Sex-reversed Males Research Articles

Transcriptome analysis of four types of gonadal tissues in largemouth bass (Micropterus salmoides) to reveal its sex-related genes.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15days post-hatching (dph) were fed diets containing 20mg/kg of 17α-methyltestosterone (17α-MT) or 30mg/kg of 17β-estradiol (17β-E2) for 60days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17β-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17β-E2 were capable of inducing sex reversal of LMB at 15dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

Evaluating the growth performance of all male sex reversal and mixed ser tilapia (Oreochromis niloticus) cultured in earthen ponds in Binh Phuo province, Vietnam

The study was conducted to evaluate the growth performance, survival rate and yield of all male sex reversed and mixed sex Nile tilapia (Oreochromis niloticus) cultured in earthen ponds for 180 days. The reversed sex and mixed sex fingerlings (mean weight 7.43 ± 0.35 g) was randomly stocked in six earthen ponds (1,000 m2/pond). The stocking density maintained was 5 fish/m2. The stocked fish were fed a commercial pellet feed containing 35% crude protein. The feeding rate was adjusted according the size of fish, 5% at the beginning to 3% at harvest. Water environment parameters including dissolved oxygen (DO) (4.3 ± 0.8 mg/L), temperature (30 ± 0.7°C), NH3 (0.18 ± 0.2 mg/L), and pH (6.9 ± 0.5) were always within the appropriate range for the normal growth and development of tilapia. The growth rate of sex reversed tilapia was significantly higher (P < 0.05) than that of mixed tilapia throughout the experiment. The harvest weight and length of reversed sex tilapia were 410.5 ± 5.15 g and 25.48 ± 0.48 cm, respectively, and were significantly higher than that of mixed sex tilapia (P < 0.05). The survival rates and feed conversion ratio (FCR) of mono and mixed sex were 90.1% and 89.9%, respectively, but no significant differences were observed (P > 0.05). The relationship of fish lengths and weights expressed by power function revealed that the slope of the length weight regression lines was normal for reversed sex (2.72) and mixed sex Tilapia (2.93) with the high correlation coefficient (> 0.9). The findings of this study demonstrate that reversed sex tilapia has better growth performance compared to mixed sex tilapia. Therefore, reversed sex fingerlings should be used in commercial farming to increase tilapia production.

Transient square pulse-electric field induction of monosex-male reversal of the eggs of Nile tilapia (Oreochromis niloticus L., Chitralada)

We present an alternative technique of monosex-male reversal for Nile tilapia using transient square pulse-electric field inductions to enhance sex reversal rate of tilapia eggs by methyltestosterone (Oreochromis niloticus L., Chitralada). Male tilapia is preferred for aquaculture because of better size and growth rate. The egg membrane was permeabilised by an electric field pulse allowing a minimalized androgen hormone dose of 17alpha-methyltestosterone (MT) (μg l−1 concentration) to achieve monosex sex-male reversal. The technique achieved large-scale inductions of 2000 eggs rather than for individual eggs and was aimed at commercialization (Thailand petty patent no. 13516). The eggs of O. niloticus obtained from our parent breeding stocks were carefully selected to be in the segmentation-pharyngula stage (2–3 dpf - day post fertilization) for electrical inductions in a pulsed-electric field generated between narrow parallel-plate electrodes. The electrodes were powered with an adjustable signal pulse-generator (SPG) developed for on-site operations. The induction medium was prepared using HEPES buffer mixed with a minimized concentration of MT. We experimentally optimized inductions by adjusting the number of square wave pulses, pulse amplitude, pulse durations (period) and a mark-space ratio (pulse width: negative half of period) for the optimum results. A dose of 350 V (electric field intensities of 87.50 kV m−1) of 3 square pulses with 100 μs pulse-period (50:50 mark space ratio) gave the maximum rate of male-sex reversal rate (MSR) of 89.42 ± 2.28 (SD)%, hatching rate (HR) of 93.33 ± 0.58% and survival rate (SR) of 87.49 ± 1.71%, respectively. The off-spring rate (OSR) or sex ratio of the controls were 59.71 ± 3.00% male and 46.33 ± 1.53% female from a hatching rate (HR) of 91.33 ± 1.53% and a survival rate (SR) of 85.08 ± 4.16%, respectively.

