Early Gonadal Differentiation Research Articles

Completely blocking the feminization by replacing Limnodilus spp. with Artemia salina to feeding XY Zig-zag eel (Mastacembelus armatus)

Zig-zag eel (Mastacembelus armatus), with male growing faster and larger than female, is a popular farmed fish in China due to its delicious taste and rich nutritional value. The culture of mono-sex M. armatus has great value and is the best choice for aquaculture industry. However, the high feminization rate in aquaculture severely limits the application of sex control in the breeding process of M. armatus. In this study, by analyzing the early gonadal development and sex differentiation process, as well as the expression profiles of male and female pathway genes in the early XX and XY gonads, we determined that the critical period for sex differentiation and gonadal differentiation in M. armatus was 10–30 dpf. In addition, we confirmed for the first time that the feminization of M. armatus is related to feeding Limnodilus spp., as only Limnodilus spp. are fed during 10–30 dpf. Replacing Limnodilus spp. with Artemia salina to feed M. armatus fry can completely block the feminization. These results lay a solid foundation for understanding the sex determination mechanism of M. armatus, and the method of blocking the feminization of M. armatus can be used for the development of all-male breeding.

Reproductive toxicity of DDT in the Japanese medaka fish model: Revisiting the impacts of DDT+ on female reproductive health

The organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) is an endocrine-disrupting compound (EDC) that has been banned by most countries for decades. However, it continues to be detected in nearly all humans and wildlife due to its biological and environmental persistence. The ovarian dysgenesis syndrome hypothesis speculates that exposure to EDCs during sensitive developmental windows such as early gonadal differentiation lead to reproductive disorders later in life. Yet, mechanisms by which DDT affects developing gonads remain unclear due to the inherent challenge of getting developmental exposure data from adults presenting with reproductive disease. The Japanese medaka (Oryzias latipes) is a valuable fish model for sex-specific toxicological studies due to its chromosomal sex determination, external embryonic development, short generation time, and extensively mapped genome. It is well documented that medaka exposed to DDT and its metabolites and byproducts (herein referred to as DDT+) at different developmental time points experience permanent alterations in gonadal morphology, reproductive success, and molecular and hormonal signaling. However, the overwhelming majority of studies focus primarily on functional and morphological outcomes in males and females and have rarely investigated long-term transcriptional or molecular effects. This review summarizes previous experimental findings and the state of our knowledge concerning toxic effects DDT + on reproductive development, fertility, and health in the valuable medaka model. It also identifies gaps in knowledge, emphasizing a need for more focus on molecular mechanisms of ovarian endocrine disruption using enhanced molecular tools that have become increasingly available over the past few decades. Furthermore, DDT forms a myriad of over 45 metabolites and transformation products in biota and the environment, very few of which have been evaluated for environmental abundance or health effects. This reinforces the demand for high throughput and economical in vivo models for predictive toxicology screening, and the Japanese medaka is uniquely positioned to meet this need.

A time-course transcriptome analysis revealing the potential molecular mechanism of early gonadal differentiation in the Chinese giant salamander

The Chinese giant salamander (CGS) Andrias davidianus is the largest extant amphibian and has recently become an important species for aquaculture with high economic value. Meanwhile, its wild populations and diversity are in urgent need of protection. Exploring the mechanism of its early gonadal differentiation will contribute to the development of CGS aquaculture and the recovery of its wild population. In this study, transcriptomic and phenotypic research was conducted on the critical time points of early gonadal differentiation of CGS. The results indicate that around 210 days post-hatching (dph) is the critical window for female CGS's gonadal differentiation, while 270 dph is that of male CGS. Besides, the TRPM1 gene may be the crucial gene among many candidates determining the sex of CGS. More importantly, in our study, key genes involved in CGS's gonadal differentiation and development are identified and their potential pathways and regulatory models at early stage are outlined. This is an initial exploration of the molecular mechanisms of CGS's early gonadal differentiation at multiple time points, providing essential theoretical foundations for its captive breeding and offering unique insights into the conservation of genetic diversity in wild populations from the perspective of sex development.

