Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica).

BACKGROUND: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. RESULTS: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). CONCLUSIONS: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

Overview of chicken embryo genes related to sex differentiation.

Sex determination in chickens at an early embryonic stage has been a longstanding challenge in poultry production due to the unique ZZ:ZW sex chromosome system and various influencing factors. This review has summarized the genes related to the sex differentiation of chicken early embryos (mainly Dmrt1, Sox9, Amh, Cyp19a1, Foxl2, Tle4z1, Jun, Hintw, Ube2i, Spin1z, Hmgcs1, Foxd1, Tox3, Ddx4, cHemgn and Serpinb11 in this article), and has found that these contributions enhance our understanding of the genetic basis of sex determination in chickens, while identifying potential gene targets for future research. This knowledge may inform and guide the development of sex screening technologies for hatching eggs and support advancements in gene-editing approaches for chicken embryos. Moreover, these insights offer hope for enhancing animal welfare and promoting conservation efforts in poultry production.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Regulation of sexual differentiation initiation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Transcriptomic analysis reveals the molecular basis of photoperiod-regulated sex differentiation in tropical pumpkins (Cucurbita moschata Duch.).

BACKGROUND: Photoperiod, or the length of the day, has a significant impact on the flowering and sex differentiation of photoperiod-sensitive crops. The "miben" pumpkin (the main type of Cucurbita moschata Duch.) is well-known for its high yield and strong disease resistance. However, its cultivation has been limited due to its sensitivity to photoperiod. This sensitivity imposes challenges on its widespread cultivation and may result in suboptimal yields in regions with specific daylength conditions. As a consequence, efforts are being made to explore potential strategies or breeding techniques to enhance its adaptability to a broader range of photoperiods, thus unlocking its full cultivation potential and further promoting its valuable traits in agriculture. RESULTS: This study aimed to identify photoperiod-insensitive germplasm exhibiting no difference in sex differentiation under different day-length conditions. The investigation involved a phenotypic analysis of photoperiod-sensitive (PPS) and photoperiod-insensitive (PPIS) pumpkin materials exposed to different day lengths, including long days (LDs) and short days (SDs). The results revealed that female flower differentiation was significantly inhibited in PPS_LD, while no differences were observed in the other three groups (PPS_SD, PPIS_LD, and PPIS_SD). Transcriptome analysis was carried out for these four groups to explore the main-effect genes of sex differentiation responsive to photoperiod. The main-effect gene subclusters were identified based on the principal component and hierarchical cluster analyses. Further, functional annotations and enrichment analysis revealed significant upregulation of photoreceptors (CmCRY1, F-box/kelch-repeat protein), circadian rhythm-related genes (CmGI, CmPRR9, etc.), and CONSTANS (CO) in PPS_LD. Conversely, a significant downregulation was observed in most Nuclear Factor Y (NF-Y) transcription factors. Regarding the gibberellic acid (GA) signal transduction pathway, positive regulators of GA signaling (CmSCL3, CmSCL13, and so forth) displayed higher expression levels, while the negative regulators of GA signaling, CmGAI, exhibited lower expression levels in PPS_LD. Notably, this effect was not observed in the synthetic pathway genes. Furthermore, genes associated with ethylene synthesis and signal transduction (CmACO3, CmACO1, CmERF118, CmERF118-like1,2, CmWIN1-like, and CmRAP2-7-like) showed significant downregulation. CONCLUSIONS: This study offered a crucial theoretical and genetic basis for understanding how photoperiod influences the mechanism of female flower differentiation in pumpkins.

Doublesex is essential for masculinization but not feminization in Lygus hesperus.

In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G0 and strongly biased heterozygous G1 offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx, whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest.

Characterization of the human fetal gonad and reproductive tract by single-cell transcriptomics.

During human fetal development, sex differentiation occurs not only in the gonads but also in the adjacent developing reproductive tract. However, while the cellular composition of male and female human fetal gonads is well described, that of the adjacent developing reproductive tract remains poorly characterized. Here, we performed single-cell transcriptomics on male and female human fetal gonads together with the adjacent developing reproductive tract from first and second trimesters, highlighting the morphological and molecular changes during sex differentiation. We validated different cell populations of the developing reproductive tract and gonads and compared the molecular signatures between the first and second trimesters, as well as between sexes, to identify conserved and sex-specific features. Together, our study provides insights into human fetal sex-specific gonadogenesis and development of the reproductive tract beyond the gonads.

Chromosome-Level Assembly of Artemia franciscana Sheds Light on Sex Chromosome Differentiation.

