Barbara Nicol

The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids

All salmonid species investigated to date have been characterized with a male heterogametic sex‐determination system. However, as these species do not share any Y‐chromosome conserved synteny, there remains a debate on whether they share a common master sex‐determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex‐determining gene, sdY (sexually dimorphic on the Y‐chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male‐specific Y‐chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex‐determining locus and also strongly suggest that sdY may be this conserved master sex‐determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex‐determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male‐specific Y‐chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species.

Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

Expression Profiling of Wnt Signaling Genes during Gonadal Differentiation and Gametogenesis in Rainbow Trout

Wnt signaling plays major roles in various processes, including ovarian differentiation and development in mammals. In order to explore its potential implication during gonadal development in a nonmammalian vertebrate species, expression of Wnt signaling genes was investigated in rainbow trout during gonadal differentiation and gametogenesis. Multiple Wnt pathway genes were expressed and exhibited distinct expression patterns. In ovary, tcf7 was highly expressed during early differentiation, whereas no sexually dimorphic expression of rspo1 was detected. During later ovarian development, wnt11 was highly expressed in granulosa cells and oocytes suggesting an implication in folliculogenesis and oogenesis, whereas wnt9b was principally detected in granulosa cells. In testis, Wnt pathway genes were mostly expressed during early spermatogenesis. Overall, these present results suggest that Wnt signaling is implicated in multiple processes of male and female gonadal development and provide basis for future studies on Wnt signaling functions in teleost fish gonads.

Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout (Oncorhynchus mykiss).

The Wnt/β‐catenin pathway is crucial for ovarian differentiation in mammals, and WNT4 is an important protein that regulates this process. While the role of Wnt4 in gonadal differentiation is relatively well characterized in mammals, little is known regarding its role in teleost fish. Therefore, we investigated the potential activity of wnt4 in gonadal differentiation in rainbow trout (Oncorhynchus mykiss), focusing on the teleost and salmonid gene duplications. Phylogenetic and synteny analyses demonstrated that teleost fish possess two wnt4 genes, wnt4a and wnt4b, as a consequence of the teleost‐specific whole‐genome duplication (3R). In rainbow trout, we also identified an additional wnt4 gene, which is a wnt4a paralog that likely resulted from the salmonid‐specific whole‐genome duplication (4R). These two Wnt4a proteins (Wnt4a1 and Wnt4a2) share a high identity (>80%) with other vertebrate Wnt4 proteins, whereas Wnt4b is clearly more divergent (60% identity). During embryogenesis and adulthood, the wnt4a1/2 transcripts were expressed in various tissues, including the ovaries and testes. In contrast, wnt4b expression was restricted to the nervous system, suggesting a sub‐ or a neo‐functionalization of this divergent paralog. During early gonadal differentiation in both males and females, the wnt4a1/2 transcripts were detected in the somatic cells surrounding the germ cells, with a slight sexual dimorphism in favor of males. These results demonstrate that, unlike mammals, rainbow trout do not display an ovary‐predominant wnt4 expression profile during early gonadal differentiation. Mol. Reprod. Dev. 79:51–63, 2012.

Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice

Background Mice exposed to high levels of arsenic in utero have increased susceptibility to tumors such as hepatic and pulmonary carcinomas when they reach adulthood. However, the effects of in utero arsenic exposure on general physiological functions such as reproduction and metabolism remain unclear. Objectives We evaluated the effects of in utero exposure to inorganic arsenic at the U.S. Environmental Protection Agency (EPA) drinking water standard (10 ppb) and at tumor-inducing levels (42.5 ppm) on reproductive end points and metabolic parameters when the exposed females reached adulthood. Methods Pregnant CD-1 mice were exposed to sodium arsenite [none (control), 10 ppb, or 42.5 ppm] in drinking water from gestational day 10 to birth, the window of organ formation. At birth, exposed offspring were fostered to unexposed dams. We examined reproductive end points (age at vaginal opening, reproductive hormone levels, estrous cyclicity, and fertility) and metabolic parameters (body weight changes, hormone levels, body fat content, and glucose tolerance) in the exposed females when they reached adulthood. Results Arsenic-exposed females (10 ppb and 42.5 ppm) exhibited early onset of vaginal opening. Fertility was not affected when females were exposed to the 10-ppb dose. However, the number of litters per female was decreased in females exposed to 42.5 ppm of arsenic in utero. In both 10-ppb and 42.5-ppm groups, arsenic-exposed females had significantly greater body weight gain, body fat content, and glucose intolerance. Conclusion Our findings revealed unexpected effects of in utero exposure to arsenic: exposure to both a human-relevant low dose and a tumor-inducing level led to early onset of vaginal opening and to obesity in female CD-1 mice. Citation Rodriguez KF, Ungewitter EK, Crespo-Mejias Y, Liu C, Nicol B, Kissling GE, Yao HH. 2016. Effects of in utero exposure to arsenic during the second half of gestation on reproductive end points and metabolic parameters in female CD-1 mice. Environ Health Perspect 124:336–343; http://dx.doi.org/10.1289/ehp.1509703

Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice

ABSTRACT Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonads identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.

