Mechanism Of Sex Determination Research Articles

The role of Wolbachia and the environment on sex determination of the Indian lac insect, Kerria lacca (Coccoidea: Tachardiidae)

• In Kerria lacca , presence of endosymbiont Wolbachia is developmental-stage dependent. • Administration of antibiotic enhances the sex ratio of the lac insect multifold. • The lac insect sex ratio is influenced by environmental factors. • Mode of metamorphosis together with Wolbachia dynamics explains sex ratio changes. The Indian lac insect, Kerria lacca (Kerr) exhibits a highly variable sex ratio, apparently influenced by various environmental factors, in the absence of a chromosomal sex determination mechanism. The endosymbiont Wolbachia, well known as sex ratio distorter, was reported in this species but its role in the sex determination of the host has hitherto remained unexplored. Distinct variation in the presence of Wolbachia was observed in relation to the developmental stages and the sexes of K. lacca as well as the host plant species; adult males of W + whereas adult females are W -. The post-settlement environmental factors such as host plant, season and genetic makeup affect the sex ratio in this insect, probably mediated through Wolbachia dynamics. Denser settlement of the crawlers led to comparatively higher Wolbachia presence with an elevated sex ratio. Suppression of Wolbachia through antibiotic administration resulted in a nearly three-fold increase in the sex ratio. Distinct transitions in the proportion of insects with heterochromatic genome (cytological males) have been observed during the first instar on different host plants, indicating that changes in the sex ratio occur during early development phase. Environmental factors appear to influence Wolbachia , which in turn brings about sex ratio changes, probably mediated through changes in juvenile hormone levels, as the male and female lac insects show distinct modes of metamorphosis.

Fate Decisions of Chicken Primordial Germ Cells (PGCs): Development, Integrity, Sex Determination, and Self-Renewal Mechanisms.

Primordial germ cells (PGCs) are precursor cells of sperm and eggs. The fate decisions of chicken PGCs in terms of their development, integrity, and sex determination have unique features, thereby providing insights into evolutionary developmental biology. Additionally, fate decisions in the context of a self-renewal mechanism have been applied to establish culture protocols for chicken PGCs, enabling the production of genome-edited chickens and the conservation of genetic resources. Thus, studies on the fate decisions of chicken PGCs have significantly contributed to both academic and industrial development. Furthermore, studies on fate decisions have rapidly advanced owing to the recent development of essential research technologies, such as genome editing and RNA sequencing. Here, we reviewed the status of fate decisions of chicken PGCs and provided insight into other important research issues that require attention.

Intersex is required for female sexual development in Hermetia illucens.

Sex-determination pathways are extremely diverse. Understanding the mechanism of sex determination in insects is important for genetic manipulation of the pest population and for breeding of economically valuable insects. Although sex determination has been well characterized in the model species Drosophila melanogaster, little is known about this pathway in Stratiomyidae. In the present study, we first identified the Drosophila intersex (ix) homolog in Hermetia illucens, also known as the black soldier fly, which belongs to the Stratiomyidae family and which is an important insect for the conversion of various organic wastes. Phylogenetic analyses and multiple sequence alignment revealed that Hiix is conserved compared with Drosophila. We showed that Hiix is highly expressed in internal genitalia. Disruption of the Hiix gene using CRISPR/Cas9 resulted in female-specific defects in external genitalia and abnormal and undersized ovaries. Taken together, our study furthers our understanding of sex determination in insects and could facilitate breeding of H.illucens.

Genome-wide identification and analysis of the molecular evolution and expression of type-A response regulator genes in Populus deltoids

