Gonadal Gene Research Articles

Identification of the molecular sex‐differentiation period in the siberian sturgeon

Sexual development prior to gonadal sex differentiation is regulated by various molecular mechanisms. In fish, a "molecular sex-differentiation period" has been identified in species for which sex can be ascertained prior to gonadal sex differentiation. The present study was designed to identify such a period in a species for which no genetic sex markers or monosex populations are available. Siberian sturgeons undergo a slow sex-differentiation process over several months, so gonad morphology and gene expression was tracked in fish from ages 3-27 months to identify the sex-differentiation period. The genes amh, sox9, and dmrt1 were selected as male gonad markers; cyp19a1a and foxl2a as female gonad markers; and cyp17a1 and ar as markers of steroid synthesis and steroid receptivity. Sex differentiation occurred at 8 months, and was preceded by a molecular sex-differentiation period at 3-4 months, at which time all of the genes except ar showed clear expression peaks. amh and sox9 expression seemed to be involved in male sexual development whereas dmrt1, a gene involved in testis development in metazoans, unexpectedly showed a pattern similar to those of the genes known to be involved in female gonadal sex differentiation (cyp19a1 and foxl2a). In conclusion, the timing of and gene candidates involved with molecular sex differentiation in the Siberian sturgeon were identified.

Acclimation of killifish to thermal extremes of hot spring: Transcription of gonadal and liver heat shock genes

In this study, we explored the hypothesis that killifish acclimate to thermal extremes through regulation of genes involved in stress and metabolism. We examined the liver and gonadal transcription of heat shock proteins (hsp70, hsp90a, hsp90b), glucokinase (gck), and high mobility group b1 (hmgb1) protein in wild killifish species from hot springs and rivers using quantitative real-time PCR. Moreover, we exposed a river killifish species to a long-term thermal regime of hot spring (37–40°C) and examined the liver transcription of the heat shock genes. Our results showed that hot spring killifish showed a significant, strong upregulation of liver hsp90a. Moreover, the testicular transcript levels of hsp90a, hsp90b, and hsp70 were higher in hot spring killifish than the river ones. The results of the common garden experiments showed that the transcripts of hsp70, hsp90b, and hmgb1 were mildly induced (> twofold) at the time when temperature reached to 37–40°C, while the transcripts of hsp90a were strongly induced (17-fold increase). The level of hsp90a was dramatically more upregulated when fish were maintained in thermal extreme (42-fold change higher than in ambient temperature). Moreover, a significant downregulation of gck transcripts was observed at the time when temperature was raised to 37–40°C (80-fold decrease) and during exposure to long-term thermal extreme (56-fold decrease). It can be concluded that the regulation of heat shock genes particularly hsp90a might be a key factor of the acclimation of fish to high temperature environments like hot springs.

Adverse outcome pathway: Framework, application, and challenges in chemical risk assessment

The environmental estrogen 17α-ethinylestradiol (EE2) will depress or completely inhibit egg production in many common model teleosts at low concentrations (≤0.5 ng/L; Runnalls et al., 2015). This inhibition is not seen in the estuarine killifish, or mummichog (Fundulus heteroclitus), even when exposed to 100 ng/L EE2. This relative insensitivity to EE2 exposure indicates species-specific mechanisms for compensating for exogenous estrogenic exposure. This review compares various reproductive responses elicited by EE2 in mummichog to other common model teleosts, such as zebrafish (Danio rerio) and fathead minnow (Pimephales promelas), identifying key endpoints where mummichog differ from other studied fish. For example, EE2 accumulates primarily in the liver/gall bladder of mummichog, which is different than zebrafish and fathead minnow in which accumulation is predominantly in the carcass. Despite causing species-specific differences in fecundity, EE2 has been shown to consistently induce hepatic vitellogenin in males and cause feminization/sex reversal during gonadal differentiation in larval mummichog, similar to other species. In addition, while gonadal steroidogenesis and plasma steroid levels respond to exogenous EE2, it is generally at higher concentrations than observed in other species. In mummichog, production of 17β-estradiol (E2) by full grown ovarian follicles remains high; unlike other teleost models where E2 synthesis decreases as 17α,20β-dihydroxy-4-prenen-3-on levels increase to induce oocyte maturation. New evidence in mummichog indicates some dissimilarity in gonadal steroidogenic gene expression responses compared to gene expression responses in zebrafish and fathead minnow exposed to EE2. The role of ovarian physiology continues to warrant investigation regarding the tolerance of mummichog to exogenous EE2 exposure. Here we present a comprehensive review, highlighting key biological differences in response to EE2 exposure between mummichog and other commonly used model teleosts.

NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and β-catenin accumulation.

BackgroundAdrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A.ResultsHere we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3β was aberrantly phosphorylated during G1 phase and β-catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells.ConclusionCentrosomal DNA-PK signaling triggers the accumulation of β-catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.

Differentiation and morphogenesis of the ovary and expression of gonadal development-related genes in the protogynous hermaphroditic ricefield eel Monopterus albus.

The ovarian differentiation, morphogenesis and expression of some putative gonadal development-related genes were analysed in the ricefield eel Monopterus albus, a protogynous hermaphroditic teleost with a single elongate ovary. At c. 1 day post-hatching (dph), the gonadal ridge was colonized with primordial germ cells (PGCs) at the periphery and transformed into the gonadal primordium, which appeared to contain two germinal epithelia. At c. 7 dph, four ovarian cavities appeared in the gonadal tissue with two in each germinal epithelial compartment, and the indifferent gonad might have begun to differentiate into the ovary. The oocytes at the leptotene stage in meiosis I appeared at c. 14 dph, and oocytes at the diplotene stage at c. 30 dph. As development proceeded, the connective tissue separating the two germinal epithelia disappeared, and two of the four ovarian cavities collapsed into one. At 60 dph, the gonad had already taken the shape as observed in the adults. One outer and two inner ovarian cavities could be easily recognized, with slightly basophilic primary growth oocytes usually residing close to the outer ovarian cavity. The expression of cyp19a1a and erb in the early gonad was detected at 6 dph. The abundant expression of foxl2 coincided with the up-regulation of cyp19a1a at 8 dph onwards. The expression of dmrt1 isoforms was not detectable until 8 dph for dmrt1a and dmrt1b and until 33 dph for dmrt1d. The earlier appearance of cyp19a1a compared to dmrt1 transcripts in the indifferent gonad may contribute to the initial differentiation of the gonad towards the ovary in M. albus.

Intersex occurrence in rainbow trout (Oncorhynchus mykiss) male fry chronically exposed to ethynylestradiol.

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization – dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes.

Effects of benzophenone-3 exposure on endocrine disruption and reproduction of Japanese medaka (Oryzias latipes)—A two generation exposure study

Benzophenone-3 (BP-3) has been widely used in sunscreens and cosmetics to protect human skin from the harmful effects of UV irradiation. While BP-3 has been frequently detected in surface waters, sediments and biota, only limited information is available on its in vivo toxicity, particularly in fish. In the present study the endocrine disrupting capacity of BP-3 and its underlying mechanisms were investigated using Japanese medaka (Oryzias latipes). Adult Japanese medaka pairs (F0) were exposed to 0, 4.7, 8.4, 26, or 90μg/L (or 0, 15, 50, 150, or 500μg/L of BP-3 based on nominal concentration) for 14 d and its effects on sex steroid hormones, and transcription of various associated genes were determined. Following additional 14 d of exposure, the F1 eggs reproduced were counted and were further exposed to 0, 5.4, 12, or 30μg/L of BP-3 (or 0, 15, 50, or 150μg/L based on nominal concentrations) until 30 d after hatching. Chemical analysis of the exposed media confirmed transformation of BP-3 to benzophenone-1 (BP-1), a more potent estrogen agonist. After 14 d of the adult fish exposure, plasma concentrations of testosterone (T) significantly increased in male fish. The 17β-estradiol (E2) to T (E2/T) ratio showed significant decreases in both male and female fish. Overall down-regulation of gonadal steroidogenic genes such as star, cyp11a, cyp17, hsd3b, hsd17b3, and cyp19a was also observed. After 28 d of exposure, the daily average egg reproduction per female was significantly reduced at 26μg/L of BP-3. However, hatchability of F1 eggs was not affected by continuous exposure. After continued exposure until 30 dph, juvenile fish showed concentration-dependent decrease of condition factor, but mortality was not affected. Our observation clearly indicates that endocrine balance and reproduction performance in fish could be affected by μg/L level exposure to BP-3. Consequences of longer term exposure over multi-generations warrant further investigation.

Development of retroviral vectors for tissue-restricted expression in chicken embryonic gonads.

The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.

Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes.