A computational analysis of the molecular mechanisms underlying the effects of ibuprofen and dibutyl phthalate on gene expression in fish

The impact of emerging pollutants such as ibuprofen and dibutyl phthalate on aquatic species is a growing concern and the need for proper assessment and evaluation of these toxicants is imperative. The objective of this study was to examine the toxicogenomic impacts of ibuprofen and dibutyl phthalate on Clarias gariepinus, a widely distributed African catfish species. Results showed that exposure to the test compounds caused significant changes in gene expression, including upregulation of growth hormone, interleukin, melatonin receptors, 17β-Hydroxysteroid Dehydrogenase, heat shock protein, doublesex, and mab-3 related transcription factor. On the other hand, expression of forkhead Box Protein L2 and cytochrome P450 was downregulated, revealing a potential to induce female to male sex reversal. The binding affinities and hydrophobic interactions of the test compounds with the reference genes were also studied, showing that ibuprofen had the lowest binding energy and the highest affinity for the docked genes. Both compounds revealed a mutual molecular interaction with amino acids residues within the catalytic cavity of the docked genes. These results provide new insights into the toxic effects of ibuprofen and dibutyl phthalate on Clarias gariepinus, contributing to a better understanding of the environmental impact of these pollutants.

Temperature Incubation During the Embryo-Larval Stage for Inducing Sex Reversal of Nile Tilapia Red NIFI Strain

This research aims to achieve male sex reversal in Nile Tilapia (Orechromis niloticus) of the red NIFI strain through high-temperature incubation during the embryonic or pre-swimming larval phase. Larvae resulting from the natural mating of red NIFI strain broodstock were incubated under temperature treatments of 31oC, 33oC, 35oC, and a control group (<30oC), each with three replicates. Temperature regulation was facilitated using a heater. During the initial 3 hours of incubation, the heater was turned off for 6 hours, followed by another 3 hours of incubation with the heater on. This pattern was repeated daily until the larvae began to swim. The treated larvae were then reared for 90 days. Thirty specimens from each replicate were identified for gender using the squash acetocarmine method. The study results indicated that temperature differences significantly influenced (p<0.05) the sex ratio of Nile tilapia in the red NIFI strain. The highest proportion of males was achieved at 33oC, at 73.33%, and 35oC, at 64.43%. There were no significant differences (p>0.05) in fry survival rates at 7 days and after 90 days of rearing.

Male refractory hypospadias with sexual reversal: a case report

BackgroundHypospadias is one of the most prevalent urogenital malformations in clinic. However, some hypospadias may have a more complex disorder of sex development. Usually, hypospadias in these patients is severe. Among them, the 46,XX male sex reversal syndrome is a rare disorder of sex development, and this may be the main reason for this type of hypospadias being difficult to repair.Case presentationWe present a Han nationality 19-year-old male with failure of repeated repair of hypospadias. No sperm was found on semen analysis. Lingual mucosal graft was carried out for this patient. It still did not succeed after using lingual mucosal graft repair. Karyotype analysis of this patient confirmed 46,XX karyotype.ConclusionHypospadias with 46,XX male sex reversal syndrome is hard to repair. Chromosome karyotype examination in patients with hypospadias is suggested. Genetic testing is recommended. In the future, further research is needed on the pathogenesis of disease and how to treat and prevent it.

Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

Sex-Associated SNP Confirmation of Sex-Reversed Male Farmed Japanese Flounder Paralichthys olivaceus.