Since the commercialization of brine shrimp (genus Artemia) in the 1950s, this lineage, and in particular the model species Artemia franciscana, has been the subject of extensive research. However, our understanding of the genetic mechanisms underlying various aspects of their reproductive biology, including sex determination, is still lacking. This is partly due to the scarcity of genomic resources for Artemia species and crustaceans in general. Here, we present a chromosome-level genome assembly of A. franciscana (Kellogg 1906), from the Great Salt Lake, United States. The genome is 1 GB, and the majority of the genome (81%) is scaffolded into 21 linkage groups using a previously published high-density linkage map. We performed coverage and FST analyses using male and female genomic and transcriptomic reads to quantify the extent of differentiation between the Z and W chromosomes. Additionally, we quantified the expression levels in male and female heads and gonads and found further evidence for dosage compensation in this species.

Determination of the timing of early gonadal differentiation in silver pomfret, Pampus argenteus.

Silver pomfret is a species of global significance due to its high nutritional in fisheries sector. To accurately ascertain the timing of sex differentiation mechanism and mRNA level in this species, this study examined gonad morphology and patterns of gene expression related to sex differentiation in males and females from 51 to 180 days post hatch (dph), the temperature of water was maintained at 26 ± 1 â„ƒ. Distinct morphological differentiation of the silver pomfret ovaries, marked by the emergence of primary oocytes, became apparent from 68 dph. By 108 dph, the testes began to differentiate, as evidenced by the appearance of the efferent duct. Early oocytes exhibited a diameter ranged from 0.077 mm to 0.682 mm, with an average diameter of 0.343 ± 0.051 mm. The proportions of various types of germ cells within the testes were subjected to analysis. The localization of Vasa during the early stages of sexual differentiation was a subject to analysis as well. Vasa was predominantly localized within the cytoplasm of gonocyte, peri-nucleolus stage oocytes, primary oocytes and type A spermatogonocytes, indicating that Vasa is involved in the early gonadal differentiation of silver pomfret. The study investigated the expression patterns of dmrt1, gsdf, amh, foxl2, cyp19a1a, cyp11a, sox3 and vasa, all of which are involved in the sex differentiation of teleosts. Among these genes, amh, gsdf, sox3, foxl2, vasa were indentified as crucial contributors to the early gonadal development of silver pomfret. Significant sex-related differences were observed in the expression patterns of amh, dmrt1, gsdf, cyp11a, sox3, cyp19a1a, vasa. This study provides novel insights into the timing of physiological changes associated with the sexual differentiation of silver pomfret. Collectively, the present data indicates that the differentiation of ovaries and testes take place approximately at 68 dph in females and 108 dph in males.

Let's talk about sex: Mechanisms of neural sexual differentiation in Bilateria.

In multicellular organisms, sexed gonads have evolved that facilitate release of sperm versus eggs, and bilaterian animals purposefully combine their gametes via mating behaviors. Distinct neural circuits have evolved that control these physically different mating events for animals producing eggs from ovaries versus sperm from testis. In this review, we will describe the developmental mechanisms that sexually differentiate neural circuits across three major clades of bilaterian animals-Ecdysozoa, Deuterosomia, and Lophotrochozoa. While many of the mechanisms inducing somatic and neuronal sex differentiation across these diverse organisms are clade-specific rather than evolutionarily conserved, we develop a common framework for considering the developmental logic of these events and the types of neuronal differences that produce sex-differentiated behaviors. This article is categorized under: Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development.

hsd17b1 is a key gene for ovarian differentiation of the Siberian sturgeon.

This is the first work using gonads from undifferentiated, genetically-sexed Siberian sturgeon describing expression changes in genes related to steroid synthesis and female and male sex differentiation. One factor identified as relevant for ovarian differentiation was the gene coding for the enzyme Hsd17b1, which converts estrone into estradiol-17ß. hsd17b1 was highly activated in female gonads at 2.5 months of age, around the onset of sex differentiation, preceding activation of two other genes involved in estrogen production (cyp19a1 and foxl2). hsd17b1 was also strongly repressed in males. Two known foxl2 paralogs are found in Siberian sturgeon-foxl2 and foxl2l-but only foxl2 appeared to be associated with ovarian differentiation. With regard to the male pathway, neither 11-oxygenated androgens nor classic male genes (amh, dmrt1, sox9, and dhh) were found to be involved in male sex differentiation, leaving open the question of which genes participate in early male gonad development in this ancient fish. Taken together, these results indicate an estrogen-dependence of female sex differentiation and 11-oxygenated androgen-independence of male sex differentiation.

Temperature-Induced Sex Differentiation in River Prawn (Macrobrachium nipponense): Mechanisms and Effects.

