Genome-Wide Identification and Expression Analysis of the STAT Family in Reeve's Turtle (Mauremys reevesii).

The Stat (signal transducer and activator of transcription) gene family plays a vital role in regulating immunity and the processes of cellular proliferation, differentiation, and apoptosis across diverse organisms. Although the functions of Stat genes in immunity have been extensively documented in many mammals, limited data are available for reptiles. We used phylogenetic analysis to identify eight putative members of the Stat family (Stat1-1, Stat1-2, Stat2, Stat3, Stat4, Stat5b, Stat6-1, and Stat6-2) within the genome of M. reevesii, a freshwater turtle found in East Asia. Sequence analysis showed that the Stat genes contain four conserved structural domains protein interaction domain, coiled-coil domain, DNA-binding domain, and Src homology domain 2. In addition, Stat1, Stat2, and Stat6 contain TAZ2bind, Apolipo_F, and TALPID3 structural domains. The mRNA levels of Stat genes were upregulated in spleen tissues at 4, 8, 12, and 16 h after administration of lipopolysaccharide, a potent activator of the immune system. Stat5b expression at 12-h LPS post-injection exhibited the most substantial difference from the control. The expression of Stat5b in spleen tissue cellular was verified by immunofluorescence. These results suggest that Stat5b plays a role in the immune response of M. reevesii and may prove to be as a positive marker of an immune response in future studies.

Metabolic consequences of sex reversal in two lizard species: a test of the like-genotype and like-phenotype hypotheses.

Vertebrate sex is typically determined genetically, but in many ectotherms sex can be determined by genes (genetic sex determination, GSD), temperature (temperature-dependent sex determination, TSD), or interactions between genes and temperature during development. TSD may involve GSD systems with either male or female heterogamety (XX/XY or ZZ/ZW) where temperature overrides chromosomal sex determination to cause a mismatch between genetic sex and phenotypic sex (sex reversal). In these temperature-sensitive lineages, phylogenetic investigations point to recurrent evolutionary shifts between genotypic and temperature-dependent sex determination. These evolutionary transitions in sex determination can occur rapidly if selection favours the reversed sex over the concordant phenotypic sex. To investigate the consequences of sex reversal on offspring phenotypes, we measured two energy-driven traits (metabolism and growth) and 6 month survival in two species of reptile with different patterns of temperature-induced sex reversal. Male sex reversal occurs in Bassiana duperreyi when chromosomal females (female XX) develop male phenotypes (maleSR XX), while female sex reversal occurs in Pogona vitticeps when chromosomal males (male ZZ) develop female phenotypes (femaleSR ZZ). We show metabolism in maleSR XX was like that of male XY; that is, reflective of phenotypic sex and lower than genotypic sex. In contrast, for Pogona vitticeps, femaleSR ZZ metabolism was intermediate between male ZZ and female ZW metabolic rate. For both species, our data indicate that differences in metabolism become more apparent as individuals become larger. Our findings provide some evidence for an energetic advantage from sex reversal in both species but do not exclude energetic processes as a constraint on the distribution of sex reversal in nature.

Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs.

Lizards are considered vulnerable to climate change because many operate near their thermal maxima. Exposure to higher temperatures could reduce activity of these animals by forcing them to shelter in thermal refugia for prolonged periods to avoid exceeding lethal limits. While rising temperatures should reduce activity in tropical species, the situation is less clear for temperate-zone species where activity can be constrained by both low and high temperatures. Here, we measure the effects of natural variation in environmental temperatures on activity in a temperate grassland lizard and show that it is operating near its upper thermal limit in summer even when sheltering in thermal refuges. As air temperatures increased above 32 °C, lizard activity declined markedly as individuals sought refuge in cool microhabitats while still incurring substantial metabolic costs. We estimate that warming over the last two decades has required these lizards to increase their energy intake up to 40% to offset metabolic losses caused by rising temperatures. Our results show that recent increases in temperature are sufficient to exceed the thermal and metabolic limits of temperate-zone grassland lizards. Extended periods of high temperatures could place natural populations of ectotherms under significantly increased environmental stress and contribute to population declines and extinction.

Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Polygenic sex determination in vertebrates - is there any such thing?

