Sex Determination in Homo sapiens as a Multi-Step Process: Potential for Development of Variants and Sex Differences in Disease Risk

Reproduction via cis-binary mechanisms appears to have evolved fairly early in the evolution of complex organisms, and a system committed to prior to evolution of humans. While the evolution of a chromosomal-specific approach has been a successful strategy for survival of a large variety of species including humans, the fidelity of sex determination leading to 100% cis-binary outcomes is not achieved in many species, with evidence for homosexual or bisexual behaviour evident in more than 1500 species. Thus, such outcomes indicates that sex determination is a multi-step process and not a single event, and as such, could lead to the appearance of variants during the process which developed much earlier than humans. Variants could arise either due to intrinsic variation in the steps of determination, or also be influenced by environmental factors of a biological or psychological nature. In contrast to homosexual variants which do not require interventions such as hormone therapy or surgery, expression of gender dysphoria, is more based in psychology, but also has biological underpinnings and can be influenced by such hormonal interventions and surgery. While the numbers of those with gender dysphoria is small (~0.6% - 1.0% of population), the attention given to this issue raises the possibility of biological and psychological environmental factors impacting the emergence of some of those expressing gender dysphoria. Furthermore, transitioning from male-to-female or female-to-male can have consequences regarding disease risks latter in life, including the appearance of autoimmune diseases. This review will attempt to review some of the evidence regarding sex determination, discuss why the system has potentially not been improved upon during evolution, how a potential role for sex chromosome function on neurodevelopment may be central to variation in humans, and how commitment to the current strategy is likely integrated into other sex-related events such as puberty, pregnancy, and menopause to ensure species survival. It will also discuss how variants in sex determination could contribute to sex differences in disease risk and how epigenetic modifications could play a role in such risk. .

Silencing transformer and transformer-2 in Zeugodacus cucurbitae causes defective sex determination with inviability of most pseudomales

transformer is a switch gene for sex determination in many insects, which cooperates with transformer-2 that is expressed in both sexes to regulate female differentiation, particularly in dipterans. Zeugodacus cucurbitae(Coquillett) is a very destructive pest worldwide, however, its sex determination pathway remains largely uncharacterized. Here, we show that the female sex ratio is sharply reduced with knockdown of either transformer or transformer-2 by RNA interference in early embryos of Z. cucurbitae. Most of the males grown from the embryos with transient transformer and transformer-2 suppression mated with wild-type females and produced mixed sex progeny, with one exception that produced only female progeny, and all of the few remaining males failed to mate with wild-type females and produced no progeny. The exceptional male and those males with mating failure were XX pseudomales as determined by the detection of Y chromosome-linked Maleness-on-the-Y, indicating that most XX pseudomales are not viable. The phenotypes of transformer and transformer-2 suggest that they play a key role in regulating sex determination and are required for female sexual development of Z. cucurbitae. Our results will be beneficial to the understanding of sex determination in Z. cucurbitae and can facilitate the development of genetic sexing strains for its biological control.

Maleness-on-the-Y(MoY) orthologue is a key regulator of male sex determination in Zeugodacus cucurbitae(Diptera:Tephritidae)

The initiation of sex differentiation in insects is regulated by primary sex determination signals.In the Medfly Ceratitis capitata and other tephritids,Maleness-on-the-Y(MoY) is the master gene for male sex determination.However,the primary signal in Zeugodacus cucurbitae(Coquillett),a very destructive tephritid pest across the world,remains ambiguous.In this study,we have isolated and characterized the Medfly MoY homolog in Z.cucurbitae,ZcMoY.ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species.ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults.Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs,giving rise to intersexes and feminization of XY individuals.The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Z.cucurbitae males.Our findings will help the understanding of sex determination in Z.cucurbitae and facilitate the development of genetic sexing strains in its biological control.

The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

Sex determination and dif ferentiation in Aurelia sp.1: the absence of temperature dependence

Cnidarians, being regarded as ‘basal' metazoan animals, are considered to have relatively high plasticity in terms of sex reversal. In this study we used an experimental approach to demonstrate sexual diff erentiation and plasticity in benthic polyps and pelagic medusae of A urelia sp.1 maintained at dif ferent temperatures. Results indicated that in A urelia sp.1, sex diff erentiation has been determined at the polyp stage and that all medusae originating from a given polyp are, phenotypically, of the same sex. In addition, the sex of polyps budding from the same clone(either male or female) at dif ferent temperatures appears to be the same as that of the parent. The sex of medusae that had originated from a known-sex polyp was observed to remain the same as that of the parent, irrespective of differences in strobilation or rearing temperatures. These results indicate that the mechanism of sex determination of Aurelia sp.1. is not influenced by prevailing temperature regimes. A comparison of variability in terms of sexual plasticity of A urelia sp.1 with that of Hydrozoa and Anthozoa suggests that species characterized by a free-swimming medusa life stage have a high dispersal potential, which probably results in a lower rate of sex reversal.

