Evolution Of Sex-determining Mechanisms Research Articles

Identification, Expression and Evolutional Analysis of Two cyp19-like Genes in Amphioxus

The mechanism of sex determination and differentiation in animals remains a central focus of reproductive and developmental biology research, and the regulation of sex differentiation in amphioxus remains poorly understood. Cytochrome P450 Family 19 Subfamily A member 1 (CYP19A1) is a crucial sex differentiation gene that catalyzes the conversion of androgens into estrogens. In this study, we identified two aromatase-like genes in amphioxus: cyp19-like1 and cyp19-like2. The cyp19-like1 is more primitive and may represent the ancestral form of cyp19 in zebrafish and other vertebrates, while the cyp19-like2 is likely the result of gene duplication within amphioxus. To gain further insights into the expression level of these two aromatase-like, we examined their expression in different tissues and during different stages of gonad development. While the expression level of the two genes differs in tissues, both are highly expressed in the gonad primordium and are primarily localized to microsomal membrane systems. However, as development proceeds, their expression level decreases significantly. This study enhances our understanding of sex differentiation mechanisms in amphioxus and provides valuable insights into the formation and evolution of sex determination mechanisms in vertebrates.

Direct male development in chromosomally ZZ zebrafish.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish (Danio rerio), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome, or fewer than two Z chromosomes, is essential to initiate oocyte development; and without the W factor, or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

Latitudinal Variation in the Pattern of Temperature-Dependent Sex Determination in the Japanese Gecko, Gekko japonicus.

Simple SummaryIn egg-laying lizards, sex is determined by genetic factors in species with sex chromosomes or egg incubation temperatures in species without sex chromosomes, i.e., temperature-dependent sex determination (TSD). Surprisingly, recent studies find sex chromosomes and TSD co-occur in the same species in some lizards. The Japanese gecko from Japan may be this case. However, Japanese gecko with TSD from a Chinese population does not have sex chromosomes, suggesting that the pattern of TSD in this gecko may vary among populations. We incubated gecko eggs from three populations in China at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the sex determination pattern. We found that the temperature yielding an equal number of sons and daughters of the low-latitude population was lower than that of the two high-latitude populations. Moreover, the low-latitude population had a narrower temperature range producing mixed sex offspring at lower temperatures, but a wider range at higher temperatures. Sex ratio was almost 1:1 for the low-latitude population when incubated from 26 to 30 °C. Conversely, more male offspring were produced at 28 or 30 °C in the two high-latitude populations. Our study may provide an interesting system to explore the evolution of sex determination mechanisms in animals.Identifying latitudinal variation in the pattern of temperature-dependent sex determination (TSD) may provide insight into the evolution of sex determining system in vertebrates, but such studies remain limited. Here, we quantified TSD patterns of three geographically separated populations of the Japanese gecko (Gekko japonicus) along the latitudinal cline of China. We incubated gecko eggs from the three populations at constant temperatures of 24, 26, 28, 30, and 32 °C to quantify the TSD pattern. Our study demonstrated that G. japonicus exhibited a FMF pattern of TSD, with the low and high incubation temperatures yielding significantly female-biased hatchlings, and the medium temperatures producing male-biased hatchlings. More interestingly, we found latitudinal variations in the TSD pattern in terms of pivotal temperatures (Tpivs), transitional range of temperatures (TRT), and the sex ratios at the medium temperatures. The Tpivs for the low-latitude population were lower than those for the two high-latitude populations. The low-latitude population has a narrower FM TRT, but a wider MF TRT. The sex ratio is almost 50:50 for the low-latitude population when eggs were incubated from 26 to 30 °C. Conversely, the sex ratio is male-biased for the two high-latitude populations at 28 or 30 °C. Therefore, G. japonicus may provide an interesting system to explore the evolution of TSD in reptiles given the diversity of TSD patterns among populations.