Genetic sex determination (SD) in most vertebrates is controlled by a single master sex gene, which ensures a 1:1 sex ratio. However, more complex systems abound, and several have been ascribed to polygenic SD (PSD), in which many genes at different loci interact to produce the sexual phenotype. Here we examine claims for PSD in vertebrates, finding that most constitute transient states during sex chromosome turnover, or aberrant systems in species hybrids. To avoid confusion about terminology, we propose a consistent nomenclature for genetic SD systems.

Three dimensions of thermolabile sex determination.

The molecular mechanism of temperature-dependent sex determination (TSD) is a long-standing mystery. How is the thermal signal sensed, captured and transduced to regulate key sex genes? Although there is compelling evidence for pathways via which cells capture the temperature signal, there is no known mechanism by which cells transduce those thermal signals to affect gene expression. Here we propose a novel hypothesis we call 3D-TSD (the three dimensions of thermolabile sex determination). We postulate that the genome has capacity to remodel in response to temperature by changing 3D chromatin conformation, perhaps via temperature-sensitive transcriptional condensates. This could rewire enhancer-promoter interactions to alter the expression of key sex-determining genes. This hypothesis can accommodate monogenic or multigenic thermolabile sex-determining systems, and could be combined with upstream thermal sensing and transduction to the epigenome to commit gonadal fate.

Polyandry and non-random fertilisation maintain long-term genetic diversity in an isolated island population of adders (Vipera berus).

Conservation genetic theory suggests that small and isolated populations should be subject to reduced genetic diversity i.e., heterozygosity and allelic diversity. Our 34 years study of an isolated island population of adders (Vipera berus) in southern Sweden challenges this notion. Despite a lack of gene flow and a yearly mean estimated reproductive adult population size of only 65 adult adders (range 12-171), the population maintains high levels of heterozygosity and allelic diversity similar to that observed in two mainland populations. Even a 14-year major "bottleneck" i.e., a reduction in adult adder numbers, encompassing at least four adder generations, did not result in any reduction in the island adders' heterozygosity and allelic diversity. Female adders are polyandrous, and fertilisation is non-random, which our empirical data and modelling suggest are underpinning the maintenance of the population's high level of heterozygosity. Our empirical results and subsequent modelling suggest that the positive genetic effects of polyandry in combination with non-random fertilisation, often overlooked in conservation genetic analyses, deserve greater consideration when predicting long-term survival of small and isolated populations.

Meiotic chromosome dynamics and double strand break formation in reptiles.

During meiotic prophase I, tightly regulated processes take place, from pairing and synapsis of homologous chromosomes to recombination, which are essential for the generation of genetically variable haploid gametes. These processes have canonical meiotic features conserved across different phylogenetic groups. However, the dynamics of meiotic prophase I in non-mammalian vertebrates are poorly known. Here, we compare four species from Sauropsida to understand the regulation of meiotic prophase I in reptiles: the Australian central bearded dragon (Pogona vitticeps), two geckos (Paroedura picta and Coleonyx variegatus) and the painted turtle (Chrysemys picta). We first performed a histological characterization of the spermatogenesis process in both the bearded dragon and the painted turtle. We then analyzed prophase I dynamics, including chromosome pairing, synapsis and the formation of double strand breaks (DSBs). We show that meiosis progression is highly conserved in reptiles with telomeres clustering forming the bouquet, which we propose promotes homologous pairing and synapsis, along with facilitating the early pairing of micro-chromosomes during prophase I (i.e., early zygotene). Moreover, we detected low levels of meiotic DSB formation in all taxa. Our results provide new insights into reptile meiosis.

Diversity of reptile sex chromosome evolution revealed by cytogenetic and linked-read sequencing.

Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline, combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa, pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes, as well as translocations between different ancestral macro- and microchromosomes. We discuss the evolutionary drivers of the slow differentiation and turnover of reptile sex chromosomes.

Truncated jarid2 and kdm6b transcripts are associated with temperature-induced sex reversal during development in a dragon lizard.

