Female Sexual Differentiation Research Articles

Intersex (ix) mutations of Drosophila melanogaster cause nonrandom cell death in genital disc and can induce tumours in genitals in response to decapentaplegic (dpp(disk)) mutations.

In Drosophila melanogaster, the intersex (ix) is a terminally positioned gene in somatic sex determination hierarchy and function with the female specific product of double sex (DSX(F)) to implement female sexual differentiation. The null phenotype of ix is to transform diplo-X individuals into intersexes while leaving haplo-X animals unaffected. This study on the effect of different intersex mutations on genital disc development provides the following major results: (i) similar range of a characteristic array of morphological structures (from almost double sex terminalia to extreme reduction of terminal appendages) was displayed by the terminalia of XX ix(1)/ix(1) , XX ix(2)/ix(2) and XX ix(5)/ix(5) individuals; (ii) an increased number of apoptotic cells were found to occur in a localized manner in mature third instar larval genital discs of ix individuals; (iii) ix mutations can induce high frequency of neoplastic tumours in genitals in the presence of decapentaplegic (dpp(disk)) mutations; and (iv) heteroallelic combinations of dpp(disk) mutations can also induce tumours in intersex genitals with variable expressivity. On the basis of these findings, we suggest that: (i) loss of function of ix causes massive cell death in both male and female genital primordia of genital discs, resulting phenotype mimicking in male and female characteristics in genitals; and (ii) at the discs, the apoptotic cells persist as 'undead' cells that can induce oncogenic transformation in the neighbouring disc cells when dpp signalling is blocked or reduced by dpp(disk) mutations.

Artificial masculinization in tilapia involves androgen receptor activation

Estrogens have a pivotal role in natural female sexual differentiation of tilapia while lack of steroids results in testicular development. Despite the fact that androgens do not participate in natural sex differentiation, synthetic androgens, mainly 17-α-methyltestosterone (MT) are effective in the production of all-male fish in aquaculture. The sex inversion potency of synthetic androgens may arise from their androgenic activity or else as inhibitors of aromatase activity. The current study is an attempt to differentiate between the two alleged activities in order to evaluate their contribution to the sex inversion process and aid the search for novel sex inversion agents. In the present study, MT inhibited aromatase activity, when applied in vitro as did the non-aromatizable androgen dihydrotestosterone (DHT). In comparison, exposure to fadrozole, a specific aromatase inhibitor, was considerably more effective. Androgenic activity of MT was evaluated by exposure of Sciaenochromis fryeri fry to the substance and testing for the appearance of blue color. Flutamide, an androgen antagonist, administered concomitantly with MT, reduced the appearance of the blue color and the sex inversion potency of MT in a dose-dependent manner. In tilapia, administration of MT, fadrozole or DHT resulted in efficient sex inversion while flutamide reduced the sex inversion potency of all three compounds. In the case of MT and DHT the decrease in sex inversion efficiency caused by flutamide is most likely due to the direct blocking of the androgen binding to its cognate receptor. The negative effect of flutamide on the efficiency of the fadrozole treatment may indicate that the masculinizing activity of fadrozole may be attributed to excess, un-aromatized, androgens accumulated in the differentiating gonad. The present study shows that when androgen receptors are blocked, there is a reduction in the efficiency of sex inversion treatments. Our results suggest that in contrast to natural sex differentiation, during sex inversion treatments, androgens, either endogenous or exogenous, participate in inducing testicular differentiation.

Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling

Research on endocrine disruption in fish has been dominated by studies on estrogen-active compounds which act as mimics of the natural estrogen, 17β-estradiol (E2), and generally exert their biological actions by binding to and activation of estrogen receptors (ERs). Estrogens play central roles in reproductive physiology and regulate (female) sexual differentiation. In line with this, most adverse effects reported for fish exposed to environmental estrogens relate to sexual differentiation and reproduction. E2, however, utilizes a variety of signaling mechanisms, has multifaceted functions and targets, and therefore the toxicological and ecological effects of environmental estrogens in fish will extend beyond those associated with the reproduction. This review first describes the diversity of estrogen receptor signaling in fish, including both genomic and non-genomic mechanisms, and receptor crosstalk. It then considers the range of non-reproductive physiological processes in fish that are known to be responsive to estrogens, including sensory systems, the brain, the immune system, growth, specifically through the growth hormone/insulin-like growth factor system, and osmoregulation. The diversity in estrogen responses between fish species is then addressed, framed within evolutionary and ecological contexts, and we make assessments on their relevance for toxicological sensitivity as well as ecological vulnerability. The diversity of estrogen actions raises questions whether current risk assessment strategies, which focus on reproductive endpoints, and a few model fish species only, are protective of the wider potential health effects of estrogens. Available – although limited – evidence nevertheless suggests that quantitative environmental threshold concentrations for environmental protection derived from reproductive tests with model fish species are protective for non-reproductive effects as well. The diversity of actions of estrogens across divergent physiological systems, however, may lead to and underestimation of impacts on fish populations as their effects are generally considered on one functional process only and this may underrepresent the impact on the different physiological processes collectively.

The discovery of Foxl2 paralogs in chondrichthyan, coelacanth and tetrapod genomes reveals an ancient duplication in vertebrates

The Foxl2 (forkhead box L2) gene is an important member of the forkhead domain family, primarily responsible for the development of ovaries during female sex differentiation. The evolutionary studies conducted previously considered the presence of paralog Foxl2 copies only in teleosts. However, to search for possible paralog copies in other groups of vertebrates and ensure that all predicted copies were homolog to the Foxl2 gene, a broad evolutionary analysis was performed, based on the forkhead domain family. A total of 2464 sequences for the forkhead domain were recovered, and subsequently, 64 representative sequences for Foxl2 were used in the evolutionary analysis of this gene. The most important contribution of this study was the discovery of a new subgroup of Foxl2 copies (ortholog to Foxl2B) present in the chondrichthyan Callorhinchus milii, in the coelacanth Latimeria chalumnae, in the avian Taeniopygia guttata and in the marsupial Monodelphis domestica. This new scenario indicates a gene duplication event in an ancestor of gnathostomes. Furthermore, based on the analysis of the syntenic regions of both Foxl2 copies, the duplication event was not exclusive to Foxl2. Moreover, the duplicated copy distribution was shown to be complex across vertebrates, especially in tetrapods, and the results strongly support a loss of this copy in eutherian species. Finally, the scenario observed in this study suggests an update for Foxl2 gene nomenclature, extending the actual suggested teleost naming of Foxl2A and Foxl2B to all vertebrate sequences and contributing to the establishment of a new evolutionary context for the Foxl2 gene.

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1KOSox9cKO ), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1KOSox9cKO mice. Moreover, since testis development occurs in XY Rspo1KOSox9cKO mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9cKO mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.

Swyer’s Syndrome: In a Fifty-Year-Old Female

Disorders of sexual differentiation may be classified into two categories; 1-disorders of gonadal differentiation; and 2-disorders of genital development. Here we present a case with complete 46, XY gonadal dysgenesis (Swyer’s syndrome) which is one of the female phenotype sexual differentiation disorders resulting from abnormal gonadal differentiation. It is well known that the risk of gonadal neoplasia is increased in gonadal dysgenesis. Swyer’s syndrome should be differentiated from syndrome of complete androgen insensitivity (Testicular feminization) and from Leydig cell agenesis. In the latter two disorders phenotype is female, karyotype 46, XY, the gonads are testes, while uterus and tubes are absent. On the contrary, the patients with Swyer’s syndrome have uterus and cervix which are hypoplastic.

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

BackgroundThe WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.MethodsWe sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.ResultsOnly one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.ConclusionAlthough we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.

Novel female-specific splice form of dsx in the silkworm, Bombyx mori

The Bombyx mori doublesex (Bmdsx), a homologue of doublesex of Drosophila, is the bottom most gene of the sex determination cascade. Bmdsx plays a very crucial role in somatic sexual development. Its pre-mRNA sex-specifically splices to generate two splice variants; one encodes female-specific and the other encodes male-specific polypeptides which differ only at their C-termini. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and are skipped in males. In the present study, we have identified a third splice form of the Bmdsx which is specific only to females and differs from the previously reported Bmdsxf isoform by the presence of 15 bp sequence. This new female splice form is generated as a result of alternative 5' splice site selection in the third exon adding additional 15 bp sequence in exon 3 which results in alteration of the reading frame leading to incorporation of an early stop codon. Thus the protein encoded by this splice form is 20 aa shorter than the known BmDsxF. Initial results obtained from the study of dsx homologues in Saturniid silkmoths suggest that both the female-specific Dsx proteins are essential for female sexual differentiation. It remains to be seen whether female-specific multiple splice forms of dsx are characteristic feature of only silkmoths or widespread among lepidopterans. The findings that sex determination mechanism is unique in lepidopterans offer an opportunity to develop genetic sexing methods in beneficial as well as economically destructive lepidopteran pests.

Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae)

doublesex ( dsx) is the bottom most gene of the sex-determination cascade of Drosophila melanogaster. The pre-mRNA of dsx splices to produce male- and female-specific transcripts which code for the male- and female-specific proteins, respectively. dsx homologues have been characterized from different (many in Diptera, two in Hypmenoptera and only one in Lepidoptera) insect species. Sex-specific splice forms of dsx pre-mRNA in all these species code for one male- and one female-specific DSX proteins, which regulate the downstream target genes responsible for sex-specific characters. In the present study we have cloned and characterized the dsx homologues from two saturniid silkmoths, Antheraea assama and Antheraea mylitta. The divergence time between Saturniidae and Bombycidae to which the domesticated silkworm, Bombyx mori belongs is estimated to be around 160.9 MY. Interestingly, the dsx pre-mRNA of these wild silkmoths sex-specifically splices to generate multiple splice variants. On the basis of their open reading frame (ORF) and conceptual translation, two female-specific (DSX F1 and DSX F2) and one male-specific (DSX M) proteins could be inferred, in both the moths. Presence or absence of a 15 bp stretch within the ORF of the two groups of female-specific transcripts resulted in the production of two distinct female-specific DSX proteins. The sex-specific DSX proteins have common amino-terminal sequence but sex-specific carboxy termini. The two female-specific DSX proteins (DSX F1 and DSX F2) share common DNA binding domain (DM domain) and oligomerization domain (OD domain) and differ only at their extreme C-termini by 21aa. Functional analysis of dsx transcripts in A. assama by dsRNA mediated knock-down resulted in complete abolition of expression of vitellogenin and hexamerin genes, the direct targets of the DSX proteins, irregular differentiation of gonads, and drastic reduction in fecundity and hatchability. Together, these results suggest the involvement of both the female-specific DSX proteins in the process of female sexual differentiation. Further, conservation of the 4th exon sequence, especially the PESS sequence responsible for the sex-specific splicing of Bmdsx in the female-specific transcripts of Aadsx and Amydsx, indicated the existence of a common mechanism of sex-specific splicing of dsx homologues in silkmoths. To our knowledge this is the first report of existence of multiple splice forms of dsx pre-mRNA encoding two female-specific DSX proteins.

Ontogenesis of gonadal aromatase gene expression in atlantic silverside (Menidia menidia) Populations with genetic and temperature‐dependent sex determination

Cytochrome P450 aromatase (P450arom), an enzyme that converts testosterone to 17beta-estradiol, is an important mediator of sex determination in teleosts with genetic sex determination (GSD) and temperature-dependent sex determination (TSD). We compared the ontogenetic expression of P450arom in two populations of Atlantic silversides, Menidia menidia, which exhibit TSD (South Carolina) or GSD (Nova Scotia, Canada) using quantitative, real-time polymerase chain reaction (qRT-PCR). Embryos and newly hatched larvae were reared at an intermediate sex ratio-producing temperature (21 degrees C), and older larvae and juveniles were reared at temperatures that feminize (15 degrees C) and masculinize (28 degrees C) to assess the temperature response of P450arom during development. Before sex determination, embryos and newly-hatched larvae displayed negligible P450arom expression, indicating minimal upregulation of this gene before sex determination. Gene expression increased in both populations during sex differentiation. Nova Scotia fish with GSD exhibited presumptive male- and female-like expression levels during early sex differentiation that were not influenced by temperature. South Carolina fish displayed low levels of expression at 28 degrees C with significantly heightened expression in some individuals at 15 degrees C, indicating that P450arom is temperature sensitive in the population with TSD. Populations also differed in the timing and maximal levels of P450arom expression, with fish from Nova Scotia exhibiting both the highest and earliest increase in expression in presumptive females. Our results support the hypothesis that P450arom is involved in female sex differentiation in this species, but is only responsive to temperature in M. menidia populations that exhibit TSD.

Model‐based prediction of defective DNA mismatch repair using clinicopathological variables in sporadic colon cancer patients

: Colon cancers with defective DNA mismatch repair (MMR) have a favorable prognosis and may lack benefit from 5-fluorouracil-based adjuvant chemotherapy. The authors developed models to predict MMR deficiency in sporadic colon cancer patients using routine clinical and pathological data. : TNM stage II and III colon carcinomas (n = 982) from 6 5-fluorouracil-based adjuvant therapy trials were analyzed for microsatellite instability and/or MMR protein expression. Tumor-infiltrating lymphocytes (TILs) were quantified (n = 326). Logistic regression and a recursive partitioning and amalgamation analysis were used to identify predictive factors for MMR status. : Defective MMR was detected in 147 (15%) cancers. Tumor site and histologic grade were the most important predictors of MMR status. Distal tumors had a low likelihood of defective MMR (3%; 13 of 468); proximal tumors had a greater likelihood (26%; 130 of 506). By using tumor site, grade, and sex, the logistic regression model showed excellent discrimination (c statistic = 0.81). Proximal site, female sex, and poor differentiation showed a positive predictive value (PPV) of 51% for defective MMR. In a patient subset (n = 326), a model including proximal site, TILs (>2/high-power field), and female sex showed even better discrimination (c statistic = 0.86), with a PPV of 81%. : Defective MMR is rare in distal, sporadic colon cancers, which should generally not undergo MMR testing. Proximal site, poor differentiation, and female sex detect 51% of tumors with defective MMR; substituting TILs for grade increases the PPV to 81%. These data can increase the efficiency of MMR testing to assist in clinical decisions. Cancer 2010. (c) 2010 American Cancer Society.

Normal and abnormal female sexual differentiation

At fertilization, genetic sex determines the orientation of sex differentiation. The genital and urinary systems are closely related embryologically and their common origin is the mesoderm. Primordial germ cells migrate through extra-embryonic tissues into the primitive gonad. Differentiation of the gonads precedes the differentiation of the internal tract and then the differentiation of the external genitalia. Although no gene for female orientation has been identified, genes involved in the different steps of genital tract development in females have been discovered. Some are also implicated in sexual differentiation defects in women, shedding light on the mechanisms that underlie congenital genitourinary malformations. To date, many questions remain unanswered.

Induced meiotic gynogenesis and sex differentiation in summer flounder ( Paralichthys dentatus)

Meiogynogenesis and temperature manipulation were used to produce XX male summer flounder broodstock for future production of monosex (all female) populations. Meiogynogens were produced by fertilizing eggs with UV-irradiated (70 mJ/cm 2) black sea bass sperm and applying 6-minute pressure shocks (58,600 kPa), two min post-fertilization. From 4 females, 132,000 eggs were produced, of which 95.6 ± 1.8% were viable, 51.0 ± 13.0% fertilized, and 15.9 ± 8.3% hatched. Following metamorphosis, meiogynogens and controls were raised under a low temperature regime (12 °C gradually increased to 20 °C), 21, and 26 °C for up to 376 days post hatch (DPH). Female sex differentiation was greater in meiogynogens (62.5%) and control fingerlings (22.6%) raised under a low temperature regime compared to those raised at the higher rearing temperatures: 0% at 21 °C, and 0 and 3.9% at 26 °C in meiogynogens and controls, respectively. These results suggest that temperature, during the critical phase preceding gonadal development, influences sex differentiation in summer flounder.

Gonadal Sex Differentiation in Cultured Juvenile Flounder, Paralichthys orbignyanus (Valenciennes, 1839)

Gonadal Sex Differentiation in Cultured Juvenile Flounder, <i>Paralichthys orbignyanus</i> (Valenciennes, 1839)

Ceratitis capitata transformer-2 gene is required to establish and maintain the autoregulation of Cctra, the master gene for female sex determination

In Drosophila melanogaster, transformer-2 (TRA-2) which is a non-sex-specific auxiliary splicing factor, is required to promote female sexual differentiation by interaction with the female-specific TRA. The two proteins positively regulate the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs, which in turn regulate phenotypic and behavioural sexual dimorphism. In the Mediterranean fruitfly Ceratitis capitata, the female-specific CcTRA is similarly required not only for Ccdsx splicing, but also to exert a novel autoregulatory function that consists of promoting female-specific splicing of Cctra pre-mRNA. This study reports the isolation and functional analysis of the C. capitata homologue of the Drosophila transformer-2 gene (Cctra-2). Transient RNAi against Cctra-2 during embryonic development causes the full sex reversal of XX flies in adult fertile pseudo-males, as well as changes in the splicing pattern of Cctra, Ccdsx and Ccfruitless (Ccfru). We propose that: 1) Cctra-2, as in Drosophila, is necessary for promoting Ccdsx and putative Ccfru pre-mRNA female-specific splicing and that 2) unlike in Drosophila, Cctra-2 appears to be necessary for establishing female sex determination in early XX embryos and for maintaining the positive feedback regulation of Cctra during development.

Clinicopathological features of 45,X/46,Xidic(Y) mosaicism and therapeutic implications: case report

45,X/46,Xidic(Y) mosaicism demands careful and thorough study because of both its variable clinical features and its potential complications. The present case relates to a three-year-old girl with the mosaic karyotype 46,X,idic(Y)(q11.2)[23]/45,X[6]. She had no signs of virilization or Turner's syndrome phenotype, but she was referred to our hospital because she presented reduced growth rate, abnormal facies and a melanotic nevus. After examination, she underwent prophylactic gonadectomy because of the risk of gonadoblastoma. Cytogenetic analysis on the streak gonads and blood showed significant differences in the 45,X cell line between these two tissues. The presence of the sex-determining region Y (SRY) gene did not determine male differentiation, which meant in the present case that the predominance of the X cell line in the gonadal tissue was probably due to the determining factor for female sexual differentiation.

Session 36: Female sexual differentiation (Clinical tutorial)

Session 36: Female sexual differentiation (Clinical tutorial)

Cadmium exposures during early development: DO they lead to reproductive impairment in fathead minnows?

The primary objective of this study was to determine whether cadmium (Cd) exposures during embryonic and larval development alter the reproductive performance, reproductive physiology, and sex ratio of fathead minnows (Pimephales promelas). Embryonic exposures were conducted by exposing adult female fathead minnows to 0, 25, or 100 microg/L Cd for 8 d prior to breeding. Larval exposures were conducted by exposing the larvae to waterborne Cd at 0, 12.5, 25, or 50 microg/L from 8 to 29 d posthatch (the time period associated with female sexual differentiation). Minnows from each exposure period were raised to maturity, at which time their reproductive success, secondary sexual characteristics, gonadosomatic index (GSI), plasma 11-ketotestosterone (11-KT) concentrations, and sex ratios were assessed. Results from the embryonic study reveal that Cd exposures alter the secondary sexual characteristics of male fathead minnows but do not alter reproductive performance, GSI, 11-KT concentrations, or sex ratios. Larval exposures, during the period of female sexual differentiation, significantly reduce the frequency of adult spawning and increase clutch size but do not alter fecundity, secondary sexual characteristics, GSI, or 11-KT. Subtle alterations in sex ratio were observed, indicating that larval Cd exposures may increase the proportion of females in an exposed population.

In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development

In humans, sexual differentiation of the external genitalia is established at 7-12 weeks post conception (wpc). During this period, maintaining the appropriate intrauterine hormone environment is critical. In contrast to other species, this regulation extends to the human fetal adrenal cortex, as evidenced by the virilization that is associated with various forms of congenital adrenal hyperplasia. The mechanism underlying these clinical findings has remained elusive. Here we show that the human fetal adrenal cortex synthesized cortisol much earlier than previously documented, an effect associated with transient expression of the orphan nuclear receptor nerve growth factor IB-like (NGFI-B) and its regulatory target, the steroidogenic enzyme type 2 3beta-hydroxysteroid dehydrogenase (HSD3B2). This cortisol biosynthesis was maximal at 8-9 wpc under the regulation of ACTH. Negative feedback was apparent at the anterior pituitary corticotrophs. ACTH also stimulated the adrenal gland to secrete androstenedione and testosterone. In concert, these data promote a distinctive mechanism for normal human development whereby cortisol production, determined by transient NGFI-B and HSD3B2 expression, provides feedback at the anterior pituitary to modulate androgen biosynthesis and safeguard normal female sexual differentiation.

Evolutionary Conservation of Ceratitis capitata transformer Gene Function

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.