Expression of Sex-Related Genes in Chicken Embryos During Male-to-Female Sex Reversal Exposure to Diethylstilbestrol

Sex emerges out of a delicate dance between a variety of promale, anti-male, and possibly profemale genes. To investigate the role that sex-related genes play in sex determination and gonadal differentiation of fowl, we constructed a male-to- female sex-reversal model of chick induced by diethylstilbestrol （DES） at onset of incubation （E0）. The results of semi- quantitative PCR showed that the expression of Sf1, the orphan nuclear receptor steroidogenic factor-1 gene, was put forward from E7d to E5d and up-regulated during E5-7d; the Dmrt1, the double sex and the Mab-3 related to transcription factor 1 gene, was down-regulated during E3-7d. Meanwhile, anti-Müllerian hormone gene （Amh） expressed at a similar level in the genetic females and sex-reversal females before E7d, while no expression products of the three female-specific genes Wpkci, Fet1 and Foxl2 were detected in male-to-female embryos. These findings suggest that the expression of some certain sex-related genes, induced by the exogenous estrogen during period of sex determination and gonadal differentiation, results in the male-to-female sex reversal. Moreover, high activity of Sf1 gene during E5-7d might be related to the profemale process, while low activity of Dmrt1 gene during E3-5d might be anti-male. The expression activity of Amh gene might only contribute to the promale process after E7d, however, it is possibly not an anti-female gene in chick embryos.

Single-cell-resolution transcriptome map revealed novel genes involved in testicular germ cell progression and somatic cells specification in Chinese tongue sole with sex reversal

Female-to-male sex reversals(pseudomales)are common in lower vertebrates and have been found in natural populations,which is a concern under rapid changes in environmental conditions.Pseudomales can exhibit altered spermatogenesis.However,the regulatory mechanisms underlying pseudomale spermatogenesis remain unclear.Here,we characterized spermatogenesis in Chinese tongue sole(Cynoglossus semilaevis),a species with genetic and environmental sex determination,based on a highresolution single-cell RNA-seq atlas of cells derived from the testes of genotypic males and pseudomales.We identified five germ cell types and six somatic cell types and obtained a single-cell atlas of dynamic changes in gene expression during spermatogenesis in Chinese tongue sole,including alterations in pseudomales.We detected decreased levels of Ca^(2+)signaling pathway-related genes in spermatogonia,insufficient meiotic initiation in spermatocytes,and a malfunction of somatic niche cells in pseudomales.However,a cluster of CaSR genes and MAPK signaling factors were upregulated in undifferentiated spermatogonia of pseudomales.Additionally,we revealed that Z chromosome-specific genes,such as piwil2,dhx37,and ehmt1,were important for spermatogenesis.These results improve our understanding of reproduction after female-to-male sex-reversal and provide new insights into the adaptability of reproductive strategies in lower vertebrates.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system

In reptiles,such as the red-eared slider turtle(Trachemys scripta elegans),gonadal sex determination is highly dependent on the environmental temperature during embryonic stages.This complex process,which leads to differentiation into either testes or ovaries,is governed by the finely tuned expression of upstream genes,notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2.Recent studies have identified epigenetic regulation as a crucial factor in testis development,with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T.s.elegans.However,whether KDM6B alone can induce testicular differentiation remains unclear.In this study,we found that overexpression of Kdm6b in T.s.elegans embryos induced the male development pathway,accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C,a temperature typically resulting in female development.Notably,this sex reversal could be entirely rescued by Dmrt1 knockdown.These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway,underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

ASER:Animal Sex Reversal Database

Sex reversal,representing extraordinary sexual plasticity during the life cycle,not only triggers reproduction in animals but also affects reproductive and endocrine system-related diseases and cancers in humans.Sex reversal has been broadly reported in animals;however,an integrated resource hub of sex reversal information is still lacking.Here,we constructed a comprehensive database named ASER(Animal Sex Reversal)by integrating sex reversal-related data of 18 species from teleostei to mammalia.We systematically collected 40,018 published papers and mined the sex reversal-associated genes(SRGs),including their regulatory networks,from 1611 core papers.We annotated homologous genes and computed conservation scores for whole genomes across the 18 species.Furthermore,we collected available RNA-seq datasets and investigated the expression dynamics of SRGs during sex reversal or sex determination processes.In addition,we manually annotated 550 in situ hybridization(ISH),fluorescence in situ hybridization(FISH),and im-munohistochemistry(IHC)images of SRGs from the literature and described their spatial expression in the gonads.Collectively,ASER provides a unique and integrated resource for researchers to query and reuse organized data to explore the mechanisms and applications of SRGs in animal breeding and human health.The ASER database is publicly available at http://aser.ihb.ac.cn/.