Sturgeon (Acipenser gueldenstaedtii) Acclimatization to Optimal Growth Conditions—A Case Study of Adaptation, Nutrition, Reproduction, and Sex Determination

The development of new agricultural industries is an important challenge for a region’s economic establishment. The connection between a research department at MIGAL Institute and biotechnology in aquaculture is an example (case study) of the interrelationships that also exist in other departments. This article describes the research conducted by the institute supporting the development of the aquaculture industry in Israel through the introduction of a relatively new fish species—the Russian sturgeon (Acipenser gueldenstaedtii), with the relevant information to be used for acclimatization to aquaculture conditions in northern Israel. Many aspects related to bringing a new species to Israel and working with this new industry to study its adaptation to existing conditions in the north of the country influenced the economic success of this new type of precision agriculture. This article describes the research on various aspects affecting the successful acclimatization of Russian sturgeon in northern Israel: introduction, food development, reproduction, hormone systems that control reproduction and growth, gonadal development, and molecular markers for sex determination, differentiation, and economic assessment of caviar production.

Sex Chromatin in Peripheral Blood Neutrophils and Sex Determination

The objective of this paper is to review in what forms sex chromatin can appear in peripheral blood neutrophils and how sex determination can be done using sex chromatin appendages. Sex chromatin is an approximately 1 micron clump of chromatin seen usually at the periphery of female nuclei in certain tissues and called “Barr body” and as a drumstick in polymor phonuclear neutrophils nuclei in the blood smears. Sex chromatin is derived from one of the two X chromosomes in the female which replicates its deoxyribonucleic acid much later than the other and is thus positively heteropyknotic. In 1954, Davidson and Smith were the first to identify and report the presence of neutrophil drumsticks and nonspecific appendages and their differences in sexes. The inactive X chromosome in neutrophils appears in one of the five forms: drumsticks, racquet forms, sessile nodules, small clubs and minor lobes. Only drumstick appendage is sex-specific and considered for sex diagnosis. For sex determination, drumsticks are significantly higher in females than in males (p < 0.001).

Evolution of sex determination in crustaceans

Sex determination(SD)involves mechanisms that determine whether an individual will develop into a male,female,or in rare cases,hermaphrodite.Crustaceans harbor extremely diverse SD systems,including hermaphroditism,environmental sex determination(ESD),genetic sex determination(GSD),and cytoplasmic sex determination(e.g.,Wolbachia controlled SD systems).Such diversity lays the groundwork for researching the evolution of SD in crustaceans,i.e.,transitions among different SD systems.However,most previous research has focused on understanding the mechanism of SD within a sin-gle lineage or species,overlooking the transition across different SD systems.To help bridge this gap,we summarize the understanding of SD in various clades of crustaceans,and discuss how different SD systems might evolve from one another.Furthermore,we review the genetic basis for transitions between different SD systems(i.e.,Dmrt genes)and propose the microcrustacean Daphnia(clade Branchiopoda)as a model to study the transition from ESD to GSD.

Genetic Approaches for Sex Determination of Chinese Female with Male Pseudohermaphroditism

Male pseudohermaphroditism is a rare disorders of sex development(DSD)that is manifested by a female-like appearance or incompletely differentiated external genitalia in an individual with a Y chromosome.In this paper,we report our investigation of the case of a 33-year-old Chinese female who was diagnosed with a malignant mixed germ-cell tumor of the ovary.To confirm the sex of the female,we utilized genetic approaches to detect amelogenin and Y-STR loci.Y chromosome microdeletion was performed to identify existing deletions in the AZF regions and SRY.Chromosome karyotyping and whole-exome sequencing(WES)were then applied to reveal the deletion of sex chromosome segments and pathogenic variations in DNA sequences.Using DNA-STR genotyping,we detected both AMEL-X and AMEL-Y fragments.We also found haplotype Y-STR loci and detected all alleles.Furthermore,no microdeletion was detected in the AZF regions and SRY.The chromosome karyotyping was 46,XY.WES revealed a transversion mutation of 58T→C in the androgen receptor exon 1,which could be the pathogenic variant in this case of abnormal sexual development.Sex determination in forensic DNA typing is based on the amelogenin system.It is important that forensic biologists should master various genetic approaches to overcome the issue of gender ambiguity caused by DSDs.

