Vertebrate Sex Determination Research Articles

WGBS of embryonic gonads revealed that long non-coding RNAs in the MHM region might be involved in cell autonomous sex identity and female gonadal development in chickens

ABSTRACT DNA methylation plays a key role in sex determination and differentiation in vertebrates. However, there are few studies on DNA methylation involved in chicken gonad development, and most focused on male hypermethylated regions (MHM). It is unclear whether there are specific differentially methylated regions (DMRs) in chicken embryonic gonads regulating sex determination and differentiation. Here, the DNA methylation maps showed that the difference of DNA methylation level between sexes was much higher at embryonic day 10 (E10) than that at embryonic day 6 (E6), and the significant differentially methylated regions at both stages were mainly distributed on the Z chromosome, including MHM1 and MHM2. The results of bisulphite sequencing PCR (BSP) and qRT-PCR showed hypomethylation of female MHM and upregulation of long non-coding RNAs (lncRNAs) whose promoter in the MHM region was consistent with the sequencing results, and similar results were in brain and muscle. In female sex-reversed gonads, the methylation pattern of MHM remained unchanged, and the expression levels of the three candidate lncRNAs were significantly decreased compared with those in females, but were significantly increased compared to males. The fluorescence in situ hybridization (FISH) results also showed that these lncRNAs were highly expressed in female embryonic gonads. The results of methyltransferase inhibitor and dual-luciferase reporter assay suggest that lncRNA expression may be regulated by DNA methylation within their promoters. Therefore, we speculated that MHM may be involved in cell-autonomous sex identity in chickens, and that lncRNAs regulated by MHM may be involved in female sexual differentiation.

Comparative analysis of gonadal transcriptomes between turtle and alligator identifies common molecular cues activated during the temperature-sensitive period for sex determination

The mode of sex determination in vertebrates can be categorized as genotypic or environmental. In the case of genotypic sex determination (GSD), the sexual fate of an organism is determined by the chromosome composition with some having dominant genes, named sex-determining genes, that drive the sex phenotypes. By contrast, many reptiles exhibit environmental sex determination (ESD), whereby environmental stimuli drive sex determination, and most notably temperature. To date, temperature-dependent sex determination (TSD) has been found in most turtles, some lizards, and all crocodylians, but commonalities in the controlling processes are not well established. Recent innovative sequencing technology has enabled investigations into gonadal transcriptomic profiles during temperature-sensitive periods (TSP) in various TSD species which can help elucidate the controlling mechanisms. In this study, we conducted a time-course analysis of the gonadal transcriptome during the male-producing temperature (26℃) of the Reeve’s turtle (Chinese three-keeled pond turtle) Mauremys reevesii. We then compared the transcriptome profiles for this turtle species during the TSP with that for the American alligator Alligator mississippiensis to identify conserved reptilian TSD-related genes. Our transcriptome-based findings provide an opportunity to retrieve the candidate molecular cues that are activated during TSP and compare these target responses between TSD and GSD turtle species, and between TSD species.

The use of in silico extreme pathway (ExPa) analysis to identify conserved reproductive transcriptional-regulatory networks in humans, mice, and zebrafish

Vertebrate sex determination and differentiation are coordinated by the activations and maintenance of reproductive transcriptional-regulatory networks (TRNs). There is considerable interest in studying the conserved design principles and functions of reproductive TRNs given that their intricate regulation is susceptible to disruption by gene mutations or exposures to exogenous endocrine disrupting chemicals (or EDCs). In this manuscript, the Boolean rules describing reproductive TRNs in humans, mice, and zebrafish, were represented as a pseudo-stoichiometric matrix model. This model mathematically described the interactions of 35 transcription factors with 21 sex determination and differentiation genes across the three species. The in silico approach of Extreme Pathway (ExPa) analysis was used to predict the extent of TRN gene activations subject to the species-specific transcriptomics data, from across various developmental life-stages. A goal of this work was to identify conserved and functional reproductive TRNs across the three species. ExPa analyses predicted the sex differentiation genes, DHH, DMRT1, and AR, to be highly active in male humans, mice, and zebrafish. Whereas FOXL2 was the most active gene in female humans and mice; and CYP19A1A in female zebrafish. These results agree with the expectation that regardless of a lack of sex determination genes in zebrafish, the TRNs responsible for canalizing male vs. female sexual differentiation are conserved with mammalian taxa. ExPa analysis therefore provides a framework with which to study the TRNs that influence the development of sexual phenotypes. And the in silico predicted conservation of sex differentiation TRNs between mammals and zebrafish identifies the piscine species as an effective in vivo model to study mammalian reproductive systems under normal or perturbed pathologies.

