Complete Sex Reversal Research Articles

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis

Research questionPremature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1–3.7% women. Genetic factors explain 20–30% of POI cases, but most causes remain unknown despite genomic advancements. DesignWe used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity. ResultsDespite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea – the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO. ConclusionsThis study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehogsignal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

Insulin-like Androgenic Gland Hormone Induced Sex Reversal and Molecular Pathways in Macrobrachium nipponense: Insights into Reproduction, Growth, and Sex Differentiation.

This study investigated the potential to use double-stranded RNA insulin-like androgenic gland hormone (dsIAG) to induce sex reversal in Macrobrachium nipponense and identified the molecular mechanisms underlying crustacean reproduction and sex differentiation. The study aimed to determine whether dsIAG could induce sex reversal in PL30-male M. nipponense during a critical period. The sex-related genes were selected by performing the gonadal transcriptome analysis of normal male (dsM), normal female (dsFM), neo-female sex-reversed individuals (dsRM), and unreversed males (dsNRM). After six injections, the experiment finally resulted in a 20% production of dsRM. Histologically, dsRM ovaries developed slower than dsFM, but dsNRM spermathecae developed normally. A total of 1718, 1069, and 255 differentially expressed genes were identified through transcriptome sequencing of the gonads in three comparison groups, revealing crucial genes related to reproduction and sex differentiation, such as GnRHR, VGR, SG, and LWS. Principal Component Analysis (PCA) also distinguished dsM and dsRM very well. In addition, this study predicted that the eyestalks and the "phototransduction-fly" photoperiodic pathways of M. nipponense could play an important role in sex reversal. The enrichment of related pathways and growth traits in dsNRM were combined to establish that IAG played a significant role in reproduction, growth regulation, and metabolism. Finally, complete sex reversal may depend on specific stimuli at critical periods. Overall, this study provides valuable findings for the IAG regulation of sex differentiation, reproduction, and growth of M. nipponense in establishing a monoculture.

Disorders of Sexual Development (DSD) in Piauí: an epidemiological study

Disorders of Sex Development (DSD) are congenital conditions in which the chromosomal, gonadal, or anatomical development of sex is atypical. DSD can occur due to variations in sex chromosomes or autosomes, in genes related to gonadal or genital development, or in alterations in hormone production by the reproductive organs. These conditions can range from common abnormalities such as cryptorchidism and hypospadias to rare and complex conditions such as complete sex reversal. This study analyzed the clinical and cytogenetic profile of individuals suspected of DSD who underwent karyotype testing in the state of Piauí from 2020 to 2022. Out of the 250 patients evaluated, 21% were suspected of having DSD, with a predominance of females (60%). Turner syndrome was the most common clinical suspicion, followed by ambiguous genitalia, Klinefelter syndrome, and micropenis. The results emphasize the importance of early diagnosis and psychological support for parents and individuals with DSD, aiming for appropriate treatment and the promotion of a healthy and satisfying life. Additionally, the study highlighted the need for the implementation of molecular tests to reach a conclusive diagnosis when DSD is not of chromosomal origin.

Overview of Avian Sex Reversal.

Sex determination and differentiation are processes by which a bipotential gonad adopts either a testicular or ovarian cell fate, and secondary sexual characteristics adopt either male or female developmental patterns. In birds, although genetic factors control the sex determination program, sex differentiation is sensitive to hormones, which can induce sex reversal when disturbed. Although these sex-reversed birds can form phenotypes opposite to their genotypes, none can experience complete sex reversal or produce offspring under natural conditions. Promising evidence indicates that the incomplete sex reversal is associated with cell autonomous sex identity (CASI) of avian cells, which is controlled by genetic factors. However, studies cannot clearly describe the regulatory mechanism of avian CASI and sex development at present, and these factors require further exploration. In spite of this, the abundant findings of avian sex research have provided theoretical bases for the progress of gender control technologies, which are being improved through interdisciplinary co-operation and will ultimately be employed in poultry production. In this review, we provide an overview of avian sex determination and differentiation and comprehensively summarize the research progress on sex reversal in birds, especially chickens. Importantly, we describe key issues faced by applying gender control systems in poultry production and chronologically summarize the development of avian sex control methods. In conclusion, this review provides unique perspectives for avian sex studies and helps scientists develop more advanced systems for sex regulation in birds.

Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish.

Although evolutionary fates and expression patterns of duplicated genes have been extensively investigated, how duplicated genes co-regulate a biological process in polyploids remains largely unknown. Here, we identified two gsdf (gonadal somatic cell-derived factor) homeologous genes (gsdf-A and gsdf-B) in hexaploid gibel carp (Carassius gibelio), wherein each homeolog contained three highly conserved alleles. Interestingly, gsdf-A and gsdf-B transcription were mainly activated by dmrt1-A (dsx- and mab-3-related transcription factor 1) and dmrt1-B, respectively. Loss of either gsdf-A or gsdf-B alone resulted in partial male-to-female sex reversal and loss of both caused complete sex reversal, which could be rescued by a nonsteroidal aromatase inhibitor. Compensatory expression of gsdf-A and gsdf-B was observed in gsdf-B and gsdf-A mutants, respectively. Subsequently, we determined that in tissue culture cells, Gsdf-A and Gsdf-B both interacted with Ncoa5 (nuclear receptor coactivator 5) and blocked Ncoa5 interaction with Rora (retinoic acid-related orphan receptor-alpha) to repress Rora/Ncoa5-induced activation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a). These findings illustrate that Gsdf-A and Gsdf-B can regulate male differentiation by inhibiting cyp19a1a transcription in hexaploid gibel carp and also reveal that Gsdf-A and Gsdf-B can interact with Ncoa5 to suppress cyp19a1a transcription in vitro. This study provides a typical case of cooperative mechanism of duplicated genes in polyploids and also sheds light on the conserved evolution of sex differentiation.

Development and gene expression analysis of gonad during 17α-methyltestosterone-induced sex reversal in mandarin fish (Siniperca chuatsi)

Mandarin fish (Siniperca chuatsi) is one of the most important commercial fish in aquaculture in the world. As females grow faster than males, the cultivation of all-female mandarin fish has great economic value and commercial prospects. However, little is known about the molecular mechanism of the early gonad development and sex reversal of mandarin fish. In this study, histological methods and transcriptome sequencing techniques were used to study the early gonadal development and gene expression pattern of genetically female (XX) mandarin fish during MT-induced masculinization. The histological results showed that the labile period of sex differentiation of XX mandarin fish was before 36 dpf and MT treatment before this period could induce complete sex reversal. The transcriptome results showed that the expression levels of a few female-related genes were down-regulated by MT during the morphological differentiation stage to cytological differentiation stage of XX gonad, such as cyp19a1a, foxl2, foxl3, hsd17b1, figla, gdf9, bmp15 and β-catenin, suggesting that the changes of these genes may determine the direction of sex differentiation in mandarin fish. In addition, MT also activated the expression of some male-related genes, such as dmrt1, hsd17b3, hsd11b2, cyp11a1, cyp11b, amh, amhr2, sox9 and gsdf. Some of these genes were activated during spermatogenesis stage, suggesting that the up-regulation of these male-related genes may be the result of sex differentiation rather than the cause. The qRT-PCR results of eight randomly selected differentially expressed genes were basically consistent with the transcriptome results, which proved that the transcriptome data were reliable. These results indicated that the expression of genes related to estrogen synthesis in early development stage may play a decisive role in the direction of sex differentiation in mandarin fish.

22q13 Duplication in Newborn With Dysmorphic Features: The Role of SOX10 in Disorders of Sex Development