Macrobrachium nipponense is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually results in higher yield. To investigate the impact of temperature on sexual differentiation in M. nipponense, two temperature treatments (26 °C and 31 °C) were conducted. The results showed that compared to the 31 °C treatment (3.20 ± 0.12), the 26 °C treatment displayed a lower female/male ratio (2.20 ± 0.11), which implied that a lower temperature could induce masculinization in M. nipponense. The temperature-sensitive sex differentiation phase was 25-35 days post hatching (DPH) at 26 °C while 15-20 DPH at 31 °C. Transcriptome and qPCR analysis revealed that a lower temperature up-regulated the expression of genes related to androgen secretion, and down-regulated the expressions of genes related to oogonia differentiation. Thirty-one temperature-regulated sex-differentiation genes were identified and the molecular mechanism of temperature-regulated sex differentiation was suggested. The finding of this study indicates that temperature regulation can be proposed as an innovative strategy for improving the culture yield of M. nipponense.

Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes).

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.

Rhodopsin orphan GPCR20 interacts with neuropeptides and directs growth, sexual differentiation, and egg production in female Schistosoma mansoni.

IMPORTANCE: Schistosomes cause schistosomiasis, one of the neglected tropical diseases as defined by the WHO. For decades, the treatment of schistosomiasis relies on a single drug, praziquantel. Due to its wide use, there is justified fear of resistance against this drug, and a vaccine is not available. Besides its biological relevance in signal transduction processes, the class of G protein-coupled receptors (GPCRs) is also well suited for drug design. Against this background, we characterized one GPCR of Schistosoma mansoni, SmGPCR20, at the molecular and functional level. We identified two potential neuropeptides (NPPs) as ligands, SmNPP26 and SmNPP40, and unraveled their roles, in combination with SmGPCR20, in neuronal processes controlling egg production, oogenesis, and growth of S. mansoni females. Since eggs are closely associated with the pathogenesis of schistosomiasis, our results contribute to the understanding of processes leading to egg production in schistosomes, which is under the control of pairing in this exceptional parasite.

Epigenetic mechanisms underlying sex differences in the brain and behavior.

Sex differences are found across brain regions, behaviors, and brain diseases. Sexual differentiation of the brain is initiated prenatally but it continues throughout life, as a result of the interaction of three major factors: gonadal hormones, sex chromosomes, and the environment. These factors are thought to act, in part, via epigenetic mechanisms which control chromatin and transcriptional states in brain cells. In this review, we discuss evidence that epigenetic mechanisms underlie sex-specific neurobehavioral changes during critical organizational periods, across the estrous cycle, and in response to diverse environments throughout life. We further identify future directions for the field that will provide novel mechanistic insights into brain sex differences, inform brain disease treatments and women's brain health in particular, and apply to people across genders.

Temperature- and genotype-dependent stress response and activation of the hypothalamus-pituitary-interrenal axis during temperature-induced sex reversal in pejerrey Odontesthes bonariensis, a species with genotypic and environmental sex determination.

In the pejerrey Odontesthes bonariensis (Atheriniformes, Atherinopsidae), exposure to high and low temperatures during the critical period of sex determination (CPSD) induce testicular and ovarian differentiation, respectively, regardless of the presence or not of the sex determining gene amhy, which is crucial for testis formation only at intermediate, sexually neutral temperatures. In this study we explored the existence of genotype-specific signaling of Crh (Corticotropin Releasing Hormone) family genes and their associated carrier protein, receptors, and other stress-related genes in response to temperature during the CPSD and the potential involvement of the central nervous system via the hypothalamus-pituitary-interrenal (HPI) axis in the sex determination of this species. The Crh family genes crhb, uts1, ucn3, the receptor crhr1 and the stress-related genes gr1, gr2, nr3c2 were transiently upregulated in the heads of pejerrey larvae during the CPSD by high temperature alone or in combination with other factors. Only crhr2 transcript abundance was not influenced by temperature but independently by time and genotype. In most cases, mRNA abundance was higher in the XX heads compared to that of XY individuals. The mRNAs of some of these genes were localized in the hypothalamus of pejerrey larvae during the CPSD. XX larvae also showed higher whole-body cortisol titers than the XY, downregulation of cyp19a1a and upregulation of the testis-related genes amhy/amha in trunks (gonads) and were 100% masculinized at the high temperature. In contrast, at the low temperature, crhbp and avt were upregulated in the heads, particularly the former in XY larvae. cyp19a1a and amhy/amha were up- and downregulated, respectively, in the gonads, and fish were 100% feminized. Signaling via the HPI axis was observed simultaneously with the first molecular signs of ongoing sex determination/differentiation in the gonads. Overall, the results strongly suggest a temperature-dependent, genotype-specific regulatory action of the brain involving the Crh family of stress-related genes on the process of environmental sex determination of pejerrey.

Sex-specific transcriptome of the chicken chorioallantoic membrane.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers.

Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y).

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.