Female Japanese flounder Paralichthys olivaceus grow more rapidly than the male. The goal of all-female commercial production requires an efficient method of genetic sex identification. We conducted genome-wide association analysis of female and male farmed Japanese flounder (n = 24 per phenotypic sex) and found all regions of chromosome 24 to be significantly associated with phenotypic sex, suggesting it as the sex chromosome. Genetic sex was identified based on single nucleotide polymorphisms (SNP) on chromosome 24 (n = 3568) using multidimensional scaling analysis, and individuals were clearly separated according to sex by the first dimension. The 61 SNPs most highly associated with sex were selected, and an amplicon-based SNP panel was developed. This was used to determine genetic sex of 39 females and 40 males. Eleven phenotypic males were assigned as female with XX genotype, suggesting sex reversal. Genetic sex was also assessed based on the indel of the amh gene promoter, which is the major candidate sex gene of Japanese flounder. We found four SNPs perfectly associated with genotypic sex in the sex-associated SNP panel, one of which was located in exon 2 of the amh gene. Along with the indel of the amh gene promoter, the sex-associated SNP panel will be of value in identifying genetic sex of farmed Japanese flounder. Molecular sexing will facilitate all-female production by breeding sex-reversed males.

A Cryptic Sex-Linked Locus Revealed by the Elimination of a Master Sex-Determining Locus in Medaka Fish.

AbstractSex chromosomes rapidly turn over in several taxonomic groups. Sex chromosome turnover is generally thought to start with the appearance of a new sex-determining gene on an autosome while an old sex-determining gene still exists, followed by the fixation of the new one. However, we do not know how prevalent the transient state is, where multiple sex-determining loci coexist within natural populations. Here, we removed a Y chromosome with a master male-determining gene DMY from medaka fish using high temperature-induced sex-reversed males. After four generations, the genomic characteristics of a sex chromosome were found on one chromosome, which was an autosome in the original population. Thus, the elimination of a master sex-determining locus can reveal a cryptic locus with a possible sex-determining effect, which can be the seed for sex chromosome turnover. Our results suggest that populations that seem to have a single-locus XY system may have other chromosomal regions with sex-determining effects. In conclusion, the coexistence of multiple sex-determining genes in a natural population may be more prevalent than previously thought. Experimental elimination of a master sex-determining locus may serve as a promising method for finding a locus that can be a protosex chromosome.

Medaka factor in the germline alpha (figα) is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor, figα, is one of the earliest marker genes of oocyte differentiation in vertebrates. In the present study, we made figα knockout medaka by CRISPR/Cas9, expecting aborted progress of oogenesis, to see if differentiation of somatic ovarian tissues is affected. Figα knockout male gonads differentiated normally into testes with functional sperm. The females, on the other hand, were sterile; there are oocytes only up to pachytene. No growing oocytes in diplotene were found. The phenotype was already apparent at 10 days after hatching, when diplotene oocytes start to develop in the control. Furthermore, several putative target genes of figα were not expressed in the mutant female gonads. Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred. Figα knockout female gonads differentiated morphologically into ovaries, suggesting that in medaka, figα knockout prevents oogenesis progress into diplotene stage, and ovarian differentiation do not need developed oocytes beyond pachytene stage. This ovarian phenotype reminded us of "pachytene checkpoint", which blocks meiotic progress into diplotene when chromosomal abnormalities are present. Therefore, we further analyzed phenotype of oocytes in the mutant ovaries. The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage. The number of apoptotic oocytes in the knockout ovaries was small. Finally, we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining. Taken together, figα knockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than "pachytene checkpoint" and arrested oocytes remained in the ovary. This is in contrast to figα knockout mice where all oocytes underwent apoptosis quickly after birth.