Master-Key Regulators of Sex Determination in Fish and Other Vertebrates-A Review.

In vertebrates, mainly single genes with an allele ratio of 1:1 trigger sex-determination (SD), leading to initial equal sex-ratios. Such genes are designated master-key regulators (MKRs) and are frequently associated with DNA structural variations, such as copy-number variation and null-alleles. Most MKR knowledge comes from fish, especially cichlids, which serve as a genetic model for SD. We list 14 MKRs, of which dmrt1 has been identified in taxonomically distant species such as birds and fish. The identification of MKRs with known involvement in SD, such as amh and fshr, indicates that a common network drives SD. We illustrate a network that affects estrogen/androgen equilibrium, suggesting that structural variation may exert over-expression of the gene and thus form an MKR. However, the reason why certain factors constitute MKRs, whereas others do not is unclear. The limited number of conserved MKRs suggests that their heterologous sequences could be used as targets in future searches for MKRs of additional species. Sex-specific mortality, sex reversal, the role of temperature in SD, and multigenic SD are examined, claiming that these phenomena are often consequences of artificial hybridization. We discuss the essentiality of taxonomic authentication of species to validate purebred origin before MKR searches.

Genomic imprinting-like monoallelic paternal expression determines sex of channel catfish

The X and Y chromosomes of channel catfish have the same gene contents. Here, we report allelic hypermethylation of the X chromosome within the sex determination region (SDR). Accordingly, the X-borne hydin-1 gene was silenced, whereas the Y-borne hydin-1 gene was expressed, making monoallelic expression of hydin-1 responsible for sex determination, much like genomic imprinting. Treatment with a methylation inhibitor, 5-aza-dC, erased the epigenetic marks within the SDR and caused sex reversal of genetic females into phenotypic males. After the treatment, hydin-1 and six other genes related to cell cycle control and proliferative growth were up-regulated, while three genes related to female sex differentiation were down-regulated in genetic females, providing additional support for epigenetic sex determination in catfish. This mechanism of sex determination provides insights into the plasticity of genetic sex determination in lower vertebrates and its connection with temperature sex determination where DNA methylation is broadly involved.

Sex-linked markers in an Australian frog Platyplectrum ornatum (Limnodynastidae) with a small genome and homomorphic sex chromosomes

Amphibians have highly diverse sex-determining modes leading to a notable interest in vertebrate sex determination and sex chromosome evolution. The identification of sex-determining systems in amphibians, however, is often difficult as a vast majority consist of homomorphic sex chromosomes making them hard to distinguish. In this study, we used Diversity Array Technology sequencing (DArTseq) to identify the sex-determining system in the ornate burrowing frog from Australia, Platyplectrum ornatum. We applied DArTseq to 44 individuals, 19 males and 25 females, collected from two locations to develop sex-linked markers. Unexpectedly, these 44 individuals were classified into two distinct population clusters based on our SNP analyses, 36 individuals in cluster 1, and 8 individuals in cluster 2. We then performed sex-linkage analyses separately in each cluster. We identified 35 sex-linked markers from cluster 1, which were all associated with maleness. Therefore, P. ornatum cluster 1 is utilising a male heterogametic (XX/XY) sex-determining system. On the other hand, we identified 210 sex-linked markers from cluster 2, of which 89 were male specific, i.e., identifying XX/XY sex determining system and 111 were female specific, i.e., identifying ZZ/ZW sex determining system, suggesting existence of either male or female heterogametic sex determining system in cluster 2. We also performed cytogenetic analyses in 1 male and 1 female from cluster 1; however, we did not detect any visible differentiation between the X and Y sex chromosomes. We also mapped sex-linked markers from the two clusters against the P. ornatum genome and our comparative analysis indicated that the sex chromosomes in both clusters shared homologies to chromosome 10 (autosome) of Rana temporaria and ZWY sex chromosome of Xenopus tropicalis. Our preliminary data suggest that it is plausible that the cluster 2 has a potential to be either male or female heterogamety in sex determination, requiring further investigation.