Sexual plasticity in bony fishes:Analyzing morphological to molecular changes of sex reversal

Sex reversal is one of the characteristic properties of sexual plasticity in bony fishes wherein both natural and induced sex change happens at various stages of life cycle in different species.Sex determination in gonochoristic species is genetically regulated,wherein the same sex is retained throughout their life span whereas hermaphrodites change their sex during development or adulthood.In sequential hermaphrodites,serial sex change occurs at different points of life cycle.Concurrently,synchronous hermaphrodites function as both the sexes during spawning.Other variables like temperature,pH and social factors can trigger sex reversal in teleost.Sex reversal through gene mutations and chemicals/hormones,including sex steroids,can be induced mostly at early developmental stages but natural sex reversal can occur at any time.Sex reversal mechanism shows morphological to molecular changes,which are ideal for identification of sex-specific gene markers.In fact,gonadal transdifferentiation occurs at the molecular level through differential expression of transcription factors and steroidogenic enzyme genes vis-a-vis hormones,thereby imparting phenotypic or structural changes.In addition,brain shows sexual dimorphism which is mostly consequential to gonadal sex development and occasionally either causative.The major breakthrough in this line is the identification of sex determining genes such as dmy/dmrt1Yb,gsdfY,sox3 in the Japanese medaka and amhY in Patagonian pejerrey.Incidentally,the induction of mono-sex population by favouring one sex due to sex-specific differences in growth is an important economic boom for aquaculture.This review comprehensively highlights key molecular factors involved in natural and induced sex reversal conditions to illustrate teleostean sexual plasticity and its application perspectives.

Sex reversal and the androgenic gland (AG) in Macrobrachium rosenbergii: A review

The male giant freshwater prawn(Macrobrachium rosenbergii)generally exhibit faster growth rates compared to females of similar age.Consequently,the practice of culturing monosex(all-male)prawn can increase both the production yield and income.Manually selecting males for culture during the grow-out period has long been practiced in commercial prawn farming,but is labor-intensive and generally unsuccessful in producing a male monoculture.The androgenic gland(AG)is essential for sexual differentiation in prawn and development of male primary and secondary sexual characteristics.Microsurgical removal of the AG(andrectomy)from male prawn at an early development stage will transform the male into a fully-functioning neo-female.The M.rosenbergii androgenic gland-specific insulin-like hormone(Mr-IAG)plays critical roles in male spermatogenesis and male sexual development.RNA interference(RNAi)is a highly specific post-transcriptional interference or silencing of gene expression in cells.Silencing Mr-IAG had induced full and functional sex reversal of male to neo-female(genetic male with a female phenotype).Microsurgical removal of AG and RNAi are two different approaches to producing neo-females and both methods target the manipulation of AG for efficient sex-reversal.Neo-females can mate with normal males to produce all-male prawn progeny,which can be used for commercial prawn farming and to improve production efficiency and profitability.This article reviewed recent research related to the development and functions of AG and the testis and their interactions with morphological differentiation at various stages for male M.rosenbergii,aiming to increase understanding on this topic and facilitate discovery of new techniques for advancing M.rosenbergii aquaculture.

Male sex determination： insights into molecular mechanisms

Disorders of sex development often arise from anomalies in the molecular or cellular networks that guide the differentiation of the embryonic gonad into either a testis or an ovary, two functionally distinct organs. The activation of the Y-linked gene Sry （sex- determining region Y） and its downstream target Sox9 （Sry box-containing gene 9） triggers testis differentiation by stimulating the differentiation of Sertoli cells, which then direct testis morphogenesis. Once engaged, a genetic pathway promotes the testis development while actively suppressing genes involved in ovarian development. This review focuses on the events of testis determination and the struggle to maintain male fate in the face of antagonistic pressure from the underlying female programme.