Cytokinins have vital effects on various plant development-related processes. Type-A response regulators (RRs) are among the most important regulators in the cytokinin signaling pathway. Specifically, RR genes are the primary cytokinin-responsive genes, encode negative regulators of cytokinin signaling. Similar sex-determining mechanisms involving a type-A RR homolog have recently been identified in sister genera Populus and Salix. Here, we aimed to identify and characterize type-A RR gene family members in Populus deltoides. On the basis of the conserved receiver domain and phylogenetic analysis, we identified 9, 11, and 14 type-A RR genes were identified in P. deltoides, Populus trichocarpa, and Salix suchowensis, respectively. The exon-intron organization and chromosomal locations of these genes in P. deltoides as well as the characteristics of the encoded proteins were analyzed in detail. Gene duplication events, molecular evolution, and phylogenetic relationships were analyzed for all identified type-A RR genes along with 10 Arabidopsis thaliana homologs. These results indicated that the expansion of the type-A RR gene family was primarily due to whole-genome and segmental duplication events. The collinear type-A ARR pairs in A. thaliana were all derived from the α-whole-genome duplication (WGD) event, whereas those in the three Salicaceae species were divided into two groups, one comprising 10 pairs derived from the p-WGD event and the other consisting of 11 pairs derived from the γ-WGD event or a more ancient duplication event. All of the duplicated paralogous gene pairs had undergone purifying selection. Although different type-A RR paralogs experienced different selection pressures, convergent evolution was detected within the receiver domain-encoding sequence, indicative of the importance of this domain for the phosphorelay-related function of the encoded proteins. Three type-A RR genes (PdRR9, PtRR11, and SsRR12) associated with sex determination in Salicaceae species formed a small subclade that lacked A. thaliana counterparts. Evaluation of gene expression in vegetative tissues and at different floral developmental stages in P. deltoides revealed the specific spatiotemporal expression of each PdRR gene, with two (PdRR8 and PdRR9) of the nine PdRR genes exhibiting the opposite sex-biased expression patterns. Diverse expression levels were detected between all eight paralogous PdRR pairs. These findings support future research to elucidate the functional divergence of type-A RR genes.

On the origin of patterns of temperature-dependent sex determination.

Species with temperature-dependent sex determination (TSD) exhibit significant variation in the relationship between incubation temperatures and the sex ratios they produce, making this an ideal system for comparing processes producing variation above and below the species level. Furthermore, a deeper mechanistic understanding of TSD macro- and microevolution may help reveal the currently unknown adaptive significance of this variation or of TSD as a whole. Here, we probe these topics by examining the evolutionary dynamics of this sex-determining mechanism in turtles. Our ancestral state reconstructions of discrete patterns of TSD suggest that producing females at cool incubation temperatures is derived and potentially adaptive. However, the ecological irrelevance of these cool temperatures and a strong genetic correlation across the sex-ratio reaction norm in Chelydra serpentina both contradict this interpretation. We further find the phenotypic consequence of this genetic correlation in C. serpentina reflected across all turtle species, suggesting that a single genetic architecture underlies both intra- and interspecific variation in TSD in this clade. This correlated architecture can explain the macroevolutionary origin of discrete TSD patterns without assigning cool-temperature female production an adaptive value. However, this architecture may also constrain adaptive microevolutionary responses to ongoing climate change.

Characterization, expression profiling, and estradiol response analysis of DMRT3 and FOXL2 in clam Cyclina sinensis

The clam Cyclina sinensis is one of the important economical aquaculture shellfish in China. However, the mechanisms of sex determination and differentiation in C. sinensis have not been fully studied. In this study, full-length cDNAs of DMRT3 and FOXL2 were cloned and functionally characterized. The ORF region of CsDMRT3 consists of 1137 nucleotides, which encode 378 amino acids contains a conserved DM domain of DMRT family. The ORF region of CsFOXL2 is 1245 bp, encodes 414 amino acids, and contains a conserved FH domain. Tissue-specific expression results showed that the higher expression level of CsDMRT3 and CsFOXL2 was found in the ovary and testis of C. sinensis. The expression levels of CsDMRT3 and CsFOXL2 also peaked at the maturation stage of male and female gonadal development, respectively. Moreover, the expression levels of CsDMRT3 and CsFOXL2 were significantly higher in the trochophore and D-larval stages than in other stages. The transcript levels of CsDMRT3 reached the highest level at 11 months of age, while the CsFOXL2 reached the highest level at 7 months of age. In estradiol-treated experiments, the expression levels of CsDMRT3 and CsFOXL2 in the gonads were highest at 5 µg/L estradiol treatment, and histologically, it was observed that the oocytes diameters became larger with increasing estradiol concentration. These results suggest that CsDMRT3 and CsFOXL2 play an important role in gonadal development and sex differentiation of C. sinensis.

Sex Chromosomes and Sex Determination in Dioecious Agricultural Plants

Unlike in animals, dioecy among flowering plants is a rare phenomenon. The vast majority of angiosperm species have a bisexual flower that combines male (androecium) and female (gynoecium) reproductive organs. However, about a quarter of species have dioecious flowers, which can be located within the same plant (monoecious) or on different plants (dioecious). The flower formation in dioecious plants is determined by various genetic mechanisms. They become more complex from the work of a single gene to the functioning of full-fledged heteromorphic sex chromosomes, which can directly affect sex differentiation or participate in the balance determination of sex (where the formation of male or female flower organs depends on the ratio of X chromosomes to autosomes, for example). In recent years, the development of sequencing techniques, bioinformatics, and molecular biology has led to an increase in interest in the sex determination mechanisms among plants. It is noteworthy that a significant number of dioecious plants have economic value. At the same time, dioeciousness often complicates the growing process. This fact increases the relevance of studies on dioecious crops. In this review, we attempt to summarize the current information on sex chromosomes and the mechanisms of sex determination in dioecious plants, concentrating on species with agricultural importance.