Sex ratio variation and sex determination in the mallee dragon Ctenophorus fordi

Recent evidence suggests that many Australian agamids show temperature-dependent sex determination(TSD)with variation in sex determining mechanisms among closely related taxa.However,as shown in other vertebrates,sex ratios can also be influenced by genetic or phenotypic differences among females in their propensity to produce sons or daughters,and these influences might confound any thermal effects of incubation per se.To address these issues,we investigated the determinants of sex ratios in the mallee dragon Ctenophorus fordi,together with a detailed analysis of karyotypes.There was no detectable variation in sex ratios arising from variation among females,clutches or incubation temperatures,which might indicate genetic sex determination for this species.However,there was no evidence of cytologically distinct sex chromosomes using standard banding techniques.The sex ratio pattern in C.fordi strongly contrasts with the results for the congener Ctenophorus pictus,where sex ratios show variation among females.Thus,Australian agamids offer promising opportunities to address fundamental issues in sex ratio biology.

Sex determination and maintenance： the role DMRT1 and FOXL2

In many species, including mammals, sex determination is genetically based. The sex chromosomes that individuals carry determine sex identity. Although the genetic base of phenotypic sex is determined at the moment of fertilization, the development of testes or ovaries in the bipotential early gonads takes place during embryogenesis. During development, sex determination depends upon very few critical genes. When one of these key genes functions inappropriately, sex reversal may happen. Consequently, an individual＇s sex phenotype may not necessarily be consistent with the sex chromosomes that are present. For some time, it has been assumed that once the fetal choice is made between male and female in mammals, the gonadal sex identity of an individual remains stable. However, recent studies in mice have provided evidence that it is possible for the gonadal sex phenotype to be switched even in adulthood. These studies have shown that two key genes, doublesex and mad-3 related transcription factor 1 （Dmrtl） and forkhead box L2 （Foxl2）, function in a Yin and Yang relationship to maintain the fates of testes or ovaries in adult mammals, and that mutations in either gene might have a dramatic effect on gonadal phenotype. Thus, adult gonad maintenance in addition to fetal sex determination may both be important for the fertility.

Sex determination mechanisms and sex control approaches in aquaculture animals

Aquaculture is one of the most efficient modes of animal protein production and plays an important role in global food security.Aquaculture animals exhibit extraordinarily diverse sexual phenotypes and underlying mechanisms,providing an ideal system to perform sex determination research,one of the important areas in life science.Moreover,sex is also one of the most valuable traits because sexual dimorphism in growth,size,and other economic characteristics commonly exist in aquaculture animals.Here,we synthesize current knowledge of sex determination mechanisms,sex chromosome evolution,reproduction strategies,and sexual dimorphism,and also review several approaches for sex control in aquaculture animals,including artificial gynogenesis,application of sex-specific or sex chromosome-linked markers,artificial sex reversal,as well as gene editing.We anticipate that better understanding of sex determination mechanisms and innovation of sex control approaches will facilitate sustainable development of aquaculture.

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish

Polyploids in vertebrates are generally associated with unisexual reproduction,but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown.Here,we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus.The synthetic octoploids were revealed to have more than 200 chromosomes,in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C.auratus into the unisexual gynogenetic hexaploid C.gibelio.Significantly,a few synthetic octoploid males were found to be fertile,and one octoploid male was confirmed to regain sexual reproduction ability,which exhibits characteristics that are the same to sexual reproduction tetraploid males,such as 1:1 sex ratio occurrence,meiosis completion and euploid sperm formation in spermatogenesis,as well as normal embryo development and gene expression pattern during embryogenesis.Therefore,the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

Chromosome-level Genomes Reveal the Genetic Basis of Descending Dysploidy and Sex Determination in Morus Plants

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation.Morus notabilis,a dioecious mulberry species,has the fewest chromosomes among Morus spp.,but the genetic basis of sex determination and karyotype evolution in this species has not been identified.In this study,three high-quality genome assemblies were generated for Morus spp.[including dioecious M.notabilis(male and female)and Morus yunnanensis(female)]with genome sizes of 301–329 Mb and were grouped into six pseudochromosomes.Using a combination of genomic approaches,we found that the putative ancestral karyotype of Morus species was close to 14 protochromosomes,and that several chromosome fusion events resulted in descending dysploidy(2n=2x=12).We also characterized a~6.2-Mb sex-determining region on chromosome 3.Four potential male-specific genes,a partially duplicated DNA helicase gene(named MSDH)and three Ty3_Gypsy long terminal repeat retrotransposons(named MSTG1/2/3),were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation.Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry.In addition,genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified.Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.