Identification of sex-linked marker and candidate sex determination gene in ornamental fish, African scat (Scatophagus tetracanthus)

The aquarium industry greatly threatens biological diversity via biological invasion. Mono-sex culturing is an important technique to reduce the possible biological invasion introduced by the aquarium fish, which escape into their non-native environment. African scat (Scatophagus tetracanthus) is a valuable aquarium fish species worldwide. Herein, its sex-linked marker and candidate sex determination (SD) gene were identified through genome sequencing. The whole genome of the male African scat was sequenced using the next-generation sequencing technology. The assembled genome is 630.96 Mb with a scaffold N50 of 38,217 bp. Phylogenetic analysis revealed that African scat diverged from spotted scat (S. argus) approximately 10.5 million years ago. One sex-linked marker (Marker-1) was detected in African scat. Marker-1 amplified a single band of 873 bp in female African scat, and double bands of 873 bp and specific 772 bp in male African scat. Next, Marker-1 was validated in 60 cultured African scat with 100% consistency. Sequence analysis revealed that the 873 and 772 bp fragments were both Dmrt1 homologous sequences. These results imply that the African scat possesses the XY sex-determination system (XX females, XY males), with Dmrt1 as its sex-linked gene. Dmrt1 cDNA sequences on the X and Y chromosomes were also cloned and labeled as Dmrt1∆X and Dmrt1Y, respectively. The amino acid (aa) sequences coded by Dmrt1∆X and Dmrt1Y were 143 aa and 300 aa, respectively. Dmrt1Y mRNA was specifically expressed in the testis, with no expression in the ovary. Therefore, Dmrt1Y is male-specific and a candidate SD gene. The genome data and sex-linked marker of African scat will be helpful for its commercial management to prevent its biological invasion. Besides, the identification of its candidate SD gene contributes to the basic research of SD in vertebrates.

Effects of semi-constant temperature on embryonic and hatchling phenotypes of six-tubercled Amazon River turtles, Podocnemis sextuberculata

PurposeWe evaluated how constant incubation temperatures affect life-history traits pre-hatching and post-hatching of the six-tubercled Amazon River turtle, Podocnemis sextuberculata. MethodsWe incubated eggs from natural nests at ten semi-constant temperatures between 22.26 ± 1.01 °C and 37.37 ± 0.38°C (2013) and at six temperatures between 25.75 ± 0.22 °C and 36.17 ± 0.15°C (2016). In 2013, we raised hatchling for 90 days to evaluate effects of temperature on early hatchling growth. We evaluated maternal effects in 2016. ResultsP. sextuberculata displays temperature-dependent sex determination and produces males at colder and females at warmer temperatures (TSD Ia). The estimated pivotal temperature was 33.73 ± 0.15 °C and the transitional range of temperatures (TRT) 1.16 ± 0.59 °C. Semi-constant temperatures below 26 °C and above 38 °C were lethal. Intermediate temperatures (32.25 °C and 31.5 °C, respectively) were optimal for hatching success and produced larger hatchlings that grew slower early in life compared to colder or warmer conditions, which produced smaller hatchlings. Warmer incubation temperatures within the optimal range (28°C–37 °C) accelerated embryonic development. In contrast, comparisons of 30, 60 and 90 days-old suggests that warmer incubation temperatures reduced growth and mass gain rates post-hatching, such that incubation temperature effects on body size at emergence disappeared by 3 months of age. ConclusionsSix-tubercled Amazon River turtles showed the highest pivotal temperature reported for any turtle. The relatively narrow TRT may limit the evolutionary potential of this vulnerable turtle in the face of global warming. Future incubation experiments at a finer scale (33°C–36 °C) are warranted to refine the sex-ratio reaction norm. Field studies that monitor natural nests are imperative to evaluate conservation measures and the effect of female-biased illegal hunting and climate change. By providing data about the thermal biology of an understudied lineage of non-model species, our study helps fill gaps in our understanding of the evolution of vertebrate sex determination and its potential adaptive value.