Background: The 46,XX testicular disorder of sex development (DSD), also known as 46,XX male reversal, is a rare form of DSD and clinical phenotype shows complete sex reversal from female to male. The sex-determining region Y (SRY) gene can be identified in most 46,XX testicular DSD patients; however, approximately 20% are SRY-negative. Here we present a 2 week old with discrepant prenatal karyotype and infant phenotype. Case: This 2-week-old had dysmorphic features (bitemporal narrowing, broad and flat nasal bridge, bilateral epicanthal folds) and multiple congenital anomalies; IUGR, hypertelorism, cleft lip and palate, ASD, small kidneys, sacral dimple. Physical exam revealed palpable inguinal masses and microphallus without hypospadias. Postnatal karyotype showed a 46,XX chromosome complement with duplication of 22q13-qter. He was negative for SRY gene by FISH. Microarray analysis confirmed the duplication encompassing the SOX10 gene. Lab evaluation showed LH 10.81 mIU/ml, FSH 3.21 mIU/ml and total testosterone 241 ng/dl. These values are consistent with activation of the hypothalamic–pituitary–gonadal axis during the neonatal period in males. Conclusion: Our case along with previous cases supports the existence of a gene on chromosome 22q that can trigger testis determination in the absence of SRY. Potential mechanisms responsible for ovotesticular disorder in the XX (SRY−) individual could involve activation of testis specifying genes in the absence of SRY and/or inadequate expression of pro-ovary/anti-testis genes such as SOX 8 and SOX9. SOX10, a gene closely related to SOX9 and SOX8, and its overexpression has been suggested as a candidate for 46,XX DSD. Over-expression of SOX10 in mice resulted in the XX DSD. The spectrum of sex reversal phenotypes and gonadal asymmetryseen in the Sox10 transgenic mice closely mirrors the range of gonadal and reproductive tract anomalies seen in cases of partial duplication of human chromosome 22q13. Although SRY-negative 46,XX testicular DSD is a rare condition, an effort to make an accurate diagnosis is important for the provision of proper genetic counseling and for guiding patients in their long-term management.

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic–pituitary–gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (Rutilus rutilus).

Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site-associated DNA sequencing (RAD-seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from nonpolluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex-reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied nondestructively using a fin clip or skin swab, with animal welfare benefits.

High Molecular Diagnosis Rate in Undermasculinized Males with Differences in Sex Development Using a Stepwise Approach.

Differences of sex development (DSDs) are a constellation of conditions that result in genital ambiguity or complete sex reversal. Although determining the underlying genetic variants can affect clinical management, fewer than half of undermasculinized males ever receive molecular diagnoses. Next-generation sequencing (NGS) technology has improved diagnostic capabilities in several other diseases, and a few small studies suggest that it may improve molecular diagnostic capabilities in DSDs. However, the overall diagnostic rate that can be achieved with NGS for larger groups of patients with DSDs remains unknown. In this study, we aimed to implement a tiered approach to genetic testing in undermasculinized males seen in an interdisciplinary DSD clinic to increase the molecular diagnosis rate in this group. We determined the diagnosis rate in patients undergoing all clinically available testing. Patients underwent a stepwise approach to testing beginning with a karyotype and progressing through individual gene testing, microarray, panel testing, and then to whole-exome sequencing (WES) if no molecular cause was found. Deletion/duplication studies were also done if deletions were suspected. Sixty undermasculinized male participants were seen in an interdisciplinary DSD clinic from 2008 to 2016. Overall, 37/60 (62%) of patients with Y chromosomes and 46% of those who were 46XY received molecular diagnoses. Of the 46,XY patients who underwent all available genetic testing, 18/28 (64%) achieved molecular diagnoses. This study suggests that the addition of WES testing can result in a higher rate of molecular diagnoses compared to genetic panel testing.

Dosis y número de aplicaciones de etefón en la reversión sexual de flores de calabacita

Ethylene can change the flowering pattern of some cucurbits, favoring the emergence of female flowers. The objective was to determine the influence of doses and number of ethephon applications in sex reversal of zucchini (Cucurbita pepo L.). The experiment was conducted in a 4 x 4 factorial arrangement, plus a control (natural flowering), the first factor being the ethephon doses (125, 250, 375 and 500 mg∙L-1) and the second the number of applications (1, 2, 3 and 4). Ethephon influenced the sexual expression of zucchini, but not enough to achieve complete sex reversal. The 500 mg∙L-1 dose provided the largest feminization for all applications. Ethephon might be a useful tool for zucchini breeding programs.