Transcriptome Analyses Reveal Expression Profiles of Morphologically Undifferentiated and Differentiated Gonads of Yangtze Sturgeon Acipenser dabryanus.

Sturgeon is known as a primitive fish with the ZZ/ZW sex determination system and is highly prized for its valuable caviar. Exploring the molecular mechanisms underlying gonadal differentiation would contribute to broadening our knowledge on the genetic regulation of sex differentiation of fish, enabling improved artificial breeding and management of sturgeons. However, the mechanisms are still poorly understood in sturgeons. This study aimed to profile expression patterns between female and male gonads at morphologically undifferentiated and early differentiated stages and identify vital genes involved in gonadal sex differentiation of sturgeons. The sexes of Yangtze sturgeon (Acipenser dabryanus) juveniles were identified via the sex-specific DNA marker and histological observation. Transcriptome analyses were carried out on female and male gonads at 30, 80 and 180 days post-hatching. The results showed that there was a total of 17 overlapped DEGs in the comparison groups of between female and male gonads at the three developmental stages, in which there were three DEGs related to ovarian steroidogenesis, including hsd17b1, foxl2 and cyp19a1. The three DEGs were highly expressed in the female gonads, of which the expression levels were gradually increased with the number of days after hatching. No well-known testis-related genes were found in the overlapped DEGs. Additionally, the expression levels of hsd17b1 and cyp19a1 mRNA were decreased with the knockdown of foxl2 mRNA via siRNA. The results further suggested that foxl2 should play a crucial role in the ovarian differentiation of sturgeons. In conclusion, this study showed that more genes involved in ovarian development than testis development emerged with sexually dimorphic expression during early gonadal sex differentiation, and it provided a preliminary understanding of the molecular regulation on gonadal differentiation of sturgeons.

Determination of the timing of early gonadal differentiation in silver pomfret, Pampus argenteus

Silver pomfret is a species of global significance due to its high nutritional in fisheries sector. To accurately ascertain the timing of sex differentiation mechanism and mRNA level in this species, this study examined gonad morphology and patterns of gene expression related to sex differentiation in males and females from 51 to 180 days post hatch (dph), the temperature of water was maintained at 26 ± 1 ℃. Distinct morphological differentiation of the silver pomfret ovaries, marked by the emergence of primary oocytes, became apparent from 68 dph. By 108 dph, the testes began to differentiate, as evidenced by the appearance of the efferent duct. Early oocytes exhibited a diameter ranged from 0.077 mm to 0.682 mm, with an average diameter of 0.343 ± 0.051 mm. The proportions of various types of germ cells within the testes were subjected to analysis. The localization of Vasa during the early stages of sexual differentiation was a subject to analysis as well. Vasa was predominantly localized within the cytoplasm of gonocyte, peri-nucleolus stage oocytes, primary oocytes and type A spermatogonocytes, indicating that Vasa is involved in the early gonadal differentiation of silver pomfret. The study investigated the expression patterns of dmrt1, gsdf, amh, foxl2, cyp19a1a, cyp11a, sox3 and vasa, all of which are involved in the sex differentiation of teleosts. Among these genes, amh, gsdf, sox3, foxl2, vasa were indentified as crucial contributors to the early gonadal development of silver pomfret. Significant sex-related differences were observed in the expression patterns of amh, dmrt1, gsdf, cyp11a, sox3, cyp19a1a, vasa. This study provides novel insights into the timing of physiological changes associated with the sexual differentiation of silver pomfret. Collectively, the present data indicates that the differentiation of ovaries and testes take place approximately at 68 dph in females and 108 dph in males.

CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate

The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms’ tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Characterization of Early Gonadal Differentiation and Induction of Sex Reversal in the Rock Bream Oplegnathus fasciatus.