ROSIE, a database of reptilian offspring sex ratios and sex-determining mechanisms, beginning with Testudines

In contrast to genotypic sex determination (GSD), temperature-dependent sex determination (TSD) in amniotic vertebrates eludes intuitive connections to Fisherian sex-ratio theory. Attempts to draw such connections have driven over 50 years of research on the evolution of sex-determining mechanisms (SDM), perhaps most prominently among species in the order Testudines. Despite regular advancements in our understanding of this topic, no efforts have been published compiling the entirety of data on the relationships between incubation temperature and offspring sex in any taxonomic group. Here, we present the Reptilian Offspring Sex and Incubation Environment (ROSIE) database, a comprehensive set of over 7,000 individual measurements of offspring sex ratios in the order Testudines as well as SDM classifications for 149 species. As the name suggests, we plan to expand the taxonomic coverage of ROSIE to include all non-avian reptiles and will regularly release updates to maintain its comprehensive nature. This resource will enable crucial future research probing the ecology and evolution of SDM, including the presumed sensitivity of TSD to rapid environmental change.

Phenotypic/genotypic sex mismatches and temperature-dependent sex determination in a wild population of an Old World atherinid, the cobaltcap silverside Hypoatherina tsurugae.

Several New World atheriniforms have been recognized as temperature-dependent sex determined (TSD) and yet possess a genotypic sex determinant (amhy) which is primarily functional at mid-range temperatures. In contrast, little is known about the sex determination in Old World atheriniforms, even though such knowledge is crucial to understand the evolution of sex determination mechanisms in fishes and to model the effects of global warming and climate change on their populations. This study examined the effects of water temperature on sex determination of an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae, in which we recently described an amhy homologue. We first assessed the occurrence of phenotypic/genotypic sex mismatches in wild specimens from Tokyo Bay for three years (2014-2016) and used otolith analysis to estimate their birth dates and approximate thermal history during the presumptive period of sex determination. Phenotypic sex ratios became progressively biased towards males (47.3%-78.2%) during the period and were associated with year-to-year increases in the frequency of XX-males (7.3%-52.0%) and decreases in XY/YY-females (14.5%-0%). The breeding season had similar length but was delayed by about 1month per year between 2014 and 2016, causing larvae to experience higher temperatures during the period of sex determination from year to year. Larval rearing experiments confirmed increased likelihood of feminization and masculinization at low and high temperatures, respectively. The results suggest that cobaltcap silverside has TSD, or more specifically the coexistence of genotypic and environmental sex determinants, and that it affects sex ratios in wild populations.

Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation.

Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Müllerian hormone (amh) as the MSD gene in Northern Pike (Esox lucius), using genomic and expression evidence as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study reveals an unexpectedly low level of differentiation between a pair of sex chromosomes harboring an old MSD gene in a wild teleost fish population, and highlights both the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade.

Evolution of sex determination mechanisms and sex chromosomes

The evolution of sex determination mechanisms and sex chromosomes has always been a central research topic in evolutionary biology. Because the sex determination process occurs during the early stage of development, and the regulation of sex-linked genes often involves non-coding RNAs and epigenetic modifications, this topic is an interdisciplinary hotspot with developmental biology and molecular biology. This review elaborates on the importance and origin of sex, numerous ways of sex determination, and the mechanisms of sex chromosome evolution. We will summarize the reported sex-determining genes and introduce the population genetic models under which the sex chromosomes evolve without homologous recombination. To date, only a few sex-determining genes have been discovered, but they have already exhibited a great diversity beyond biologists’ expectation. Future research directions will focus on identifying more upstream sex-determining genes for plants and animals and their downstream sex-determining pathways. New genome research and gene knockout techniques will develop other appropriate model organisms in this area, and to ultimately address the basic biological questions that why different organisms need to evolve such a great variety of sex-determining ways and how they transit to each other.

Effects of Temperature on the Expression of Two Ovarian Differentiation-Related Genes foxl2 and cyp19a1a.