BackgroundBlack pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis.ResultsThe gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera.ConclusionsThe study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-491) contains supplementary material, which is available to authorized users.

Sex hormone‐binding globulins characterization and gonadal gene expression during sex differentiation in the rainbow trout, Oncorhynchus mykiss

Sex hormone-binding globulin (SHBG) binds androgens and estrogens in the blood of many vertebrates, including teleost fish. In mammals, SHBG is synthetized in the liver and secreted into the blood. In fish, shbga also exhibits a hepatic expression. In salmonids, in which the gene has been duplicated, the recently discovered shbgb gene exhibits a predominantly ovarian expression. The present work aimed at gaining new insight into shbgb gene structure and expression during gonadal sex differentiation, a steroid-sensitive process, and Shbgb protein structure and binding characteristics; specifically, rainbow trout (Oncorhynchus mykiss) shbgb was analyzed. shbgb structure was analyzed in silico while expression was characterized during gonadal sex differentiation using all-male and all-female populations. We observed that shbgb gene and cognate-protein structures are similar to homologs previously described in zebrafish and mammals. The shbgb gene is predominantly expressed in differentiating female gonads, with increased expression around the end of ovarian differentiation. In the ovary, shbgb mRNA was detected in a subset of somatic cells surrounding the ovarian lamellae. Furthermore, Shbgb binds steroids with a higher selectivity than Shbga, exhibiting a higher affinity for estradiol compared to Shbga. In conclusion, Shbgb binding characteristics are clearly different from those of Shbga. Shbgb is expressed in the differentiating ovary during a period when the synthesis and action of testosterone and estradiol must be tightly regulated. This strongly suggests that Shbgb participates in the regulation of steroid metabolism and/or mediation, that is, needed during early gonadal development in rainbow trout.

Transgenic mouse models in the study of reproduction: insights into GATA protein function

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent.

Sexually dimorphic transcription of estrogen receptors in cod gonads throughout a reproductive cycle

The role of sex steroid regulation in gonadal maturation is a very complex process that is far from being fully understood. Hence, we have investigated seasonal changes in gonadal expression of estrogen receptors (ERs) in Atlantic cod (Gadus morhua L.), a batch spawner, throughout the annual reproductive cycle. Three nuclear ER partial cDNA sequences (esr1, esr2a, and esr2b) were cloned and all esr transcripts were detected mainly in liver and gonads of fish of both sexes. In situ hybridization of esrs along with germ cell (vasa) and gonadal somatic cell markers (gonadal soma-derived factor (gsdf), 3β-hydroxysteroid dehydrogenase (3βhsd), and anti-Müllerian hormone (amh) for testicular, or gsdf for ovarian somatic cells) showed that all three esrs were preferentially localized within interstitial fibroblasts composed of immature and mature Leydig cells in testis, whereas they were differentially expressed in both follicular cells and oocytes in ovary. Quantitative real-time PCR analysis revealed a sexually dimorphic expression pattern of the three esr paralogs in testis and ovary. A significant increase in esr2a expression was identified in testis and of esr2b in ovary, whereas esr1 transcripts were elevated in both testis and ovary in February and March before the spawning period. The localization and sexually dimorphic expression of esr genes in gonads indicate a direct function of estrogen via ERs in gonadal somatic cell growth and differentiation for Leydig cell in testis and follicular cells in ovary throughout the annual reproductive cycle in Atlantic cod.

A microarray-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus.

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Gonad Differentiation in Zebrafish Is Regulated by the Canonical Wnt Signaling Pathway1

Zebrafish males undergo a "juvenile ovary-to-testis" gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a "pro-female" pathway that regulates gonad differentiation in zebrafish.

Expression of SOX3 and SOX9 Genes in Gonads of Blue Gourami

In vertebrates, SOX (SRY-related HMG box) genes are thought to be due to major gene duplication events, initially occurring during early stages of metazoan evolution and later during the transition between non-vertebrate chordates and vertebrates. The aim of this study is to examine SOX3 and SOX9 transcription in oogensis and spermatogenesis in a fish model, the blue gourami (Trichogaster trichopterus). In females during oogenesis, SOX9 mRNA levels were lower compared to SOX3 mRNA levels. In males, SOX9 mRNA levels were higher in testes compared to SOX3 mRNA levels, however no significant differences between SOX3 and SOX9 mRNA levels were observed in the gonads of males that were kept under non-reproductive conditions compared to males kept with females under reproductive conditions and that were nest-builders.

NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and ß-catenin accumulation

Adrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A. Here we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3β was aberrantly phosphorylated during G1 phase and β-catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells. Centrosomal DNA-PK signaling triggers the accumulation of β-catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.

The Effect of 17α-Ethynylestradiol on Steroidogenesis and Gonadal Cytokine Gene Expression Is Related to the Reproductive Stage in Marine Hermaphrodite Fish

Pollutants have been reported to disrupt the endocrine system of marine animals, which may be exposed through contaminated seawater or through the food chain. Although 17α-ethynylestradiol (EE2), a drug used in hormone therapies, is widely present in the aquatic environment, current knowledge on the sensitivity of marine fish to estrogenic pollutants is limited. We report the effect of the dietary intake of 5 µg EE2/g food on different processes of testicular physiology, ranging from steroidogenesis to pathogen recognition, at both pre-spermatogenesis (pre-SG) and spermatogenesis (SG) reproductive stages, of gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. A differential effect between pre-SG and SG specimens was detected in the sex steroid serum levels and in the expression profile of some steroidogenic-relevant molecules, vitellogenin, double sex- and mab3-related transcription factor 1 and some hormone receptors. Interestingly, EE2 modified the expression pattern of some immune molecules involved in testicular physiology. These differences probably reflect a developmental adjustment of the sensitivity to EE2 in the gilthead seabream gonad.

Conservation and Expression of PIWI-Interacting RNA Pathway Genes in Male and Female Adult Gonad of Amniotes1

The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad.

New insights into disorders of gonad development using whole genome analysis

One of the most fundamental influences on our lives is our sex. Whether we are born as male or female has an enormous impact on our behaviour, reproductive options and disease susceptibility. Consequently, if the sex of a baby is not clear it can create many intractable issues, particularly in terms of medical management. Disorders of Sex Development (DSDs) are congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. The cause of these problems is most often a breakdown of the complex network of gene regulation responsible for proper development of testes or ovaries in the embryo. In the majority of DSD patients the etiology is unknown and they cannot be given an accurate diagnosis. We aim to identify the underlying changes in gonad genes of these DSD patients, provide a diagnosis and gain insights into gonad development. We used microarrays to detect changes in copy number in DSD patients. This approach identified potential novel testis specific regulatory regions in SOX9, a known testis gene. We also showed that deletions and duplications affecting the regulatory sequences of SOX3 can cause it to be ectopically expressed in the developing gonad, allowing it to drive testis development in the absence of SRY in patients with 46,XX testicular DSD. In addition, we have identified mutations in the novel gene, MAP3K1 in patients with 46,XY DSD; implicating a new signal transduction pathway in testis development. We’ve used Massively Parallel Sequencing (MPS) in three different ways. Firstly, we developed a rapid targeted MPS approach allowing in depth analysis of up to 200 gonad genes per DSD patient. Secondly, we used whole exome capture and MPS on several DSD families, trios and single cases. Finally, we employed whole genome MPS on trios with DSD. This work demonstrates the tremendous power of whole genome approaches, especially when combing MPS data with linkage analysis of a large family. Whole genome analysis provides a rapid approach to identification of the disease-causing mutations and molecular diagnosis in patients with DSD as well as providing insights into gonad development and sex differentiation.

Highly expressed genes in gonads of the bat star Patiria miniata: gene ontology, expression differences, and gamete recognition loci

AbstractEvolutionary analysis of mating systems in broadcast‐spawning marine animals and plants has focused on sperm‐ and egg‐surface proteins and glycopeptides that mediate reproductive interactions at different stages of gamete recognition. Improved understanding of the ecology and evolution of such interactions depends on extending our knowledge to multiple genes expressed in both sperm and eggs of diverse taxonomic groups with different modes of fertilization. Here, we use readily accessible next‐generation sequencing methods and desktop bioinformatics to characterize the repertoire of highly expressed genes in testes and ovaries of the asterinid sea star Patiria miniata, including gene ontology annotations for male‐ and female‐expressed molecules, and descriptions of two genes that encode egg‐surface molecules involved in fertilization that have not previously been studied in sea stars. The results are used to contrast expression differences between the testis and ovary, and to develop hypotheses of gamete‐specific expression. We also explore differences in ovary gene expression among multiple females from northern and southern populations that show nucleotide differentiation at many non‐expressed loci and at a gamete recognition locus.