Rock bream (Oplegnathus fasciatus) is a typical fish that has a unique multiple sex chromosome system (♀X1X1X2X2/♂X1X2Y). We examined the early gonadal development in rock bream via continuous histological observations of the gonads at 40-120days post hatching (dph). The fish was identified as a typical gonochorist, and female gonads were found to differentiate earlier than male gonads. The ovarian cavity of the female was initially observed at 80 dph, whereas the efferent duct of the male was not observed until 100 dph. Immunofluorescence with the vasa-antibody revealed that germ cells were predominantly distributed around the ovarian cavity in females and on the edge of the gonad in males during the early stages of sex differentiation. Sex reversal was induced via the oral administration of letrozole (LTZ), 17α-methyltestosterone (MT), and 17β-estradiol (E2), respectively, during the labile period of gonadal development. LTZ and MT induced 100% masculinization of genotype-females, whereas E2 induced only 50-60% feminization of genotype-males. Such findings suggest that the fish retained high sexual plasticity despite the existence of the neo-Y chromosome. MT and E2 had negative effect on fish growth, whereas LTZ did not exert such side effect. LTZ and MT could accelerate gonadal development in sex-reversed genotype-males, whereas E2 inhibited gonadal development in genotype-females of rock bream. These findings provide a basis for further research on the mechanisms of sex determination and differentiation in fishes with X1X2Y sex chromosome system and provide a sex reversal protocol for rock bream.

Early gonadal differentiation is associated with the antagonistic action of Foxl2 and Dmrt1l in the Pacific oyster.

As the second largest phylum in the zoological kingdom next to arthropods, the mechanism of gonadal differentiation in mollusca is quite complex. Currently, although much has been carried out on gonadal differentiation in the Pacific oyster, there is still unknown information that needs to be further explored. Here, analysis of the Foxl2 and Dmrt1l expression in samples at different development periods of male and female gonads as well as in annual gonad samples revealed that Log10 (Foxl2/Dmrt1l) values were an effective method for sex identification in oysters. In differentiated gonadal tissue, Log10 (Foxl2/Dmrt1l) values greater than 2 were females and less than 1 for males. Subsequent sequential sampling of the same individuals verified that Log10 (Foxl2/Dmrt1l) values greater than 2 for resting gonads would develop as females and less than 1 would develop as males in the future. Relative expression analysis of Foxl2 and Dmrt1l in the annual samples revealed a negative correlation between Log10 (Foxl2) and Log10 (Dmrt1l). Double fluorescence reporter validation results showed that DMRT1L protein was able to bind the Foxl2 promoter and repress its activity with a weak dosage effect. Antagonism between Dmrt1l and Foxl2 is therefore not restricted to vertebrates, and the competing regulatory networks are of great significance in the maintenance of gonadal sex in oysters after sexual differentiation. This study provides novel ideas and insights into the study of early gonadal differentiation in the adult oyster.

De Novo Transcriptome Analysis of the Early Hybrid Triploid Loach (Misgurnus anguillicaudatus) Provides Novel Insights into Fertility Mechanism

As a new freshwater aquaculture product, triploid loaches (Misgurnus anguillicaudatus) are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility. In this study, a natural tetraploid loach (4n = 100) (♀) was crossed with a diploid loach (2n = 50) (♂), thus creating the hybrid triploid loach (3n = 75). The histological observations of triploid offspring and diploid controls at 4 days post-hatching (dph), 15dph, 22dph, and 50dph showed that most of the hybrid triploid loaches were abortive in the early gonad differentiation process. To explore its fertility mechanism, through transcriptome analyses of triploid offspring and diploid controls at four periods, 10 differentially expressed genes related to the early fertility mechanism were identified: amh, hormad1, rec8, h2b, plvap, zp3, h2a, nrb0b1, ddx4, and esr2. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, two pathways were identified that are closely related to the early fertility mechanism at 50dph: the estrogen signaling pathway and steroid biosynthesis. The findings laid a foundation for further exploration of their molecular inhibition mechanism in hybrid triploid loaches.