Sex determination in mammalian germ cells

Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model.

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates,and it has been shown to be involved the regulation of gene expression and embryo development.However,it remains unclear how DNA methylation regulates sexual development,especially in species without sex chromosomes.To determine this,we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs)undergo significant DNA methylation reprogramming during germ cell development,and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf).When DNA methyltransferase(DNMT)activity in juveniles was blocked after 9 dpf,the zebrafish developed into females.We also show that Tet3 is involved in PGC development.Notably,we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm.Furthermore,inhibiting DNMT activity can prevent the female-to-male sex transition,suggesting that methylation reprogramming is required for zebrafish sex transition.In summary,DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.

Absence of complementary sex determination in two Leptopilina species(Figitidae,Hymenoptera)and a reconsideration of its incompatibility with endosymbiont-induced thelytoky

Complementary sex determination(CSD)is a widespread sex determination mechanism in haplodiploid Hymenoptera.Under CSD,sex is determined by the allelic state of one or multiple CSD loci.Heterozygosity at one or more loci leads to female development,whereas hemizygosity of haploid eggs and homozygosity of diploid eggs results in male development.Sexual(arrhenotokous)reproduction normally yields haploid male and diploid female offspring.Under asexual reproduction(thelytoky),diploidized unfertilized eggs develop into females.Thelytoky is often induced by bacterial endosymbionts that achieve egg diploidization by gamete duplication.As gamete duplication leads to complete homozygosity,endosymbiont-induced thelytokous reproduction is presumed to be incompatible with CSD,which relies on heterozygosity for female development.Previously,we excluded CSD in four Asobara(Braconidae)species and proposed a two-step mechanism for Wolbachia-induced thelytoky in Asobara japonica.Here,we conclusively reject CSD in two cynipid wasp species,Leptopilina heterotoma and Leptopilina clavipes.We further show that thelytoky in L.clavipes depends on Wolbachia titer but that diploidization and feminization steps cannot be separated,unlike in A.japonica.We discuss what these results reveal about the sex determination mechanism of L.clavipes and the presumed incompatibility between CSD and endosymbiont-induced thelytoky in the Hymenoptera.

Early embryonic transcriptomes of Zeugodacus tau provide insight into sex determination and differentiation genes

Zeugodacus tau(Walker)is an invasive pest.The sterile insect technique is an environment-friendly method for pest control.Understanding the mechanism of sex determination will contribute to improving efficiency of this technique.In this study,we identified the transformer(tra)gene in Z.tau.One female-specific and two male-specific isoforms of tra were found in Z.tau,and the male-specific splicing pattern of tra was found to occur 5 h after egg laying.We performed transcriptome sequencing at 1 h(E1),5 h(E5),and 9 h(E9)after egg laying and obtained high-quality transcriptome libraries of early embryo development.We identified 13297 and 11713 differentially expressed genes(DEGs)from E5 versus E1 and E9 versus E1 comparisons,respectively.To explore the potential functions of the DEGs during embryonic development,Gene Ontology,Clusters of Orthologous Groups of proteins,and Kyoto Encyclopedia of Genes and Genomes analyses were performed.Twenty-six genes potentially involved in sex determination or differentiation,including Maleness-on-the-Y(MoY),were identified in Z.tau.To verify the transcriptome results,15 genes were selected for quantitative real-time PCR validation.The results were consistent with the transcriptome sequencing results.Moreover,U2 small nuclear riboprotein auxiliary factor(U2AF-50),female lethal d(fl(2)d),and virilizer(vir)were highly expressed at E5,indicating that they may be related to the sex-specific splicing of tra.Further functional analysis is needed to confirm this speculation.Our data provide an insight into the mechanism underlying sex determination and differentiation in tephritid species.

Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus

Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pathway.Aedes(Stegomyia)albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx(Aalbdsx)yields one male-(Aalbdsx^(M))and three female-specific isoforms(Aalbdsx^(F1-3));however,the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation.In this study,we demonstrated that the Aalbdsx^(M) messenger RNA(mRNA)represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage(0-2 and 4-8 h),maternal dsx^(F) and default splicing dsx^(M) were detected in both sexes;the expression of dsx^(M) then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsx^(M) to dsx^(F) in both sexes;however,the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsx^(M) in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.