De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis' maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Evolution of plant sex and molecular mechanisms underlying plants sex separation.

Unlike animals, plants exhibit more complexity of sexual morphs. The genetic mechanism underlying plant sex is a hot research topic in plant biology. In recent decades, advanced theories have been put forth on plant sex determination, but experimental proof is scarce. In recent years, vast achievements have been made to reveal the genetic mechanisms underlying sex separation of plants at the molecular level. Although the sex determination mechanisms have been clarified only in a limited number of plant species thus far, the discoveries offer us an opportunity to understand the genetic mechanisms triggering the separation of plant sexes. This paper reviewed the different aspects of the advanced studies on plant sex evolution and the molecular mechanisms underlying plant sex separation.

Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.

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.

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.

Sexually Dimorphic Gene Expression in X and Y Sperms Instructs Sexual Dimorphism of Embryonic Genome Activation in Yellow Catfish (Pelteobagrus fulvidraco).

Paternal factors play an important role in embryonic morphogenesis and contribute to sexual dimorphism in development. To assess the effect of paternal DNA on sexual dimorphism of embryonic genome activation, we compared X and Y sperm and different sexes of embryos before sex determination. Through transcriptome sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS) of X and Y sperm, we found a big proportion of upregulated genes in Y sperm, supported by the observation that genome-wide DNA methylation level is slightly lower than in X sperm. Cytokine-cytokine receptor interaction, TGF-beta, and toll-like receptor pathways play important roles in spermatogenesis. Through whole-genome re-sequencing (WGRS) of parental fish and RNA-seq of five early embryonic stages, we found the low-blastocyst time point is a key to maternal transcriptome degradation and zygotic genome activation. Generally, sexual differences emerged from the bud stage. Moreover, through integrated analysis of paternal SNPs and gene expression, we evaluated the influence of paternal inheritance on sexual dimorphism of genome activation. Besides, we screened out gata6 and ddx5 as potential instructors for early sex determination and gonad development in yellow catfish. This work is meaningful for revealing the molecular mechanisms of sex determination and sexual dimorphism of fish species.

Screening and characterization of sex-specific markers by NGS sequencing in Spinibarbus hollandi with implication of XY sex determination system

Sex-specific markers are critical for understanding sex determination mechanism and development of unisexual breeding in fish. Spinibarbus hollandi is an important commercial aquaculture species in southeastern China. Here, through whole genome sequencing of three female and six male individuals of S. hollandi, we first screened out a total of 88 candidate male-specific sequences. After PCR validation, three male-specific markers were proved to be able to realize male-specific amplification in S. hollandi. Subsequently, the three markers were also examined in another 24 female and 24 male individuals of S. hollandi, and the accuracy was 100%. Moreover, several male-specific genes that participate in sex determination process were identified by NR annotation, which might act as sex determining genes in S. hollandi. In general, the male-specific markers suggested an XY sex determination system in S. hollandi and would contribute a lot to understanding of sex determination mechanisms and development of unisexual breeding.

A transcriptional switch controls sex determination in Plasmodium falciparum

Sexual reproduction and meiotic sex are deeply rooted in the eukaryotic tree of life, but mechanisms determining sex or mating types are extremely varied and are only well characterized in a few model organisms1. In malaria parasites, sexual reproduction coincides with transmission to the vector host. Sex determination is non-genetic, with each haploid parasite capable of producing either a male or a female gametocyte in the human host2. The hierarchy of events and molecular mechanisms that trigger sex determination and maintenance of sexual identity are yet to be elucidated. Here we show that the male development 1 (md1) gene is both necessary and sufficient for male fate determination in the human malaria parasite Plasmodium falciparum. We show that Md1 has a dual function stemming from two separate domains: in sex determination through its N terminus and in male development from its conserved C-terminal LOTUS/OST-HTH domain. We further identify a bistable switch at the md1 locus, which is coupled with sex determination and ensures that the male-determining gene is not expressed in the female lineage. We describe one of only a few known non-genetic mechanisms of sex determination in a eukaryote and highlight Md1 as a potential target for interventions that block malaria transmission.