PSI-4 Investigation of a Naturally Occurring XX Male Bull

Abstract The Y chromosome–linked gene sex-determining region (SRY) is believed to be the master initiator of male sex determination in almost all eutherian mammals, functioning to upregulate expression of its direct target gene SRY-related HMG box–containing gene 9 (SOX9). XX maleness in humans is a rare syndrome with a frequency of 1 in 20,000–25,000 males. Approximately 90% of these cases carry some amount of the Y chromosome sequences due to an illegitimate recombination between X and Y chromosomes, where most of these cases have normal male genitalia. A more severe manifestation, the remaining 10% do not have any Y-chromosome sequences, and typically have ambiguous genitalia. In this study, we report a case of an infertile bull. Physical examination of this purebred Holstein bull showed a phenotypically androgynous animal with a normal penis, cryptorchidism (2 diminutive testicles, only one descended), and with a skeletal conformation and stance usually associated with female individuals. Lymphocytes from heparin sodium anticoagulated blood were cultured, harvested, and prepared according to conventional cell culture methods revealed a normal 60, XX karyotype with no numerical or structural chromosomal aberrations. PCR analyses were negative for the SRY gene, and positive for androgen receptor gene on the X chromosome. No sperm were found in seminal fluid collected in a routine breeding soundness exam at 17 months of age. The bull was slaughtered at 18 months of age, and its reproductive tract was collected. The testicle weighed 18.9 g (descended), and 42.4 g (abdominal). Neither testicle/epididymal unit was appropriate size or fully developed. Two very enlarged vesicular glands (43.36 g and 52.52 g, normally each ~ 20 g) were observed alongside what appeared to be uterine tissue. Histology showed no tubular grooves in the ampulla and an epididymis devoid of sperm. The development of a male phenotype in SRY-negative XX individuals suggests that in addition to SRY, there are autosomal or X-linked genes involved in the sex-determining pathway. In humans, a dose change of the SOX9 gene, loss of function mutations in the WNT4, FoxL2, and RSPO1 testicular suppressing genes, and genomic rearrangements within the regulatory region of the SRY-box transcription factor 3 (SOX3) gene have all been associated with XX male sex reversal. To investigate whether a mutation or genomic rearrangement was responsible for the male phenotype in the SRY-negative 60, XX bull, DNA was collected from the bull and his sire and dam for long-read trio sequencing. These data may provide evidence for either a loss of function mutation in a gene normally inhibiting testes formation in XX individuals, or a gain of function mutation in a gene downstream of SRY in the sex determining pathway.

Gonadal Development in Smallmouth Bass (Micropterus Dolomieu) Reared in the Absence and Presence of 17-α-Ethinylestradiol.

Testicular oocytes in wild adult bass (Micropterus spp.) are considered a potential indication of exposure to estrogenic compounds in municipal, agricultural, or industrial wastewater. However, our ability to interpret links between testicular oocyte occurrence in wild fish species and environmental pollutants is limited by our understanding of normal and abnormal gonadal development. We previously reported low-to-moderate testicular oocyte prevalence (7%-38%) among adult male bass collected from Minnesota waters with no known sources of estrogenic compounds. In the present study, two experiments were conducted in which smallmouth bass (Micropterus dolomieu) fry were exposed to control water or 17-α-ethinylestradiol (EE2) during gonadal differentiation, then reared in clean water for an additional period. Histological samples were evaluated at several time points during the exposure and grow-out periods, and the sequence and timing of gonadal development in the presence of estrogen were compared with that of control fish. Testicular oocytes were not observed in any control or EE2-exposed fish. Among groups exposed to 1.2 or 5.1 ng/L EE2 in Experiment 1 or 3.0 ng/L EE2 in Experiment 2, ovaries were observed in 100% of fish up to 90 days after exposure ceased, and approximately half of those ovaries had abnormal characteristics, suggesting that they likely developed in sex-reversed males. Groups exposed to 0.1, 0.4, or 1.0 ng/L in Experiment 2 developed histologically normal ovaries and testes in proportions not significantly different from 1:1. These findings suggest that, while presumably able to cause sex reversal, juvenile exposure to EE2 may not be a unique cause of testicular oocytes in wild bass, although the long-term outcomes of exposure are unknown. Environ Toxicol Chem 2022;41:1416-1428. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Identification and expression analysis of thyroid-stimulating hormone β subunit, and effects of T3 on gonadal differentiation-related gene expression in rice field eel, Monopterus albus.