Pervasive male-biased expression throughout the germline-specific regions of the sea lamprey genome supports key roles in sex differentiation and spermatogenesis

Sea lamprey undergo programmed genome rearrangement (PGR) in which ∼20% of the genome is jettisoned from somatic cells during embryogenesis. Although the role of PGR in embryonic development has been studied, the role of the germline-specific region (GSR) in gonad development is unknown. We analysed RNA-sequence data from 28 sea lamprey gonads sampled across life-history stages, generated a genome-guided de novo superTranscriptome with annotations, and identified germline-specific genes (GSGs). Overall, we identified 638 GSGs that are enriched for reproductive processes and exhibit 36x greater odds of being expressed in testes than ovaries. Next, while 55% of the GSGs have putative somatic paralogs, the somatic paralogs are not differentially expressed between sexes. Further, putative orthologs of some the male-biased GSGs have known functions in sex determination or differentiation in other vertebrates. We conclude that the GSR of sea lamprey plays an important role in testicular differentiation and potentially sex determination.

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.

Unusual Mammalian Sex Determination Systems: A Cabinet of Curiosities.

Therian mammals have among the oldest and most conserved sex-determining systems known to date. Any deviation from the standard XX/XY mammalian sex chromosome constitution usually leads to sterility or poor fertility, due to the high differentiation and specialization of the X and Y chromosomes. Nevertheless, a handful of rodents harbor so-called unusual sex-determining systems. While in some species, fertile XY females are found, some others have completely lost their Y chromosome. These atypical species have fascinated researchers for over 60 years, and constitute unique natural models for the study of fundamental processes involved in sex determination in mammals and vertebrates. In this article, we review current knowledge of these species, discuss their similarities and differences, and attempt to expose how the study of their exceptional sex-determining systems can further our understanding of general processes involved in sex chromosome and sex determination evolution.

Identification and molecular characterization of doublesex and mab-3-related transcription factor (dmrt ) in brackish water flea, <i>Diaphanosoma celebensis</i>, exposed to bisphenol analogs

Doublesex and mab-3 related transcription factor (dmrt) play crucial roles in sex determination and sex differentiation in vertebrates and invertebrates. Although dmrt genes have been identified in vertebrates, little is known about aquatic invertebrates. In this study, two dmrt genes, namely, Dc_dmrt93B and Dc_dmrt99B, were identified from brackish water flea, Diaphanosoma celebensis. Transcriptional changes were observed in the dmrt genes when the flea was exposed to bisphenol (BP), an endocrine disruptor. Sequence and phylogenetic analyses showed that both dmrt genes contained two conserved domains, namely, DM and DMA, closely clustered with those of Daphnia spp. Additionally, a significant increase in the Dc_dmrt99B mRNA expression level was observed upon exposure to intermediate concentrations of BP (bisphenol A>bisphenol S=bisphenol F, p<0.05), while the expression of Dc_dmrt93B mRNA was slightly modulated. These findings imply that the two dmrt genes may be involved in sex differentiation of D. celebensis. Furthermore, it was found that the ability of BP to modulate dmrt genes could affect development and reproduction. This study provides a basis for understanding the function of the dmrt genes and the molecular mode of action of BP in small crustaceans.