Dosage Effects of Fadrozole on Growth and Development of Sex-Reversed Genetic Female Chickens

Fadrozole, an aromatase inhibitor, can masculinize genetic female chickens and high-dose decreases the hatchability. Therefore, it is important to study the growth and development of sex-reversed females after hatch. Chick embryos from a population of CAU3 egg-type were treated with different concentrations of Fadrozole prior to the sexual differentiation at E3.0 （st18）. At hatch, the phenotypic sex and genetic sex were identified by vent sexing and genetic diagnosis with CHD1, respectively. Body weight and shank length of sex reversal were tested at 8 and 20 wk, respectively. Testicular development, oviduct and ovarian degeneration were observed and serum concentration of estradiol and testosterone were tested with radioimmunoassay （RIA） at 30 wk. The results showed that body weight and shank length of sex- reversed females were not significantly different between low-dose groups （0.1, 0.3, and 0.5 mg for F1, F2, and F3, respectively） and high-dose groups （1.0 and 1.3 mg for F4 and F5, respectively） （P〉0.05）. Left and right testes or ovotestes in F2, F3, F4, and F5 groups were heavier than that of in F1 group （P〈0.05）. While the gonad weight of treatment groups were less than that in male control （P〈0.05）, oviduct weight in F2, F3, F4, and F5 groups were significant differences compared with female control and F1 group （P〈0.05）. Egg number from onset of laying egg to 30 wk in F4 and F5 groups were less than in female control, F1 and F2 groups （P〈0.05）. Serum testosterone level in F5 group was significant higher compared with female control, F1, F2, F3, and F4 groups （P〈0.05）, but significant lower compared with male control （P〈0.05）. While concentration of serum estradiol in F5 group was significant lower compared with female control, F1, F2, and F4 groups （P〈0.05）. In conclusion, the concentration of Fadrozole do not affect postnatal growth of sex-reversed female chicken and the degree of sex-reversed females elevate with the increase of Fadrozole concentration at sex maturity.

Offspring Sex Is Not Determined by Gestation Temperature in a Viviparous Lizard(Eremias multiocellata) from the Desert Steppe of Inner Mongolia

Sex-determining systems show a striking diversity not only among species, but also among popula tions. In reptiles, sex-determina tion is a continuum, from tempera ture-dependent sex determination(TSD) to genetic sex determination(GSD).The multi-ocellated racerunner(Eremias multiocellata)is reported to be a cryptic ZZ/ZW chromosomal TSD species, with male-biased sex ratios at high temperatures in two Gansu populations. However, the generality of the sex-determining pattern in different populations of this species remains unclear. To investigate the mode of sex determina tion in a popula tion of E.multiocellata from the desert steppe of Inner Mongolia,we first identified sex chromosomes via comparative genomic hybridization(CGH). We then conducted a thermal manipulation experiment to determine the effect of gestation temperature on offspring sex ratios.From the CGH studies we found that lizards from the Inner Mongolia population possessed ZZ/ZW sex chromosomes. However, our thermal manipulation experiment showed that gestation temperature did not affect the sex ratio of neonates in this population. In combination, these results rule out TSD in the Inner Mongolia population of E. multiocellata, and suggest that there is widespread geographic variation in the sexdetermining system of this species.

Investigation of SRY Gene in Sex Reversed Syndrome Patients

SRY (sex-determining region Y chromosome) is considered as a strong candidste for the TDF (testis determining factor) and has been cloned following another candidate ZFY (zinc finger protein gene). In this study,eight cases of sex revelsal, including four 46, XX males and four 46, XY females aere examined for the presence of SRY sequence and a Y-repeated DNA locus. Our data indicated that the genownic DNA of the four classical 46,XX males had the SRY sequences. On the other hand, both SRY sequences and Ye repeated DNA sequences were present in all four 46, XY females.These resultssuavest that SAY sequences were responsible for the sexraversal of 46, XX males Whereas there may be other genetic mechanisms forthe sex reversal of 46, XY females Without the lack of SRY sequences.

Recovery of gonadal development in tiger puffer Takifugu rubripes after exposure to 17β-estradiol during early life stages

The aim of the present study was to investigate the long-term effects of 17l）-estradiol （E2） exposure on gonadal development in the tiger puffer （Taktfugu rubripes）, which has a genetic sex determination system of male homogametic XY-XX. Tiger puffer larvae were exposed to 1, 10 and 100 μg/L E2 from 15 to 100 days post-hatch （dph） and then maintained in clean seawater until 400 dph. Changes in sex ratio, gonadal structure and gonadosomatic index （GSI） were monitored at 100, 160, 270 and 400 dph. Sex-associated single nucleotide polymorphism （SNP） markers were used to analyze the genetic sex of samples, except those at 100 dph. Exposure had a positive effect on the conversion of genetically male gonads into phenotypically female gonads at 100 dph. However, gonads from 60% of genetic XY males in the 1-μg/L E2 group and 100% in the 10-μg/L E2 group developed intersexual gonads at 160 dph; gonads of all genetic XY males in the two treatment groups reverted to testis by 270 dph. While 38%, 57% and 44% of gonads of XY fish in the 100-gg/L E2 group reverted to intersexual gonads at 160, 270 and 400 dph, respectively, none reverted to testis after E2 treatment. In addition, E2 exposure inhibited gonadal growth of both genetic sexes, as indicated by the clear dose-dependent decrease in GSI at 270 and 400 dph. The results showed that exposure to E2 during the early life stages of tiger puffer disrupted gonadal development, but that fish recovered after migration to clean seawater. The study suggests the potential use of tiger puffer as a valuable indicator species to evaluate the effects of environmental estrogens on marine fish, thereby protecting valuable fishery resources.