Thyroid-stimulating hormone (TSH) is an important glycoprotein in hypothalamic-pituitary-thyroid axis, which plays a crucial role in the synthesis and release of thyroid hormones in vertebrates. Rice field eel, Monopterus albus, a protogynous hermaphroditic fish, which undergoes sex reversal from a functional female to a male, is an ideal model to investigate the regulation of sex differentiation. In this study, we obtained the cDNA sequence of thyroid-stimulating hormone β subunit (tshβ) from rice field eel, which contained a complete open reading frame and encoded a putative protein of 151 amino acids. Multiple alignment of protein sequences showed that tshβ was highly conserved in teleost. The tissue distribution indicated that tshβ showed high expression in the pituitary, moderate expression in the brain region, gonad, intestine and liver, and low expression in other peripheral tissues. During natural sex reversal, the expression of tshβ had no significant difference in the pituitary. Compared to that in the ovary, the expression of tshβ increased significantly in the gonad at late intersexual and male stages. After treatment by different doses of triiodothyronine (T3) (1μg/g, 10μg/g and 100μg/g body weight), serum T3 and free triiodothyronine (FT3) increased sharply, while the expression of tshβ were inhibited significantly in the pituitary. Although T3 had no significant effect on the levels of serum E2, it stimulated the release of serum 11-KT at high-dose group. We also detected the effects of T3 on the expression of gonadal differentiation-related genes in rice field eel. T3 treatment inhibited the expression of foxl2, cyp19a1a and dax1, while stimulated the expression of sox9a1. These results indicate that TSH may be involved in sex differentiation, and THs may play roles in the regulation of male development and sex reversal in rice field eel.

Aromatase inhibitor induces sex reversal in the protogynous hermaphroditic rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a protogynous hermaphroditic fish reared commercially as an aquaculture species in East Asia. Artificial breeding of rice field eels requires an abundance of adult females and males; however, the eels need to undergo a long period of female and intersexual stages before they develop into males, which results in a shortage of functional males in production practice. This study developed an artificial method for producing functional males through the oral administration of an aromatase inhibitor (AI, letrozole: 300 mg/kg feed) in juvenile fish. Histological analysis of gonadal sections of treated fish revealed the transition of primary ovaries to mature testes. During the initial period of the AI treatment, 2-month-old rice field eels were exclusively female. AI administration resulted in a significant reduction in 17β-estradiol levels and increase in testosterone and 11-ketotestosterone levels in vivo. Cell proliferation occurred in the outer layer of the gonadal epithelium, and oocytes degenerated in the ovaries. After 120 days of AI treatment, the ovaries were completely converted to testes. The sexually reversed fish maintained the male sex and produced functional sperm after cessation of drug treatment. Notably, artificially sex-reversed males' fecundity did not differ significantly from wild-type males. Quantitative PCR analysis revealed that the expression of cyp19a1a decreased, whereas the mRNA levels of estrogen receptors esr1, esr2a, and esr2b increased significantly during AI treatment. Moreover, the expression of foxl2 was downregulated, whereas dmrt1, amh, and sox9 were upregulated in the gonads of fish administered AI. This study revealed that endogenous estrogen synthesis plays a key role in the sex change of rice field eels, with ovaries undergoing a reversal to testes in response to estrogen insufficiency. Such artificially induced sex conversion will be of considerable value in breeding rice field eels.

Generation of an induced pluripotent stem cell line GZHMCi008-A derived from a patient with SRY-positive 46,XX testicular disorder of sex development

The testicular disorder of sex development (TDSD) is a rare condition characterized by a male appearance with a female karyotype. The most frequent cause of TDSD is misplacement of the sex determining region Y (SRY) gene on the X chromosome. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a patient with SRY-positive 46,XX TDSD. This cell line offers an unprecedented cellular model to investigate the profound manifestations like infertility of the male sex reversal patients, and serves as a useful tool to develop therapies for the disease.

Cyp17a1 is Required for Female Sex Determination and Male Fertility by Regulating Sex Steroid Biosynthesis in Fish.