Identification and Expression Analysis of Wnt2 Gene in the Sex Differentiation of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture animal in China. The Wnt gene family plays important regulatory roles in the development and growth of mammals. However, the precise function of these family genes has not been well understood in the sex differentiation of Chinese soft-shelled turtles. Here, we cloned a member of the Wnt family, Wnt2, which obtained a 1077 bp open reading frame that encoded a 358-aa protein. The putative amino acid sequences of proteins are exceeded 80% identical to other turtles. The expression level of Wnt2 peaked at the 14th stage both in female and male embryos during the early gonadal differentiation period of Chinese soft-shelled turtles, which occurred before gonadal differentiation. Wnt2 mRNA was expressed at higher levels in the brains and gonads of mature P. sinensis females compared with those in mature males. Wnt agonists significantly affected the expression level of Wnt2 during the gonadal differentiation period. After Wnt agonists (1.0 μg/μL, 2.5 μg/μL, 5.0 μg/μL) treatment, the expression level of the Wnt2 generally appeared to have an inverted-V trend over time in female embryonic gonads. The results suggested that Wnt2 may participate in the regulation of gonad development in P. sinensis during the early embryonic stages. These results could provide a theoretical basis for the reproduction process of the Chinese soft-shelled turtle.

Deletion of Wt1 during early gonadogenesis leads to differences of sex development in male and female adult mice.

Assessing the role of the WT1 transcription factor (WT1) during early gonad differentiation and its impact on adult sex development has been difficult due to the complete gonadal agenesis and embryonic lethality exhibited by Wt1KO mouse models. Here, we generated Wt1LoxP/GFP;Wt1Cre mice, the first Wt1KO mouse model that reaches adulthood with a dramatically reduced Wt1 expression during early gonadogenesis. Wt1LoxP/GFP;Wt1Cre mice lacked mature gonads and displayed genital tracts containing both male and female genital structures and ambiguous genitalia. We found that WT1 is necessary for the activation of both male and female sex-determining pathways, as embryonic mutant gonads failed to upregulate the expression of the genes specific for each genetic programme. The gonads of Wt1LoxP/GFP;Wt1Cre mice showed a lack of production of Sertoli and pre-granulosa cells and a reduced number of germ cells. NR5A1 and the steroidogenic genes expression was modulated differently in XY and XX Wt1LoxP/GFP;Wt1Cre gonads, explaining the mutant phenotypes. Further studies of the XX Wt1LoxP/GFP;Wt1Cre gonads revealed that deletion of WT1 at an early stage impaired the differentiation of several cell types including somatic cells and the ovarian epithelium. Through the characterisation of this Wt1KO mouse model, we show that the deletion of Wt1 during early gonadogenesis produces dramatic defects in adult sex development.

Timing of early gonadal differentiation and effects of estradiol-17β treatments on the sex differentiation in Largemouth bass (Micropterus salmoides).

In this study, an efficient estradiol-17β (E2)-induced feminization method was established based on the timing of early gonadal differentiation in Largemouth bass (Micropterus salmoides). Histological section resultsshowed that from 20days post-hatch (dph) to 30 dph, the germ cells gradually differentiated into oogonium and spermatic deferent, respectively. Moreover, female-biased genes Foxl2 and Cyp19a1a were up-regulated to the first peak at 20 dph, while the male-biased genes Dmrt1 were up-regulated to the first peak at 30 dph. These results indicated that the timing of early gonadal differentiation in Largemouth bass was between 20 and 30 dph. Therefore, 15 dph Largemouth bass with a body length of 15.10 ± 0.09mm were chosen, and four E2-treated diets were set as 0 (E0, control), 50mg/kg E2 (E50), 100mg/kg E2 (E100), and 200mg/kg E2 (E200). After feeding with E2-treated diets for 60days, female ratios were 55%, 100%, 100%, and 100% in E0, E50, E100, and E200 groups, respectively. No intersex fish were observed in all the groups. However, 30% of females in the E200 group possessed thinner ovaries, with smaller ovary cavity structures and a decreased number of primary oocyte cells than those in other groups. Besides, the Largemouth bass in the E0 group grew more than those in E50, E100, and E200 groups during the E2 treatments period (P < 0.05). In conclusion, our study suggested that 50-100mg/kg E2-treated diets could effectively induce the feminization of 15 dph Largemouth bass within 60days duration time, which provided valuable information for the breeding of the all-male Largemouth bass population.