Masculinization under combined administration of an androgen and an aromatase inhibitor in the Ussuri catfish, Pelteobagrus ussuriensis (Dybowski, 1872)

In this study, we used the central composite experimental design and response surface methodology to investigate the masculinization as well as larval growth, survival under combined administration of an androgen (17α-methyltestosterone, MT) and an aromatase inhibitor (letrozole, AI) in the Ussuri catfish, Pelteobagrus ussuriensis. The results showed that the masculinization was significantly promoted by MT and AI. The nonsignificant MT × AI interaction indicated that MT acted on masculinization independently of AI and vice versa, suggesting that they may function through different molecular pathways. The impact of MT was greater than that of AI, indicating that masculinization via the direct action of MT was more important than that of AI during masculinization. The optimal MT/AI dosage was obtained by simultaneously optimizing three reliable models in consideration of the growth and survival. A verification trial under the commercial setting showed that this optimal MT/AI combination had greater potential for application to culture production, albeit not complete sex reversal. Our results suggest that using a combined administration of MT and AI to masculinize the Ussuri catfish be preferred.

Challenging Dissemblance in Pauli Murray Historiography, Sketching a History of the Trans New Negro

Challenging Dissemblance in Pauli Murray Historiography, Sketching a History of the Trans New Negro

Molecular signaling pathways elicited by 17α-ethinylestradiol in Japanese medaka male larvae undergoing gonadal differentiation

Estrogenic contaminants released into water bodies are potentially affecting the reproduction of aquatic organisms. Exposure to 17α-ethinylestradiol (EE2), a synthetic estrogen agonist commonly found in sewage effluents, has been shown to cause gonadal changes in male gonochoristic fish ranging from gonadal intersex to complete sex reversal. Although these gonadal changes have been well studied in Japanese medaka Oryzias latipes, the molecular mechanisms behind them are poorly understood. Our objective was to study the signaling pathways elicited by exposure to different concentrations of EE2 in this species. Embryos and larvae were sexed by the presence of leucophores and dmy expression (only in males). Male medaka were exposed to two EE2 concentrations (30 and 300 ng/L) during their gonadal differentiation period (7–22 dpf). The transcriptome of larvae was analyzed using RNA sequencing followed by pathway analysis. Genes involved in sex differentiation and gonadal development (e.g., cldn19, ctbp1, hsd17b4) showed a female-like expression pattern in EE2-exposed males with some genes changing in expression in an EE2 concentration-dependent manner. However, not all genes known to be involved in sex differentiation and gonadal development (e.g., wnt4b) were altered by EE2. Several of the prominently affected signaling pathways involved genes associated with steroidogenesis, steroid receptor signaling and steroid metabolism, such as cyp2b3, cyp3b40, cyp1a, hsd17b4. We also report on novel genes and pathways affected that might play a role in gonadal changes, including several genes associated with FXR/RXR and LXR/RXR activation networks. This study is the first to examine the transcriptomic changes in male fish resulting from exposure to EE2 during the gonadal differentiation period, providing new insights on the signaling pathways involved in the development of gonadal changes in gonochoristic fish.

Two Korean girls with complete androgen insensitivity syndrome diagnosed in infancy.

Androgen insensitivity syndrome (AIS) is a rare genetic disease caused by various abnormalities in the androgen receptor (AR). The AR is an essential steroid hormone receptor that plays a critical role in male sexual differentiation and development and preservation of the male phenotype. Mutations in the AR gene on the X chromosome cause malfunction of the AR so that a 46,XY karyotype male has some physical characteristics of a woman or a full female phenotype. Depending on the phenotype, AIS can be classified as complete, partial or mild. Here, we report 2 cases of complete AIS in young children who showed complete sex reversal from male to female as a result of AR mutations. They had palpable inguinal masses and normal female external genitalia, a blind-end vagina and absent Müllerian duct derivatives. They were both 46,XY karyotype and AR gene analysis demonstrated pathologic mutations in both. Because AIS is inherited in an X-linked recessive manner, we performed genetic analysis of the female family members of each patient and found the same mutation in the mothers of both patients and in the female sibling of case 2. Gonadectomy was performed in both patients to avoid the risk of malignancy in the undescended testicles, and estrogen replacement therapy is planned for their adolescence. Individuals with complete AIS are usually raised as females and need appropriate care.