Gonadal Steroid Hormones Levels in a Bitch with True Bilateral Hermaphroditism

Intersexualism occurs in all species of maxnmals and the pathogenesis of abnormal development of the gonads is still unclear. Thus, the aim of this report is to describe the hormonal and histopathological findings from a case of bilateral true hermaphroditism in a mixed-breed bitch due to the few case reports published involving hormonal levels in dogs with intersexuality. Serum was obtained from the animal before surgery and was submitted to hormone measurements. Progesterone and testosterone were evaluated by radioimnunoassay and estrogen by ELISA （immune-enzymatic assay）. The values of progesterone, testosterone and estrogen concentrations were 2.26 ng/mL, 0.05 ng/mL and 9.7 pg/mL, respectively. The proportion of ovarian tissue found in the ovotestes after histopathological examination was higher in relation to seminiferous tubules, which may explain the low level of serum testosterone. Low concentration of serum testosterone may have contributed to the partial virilization of external genitalia, since the animal only had clitoral hypertrophy. Estrogen and progesterone levels found were compatible with the initial stage of estrus （preovulatory LH （luteinizing hormone） surge）, which shows that ovarian follicles found in the bilateral ovotestes contributed to the occurrence of the estrous cycle before surgery. Quantitation of serum hormones may also shed light on the cause of disturbances in sex differentiation in canine species. More studies are needed to elucidate the real cause of intersex in dogs.

Performance of Brooders,Fry and Growth of the Nile Tilapia(Oreochromis niloticus)Cultured in an Experimental Recirculating Aquaculture System

We present results of a long-term study aimed to optimize Nile tilapia O. niloticus production in a recirculating aquaculture system (RAS) in México City. Three experiments were evaluated: 1) fry production;2) production of masculinized fries;and 3) assessment of growth under three different densities (10, 30 and 75 fish/m3). The broodstock produced on average 2400 ± 600 fry every 15 days during six months, with a total production of 28,800 ± 2000. One hundred percent males were produced in 274-L raceways in an eight-week period. The obtained results in fish production in the RAS system indicated variations related to density. A daily growth rate of 3.6 g/day was registered with a density of 10 fish/m3, 0.9 g/day in 30 fish/m3 and 0.4 g/day in 75 fish/m3. This information could be useful for production purposes in this type of systems.

Production of XX neo-male mandarin fish, Siniperca chuatsi, after sexual differentiation by oral administration of 17α-methyltestosterone

A well-known fact about mandarin fish(Siniperca chuatsi)is that females grow faster than males.Mandarin fish that have all-female populations have economic advantages,but traditional neo-male fish are difficult to produce.In this study,indirect feeding of 500 mg/kg 17α-methyltestosterone(MT)to XX mandarin fish(60 dpf,days post fertilization)with completely differentiated ovaries induced secondary sex reversal.At 40 days after MT treatment,14.7%of mandarin fish with complete reversal was produced,which showed for the first time that XX mandarin fishes with differentiated ovaries still have potentially bisexual germ cells and the plasticity of sex reversal.In the MT treatment group,there was no significant difference in survival compared to the control group.The body weight of the MT treatment group was significantly lower than that of the control group 40 days after treatment(dat)(P<0.05).During MT treatment,serum 11-KT and T concentrations increased and serum E2 concentration decreased in the MT treatment group,but there was no significant difference in hormone levels between the MT treatment group and the control group.A masculinization of mandarin fish was also confirmed by the expression patterns of sex-specific genes sox9,dmrt1,cyp19a1a and foxl2.A significant amount of sox9 and dmrt1 was expressed in the fish gonads of the MT treatment group,whereas cyp19a1a and foxl2 were minimally expressed in the fish gonads.

Self-fertilization in a gonochoristic fish yellow catfish

Sex control techniques are in great demand for aquaculture because of the huge benefits of mono-sex production.In a previous study on yellow catfish(Tachysurus fulvidraco),intersex individuals were produced by administrating 17α-methyltestosterone(MT)or aromatase inhibitor letrozole(LZ).The ovarian and testicular portions of intersexual gonads developed well and were completely separated,arousing our interest in producing self-fertilizable fish and clone-like fish populations.In this study,yellow catfish fry were treated with a combination of MT and LZ at a low dose(5 mg kg^(-1)MT,300 mg kg^(-1)LZ)at a low water temperature(27.0℃,mixed sex-producing temperature,LL group)and a high temperature(33.5◦C,male-producing temperature,HL group),and with a high dose(10 mg kg^(-1) MT,600 mg kg^(-1) LZ)at a low water temperature(LH group),during 12-65 days post-hatching,the critical period of sex differentiation.Both XX and XY intersex were produced with different percentages at the same time through co-treatment with MT and LZ,with or without the help of male-producing temperature.The highest percentage of XY intersex(53%in XY,36%in XX and XY)and XX intersex(47%in XX,25%in XX and XY)was produced in LH and HL groups,respectively.Results of gonadal morphology,histology,and sperm dynamic analysis indicate that some XX and XY intersex individuals were capable of self-fertilization through artificial insemination.The reproductive performance of these intersex individuals was then investigated by injecting spawning agents,dissecting gonads,and artificially fertilizing eggs with their own sperm.The XX,XY,and YY genotypic individuals were produced by the self-fertilization of XY intersex individuals.The present study successfully produced high percentages of XX and XY intersex and conducted self-fertilization by mating the eggs and sperm of a single intersex individual.Self-fertilization in fish will show great potential in aquaculture genetics and breeding,for example,generating highly homozygous(or"clonal")genetic material,producing mono-sex populations,and identifying unknown sex determination modes.In particular,the cloning method of producing genetic materials will be greatly beneficial for maintaining advantageous traits through self-fertilization generation after generation.

Comparative assessment of natural and synthetic reproductive inhibitors in Oreochromis niloticus

Nowadays, natural sources of sex reversal agents are preferred over synthetic ones in fish farming due to their reliability and economic value. This study compared the effectiveness of 17α-methyltestosterone (MT), Carica papaya seed meal (PSM), and common carp testes (CCT) with tilapia (Oreochromis niloticus) in terms of sex reversal, hematological parameters, gonadal histology, enzymatic activity, and overall growth. A 90-day trial was conducted with 560 tilapia fry (2- 3 days old) distributed into one control and six treatment aquaria, each with two replicates. Fries were fed with a control diet (T0) or one of six experimental diets (T1-T6) containing different MT, PSM, or CCT levels for 30 days, followed by the control diet for 60 days. MT induced the highest male proportion (85% and 75% in T2 and T1, respectively), while PSM and CCT enhanced tilapia’s growth and carcass composition. Histological analysis revealed gonadal deformities in MT and PSM treatments, which might lead to sterility. The gonadosomatic index (GSI) was reduced in natural treatments as compared with synthetic ones. Hematological parameters did not show any adverse effects of PSM and CCT. Protease and amylase activities were higher in PSM and CCT than in MT, indicating better digestion and feed absorption. PSM and CCT are biodegradable, locally available, and eco-friendly alternatives to synthetic hormones to change sex in tilapia. Natural sources (plant and animal by-products) are preferable to synthetic sources as they are less expensive and control prolific breeding in tilapia.

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.

The role of sex chromosomes in mammalian germ cell differentiation： can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes？

The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

Why are sexually selected weapons almost absent in females？

In sex role reversed species, predominantly females evolve sexually selected traits, such as ornaments and/or weapons. Female ornaments are common and their function well documented in many species, whether sex role reversed or not. However, sexually selected female weapons seem totally absent except for small wing spurs in three jacana species, present in both males and females. This poor female weaponry is in sharp contrast to the situation in species with conventional sex roles： males com- monly have evolved sexually selected weapons as well as ornaments. At the same time, females in many taxa have naturally se- lected weapons, used in competition over resources or in predator defence. Why are sexually selected weapons then so rare, al- most absent, in females？ Here I briefly review weaponry in females and the function of these weapons, conclude that the near ab- sence of sexually selected weapons begs an explanation, and suggest that costs of sexually selected weapons may exceed costs of ornaments. Females are more constrained when evolving sexually selected traits compared to males, at least compared to those males that do not provide direct benefits, as trait costs reduce a female＇s fecundity. I suggest that this constraining trade-off between trait and fecundity restricts females to evolve ornaments but rarely weapons. The same may apply to paternally investing males. Whether sexually selected weapons actually are more costly than sexually selected ornaments remains to be investigated