Dynamic Gene Expression and Alternative Splicing Events Demonstrate Co-Regulation of Testicular Differentiation and Maturation by the Brain and Gonad in Common Carp.

The common carp (Cyprinus carpio) accounts for approximately 10% of the annual freshwater aquaculture production and is an ideal model to study cyprinidae reproduction. Female common carp grow faster than the males; therefore, related research presents an opportunity with high application value. Although we have a detailed understanding of common carp’s early gonadal differentiation process, information about genome-wide gene expression, regulation, and underlying molecular mechanisms during this process remain limited. Here, time-course data comprising six key stages during testicular differentiation and maturation were investigated to further understand the molecular mechanisms underlying the testicular development in cyprinid species. After integrating these time-series data sets, common carp genome, including 98,345 novel transcripts and 3,071 novel genes were re-annotated and precisely updated. Gene co-expression network analysis revealed that the ubiquitin-mediated proteolysis pathway was essential for metabolism during testicular differentiation in the endocrine system of C. carpio. Functional enrichment analyses indicated that genes mainly related to amino acid metabolism and steroid hormone synthesis were relatively highly expressed at the testicular undifferentiation stages, whereas genes associated with cell cycle and meiosis were expressed from the beginning of testicular differentiation until maturation. The dynamics of alternative splicing events demonstrated that exon skipping accounted for majority of the alternative splicing events in the testis and the brain during gonad development. Notably, several potential male-specific genes (fanci and sox30) and brain-specific genes (oxt, gad2, and tac1, etc.) were identified. Importantly, we traversed beyond the level of transcription to test for stage- and gonad-specific alternative splicing patterns between the brain and testis. This study is the first to describe a comprehensive landscape of alternative splicing events and gene expression patterns during gonadogenesis in common carp. This work is extremely valuable to elucidate the mechanisms underlying gonadal differentiation in Cyprinidae as well as other fish species.

Chlorpyrifos Effect on Vitellogenin, Ovarian Steroid in Adult and NR5A1 Expression in Fry of the Freshwater Catfish, Heteropneustes fossilis (Bloch, 1794)

Aim: To study the interference of organophosphate pesticide chlorpyrifos (CPF) in gonad function at the steroidal and gene levels during prespawning adult and fry stage respectively of the freshwater stinging catfish, Heteropneustes fossilis. Methods: The matured adult fish of early prespawning stage were exposed to different combinations of CPF (0.174 μM, 10-1th of 96 h LC50), gonadotropin (hCG; 100 IU.fish-1) and natural antioxidants (curcumin; 10 mg.L-1 and ascorbic acid; 5 mg.L-1) for 24 h to measure vitellogenin and ovarian steroid. Twenty days post-hatch, fish fry were exposed to CPF for 96 h to understand the influence on NR5A1 gene expression pattern. Results: The CPF increased vitellogenin level at liver, ovary and serum. Among steroidal cascade CPF induced highest estradiol followed by a significant increase in progesterone and 17α, 20β-dihydroxyprogesterone as compared to control fish. The gonadotropin further increased CPF induced effects on vitellogenin and ovarian steroidal level. Though, antioxidants co-incubation with CPF did not produce any significant change on either vitellogenin or in ovarian steroids content. CPF triggers NR5A1 gene expression in a fry that indicates a role in early gonad differentiation and development. Conclusion: The present study clearly indicates the major influence of CPF in gonadal physiology of both adult and fry stages of freshwater catfish, Heteropneustes fossilis.

MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle†.