Diverse Regulation but Conserved Function: SOX9 in Vertebrate Sex Determination

Sex determination occurs early during embryogenesis among vertebrates. It involves the differentiation of the bipotential gonad to ovaries or testes by a fascinating diversity of molecular switches. In most mammals, the switch is SRY (sex determining region Y); in other vertebrates it could be one of a variety of genes including Dmrt1 or dmy. Downstream of the switch gene, SOX9 upregulation is a central event in testes development, controlled by gonad-specific enhancers across the 2 Mb SOX9 locus. SOX9 is a ‘hub’ gene of gonadal development, regulated positively in males and negatively in females. Despite this diversity, SOX9 protein sequence and function among vertebrates remains highly conserved. This article explores the cellular, morphological, and genetic mechanisms initiated by SOX9 for male gonad differentiation.

Sex-Based Variation of Gene Expression in the Gonads and Fins of Russian Sturgeon (&amp;lt;i&amp;gt;Acipenser gueldenstaedtii&amp;lt;/i&amp;gt;)

Russian sturgeon (Acipenser gueldenstaedtii) is a primitive freshwater fish and a source of black caviar. The genes involved in sexual determination and differentiation are still unknown and there are no molecular markers for sex identification in this species. Studying the variation of the sex-based differences in genomic sequences and in gene expression in the sturgeon may lead to markers of sex in early stages of development and advances in aquaculture, as well as provide novel insights about the evolution of reproduction, sex determination, and sexual differentiation mechanisms in vertebrates. Previous studies by our and other groups have identified differentially expressed genes in the gonads of adult female and male sturgeon. The current study aimed to test whether these genes were also differentially expressed in non-gonadal tissue, namely fins. We measured by qRT-PCR the mRNA levels of 29 known and novel sex-related genes in the gonads and fins of males (4 years old) and females (7 years old; sexual maturation is earlier in males than in females). Six genes (ATP6, IGFRM, LIA1A, S1A, NPL1A, GAPDH and SOX9) showed higher expression in female fins. However, only ATP6 mRNA levels differed in fins of males and females of the same age (4 years old). These findings underscore the impracticality of sex identification based on gene expression in non-gonadal tissue and the need for genetic sex markers in the Russian sturgeon.

Interaction between DMRT1 and PLZF protein regulates self-renewal and proliferation in male germline stem cells

Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with XX/XY sex determination.

Phenotypic sex of an organism is determined by molecular changes in the gonads, so-called molecular sex differentiation, which should precede the rise of cellular or anatomical sex-distinguishing features. This study characterized molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with established XX/XY genotypic sex determination. Next generation sequencing was conducted on sablefish ovarian and testicular mRNAs to obtain sequences for transcripts associated with vertebrate sex determination and differentiation and early reproductive development. Gene-specific PCRs were developed to determine the distribution and ontogenetic gonadal expression of transcription, growth, steroidogenic and germline factors, as well as gonadotropin and steroid receptors. Molecular changes associated with sex differentiation were first apparent in both XY- and XX-genotype sablefish at~60mm in body length and prior to histological signs of sex differentiation. The earliest and most robust markers of testicular differentiation were gsdf, amh, dmrt1, cyp11b, star, sox9a, and fshr. Markedly elevated mRNA levels of several steroidogenesis-related genes and ar2 in differentiating testes suggested that androgens play a role in sablefish testicular differentiation. The earliest markers of ovarian differentiation were cyp19a1a, lhcgr, foxl2, nr0b1, and igf3. Other transcripts such as figla, zp3, and pou5f3 were expressed predominantly in XX-genotype fish and significantly increased with the first appearance and subsequent development of primary oocytes. This study provides valuable insight to the developmental sequence of events associated with gonadal sex differentiation in marine teleosts with XX/XY sex determination. It also implicates particular genes in processes of male and female development and establishes robust molecular markers for phenotypic sex in sablefish, useful for ongoing work related to sex control and reproductive sterilization.

Peroxisome proliferator-activated receptor alpha is involved in the temperature-induced sex differentiation of a vertebrate

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Index to Titles and Authors/Editors of Books Reviewed

Index to Titles and Authors/Editors of Books Reviewed

Parallel Evolution of Two dmrt1-Derived Genes, dmy and dm-W, for Vertebrate Sex Determination.

SummaryAnimal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medaka dmy and the clawed frog dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment of dmy and dm-W as sex-determining genes.