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

ABSTRACTRNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG – 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

Maternal provision of transformer-2 is required for female development and embryo viability in the wasp Nasonia vitripennis

In insect sex determination a primary signal starts the genetic sex determination cascade that, in most insect orders, is subsequently transduced down the cascade by a transformer (tra) ortholog. Only a female-specifically spliced tra mRNA yields a functional TRA-protein that forms a complex with TRA2, encoded by a transformer-2 (tra2) ortholog, to act as a sex specific splicing regulator of the downstream transcription factors doublesex (dsx) and fruitless (fru). Here, we identify the tra2 ortholog of the haplodiploid parasitoid wasp N. vitripennis (Nv-tra2) and confirm its function in N. vitripennis sex determination. Knock down of Nv-tra2 by parental RNA interference (pRNAi) results in complete sex reversal of diploid offspring from female to male, indicating the requirement of Nv-tra2 for female sex determination. As Nv-tra2 pRNAi leads to frequent lethality in early developmental stages, maternal provision of Nv-tra2 transcripts is apparently also required for another, non-sex determining function during embryogenesis. In addition, lethality following Nv-tra2 pRNAi appears more pronounced in diploid than in haploid offspring. This diploid lethal effect was also observed following Nv-tra pRNAi, which served as a positive control in our experiments. As diploid embryos from fertilized eggs have a paternal chromosome set in addition to the maternal one, this suggests that either the presence of this paternal chromosome set or the dosage effect resulting from the diploid state is incompatible with the induced male development in N. vitripennis caused by either Nv-tra2 or Nv-tra pRNAi. The role of Nv-tra2 in activating the female sex determination pathway yields more insight into the sex determination mechanism of Nasonia.

Establishment of monosex female production of sablefish (Anoplopoma fimbria) through direct and indirect sex control

Methods are needed for monosex production of sablefish (Anoplopoma fimbria) to capitalize on superior growth of females relative to males. This study aimed to characterize the system of sex determination and establish methods for both direct and indirect feminization of sablefish. Juvenile sablefish ~40mm fork length were fed diets containing 17β-estradiol (E2), 17α-methyltestosterone (MT), or ethanol vehicle alone (control) for 2months and then divided into subgroups either shifted to the control diet (E2- and MT-short (S) duration treatments) or maintained on steroid-treated diets for an additional 2months (E2- and MT-long (L) duration treatments). Fish were subsampled for gonadal histology at the midpoint and end of the dietary treatments and all remaining fish were individually tagged to monitor subsequent growth and gonadal development for 36months (post-treatment time points of 0, 4, 16, 24, and 36months). Control fish exhibited balanced (1:1) sex ratios. E2 treatment regardless of duration induced ovarian differentiation in 100% of the genotypic males (i.e., direct feminization), while only the MT-L treatment induced testicular differentiation in 100% of the genotypic females (i.e., generating “neomales”). A high proportion (89%) of MT-S genotypic females possessed intersex gonads, indicating short-term MT exposure was insufficient to induce complete sex reversal. Significant differences in sex-specific growth were apparent by 16months post-treatment and associated with gonadal phenotype; sablefish with ovaries outgrew those with testes, regardless of genotypic sex. Fish from the E2-S and -L and MT-L treatment groups maintained their respective feminized and masculinized states after steroid withdrawal, although MT-S treated genotypic females tended to show further masculinization post-treatment. Upon reaching sexual maturity, control males and neomales were crossed with wild-caught female broodstock. Resultant embryos from control crosses were 54.6±8.9% (mean±SD) genotypic female, while those from neomale-female crosses were 100±0.0% genotypic female. Following sex differentiation, gonadal histology demonstrated that offspring from control crosses had a balanced phenotypic sex ratio, whereas all neomale offspring developed normal ovaries. This study establishes that sablefish utilize an XX/XY system of sex determination and methods for both direct and indirect monosex female production.

Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22.

Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. We identified a gene, Musca domesticamale determiner (Mdmd), as the M-factor. Mdmd originated from a duplication of the spliceosomal factor gene CWC22 (nucampholin). Targeted Mdmd disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes transformer and doublesex The presence of Mdmd on different chromosomes indicates that Mdmd translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator.