Onset Of Sexual Differentiation Research Articles

Hsd17b1 is a key gene for ovarian differentiation of the Siberian sturgeon.

This is the first work using gonads from undifferentiated, genetically-sexed Siberian sturgeon describing expression changes in genes related to steroid synthesis and female and male sex differentiation. One factor identified as relevant for ovarian differentiation was the gene coding for the enzyme Hsd17b1, which converts estrone into estradiol-17β. hsd17b1 was highly activated in female gonads at 2.5 months of age, around the onset of sex differentiation, preceding activation of two other genes involved in estrogen production (cyp19a1 and foxl2). hsd17b1 was also strongly repressed in males. Two known foxl2 paralogs are found in Siberian sturgeon-foxl2 and foxl2l-but only foxl2 appeared to be associated with ovarian differentiation. With regard to the male pathway, neither 11-oxygenated androgens nor classic male genes (amh, dmrt1, sox9, and dhh) were found to be involved in male sex differentiation, leaving open the question of which genes participate in early male gonad development in this ancient fish. Taken together, these results indicate an estrogen-dependence of female sex differentiation and 11-oxygenated androgen-independence of male sex differentiation.

Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

Tunas (genus Thunnus) are one of the most ecologically and commercially important fish worldwide. To establish a biological basis for reproduction in this globally essential species, we have recently studied crucial reproductive aspects of the Pacific bluefin tuna (T. orientalis; PBT), as a model of tuna species, based on our closed-cycle aquaculture technology. In this study, we clarified the global expression profile of the genes regulating gonadal sex differentiation in PBT, as this developmental process is vital to sexual reproduction. Based on the results of our comparative (RNA-sequencing) and temporal (qRT-PCR) transcriptome analyses using the updated genome dataset, we propose the molecular mechanisms of gonadal sex differentiation in PBT. In female gonads, foxl2 and cyp19a1a (coding aromatase) are expressed at the onset of sex differentiation. Active aromatase-mediated estrogen biosynthesis, which includes positive regulation of cyp19a1a expression by Foxl2, induces ovarian differentiation. By contrast, dmrt1 and gsdf are upregulated in differentiating male gonads lacking active estrogen synthesis. Dmrt1 and Gsdf would mainly promote testicular differentiation. Furthermore, androgen biosynthesis is upregulated in differentiating male gonad. Endogenous androgens may also be vital to testicular differentiation. This study provides the first comprehensive data clarifying the molecular basis for gonadal sex differentiation in tunas.

Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.)

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor β superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae)

The synthetic 17α-ethinylestradiol (EE2) is a common estrogenic pollutant that has been suspected to affect the demography of river-dwelling salmonids. One possibility is that exposure to EE2 tips the balance during initial steps of sex differentiation, so that male genotypes show female-specific gene expression and gonad formation. Here we study EE2 effects on gene expression around the onset of sex differentiation in a population of European grayling (Thymallus thymallus) that suffers from sex ratio distortions. We exposed singly-raised embryos to one dose of 1 ng/L EE2, studied gene expression 10 days before hatching, at the day of hatching, and around the end of the yolk-sac stage, and related it to genetic sex (sdY genotype). We found that exposure to EE2 affects expression of a large number of genes, especially around hatching. These effects were strongly sex-dependent. We then raised fish for several months after hatching and found no evidence of sex reversal in the EE2-exposed fish. We conclude that ecologically relevant (i.e. low) levels of EE2 pollution do not cause sex reversal by simply tipping the balance at early stages of sex differentiation, but that they interfere with sex-specific gene expression.

Characterization of a male specific region containing a candidate sex determining gene in Atlantic cod

The genetic mechanisms determining sex in teleost fishes are highly variable and the master sex determining gene has only been identified in few species. Here we characterize a male-specific region of 9 kb on linkage group 11 in Atlantic cod (Gadus morhua) harboring a single gene named zkY for zinc knuckle on the Y chromosome. Diagnostic PCR test of phenotypically sexed males and females confirm the sex-specific nature of the Y-sequence. We identified twelve highly similar autosomal gene copies of zkY, of which eight code for proteins containing the zinc knuckle motif. 3D modeling suggests that the amino acid changes observed in six copies might influence the putative RNA-binding specificity. Cod zkY and the autosomal proteins zk1 and zk2 possess an identical zinc knuckle structure, but only the Y-specific gene zkY was expressed at high levels in the developing larvae before the onset of sex differentiation. Collectively these data suggest zkY as a candidate master masculinization gene in Atlantic cod. PCR amplification of Y-sequences in Arctic cod (Arctogadus glacialis) and Greenland cod (Gadus macrocephalus ogac) suggests that the male-specific region emerged in codfishes more than 7.5 million years ago.

Higher growth rate and gene expression in male zebra finch embryos are independent of manipulation of maternal steroids in the eggs

Sexual dimorphism in prenatal development is widespread among vertebrates, including birds. Its mechanism remains unclear, although it has been attributed to the effect of maternal steroid hormones. The aim of this study was to investigate how increased levels of steroid hormones in the eggs influence early embryonic development of male and female offspring. We also asked whether maternal hormones take part in the control of sex-specific expression of the genes involved in prenatal development. We experimentally manipulated hormones’ concentrations in the egg yolk by injecting zebra finch females prior to ovulation with testosterone or corticosterone. We assessed growth rate and expression levels of CDK7, FBP1 and GHR genes in 37h-old embryos. We found faster growth and higher expression of two studied genes in male compared to female embryos. Hormonal treatment, despite clearly differentiating egg steroid levels, had no effect on the sex-specific pattern of the embryonic gene expression, even though we confirmed expression of receptors of androgens and glucocorticoids at such an early stage of development. Thus, our study shows high stability of the early sex differences in the embryonic development before the onset of sexual differentiation and indicates their independence of maternal hormones in the egg.

Sexual Differentiation and size at First Maturity of the MusselChoromytilus chorus(Molina, 1782) (Mollusca, Bivalvia) in Northern Chile.

The mussel Choromytilus chorus (Molina, 1782) is a commercially and ecologically important bivalve, which is extensively distributed along the Chilean coast (20°S–54°S). However, there are no previous studies regarding the reproductive aspects of this species in the northern part of its range. Mussels were collected at Caleta Errázuriz, along the Antofagasta coast (ca. 24°S), between December 2014 and June 2015. A total of 1620 individuals were examined using histological techniques and macroscopic visual inspection. Seventeen size categories, which included individuals from 5 mm to 100 mm in length, were analyzed. The size at the onset of sexual differentiation and the size at first sexual maturity (50% mature individuals) were determined. Males comprised 49% of the total sample, and 51% were females. Mussels from 5–22 mm in valve length were undifferentiated and from 22–24 mm were sexually differentiated. The size at first sexual maturity was recorded as 38–40 mm. In vitro fertilization trials demonstrated that sexually differentiated individuals were able to produce viable gametes. The biological parameters analyzed for C. chorus were similar to those recorded for populations of this bivalve from the southern latitudes. However, the lower number of eggs per female and the smaller egg size suggests that the population parameters may be different for C. chorus between the northern and southern latitudes.

Cell-Specific mRNA Profiling of the Caenorhabditis elegans Somatic Gonadal Precursor Cells Identifies Suites of Sex-Biased and Gonad-Enriched Transcripts.

The Caenorhabditis elegans somatic gonad differs greatly between the two sexes in its pattern of cell divisions, migration, and differentiation. Despite decades of study, the genetic pathways directing early gonadal development and establishing sexual dimorphism in the gonad remain largely unknown. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in the somatic gonadal precursor cells and their daughter cells of each sex at the onset of sexual differentiation. We identified several hundred gonad-enriched transcripts, including the majority of known regulators of early gonadal development, and transgenic reporter analysis confirmed the effectiveness of this approach. Before the division of the somatic gonad precursors, few sex-biased gonadal transcripts were detectable; less than 6 hr later, after their division, we identified more than 250 sex-biased transcripts, of which about a third were enriched in the somatic gonad compared to the whole animal. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in the somatic gonadal precursor cells around the time of their first division. About 10% of male-biased transcripts had orthologs with male-biased expression in the early mouse gonad, suggesting possible conservation of gonad sex differentiation. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in the somatic gonad. Our data illustrate the power of cell-specific transcriptome analysis and suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent.

Gene expression analysis at the onset of sex differentiation in turbot (Scophthalmus maximus).

BackgroundControlling sex ratios is essential for the aquaculture industry, especially in those species with sex dimorphism for relevant productive traits, hence the importance of knowing how the sexual phenotype is established in fish. Turbot, a very important fish for the aquaculture industry in Europe, shows one of the largest sexual growth dimorphisms amongst marine cultured species, being all-female stocks a desirable goal for the industry. Although important knowledge has been achieved on the genetic basis of sex determination (SD) in this species, the master SD gene remains unknown and precise information on gene expression at the critical stage of sex differentiation is lacking. In the present work, we examined the expression profiles of 29 relevant genes related to sex differentiation, from 60 up to 135 days post fertilization (dpf), when gonads are differentiating. We also considered the influence of three temperature regimes on sex differentiation.ResultsThe first sex-related differences in molecular markers could be observed at 90 days post fertilization (dpf) and so we have called that time the onset of sex differentiation. Three genes were the first to show differential expression between males and females and also allowed us to sex turbot accurately at the onset of sex differentiation (90 dpf): cyp19a1a, amh and vasa. The expression of genes related to primordial germ cells (vasa, gsdf, tdrd1) started to increase between 75–90 dpf and vasa and tdrd1 later presented higher expression in females (90-105 dpf). Two genes placed on the SD region of turbot (sox2, fxr1) did not show any expression pattern suggestive of a sex determining function. We also detected changes in the expression levels of several genes (ctnnb1, cyp11a, dmrt2 or sox6) depending on culture temperature.ConclusionOur results enabled us to identify the first sex-associated genetic cues (cyp19a1a, vasa and amh) at the initial stages of gonad development in turbot (90 dpf) and to accurately sex turbot at this age, establishing the correspondence between gene expression profiles and histological sex. Furthermore, we profiled several genes involved in sex differentiation and found specific temperature effects on their expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2142-8) contains supplementary material, which is available to authorized users.

Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq

BackgroundDespite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29).ResultsRNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue.ConclusionsThis study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1886-5) contains supplementary material, which is available to authorized users.

Expression of aromatase in the embryonic brain of the olive ridley sea turtle (Lepidochelys olivacea), and the effect of bisphenol-A in sexually differentiated embryos.

Brain aromatase participates in several biological processes, such as regulation of the reproductive-endocrine axis, memory, stress, sexual differentiation of the nervous system, male sexual behavior, and brain repair. Here we report the isolation and expression of brain aromatase in olive ridley sea turtle (Lepidochelys olivacea) embryos incubated at male- and female-promoting temperatures (MPT and FPT, respectively), at the thermosensitive period (TSP) and the sex-differentiated period. Also, aromatase expression was assessed in differentiated embryos exposed to bisphenol-A (BPA) during the TSP. BPA is a monomer of polycarbonate plastics and is considered an endocrine-disrupting compound. Normal aromatase expression was measured in both forebrain and hindbrain, showing higher expression levels in the forebrain of differentiated embryos at both incubation temperatures. Although no significant differences were detected in the hindbrain, expression was slightly higher at MPT. BPA did not affect aromatase expression neither in forebrains or hindbrains from embryos incubated at MPT, whereas at FPT an inverted U-shape curve was observed in forebrains with significant differences at lower concentrations, whereas in hindbrains a non-significant increment was observed at higher concentrations. Our data indicate that both incubation temperature and developmental stage are critical factors affecting aromatase expression in the forebrain. Because of the timing and location of aromatase expression in the brain, we suggest that brain aromatase may participate in the imprinting of sexual trends related to reproduction and sexual behavior at the onset of sex differentiation, and BPA exposure may impair aromatase function in the female forebrain.

Interaction between the Wilms tumour factor-1 element in the promoter of Amh and a downstream enhancer is required for a strong expression of the gene in pre-pubertal sertoli cells

Amh (anti-Müllerian hormone) is a single copy gene which is expressed strongly in Sertoli cells in the foetal testis and participates in the onset of sexual differentiation. Its promoter driving the expression of a reporter gene (d2EGFP) has been used to analyse the role of certain defined putative elements and a downstream enhancer element in gene expression. These experiments were carried out in vitro using a line of pre-pubertal mouse Sertoli cells, transienly transfected with circular DNA constructs with variously mutated promoter elements. A downstream enhancer element, situated immediately 3’ of the polyadenylation (PA) signal for Amh, has been inserted in an equivalent position in the d2EGFP construct. When the Amh promoter is unmodified, the downstream enhancer (DE) is positively associated with a large increase in EGFP expression. This is at least partly the consequence of an increased rate of expression by individual cells. Experiments using variously truncated Amh promoters indicate that an upstream region (-214 to -336) may play a minor role in facilitating enhancement. However mutation of the Wilms tumour factor-1 element, situated between the tata box and the start of translation, results in an almost complete suppression of enhancement.

Sex-Biased miRNA Expression in Atlantic Halibut (Hippoglossus hippoglossus) Brain and Gonads

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

Pod1 is involved in the sexual differentiation and gonadal development of the Nile tilapia

Pod1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors that have been implicated in the regulation of sexual differentiation and gonadal development in mammals. However, to date, little is known about the role of Pod1 in nonmammalian vertebrate gonadogenesis. We cloned and characterized the Pod1 gene from tilapia. The tilapia Pod1 gene contains an open reading frame (ORF) of 525 nucleotides which potentially codes for a protein with 174 amino acids. Sequence alignment revealed that the deduced tilapia protein sequence shared high homology (79.5% to 90.5%) with the Pod1 sequences of other vertebrates. The tissue distribution of Pod1 revealed by RT-PCR showed that it had varied expression patterns in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of Pod1 during tilapia sexual differentiation and gonadal development. In the undifferentiated gonad, Pod1 was expressed in the somatic cells of both sexes. Subsequently, Pod1 expression in tilapia persisted in differentiated juvenile and adult ovary and testis. Our data indicate for the first time that Pod1 is not only necessary for the onset of sexual differentiation, but also plays an important role in gonadal development in the teleost.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Not too late to remodel (Rapid Remodeling of Airway Vascular Architecture at Birth by Amy Ni, Erin Lashnits, Li-Chin Yao, Peter Baluk, and Donald M. McDonald, Dev Dyn 239:2354–2366) When form ceases to support function, it is time to remodel. Ni et al. discover that the mantra is not limited to kitchens and homes; tracheal vasculature also follows the same principle. Using the trachea as an easily studied representative of the airways, they document that embryonic day (E) 17.5 mice have a web-like primitive tracheal vascular plexus that is reminiscent of vascular architecture in the yolk sac. During postnatal stages P0–P3, an astounding 76% the vascular network is pruned, leaving a skeleton of vasculature that grows horizontally over the ladder-like cartilage rings lining the trachea. At P4, horizontal capillaries re-grow, and arterioles and venules begin to grow vertically between the rings, acquiring most features of adult vasculature by P7. Of interest, the vasculature of kidney, brain, and liver, all fail to undergo a similar overhaul at birth, suggesting that exposure to atmospheric oxygen may contribute to the remodeling phenomenon in the trachea. While the hypothesis remains to be tested, the authors do identify other mechanisms associated with vascular regression and re-growth. Hypoxic regions and hypoxia inducible factor (HIF1-α) correlate with vessel survival after birth, and survival of tracheal vasculature from P0 to P7 is dependent upon vascular endothelial growth factor (VEGF). Future work will focus on the roles of signaling pathways and changes in oxygen tension on vascular remodeling during postnatal development and disease. Smart screen (Essential Genes for Astroglial Development and Axon Pathfinding During Zebrafish Embryogenesis by Michael J.F. Barresi, Sean Burton, Kristina Dipietrantonio, Adam Amsterdam, Nancy Hopkins, and Rolf O. Karlstrom, Dev Dyn 239:2603–2618) Astroglia are the unsung heroes of neural development. Once believed to function solely as scaffold support for neurons, it is now understood that they play wide-ranging roles during neurogenesis such as in forming the blood–brain barrier, neural patterning, and regeneration. Despite their universal importance, little is known about astroglial development. To remedy this oversight, the authors screened an existing zebrafish mutant collection, for which the affected genes were already identified. They describe 25 mutants that are placed into one or more categories: axonal pathfinding, gliogenesis, glial patterning and neurogenesis. One important observation is that defects in glial patterning correlate with perturbed axon pathfinding, suggesting axon–glial interactions are important for axon scaffold formation. Another is the discovery of a new class of mutants affecting cell number of radial glial cells, the main source of neural stem cells in vertebrate embryos. The work also identifies potential novel roles for the affected genes, many of which are familiar signaling pathway components or transcription factors (i.e., chordin, tbx16). These intriguing preliminary findings double as jumping-off points from which to further the understanding of brain development. Green eggs and sperm (The ziwi Promoter Drives Germline-Specific Gene Expression in Zebrafish by David H. Leu and Bruce W. Draper, Dev Dyn 239:2714–2721) Here Leu and Draper unveil a tool that will be of interest to germ cell researchers and the zebrafish community at-large. They show that a 4.8-kb promoter of ziwi, which encodes an Argonaut class protein involved in Piwi-interacting RNA (piRNA) metabolism, can drive germline-specific, heterologous gene expression. Expression of the Tg(ziwi:EGFP) transgene initiates at 7 days postfertilization (dpf) in bipotential gonocytes and continues to be expressed after the onset of sexual differentiation (18–21 dpf) in all testis and ovary germ cells. Demonstrating the utility of the tool as a germ cell marker, Tg(ziwi:EGFP) aids the authors in defining new substages in stage-1A oocytes. What sets this germline-specific transgene apart from others, however, is that it is reliably expressed in premeiotic oocytes. The authors discuss the potential utility of the ziwi promoter in manipulating gene expression in the germline, producing genetic mosaic germlines, and speculate how the tool could propel the future of germ cell related technologies in zebrafish.

Sex-dimorphic gene expression and ineffective dosage compensation of Z-linked genes in gastrulating chicken embryos

BackgroundConsiderable progress has been made in our understanding of sex determination and dosage compensation mechanisms in model organisms such as C. elegans, Drosophila and M. musculus. Strikingly, the mechanism involved in sex determination and dosage compensation are very different among these three model organisms. Birds present yet another situation where the heterogametic sex is the female. Sex determination is still poorly understood in birds and few key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective.ResultsBy comparing microarrays from microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses, using an enhanced annotation tool for Affymetrix probesets of the chicken genome developed in our laboratory (called Manteia), show that among these male-biased genes found on the Z chromosome, more than 20 genes play a role in sex differentiation.ConclusionsThese results corroborate previous studies demonstrating the rather inefficient dosage compensation for Z chromosome in birds and show that this sexual dimorphism in gene regulation is observed long before the onset of sexual differentiation. These data also suggest a potential role of non-compensated Z-linked genes in somatic sex differentiation in birds.

Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development

Malformations of the external genitalia are among the most common congenital anomalies in humans. The urogenital and anorectal sinuses develop from the embryonic cloaca, and the penis and clitoris develop from the genital tubercle. Within the genital tubercle, the endodermally derived urethral epithelium functions as an organizer and expresses sonic hedgehog (Shh). Shh knockout mice lack external genitalia and have a persistent cloaca. This identified an early requirement for Shh, but precluded analysis of its later role in the genital tubercle. We conducted temporally controlled deletions of Shh and report that Shh is required continuously through the onset of sexual differentiation. Shh function is divisible into two temporal phases; an anogenital phase, during which Shh regulates outgrowth and patterning of the genital tubercle and septation of the cloaca, and a later external genital phase, during which Shh regulates urethral tube closure. Disruption of Shh function during the anogenital phase causes coordinated anorectal and genitourinary malformations, whereas inactivation during the external genital phase causes hypospadias. Shh directs cloacal septation by promoting cell proliferation in adjacent urorectal septum mesenchyme. Additionally, conditional inactivation of smoothened in the genital ectoderm and cloacal/urethral endoderm shows that the ectoderm is a direct target of Shh and is required for urethral tube closure, highlighting a novel role for genital ectoderm in urethragenesis. Identification of the stages during which disruption of Shh results in either isolated or coordinated malformations of anorectal and external genital organs provides a new tool for investigating the etiology of anogenital malformations in humans.

The Plasmodium falciparum protein Pfg27 is dispensable for gametocyte and gamete production, but contributes to cell integrity during gametocytogenesis

In the human malaria parasite Plasmodium falciparum, gametocyte maturation is a process remarkably longer than in other malaria species, accompanied by expression of 2-300 sexual stage-specific proteins. Disruption of several of their encoding genes so far showed that only the abundant protein Pfg27, produced at the onset of sexual differentiation, is essential for gametocyte production. In contrast with what has been previously described, here we show that P. falciparum pfg27 disruptant lines are able to undergo all stages of gametocyte maturation, and are able to mature into gametes. A fraction of Pfg27-defective gametocytes show, however, distinct abnormalities in intra- and extra-cellular membranous compartments, such as accumulation of parasitophorous vacuole-derived vesicles in the erythrocyte cytoplasm, large intracellular vacuoles and discontinuities in their trilaminar cell membrane. This work revises current knowledge on the role of Pfg27, indicating that the protein is not required for parasite entry into sexual differentiation, and suggesting that it is instead involved in maintaining cell integrity in the uniquely long gametocytogenesis of P. falciparum.

Sexually Dimorphic MicroRNA Expression During Chicken Embryonic Gonadal Development1

MicroRNAs are a highly conserved class of small RNAs that function in a sequence-specific manner to posttranscriptionally regulate gene expression. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryogenesis, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a model, microarrays were used to profile the expression of chicken miRNAs prior to, during, and after the time of gonadal sex differentiation (Embryonic Day 5.5 [E5.5], E6.5, and E9.5). Sexually dimorphic miRNAs were identified, and the expression patterns of several were subjected to further validation by in situ hybridization and Northern blot analysis. Expression of one chicken miRNA, MIR202*, was observed to be sexually dimorphic, with upregulation in the developing testis from the onset of sexual differentiation. Additional data from deep sequencing of male and female embryonic gonad RNA samples also indicated upregulation of MIR202* in male gonads. These findings provide the first evidence of sexually dimorphic miRNA expression during vertebrate gonadal sex differentiation and suggest that MIR202* may function in regulating testicular development.

Different sox17 transcripts during sex differentiation in sea bass, Dicentrarchus labrax

Sox genes participate in several developmental processes, including sex determination and differentiation. In this study, the genomic structure of sox17 was characterized in the sea bass (sb). Two transcripts, one producing a normal protein (sb Sox17) and another producing a truncated protein (sb t-Sox17) were detected. A third, novel transcript, originated by intron retention (sb i-sox17) was also observed. Sb sox17 was widely distributed, whereas sb i-sox17 was predominantly found in skin and brain. In gonads, sb sox17 expression first increased at 150 days of age, coinciding with the onset of sex differentiation. At 250 days and onwards, sb sox17 expression was significantly higher in females, and mRNA levels correlated with those of gonadal aromatase. Thus, this study provides the first evidence for the presence of alternative splicing by intron retention in a Sox17 gene, and for sex-related differences in expression, implicating sox17 in ovarian development and function in fish.