Exposure to stress induces a series of responses and influences a wide range of biological processes including sex differentiation in fish. The present work investigated the molecular and physiological response to thermal stress throughout the early development stage covering the whole period of sex differentiation of bluegill, Lepomis macrochirus. Larvae were treated using three temperatures, 17, 24, and 32°C from 6 to 90 days posthatching (dph) in 30-L round tanks. There is no significant difference of the sex ratio and survival among the three temperature groups in the geographic population used in this study. Two ovarian differentiation-related genes foxl2 and cyp19a1a were detected at 7 dph suggesting that these genes have already played a role prior to sex differentiation. The expression of foxl2 reached the peak and was thermosensitive just prior to the onset of ovarian differentiation at 27 dph. Histological examination displayed that the proliferation of germ cells and ovarian differentiation were delayed at the low-temperature treatment (17°C) at 97 dph compared with higher temperatures. In conclusion, the water temperature regulates the sex differentiation of bluegill through modulation of the expression of foxl2 and cyp19a1a. A comparative study of the expression profile of sex differentiation-related genes in species will shed light on the evolution of sex-determination mechanisms and the impact of stress on sex differentiation.

Evolutionary Significance of Wolbachia-to-Animal Horizontal Gene Transfer: Female Sex Determination and the f Element in the Isopod Armadillidium vulgare.

An increasing number of horizontal gene transfer (HGT) events from bacteria to animals have been reported in the past years, many of which involve Wolbachia bacterial endosymbionts and their invertebrate hosts. Most transferred Wolbachia genes are neutrally-evolving fossils embedded in host genomes. A remarkable case of Wolbachia HGT for which a clear evolutionary significance has been demonstrated is the “f element”, a nuclear Wolbachia insert involved in female sex determination in the terrestrial isopod Armadillidium vulgare. The f element represents an instance of bacteria-to-animal HGT that has occurred so recently that it was possible to infer the donor (feminizing Wolbachia closely related to the wVulC Wolbachia strain of A. vulgare) and the mechanism of integration (a nearly complete genome inserted by micro-homology-mediated recombination). In this review, we summarize our current knowledge of the f element and discuss arising perspectives regarding female sex determination, unstable inheritance, population dynamics and the molecular evolution of the f element. Overall, the f element unifies three major areas in evolutionary biology: symbiosis, HGT and sex determination. Its characterization highlights the tremendous impact sex ratio distorters can have on the evolution of sex determination mechanisms and sex chromosomes in animals and plants.

Dmrt1 induces the male pathway in a turtle species with temperature-dependent sex determination.

The molecular mechanism underlying temperature-dependent sex determination (TSD) has been a long-standing mystery; in particular, the thermosensitive genetic triggers for gonadal sex differentiation are largely unknown. Here, we have characterized a conserved DM domain gene, Dmrt1, in the red-eared slider turtle Trachemys scripta (T. scripta), which exhibits TSD. We found that Dmrt1 has a temperature-dependent, sexually dimorphic expression pattern, preceding gonadal sex differentiation, and is capable of responding rapidly to temperature shifts and aromatase inhibitor treatment. Most importantly, loss- and gain-of-function analyses provide solid evidence that Dmrt1 is both necessary and sufficient to initiate male development in T. scripta Furthermore, the DNA methylation dynamics of the Dmrt1 promoter are tightly correlated with temperature and could mediate the impact of temperature on sex determination. Collectively, our findings demonstrate that Dmrt1 is a candidate master male sex-determining gene in this TSD species, consistent with the idea that DM domain genes are conserved during the evolution of sex determination mechanisms.

The evolution of mating type switching

Predictions about the evolution of sex determination mechanisms have mainly focused on animals and plants, whereas unicellular eukaryotes such as fungi and ciliates have received little attention. Many taxa within the latter groups can stochastically switch their mating type identity during vegetative growth. Here, we investigate the hypothesis that mating type switching overcomes distortions in the distribution of mating types due to drift during asexual growth. Using a computational model, we show that smaller population size, longer vegetative periods and more mating types lead to greater distortions in the distribution of mating types. However, the impact of these parameters on optimal switching rates is not straightforward. We find that longer vegetative periods cause reductions and considerable fluctuations in the switching rate over time. Smaller population size increases the strength of selection for switching but has little impact on the switching rate itself. The number of mating types decreases switching rates when gametes can freely sample each other, but increases switching rates when there is selection for speedy mating. We discuss our results in light of empirical work and propose new experiments that could further our understanding of sexuality in isogamous eukaryotes.

Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.

Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal gonads showed typical testicular size and vasculature. Key ovarian markers, including Wnt4 and Foxl2, were repressed. Sertoli cells expressing the hallmark testis-determining gene Sox9 were formed, although they did not assemble into normal testis cords. Other bipotential lineages differentiated into testicular cell types, including steroidogenic fetal Leydig cells and non-meiotic germ cells. As a consequence, male internal and external reproductive organs developed postnatally, with an absence of female reproductive tissues. These results reveal that Dmrt1 has retained its ability to act as the primary testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans.

Non-Homologous Sex Chromosomes in Two Geckos (Gekkonidae: Gekkota) with Female Heterogamety

Evaluating homology between the sex chromosomes of different species is an important first step in deducing the origins and evolution of sex-determining mechanisms in a clade. Here, we describe the preparation of Z and W chromosome paints via chromosome microdissection from the Australian marbled gecko (Christinus marmoratus) and their subsequent use in evaluating sex chromosome homology with the ZW chromosomes of the Kwangsi gecko (Gekko hokouensis) from eastern Asia. We show that the ZW sex chromosomes of C. marmoratus and G. hokouensis are not homologous and represent independent origins of female heterogamety within the Gekkonidae. We also show that the C. marmoratus Z and W chromosomes are genetically similar to each other as revealed by C-banding, comparative genomic hybridization, and the reciprocal painting of Z and W chromosome probes. This implies that sex chromosomes in C. marmoratus are at an early stage of differentiation, suggesting a recent origin.

Function and evolution of sex determination mechanisms, genes and pathways in insects

Animals have evolved a bewildering diversity of mechanisms to determine the two sexes. Studies of sex determination genes – their history and function – in non-model insects and Drosophila have allowed us to begin to understand the generation of sex determination diversity. One common theme from these studies is that evolved mechanisms produce activities in either males or females to control a shared gene switch that regulates sexual development. Only a few small-scale changes in existing and duplicated genes are sufficient to generate large differences in sex determination systems. This review summarises recent findings in insects, surveys evidence of how and why sex determination mechanisms can change rapidly and suggests fruitful areas of future research.

MULTIPLE INTERACTING LOCI CONTROL SEX DETERMINATION IN LAKE MALAWI CICHLID FISH

Several models have been proposed to suggest how the evolution of sex-determining mechanisms might contribute to speciation. Here, we describe the inheritance of sex in 19 fish species from the rapidly evolving flock of cichlids in Lake Malawi, Africa. We found that many of these species have a male heterogametic (XY) system on linkage group 7. Some species also segregate for a female heterogametic (ZW) system on linkage group 5 that is coincident with a dominant orange-blotch (OB) color pattern in females. The ZW system is epistatically dominant to the XY system when both are segregating within a family. Several lines of evidence suggest that additional sex-determining loci are segregating in some species. These results are consistent with the idea that genetic conflicts play an important role in the evolution of these species flocks and suggest that evolution of sex-determining mechanisms has contributed to the radiation of cichlid fish in East Africa.

Sex determination: the power of DMRT1

DMRT1, a mammalian gene related to sex-determining genes in flies and nematodes, is located in a region of the human genome that is important for sex development. This suggests that a common thread might run through the evolution of sex-determining mechanisms from worms to humans. New data show that DMRT1 can cause sex reversal in chickens, adding support to this hypothesis.

Segregation distortion and the evolution of sex-determining mechanisms

Segregation distorters are alleles that distort normal segregation in their own favour. Sex chromosomal distorters lead to biased sex ratios, and the presence of such distorters, therefore, may induce selection for a change in the mechanism of sex determination. The evolutionary dynamics of distorter-induced changes in sex determination has only been studied in some specific systems. Here, we present a generic model for this process. We consider three scenarios: a driving X chromosome, a driving Y chromosome and a driving autosome with a male-determining factor. We investigate how the invasion prospects of a new sex-determining factor are affected by the strength of distortion and the fitness effect of the distorting allele. Our models show that in many cases, segregation distortion does create selection pressure, allowing novel sex-determining alleles to spread. When distortion leads to female-biased sex ratios, a new masculinizing gene can invade, leading to a new male heterogametic system. When distortion leads to male-biased sex ratios, a feminizing factor can invade and cause a switch to female heterogamety. In many cases, the distorter-induced change in the sex-determining system eventually leads to loss of the distorter from the population. Hence, the presence of sex chromosomal distorters will often only be transient, and the distorters may remain unnoticed. The role of segregation distortion in the evolution of sex determination may, therefore, be underestimated.

Phylogeny of sex-determining mechanisms in squamate reptiles: are sex chromosomes an evolutionary trap?

Squamate reptiles possess two general modes of sex determination: (1) genotypic sex determination (GSD), where the sex of an individual is determined by sex chromosomes, i.e. by sex-specific differences in genotype; and (2) temperature-dependent sex determination (TSD), where sex chromosomes are absent and sex is determined by nongenetic factors. After gathering information about sex-determining mechanisms for more than 400 species, we employed comparative phylogenetic analyses to reconstruct the evolution of sex determination in Squamata. Our results suggest relative uniformity in sex-determining mechanisms in the majority of the squamate lineages. Well-documented variability is found only in dragon lizards (Agamidae) and geckos (Gekkota). Polarity of the sex-determining mechanisms in outgroups identified TSD as the ancestral mode for Squamata. After extensive review of the literature, we concluded that to date there is no known well-documented transition from GSD to TSD in reptiles, although transitions in the opposite direction are plentiful and well corroborated by cytogenetic evidence. We postulate that the evolution of sex-determining mechanisms in Squamata was probably restricted to the transitions from ancestral TSD to GSD. In other words, transitions were from the absence of sex chromosomes to the emergence of sex chromosomes, which have never disappeared and constitute an evolutionary trap. This evolutionary trap hypothesis could change the understanding of phylogenetic conservatism of sex-determining systems in many large clades such as butterflies, snakes, birds, and mammals. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156, 168–183.

Evolution of Different Y Chromosomes in Two Medaka Species,Oryzias dancenaandO. latipes

Although the sex-determining gene DMY has been identified on the Y chromosome in the medaka (Oryzias latipes), this gene is absent in most Oryzias species, suggesting that closely related species have different sex-determining genes. Here, we investigated the sex-determination mechanism in O. dancena, which does not possess the DMY gene. Since heteromorphic sex chromosomes have not been reported in this species, a progeny test of sex-reversed individuals produced by hormone treatment was performed. Sex-reversed males yielded all-female progeny, indicating that O. dancena has an XX/XY sex-determination system. To uncover the cryptic sex chromosomes, sex-linked DNA markers were screened using expressed sequence tags (ESTs) established in O. latipes. Linkage analysis of isolated sex-linked ESTs showed a conserved synteny between the sex chromosomes in O. dancena and an autosome in O. latipes. Fluorescence in situ hybridization (FISH) analysis of these markers confirmed that sex chromosomes of these species are not homologous. These findings strongly suggest an independent origin of sex chromosomes in O. dancena and O. latipes. Further analysis of the sex-determining region in O. dancena should provide crucial insights into the evolution of sex-determination mechanisms in vertebrates.

Estradiol-17β Induces Lasting Sex Reversal at Male-Producing Temperatures in Kinosternid Turtles

In turtles with temperature-dependent sex determination (TSD), estradiol applied to eggs incubated at male-producing temperatures produces female hatchlings. In other vertebrate taxa, estradiol administration during embryogenesis often induces only temporary or incomplete feminization, and sensitivity to estradiol can vary among taxonomic families. In the present study, we treated eggs of Kinosternon scorpiodes and Sternotherus odoratus incubated at male-producing temperatures with estradiol-17β and examined reproductive morphology of offspring. Furthermore, to determine whether sex reversal resulting from estradiol application in turtles with TSD is lasting, we maintained juvenile K. scorpiodes in the lab for up to three years prior to dissection. We found normal ovarian morphology and Müllerian duct development in a significantly greater proportion of the hatchling S. odoratus and juvenile K. scorpiodes that had been treated with estradiol during embryogenesis than in the untreated group. Results further suggest that estradiol-induced sex reversal seen in TSD turtles is both permanent and ubiquitous and may shed light on the evolution of sex-determining mechanisms in vertebrates.