Building an Ovary: Insights into Establishment of Somatic Cell Lineages in the Mouse

The molecular pathways that drive the differentiation of somatic cell populations in the testis and ovary have been the subjects of intensive research over the past decade. It is now clear that ovarian differentiation is a coordinate event driven by secreted factors including R-spondin1, WNT4, and follistatin and transcriptional regulators such as β-catenin and FOXL2. These factors direct bipotential somatic cell lineages toward an ovarian fate and simultaneously suppress the emergence of testis-determining processes. This review summarizes the molecular pathways responsible for establishment of the ovary and discusses the current hypotheses on the origin(s) of somatic cell lineages and how these somatic cells acquire the characteristics necessary for their function during ovarian development and maintenance.

The duplicated rainbow trout (Oncorhynchus mykiss) T-box transcription factors 1, tbx1a and tbx1b, are up-regulated during testicular development.

Tbx1 is a member of the T‐box transcription factor gene family involved in embryogenesis and organogenesis. Recently, within a pan‐genomic screen using rainbow trout (Oncorhynchus mykiss) cDNA microarrays, we identified a tbx1 homolog with testicular over‐expression during sex differentiation. Here, we characterized two very similar rainbow trout tbx1 paralogs, tbx1a and tbx1b. In adult tissues, tbx1a expression is restricted to the gonads, with high expression in the testis, while tbx1b is more widely expressed in gonads, gills, brains, muscle, and skin. During gonadal differentiation, both genes are differentially expressed in favor of testis formation shortly after hatching. These genes are expressed in somatic cells surrounding germ cells of the differentiating testis, while no or only weak expression was observed in the differentiating ovary. tbx1a and tbx1b were also both down‐regulated in the differentiating testis during feminization with estrogens and up‐regulated in the differentiating ovary during masculinization with an aromatase inhibitor. These results suggest that tbx1a and tbx1b are probably involved in the regulation of testicular differentiation in rainbow trout. Since Tbx1 is known to interact with the retinoic acid (RA) signaling pathway, we also examined the effect of RA on the rainbow trout tbx1 expression pattern. Expression of tbx1a and tbx1b was down‐regulated in RA‐treated male gonads, suggesting that tbx1 interacts with the RA signaling pathway and thus could be involved in the control of rainbow trout gonadal differentiation. Mol. Reprod. Dev. 78:172–180, 2011.

Heritable Targeted Inactivation of the Rainbow Trout (Oncorhynchus mykiss) Master Sex-Determining Gene Using Zinc-Finger Nucleases

Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.

Follistatin Is an Early Player in Rainbow Trout Ovarian Differentiation and Is Both Colocalized with Aromatase and Regulated by the Wnt Pathway

In mammals, follistatin (FST) plays an important role in early ovarian differentiation, acting downstream of the Wnt pathway. In teleost fish, fst is implicated in folliculogenesis and oocyte maturation, and an early and specific expression during ovarian differentiation has been described in rainbow trout, Oncorhynchus mykiss. By in situ hybridization, we demonstrated that during rainbow trout gonadal differentiation, fst shares a similar expression pattern with cyp19a1a, the gene encoding ovarian aromatase, a key steroidogenic enzyme needed for ovarian differentiation in fish. Expression of fst and cyp19a1a was first detected in a few scattered cells in the embryonic ovary several days before hatching. Then, after histological differentiation, fst and cyp19a1a expression was localized in clusters of cells lining the future ovarian lamellae. As FST expression is known to be induced by the Wnt/β-catenin pathway in mammals, the Wnt pathway was inhibited in vivo with the IWR-1 molecule, and we analyzed by qPCR the effects of this treatment on fst expression. We found that IWR-1 decreased fst expression in female gonads, consistent with a regulation of fst expression by the Wnt pathway in rainbow trout. Furthermore, expression of cyp19a1a was also downregulated, suggesting an implication of the Wnt pathway in ovarian differentiation.