Sex-specific markers developed by 2b-RAD and genome sequencing reveal an XX/XY sex-determination system in mud carp (Cirrhinus molitorella)

Sex-specific markers are a powerful tool for studying sex-determination mechanisms and sex-control breeding in aquaculture. Mud carp (Cirrhinus molitorella) is one of the most important edible fishes in South China and Southeast Asia, and its processed products, such as tinned foods, have a high market value. The age of first maturation for mud carp is often three years. However, the sex-determination mechanism of mud carp is still unclear, which has affected its genetic breeding and culture industrial development. In this study, we identified sex-specific markers by 2b-RAD and genome sequencing in mud carp. The whole genome of a male mud carp was sequenced, and the total size of the draft genome was 1078.7 Mb. Nine males and nine females were subjected to 2b-RAD sequencing, and 12 male-specific scaffolds were isolated. Primers were designed based on these male scaffolds, and five male-specific markers from five scaffolds successfully discriminated males from females in the broodstock populations with 100% accuracy. Furthermore, five male-specific SNPs were identified and verified by Sanger sequencing, and all of them showed female homogamety and male heterogamety. These results indicate that mud carp has a male-heterogametic XX/XY sex-determination system. To the best of our knowledge, this is the first report of practical sex-specific markers in this species. Our results will contribute to elucidating the genetic basis of sex determination and improving the efficiency of genetic breeding in mud carp.

Expression and function analyses of sex-lethal gene in Phytoseiulus persimilis

Phytoseiulus persimilis is a specialist predator of spider mites, with promising control effects. Its population generally has stable sex ratio. Under appropriate conditions, the proportion of female offspring can reach 80%. But the mechanism of sex determination in P. persimilis is still unclear. Sex-lethal (sxl) is an important gene in studying sex regulation mechanisms in insects. In the model organism Drosophila melanogaster, it has been proved to control sex determination and dose compensation. Herein, we screened one sex-lethal gene ortholog in the transcriptome of each stage of P. persimilis that fed with spider mites (Ppsxl). It contains three highly conservative RRM domains. The expression pattern and function of Ppsxl were analyzed using real-time quantitative PCR and RNA interference. Ppsxl expressed in P. persimilis males and females at different developmental stages: the expression peaked 10 h after mating in females, and it was also highly expressed in eggs that developed into males (the first egg). The adult females, 5 hours after mating, were interfered through soaking in a mixed system of dsRNA and nanomaterials Star Polycation (dsRNA and SPc were 1:1 mixed, the concentration was 500 ng/μl). When Ppsxl was interfered, the expression of Ppsxl decreased by 47%. All female individuals after interference were fertile, and the first eggs still always developed into males. However, the daily fecundity per female decreased by 13.4%, and the proportion of female offspring decreased by 22.7%. This study showed that Ppsxl gene had a certain influence on reproduction of P. persimilis, but its influence on sex determination may not be as critical as that of D. melanogaster.

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family.

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.

Identification of sex-specific splicing via comparative transcriptome analysis of gonads from sea cucumber Apostichopus japonicus

Alternative splicing (AS) is an essential post-transcriptional regulation mechanism for sex differentiation and gonadal development, which has rarely been reported in marine invertebrates. Sea cucumber (Apostichopus japonicus) is an economically important marine benthic echinoderm with a potential XX/XY sex determination mechanism, whose molecular mechanism in the gonadal differentiation has not been clearly understood. In this study, we analyzed available RNA-seq datasets of male and female gonads to explore if AS mechanism exerts an essential function in sex differentiation and gonadal development of A. japonicus. In our results, a total of 20,338 AS events from 7219 alternatively spliced genes, and 189 sexually differential alternative splicing (DAS) events from 156 genes were identified in gonadal transcriptome of sea cucumber. Gene Ontology analysis indicated that these DAS genes were significantly enriched in spermatogenesis-related GO terms. Maximal Clique Centrality (MCC) was then applied for protein-protein interaction (PPI) analysis to search for protein interactions and hub DAS gene. Among all DAS genes, we identified 10 DAS genes closely related to spermatogenesis and (or) sperm motility and a hub gene dnah1. Thus, this study revealed that alternative isoforms were generated from certain genes in female and male gonads through alternative splicing, which may provide direct evidence that alternative splicing mechanisms participate in female and male gonads. These results suggested a novel perspective for explaining the molecular mechanisms underlying gonadal differentiation between male and female sea cucumbers.