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo–maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

The conditional deletion of steroidogenic factor 1 (Nr5a1) in Sox9-Cre mice compromises testis differentiation

Steroidogenic factor 1 (NR5A1) is essential for gonadal development. To study the importance of NR5A1 during early gonadal sex differentiation, we generated Sox9-Cre-Nr5a1 conditional knockout (cKO) mice: Sox9-Cre;Nr5a1flox/flox and Sox9-Cre;Nr5a1flox/− mice. Double-immunostaining for NR5A1 and AMH revealed silenced NR5A1 in Sertoli cells and reduced AMH+ cells in the gonads of XY Sox9-Cre-Nr5a1 cKO mice between embryonic days 12.5 (E12.5) and E14.5. Double-immunostaining for SOX9 and FOXL2 further indicated an early block in Sertoli cells and ectopic granulosa cell differentiation. The number of cells expressing the Leydig cell marker 3βHSD obviously reduced in the gonads of XY Sox9-Cre;Nr5a1flox/− but not Sox9-Cre;Nr5a1flox/flox mice at E15.5. The presence of STRA8+ cells indicated that germ cells entered meiosis in the gonads of XY Sox9-Cre-Nr5a1 cKO mice. The results of qRT-PCR revealed remarkably reduced and elevated levels of testis and ovary markers, respectively, in the gonads of XY Sox9-Cre-Nr5a1 cKO mice at E12.5‒E13.5. These data suggested that the loss of Nr5a1 abrogates the testicular pathway and induces the ectopic ovarian pathway, resulting in postnatal partial/complete male-to-female gonadal sex reversal. Our findings provide evidence for the critical role of NR5A1 in murine gonadal sex determination in vivo.

Transcriptomic analysis of gonadal development in parasitic and non-parasitic lampreys (Ichthyomyzon spp.), with a comparison of genomic resources in these non-model species.

Lampreys are jawless fishes that diverged ∼550 million years ago from other vertebrates. Sequencing of the somatic and the germline genomes of the sea lamprey (Petromyzon marinus) in 2013 and 2018, respectively, has helped to improve our understanding of the genes and gene networks that control many aspects of lamprey development. However, little is known about the genetic basis of gonadal differentiation in lampreys, partly due to the prolonged period during which their gonads remain sexually indeterminate. We performed RNA-sequencing on gonadal samples from four chestnut lamprey (Ichthyomyzon castaneus) and six northern brook lamprey (I. fossor) to identify differentially expressed genes (DEG’s) and pathways associated with transcriptomic differences in: (1) larvae during early gonadal differentiation versus definitive females (i.e., with oocytes in the slow cytoplasmic growth phase); and (2) females versus definitive males undergoing spermatogonial proliferation. We compared the mapping percentages of these transcriptomes to the two available sea lamprey reference genomes and three annotation files (Ensembl and UCSC for the somatic genome and SIMRbase for the germline genome). We found that mapping the RNA-seq reads to the germline genome gave superior results and, using Trinotate, we provided new putative annotations for 8161 genes in the somatic assembly and 880 genes for the germline assembly. We identified >2000 DEG’s between stages and sexes, as well as biological pathways associated with each. Interestingly, some of the upregulated genes (e.g., DEG’s associated with spermiation) suggest that changes in gene expression can precede morphological changes by several months. In contrast, only 81 DEG’s were evident between the chestnut lamprey (that remains sexually immature during an extended post-metamorphic parasitic feeding phase) and the nonparasitic northern brook lamprey (that undergoes sexual maturation near the end of metamorphosis), but few replicates were available for comparable stages and sexes. This work lays the foundation for identifying and confirming the orthology and the function of genes involved in gonadal development in these and other lamprey species across more developmental stages.

