypophyseal feedback mechanisms

Sexual steroids have major regulatory functions in gonadal development, maturation of gametes and sexual differentiation in vertebrates. Previous studies in amphibians provided evidence that dihydrotestosterone and activity of 5-alpha reductases might play a significant role in androgen-mediated reproductive biology. To test the involvement of 5-alpha reductases in maturation of gametes in amphibians, Xenopus laevis was exposed to finasteride (FIN), a known inhibitor of 5-alpha reductase enzyme activity. In a long-term exposure from stage 46 to 66, severe disruption of spermatogenesis was observed in histological analysis of testes as detected by occurrence of empty spermatocysts, while ovaries remained unaffected. Real-time PCR analyses of male and female brain revealed an increase of LHbeta mRNA and a decrease of FSHbeta mRNA in males, suggesting a signalling on testes that could result in increased steroidogenesis and reduced Sertoli cell proliferation. Accordingly, the mRNA expression of P450 side chain cleavage enzyme and 5-alpha reductase type 2 was increased in testes, while no effects could be observed on steroidogenic genes in ovaries. A short-term exposure to testosterone, FIN and testosterone+FIN showed that transient effects of FIN targeted males selectively and, in particular, interfered with the hypothalamus-pituitary-gonad axis. Furthermore, a negative feedback of testosterone on LHbeta was observed on males and females. This study provides evidence that exposure of X. laevis to FIN, an inhibitor of 5-alpha reductases, impaired spermatogenesis and involved sex-specific hypophyseal feedback mechanisms.

A functionally conserved member of the FTZ-F1 nuclear receptor family from Schistosoma mansoni.

The fushi tarazu factor 1 (FTZ-F1) nuclear receptor subfamily comprises orphan receptors with crucial roles in development and sexual differentiation in vertebrates and invertebrates. We describe the structure and functional properties of an FTZ-F1 from the platyhelminth parasite of humans, Schistosoma mansoni, the first receptor from this family to be characterized in a Lophotrochozoan. It contains a well conserved DNA-binding domain (55-63% identity to other family members) and a poorly conserved ligand-binding domain (20% identity to that of zebrafish FF1a). However, both the ligand domain signature sequence and the activation function 2-activation domain (AF2-AD) are perfectly conserved. Phylogenetic analysis confirmed that SmFTZ-F1 is a member of nuclear receptor subfamily 5, but that it clustered with the Drosophila receptor DHR39 and has consequently been named NR5B1. The gene showed a complex structure with 10 exons and an overall size of 18.4 kb. Two major transcripts were detected, involving alternative promoter usage and splicing of the two 5' exons, but which encoded identical proteins. SmFTZ-F1 mRNA is expressed at all life-cycle stages with the highest amounts in the larval forms (miracidia, sporocysts and cercariae). However, expression of the protein showed a different pattern; low in miracidia and higher in adult male worms. The protein bound the same monomeric response element as mammalian SF-1 (SF-1 response element, SFRE) and competition experiments with mutant SFREs showed that its specificity was identical. Moreover, SmFTZ-F1 transactivated reporter gene transcription from SFRE similarly to SF-1. This functional conservation argues for a conserved biological role of the FTZ-F1 nuclear receptor family throughout the metazoa.

Influence of environmental factors on sexual differentiation in vertebrates: Evolutionary aspects

Temperature has been shown to influence sexual differentiation of the gonads in many species of ectothermic vertebrates. Other environmental factors such as humidity, pH, and the ionic ratio of K +/Ca ++ could also modify sexual differentiation. As a general rule, species sensitive to these factors do not display heteromorphic sex chromosomes. However, breeding experiments in fishes and amphibians, cytogenetic studies in amphibians, and many observations in reptiles show that in these species sex determination is genotypic. Therefore, environmental factors appear to interact with genetic factors. In reptiles, morphological differentiation of sexchromosomes appears to be of relatively recent origin, whereas temperature sensitivy of gonadal differentiation (described in many species of turtles, three species of crocodiles and two species of lizards) is probably a much more primitive character. Persistence of the phenomenon of sensitivity to environmental factors in many modern species could be due to a possible lack or to a low rate of mutations leading to a strictly genotypic mechanism of sex determination. It appears to be correlated with a relative general stability of the genome allowing conservation also of other primitive characters in these species. However, its impact on the maintenance of species has not yet been fully elucidated. Understanding of the evolutionary significance ofsensitivity of sexual differentiation to environmental factors requires a thorough knowledge of the evolution of sex chromosomes in vertebrates, of the molecular mechanisms involved in sexual differentiation of the gonads and of reproductive strategies of various species in response to environmental variations.