In teleost fish, sex steroids are involved in sex determination, sex differentiation, and fertility. Cyp17a1 (Cytochrome P450 family 17 subfamily A member 1) is thought to play essential roles in fish steroidogenesis. Therefore, to further understand its roles in steroidogenesis, sex determination, and fertility in fish, we constructed a cyp17a1 gene mutant in Nile tilapia (Oreochromis niloticus). In XX fish, mutation of the cyp17a1 gene led to a female-to-male sex reversal with a significant decline in 17β-estradiol (E2) and testosterone (T) production, and ectopic expression of male-biased markers (Dmrt1 and Gsdf) in gonads from the critical window of sex determination. Sex reversal was successfully rescued via T or E2 administration, and ovarian characteristics were maintained after termination of E2 supplementation in the absence of endogenous estrogen production in cyp17a1-/- XX fish. Likewise, deficiencies in T and 11-ketotestosterone (11-KT) production in both cyp17a1-/- XX sex-reversed males and cyp17a1-/- XY mutants resulted in meiotic initiation delays, vas deferens obstruction and sterility due to excessive apoptosis and abnormal mitochondrial morphology. However, 11-KT treatment successfully rescued the dysspermia to produce normal sperm in cyp17a1-/- male fish. Significant increases in gonadotropic hormone (gth) and gth receptors in cyp17a1-/- mutants may excessively upregulate steroidogenic gene expression in Leydig cells through a feedback loop. Taken together, our findings demonstrate that Cyp17a1 is indispensable for E2 production, which is fundamental for female sex determination and differentiation in XX tilapia. Additionally, Cyp17a1 is essential for T and 11-KT production, which further promotes spermatogenesis and fertility in XY males.

Effects of Functional Depletion of Doublesex on Male Development in the Sawfly, Athalia rosae.

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.

A supernumerary “B-sex” chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

Molecular cloning and characterization of estrogen and androgen receptors in Mandarin fish, Siniperca chuatsi

Steroid hormones are essential for the normal reproductive activity in females and males, such as early sex differentiation, gonad development and sex reversal, which works by binding with the steroid receptors. In the present study, the full-length of estrogen receptors (ERα, ERβ1 and ERβ2) and androgen receptors (ARα and ARβ) cDNAs were isolated from the mandarin fish (Siniperca chuatsi). Real time quantitative PCR (RT-qPCR) revealed that ERα and ERβ2 expressed mainly in the liver of females and males, and ERβ1 mostly distributed in ovary and testis. Additionally, the high expressions of ARα and ARβ in both females and males were detected in gonads and livers respectively. During the early sex differentiation stage, the expressions of ERs and ARs in female were higher than in male during 5–30 day after hatching (dah), and reached the peak level at 30 dah. In males, the expressions of ERβ1, ERβ2, and ARβ increased largely during 30 dah to 60 dah, and revealed significantly higher levels than in females during 60–180 dah. In female, the expression of ERβ2 in ovary increased sharply at 180 dah. Moreover, the result of gonadal histology revealed that the spermatocytes were observed at 45 dah and the spermatozoon could be found largely at 60 dah in males and oil droplets appeared for the first time in the oocytes at 180 dah. The expression levels of three ERs and ARβ were up-regulated and the ARα was down-regulated during female to male sex reversal induced by 17α-methyltestosterone (MT). Furthermore, after MT treatment for 30 and 35 days, the expressions of ERβ1, ERβ2 and ARβ were significantly up-regulated. These results suggest that ERβ1, ERβ2 and ARβ may play critical roles in spermatogenesis and masculinization, while ERβ2 and ERβ1 may be essential for vitellogenesis and ovary development in female.

A rare case of male sex reversal syndrome (46, XX) with negative SRY gene: a disorder of sexual differentiation (DSD)

BackgroundMale sex reversal syndrome is a rare genetic cause of male infertility with an overall incidence of 1/20,000–1/100,000 males. There is mismatching between the genetic make-up and the apparent clinical features. The clinical presentation of such cases is variable ranging from ambiguous genitalia at birth, failed puberty, up to normal male phenotype with infertility and hypogonadism. The exact molecular and genetic bases of this syndrome are still unclear. Most of the recorded cases were SRY positive (i.e. representing 80–90% of all cases), and they showed translocated SRY gene on the Y chromosome. Moreover, fewer cases of male sex reversal (46, XX) were SRY negative.Case presentationHerby, we report a rare case of a 35-year-old infertile male patient who presented with azoospermia, hypergonadotropic hypogonadism, and abnormal classical (46, XX) karyotype, as well as negative FISH for SRY gene. He had a previous negative biopsy and was asking for redoing micro-TESE, whoever he was discouraged as chances to find sperm is eventually nil, and instead, he was prescribed testosterone replacement therapy to correct hypogonadism.ConclusionTherefore, any case of non-obstructive azoospermia should be offered genetic testing trying to exclude non-treatable cases and for genetic counseling.