Sex determination and differentiation in reptiles are complex. In the model species, Pogona vitticeps, high incubation temperature can cause male to female sex reversal. To elucidate the epigenetic mechanisms of thermolabile sex, we used an unbiased genome-wide assessment of intron retention during sex reversal. The previously implicated chromatin modifiers (jarid2 and kdm6b) were two of three genes to display sex reversal-specific intron retention. In these species, embryonic intron retention resulting in C-terminally truncated jarid2 and kdm6b isoforms consistently occurs at low temperatures. High-temperature sex reversal is uniquely characterized by a high prevalence of N-terminally truncated isoforms of jarid2 and kdm6b, which are not present at low temperatures, or in two other reptiles with temperature-dependent sex determination. This work verifies that chromatin-modifying genes are involved in highly conserved temperature responses and can also be transcribed into isoforms with new sex-determining roles.

Developmental dynamics of sex reprogramming by high incubation temperatures in a dragon lizard.

BACKGROUND: In some vertebrate species, gene-environment interactions can determine sex, driving bipotential gonads to differentiate into either ovaries or testes. In the central bearded dragon (Pogona vitticeps), the genetic influence of sex chromosomes (ZZ/ZW) can be overridden by high incubation temperatures, causing ZZ male to female sex reversal. Previous research showed ovotestes, a rare gonadal phenotype with traits of both sexes, develop during sex reversal, leading to the hypothesis that sex reversal relies on high temperature feminisation to outcompete the male genetic cue. To test this, we conducted temperature switching experiments at key developmental stages, and analysed the effect on gonadal phenotypes using histology and transcriptomics. RESULTS: We found sexual fate is more strongly influenced by the ZZ genotype than temperature. Any exposure to low temperatures (28 °C) caused testes differentiation, whereas sex reversal required longer exposure to high temperatures. We revealed ovotestes exist along a spectrum of femaleness to male-ness at the transcriptional level. We found inter-individual variation in gene expression changes following temperature switches, suggesting both genetic sensitivity to, and the timing and duration of the temperature cue influences sex reversal. CONCLUSIONS: These findings bring new insights to the mechanisms underlying sex reversal, improving our understanding of thermosensitive sex systems in vertebrates.

Lineage diversity within a widespread endemic Australian skink to better inform conservation in response to regional-scale disturbance.

Much attention is paid in conservation planning to the concept of a species, to ensure comparability across studies and regions when classifying taxa against criteria of endangerment and setting priorities for action. However, various jurisdictions now allow taxonomic ranks below the level of species and nontaxonomic intraspecific divisions to be factored into conservation planning-subspecies, key populations, evolutionarily significant units, or designatable units. Understanding patterns of genetic diversity and its distribution across the landscape is a key component in the identification of species boundaries and determination of substantial geographic structure within species. A total of 12,532 reliable polymorphic SNP loci were generated from 63 populations (286 individuals) covering the distribution of the Australian eastern three-lined skink, Bassiana duperreyi, to assess genetic population structure in the form of diagnosable lineages and their distribution across the landscape, with particular reference to the recent catastrophic bushfires of eastern Australia. Five well-supported diagnosable operational taxonomic units (OTUs) existed within B. duperreyi. Low levels of divergence of B. duperreyi between mainland Australia and Tasmania (no fixed allelic differences) support the notion of episodic exchange of alleles across Bass Strait (ca 60 m, 25 Kya) during periods of low sea level during the Upper Pleistocene rather than the much longer period of isolation (1.7 My) indicated by earlier studies using mitochondrial sequence variation. Our study provides foundational work for the detailed taxonomic re-evaluation of this species complex and the need for biodiversity assessment to include an examination of cryptic species and/or cryptic diversity below the level of species. Such information on lineage diversity within species and its distribution in the context of disturbance at a regional scale can be factored into conservation planning regardless of whether a decision is made to formally diagnose new species taxonomically and nomenclaturally.

Evolutionary stability inferred for a free ranging lizard with sex-reversal.