Cloning and functional analysis of c/ebpα as negative regulator of dmrt1 in Chinese tongue sole (Cynoglossus semilaevis).

c/ebpα is a member of the C/EBP family of transcription factors, which are involved in cell growth and differentiation and have a conserved basic leucine zipper (bZIP) domain. However, little is known about its function in sex determination and differentiation. In the present study, c/ebpα was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of c/ebpα was 1583bp, with a 198-bp 5' UTR, a 446-bp 3' UTR, and a 939-bp open reading frame encoding a 312-amino acid peptide. qRT-PCR revealed that c/ebpα was predominantly expressed in undifferentiated gonads of male C. semilaevis at 30 dpf and 60 dpf and peaked at 60 dpf. Expression levels of c/ebpα in the testis were constantly higher than those in ovaries at all developmental stages. Moreover, a dual-luciferase assay revealed that c/ebpα could negatively regulate the male-determining gene dmrt1 in vitro. These results provide fundamental information indicating that C. semilaevis c/ebpa might be involved in early gonadal differentiation and functions as a negative regulator of dmrt1 by repressing its transcription.

A frog with three sex chromosomes that co-mingle together in nature: Xenopus tropicalis has a degenerate W and a Y that evolved from a Z chromosome.

In many species, sexual differentiation is a vital prelude to reproduction, and disruption of this process can have severe fitness effects, including sterility. It is thus interesting that genetic systems governing sexual differentiation vary among-and even within-species. To understand these systems more, we investigated a rare example of a frog with three sex chromosomes: the Western clawed frog, Xenopus tropicalis. We demonstrate that natural populations from the western and eastern edges of Ghana have a young Y chromosome, and that a male-determining factor on this Y chromosome is in a very similar genomic location as a previously known female-determining factor on the W chromosome. Nucleotide polymorphism of expressed transcripts suggests genetic degeneration on the W chromosome, emergence of a new Y chromosome from an ancestral Z chromosome, and natural co-mingling of the W, Z, and Y chromosomes in the same population. Compared to the rest of the genome, a small sex-associated portion of the sex chromosomes has a 50-fold enrichment of transcripts with male-biased expression during early gonadal differentiation. Additionally, X. tropicalis has sex-differences in the rates and genomic locations of recombination events during gametogenesis that are similar to at least two other Xenopus species, which suggests that sex differences in recombination are genus-wide. These findings are consistent with theoretical expectations associated with recombination suppression on sex chromosomes, demonstrate that several characteristics of old and established sex chromosomes (e.g., nucleotide divergence, sex biased expression) can arise well before sex chromosomes become cytogenetically distinguished, and show how these characteristics can have lingering consequences that are carried forward through sex chromosome turnovers.

Timing of gonadal development and dimorphic expression of sex-related genes in gonads during early sex differentiation in the Yellow River carp

The common carp, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying cyprinidae gonadal sex differentiation. The present study aims at investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes (cyp19a1a, foxl2, amh, dmrt1, sox9b, sycp3, dmc1) with gonadal histological changes using mono-sex female and male Yellow River carp. H&E staining, immunohistochemistry and immunofluorescence of gonads indicated that the ovaries and testis of common carp were both directly differentiated from undifferentiated gonads. Oogonia and spermatogonia were first observed at 40 dph (days post hatching) and 70 dph in female and male gonads, respectively. Female related genes cyp19a1a and foxl2 and male related genes amh, dmrt1 and sox9b increased significantly in 30–40 dph female, and male gonads, respectively. These suggest that the critical timing of sex determination of common carp takes place between 30 and 40 dph or possibly earlier. Meiotic marker gene dmc1 and sycp3 increased expression significantly in 55 dph female gonads while sycp3 increased expression significantly in 100 dph male gonads. Oocytes in meiosis and spermatocytes were first observed in 55 dph female and 100 dph male gonads, respectively. These indicate that the initiation time of gonadal sex differentiation is 50–55 dph in female common carp and 90–100 dph in male common carp. To our knowledge, this is the first study investigating the detailed process of early gonadal sex differentiation of common carp by combining the expression patterns of sex related genes with gonadal histological changes. We have identified the critical window of sex determination and the initiation times of oogenesis and spermatogenesis in the Yellow River carp. These results provide researchers with valuable information to possibly reveal the mechanisms of sex determination and differentiation of common carp.