The sex of vertebrates is typically determined genetically, but reptile sex can also be determined by developmental temperature. In some reptiles, temperature interacts with genotype to reverse sex, potentially leading to transitions from a chromosomal to a temperature-dependent sex determining system. Transitions between such systems in nature are accelerated depending on the frequency and fitness of sex-reversed individuals. The Central Bearded Dragon, Pogona vitticeps, exhibits female heterogamety (ZZ/ZW) but can have its sex reversed from ZZ male to ZZ female by high incubation temperatures. The species exhibits sex-reversal in the wild and it has been suggested that climate change and fitness of sex-reversed individuals could be increasing the frequency of reversal within the species range. Transitions to temperature-dependent sex determination require low levels of dispersal and high (>50%) rates of sex-reversal. Here, we combine genotype-by-sequencing, identification of phenotypic and chromosomal sex, exhaustive field surveys, and radio telemetry to examine levels of genetic structure, rates of sex-reversal, movement, space use, and survival of P. vitticeps in a location previously identified as a hot spot for sex-reversal. We find that the species exhibits low levels of population structure (FST ~0.001) and a modest (~17%) rate of sex-reversal, and that sex-reversed and nonsex-reversed females have similar survival and behavioural characteristics to each other. Overall, our data indicate this system is evolutionary stable, although we do not rule out the prospect of a more gradual transition in sex-determining mechanisms in the future in a more fragmented landscape and as global temperatures increase.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Early Efficacy and Safety Outcomes of Artificial Urinary Sphincter for Stress Urinary Incontinence Following Radical Prostatectomy or Benign Prostatic Obstruction Surgery: Results of a Large Multicentric Study.

BACKGROUND: Artificial urinary sphincter (AUS) is the gold standard for the management of moderate to severe stress urinary incontinence (SUI) in the male population. While outcomes of this device in postprostatectomy incontinence (PPI) are widely described, those obtained for incontinence after benign prostatic obstruction (BPO) surgery remains poorly explored. OBJECTIVE: To compare continence outcomes after AUS implantation in a PPI population with those obtained in men incontinent after BPO surgery. DESIGN, SETTING, AND PARTICIPANTS: A retrospective review of all cases of AUS implantation between 2005 and 2020 in 16 different French centers was conducted. Only patients with primary implantation whose indication was moderate to severe SUI after prostatectomy or BPO surgery were included (excluding those with a history of radiation therapy, brachytherapy, cystectomy, high-intensity focused ultrasound therapy, or neurogenic disease). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the rate of social continence (zero or one pad per day) at 3 mo. Complications were also noted within 90 d of implantation. RESULTS AND LIMITATIONS: A total of 417 patients were included in the PPI group and 50 in the BPO surgery group. Social continence rates at 3 mo were similar between the groups (79% vs 72%, p = 0.701). Complication rate was significantly higher in the BPO group (8% vs 18%, p = 0.044). The same was found for the Clavien-Dindo type 2 complication rate (20.6% vs 44.4%, p = 0.026). The retrospective nature and lack of precise definition of incontinence are the main limitations of this study. CONCLUSIONS: This multicentric study strengthens the position of AUS as gold standard for SUI after radical prostatectomy. Comparable efficacy results were found for incontinence after BPO surgery, with nevertheless a higher rate of complications. PATIENT SUMMARY: Artificial urinary sphincter represents the gold standard for the treatment of moderate to severe stress urinary incontinence. Efficacy results are comparable between postprostatectomy incontinence and incontinence after benign prostatic obstruction surgery.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Microchromosomes are building blocks of bird, reptile, and mammal chromosomes.

Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical.

Effects of natural nest temperatures on sex reversal and sex ratios in an Australian alpine skink.

Altered climate regimes have the capacity to affect the physiology, development, ecology and behaviour of organisms dramatically, with consequential changes in individual fitness and so the ability of populations to persist under climatic change. More directly, extreme temperatures can directly skew the population sex ratio in some species, with substantial demographic consequences that influence the rate of population decline and recovery rates. In contrast, this is particularly true for species whose sex is determined entirely by temperature (TSD). The recent discovery of sex reversal in species with genotypic sex determination (GSD) due to extreme environmental temperatures in the wild broadens the range of species vulnerable to changing environmental temperatures through an influence on primary sex ratio. Here we document the levels of sex reversal in nests of the Australian alpine three-lined skink (Bassiana duperreyi), a species with sex chromosomes and sex reversal at temperatures below 20 °C and variation in rates of sex reversal with elevation. The frequency of sex reversal in nests of B. duperreyi ranged from 28.6% at the highest, coolest locations to zero at the lowest, warmest locations. Sex reversal in this alpine skink makes it a sensitive indicator of climate change, both in terms of changes in average temperatures and in terms of climatic variability.