Stages Of Sexual Differentiation Research Articles

Estrogen receptor Esr1 induces early ovarian differentiation in Trachemys scripta

This study aims to explore the roles of three estrogen receptors (Esr1, Esr2, and Gper1) in early differentiation of embryonic gonads of Trachemys scripta. The expression characteristics of the receptor genes were studied first. The Esr1, Esr2, and Gper1 agonists PPT, WAY 200070, and G-1 were respectively injected into the embryos at the male-producing temperature (MPT) before initiation of gonadal differentiation. The sex reversal of the treated embryonic gonads was analyzed in terms of morphological structure of gonads, distribution pattern of germ cells, and expression of key genes and proteins involved in sex differentiation. The expression level of esr1 during the critical stage of sex differentiation was higher than those of esr2 and gper1 (very low expression) and was particularly high in the gonads at the female-producing temperature (FPT). After treatment with PPT, the MPT gonads presented obviously feminized morphology and structure, with the germ cells exhibiting a female distribution pattern. Furthermore, the mRNA expression levels of the key genes (dmrt1, amh, and sox9) for male differentiation were down-regulated significantly, while those of the key genes (foxl2 and cyp19a1) for female differentiation were up-regulated observably. The fluorescent signals of Amh and Sox9 expression almost disappeared, while Foxl2 and Arom were activated to express abundantly, which fully demonstrated the sex reversal of the gonads from male to female (sex reversal rate: 70.27%). However, the MPT gonads treated with WAY 200070 and G-1 still differentiated into testes, and the expression patterns of the key genes and proteins were similar to those in male gonads. The above results demonstrate that activation of Esr1 alone can fully initiate the early female differentiation process of gonads, suggesting that estrogen may induce early ovarian differentiation via Esr1 in Trachemys scripta. The findings provide a basis for further revealing the mechanisms of estrogen regulation in sex determination and differentiation of turtles.

The potential regulatory role of the non-coding RNAs in regulating the exogenous estrogen-induced feminization in Takifugu rubripes gonad

Estrogen plays a pivotal role in the early stage of sex differentiation in teleost. However, the underlying mechanisms of estrogen-induced feminization process are still needed for further clarification. Here, the comparative analysis of whole-transcriptome RNA sequencing was conducted between 17beta-Estradiol induced feminized XY (E-XY) gonads and control gonads (C) in Takifugu rubripes. A total of 57 miRNAs, 65 lncRNAs, and 4 circRNAs were found to be expressed at lower levels in control-XY (C-XY) than that in control-XX (C-XX), and were up-regulated in XY during E2-induced feminization process. The expression levels of 24 miRNAs, and 55 lncRNAs were higher in C-XY than that in C-XX, and were down-regulated in E2-treated XY. Furthermore, a correlation analysis was performed between miRNA-seq and mRNA-seq data. In C-XX/C-XY, 114 differential expression (DE) miRNAs were predicted to target to 904 differential expression genes (DEGs), while in C-XY/E-XY, 226 DEmiRNAs were predicted to target to 2,048 DEGs. In C-XX/C-XY, and C-XY/E-XY, KEGG pathway enrichment analysis showed that those targeted genes were mainly enriched in MAPK signaling, calcium signaling, steroid hormone biosynthesis and ovarian steroidogenesis pathway. Additionally, the competitive endogenous RNA (ceRNA) regulatory network was constructed by 24 miRNAs, 21 lncRNAs, 4 circRNAs and 5 key sex-related genes. These findings suggested that the expression of critical genes in sex differentiation were altered in E2-treated XY T. rubripes may via the lncRNA-miRNA-mRNA regulation network to facilitate the differentiation and maintenance of ovaries. Our results provide a new insight into the comprehensive understanding of the effects of estrogen signaling pathways on sex differentiation in teleost gonads.

In vivo effect of recombinant Fsh and Lh administered to meagre (Argyrosomus regius) at the initial stages of sex differentiation

Recombinant gonadotropins, follicle stimulating (rFsh) and luteinizing hormone (rLh), offer the potential to induce gametogenesis in prepubertal fish. This study aimed to determine the in vivo effect of the administration of Argyrosomus regius rFsh and rLh on the reproductive development of prepubertal meagre juveniles at the initial stages of sexual differentiation. Juvenile meagre, 9-months old with mean weight of 219 ± 3.9 g (mean ± SEM) were randomly distributed into nine groups (n = 8 per group). Experimental groups were treated weekly with an acute injection of either rFsh or rLh. Control groups were injected with saline solution. In a 3-week experiment, different groups were administered with different doses 6, 12 or 18 µg kg−1 of rFsh or rLh or saline solution. In a 6-week experiment a group was administered with 12 µg kg−1 of rFsh and a second group with saline solution. The fish were held in a single 10 m3 tank with natural photoperiod (Feb. – March) and temperature 16.1 ± 0.4 °C. At the start of the experiment (n = 8) and at the end of the 3-week experiment, fish were blood sampled and sacrificed. Blood was analysed for 17β-estradiol (E2) and 11-ketotestosterone (11-KT). Gonads and liver were dissected and weighed. Gonads were fixed in Bouińs solution and processed for histological analysis. Juvenile meagre at the start of the experiment were in the initial stages of sexual differentiation, indicated by the presence of the ovarian cavity or testes duct that was surrounded by undifferentiated embryonic germ stem cells and somatic cells. At the end of the 3-week experiment, there was no significant difference in gonadosomatic index (GSI) amongst control (initial and saline treated) and the experimental groups. After three weeks of application of rFsh, rLh or saline all fish presented a similar gonadal structure as at the start of the experiment. However, the incidence of sporadic developing germ cells (principally spermatogonia, spermatocytes, spermatids, but also perinucleolar stage oocytes) generally increased in rGth treated meagre. A mean of 44 % of meagre treated with rFsh or rLh presented sporadic isolated developing germ cells, mainly male cells. Plasma steroid levels of E2 decreased significantly from the start of the experiments to the end. At the end of the experiments there were no differences in plasma E2 amongst Control fish and rGth treated fish. Plasma 11-KT showed no change from the start of the experiment to week 3. However, a significant increase was observed in a proportion of the rFsh group after six weeks of treatment compared to the start of the experiment and the saline control group on week 6. The application of rFsh or rLh to meagre at the initial stages of sex differentiation did not stimulate steroid production until week six (11-KT) and had a limited, but evident effect on the development of sporadic isolated germ cells. However, we conclude that rGth, rFsh or rLh did not stimulate large developmental changes in sexually undifferentiated meagre gonads.

Microcystin-LR immersion caused sequential endocrine disruption and growth inhibition in zebrafish (Danio rerio) from fertilization to sexual differentiation completion

Microcystin-LR (MC-LR) is a highly toxic congener and is also one of the most commonly found. Recent studies have demonstrated that MC-LR can disrupt growth and endocrine in fish, but how it works at the stage of the sex differentiation period had not been determined to date. In this study, zebrafish (Danio rerio) embryos were exposed to MC-LR (0 and 10 μg/L), and sampled at 14, 28, and 42 days post fertilization (dpf), respectively. The results demonstrated that MC-LR caused the growth inhibition of zebrafish at 42 dpf. The expression levels of genes related to the growth hormone/insulin-like growth factor (GH/IGF) and hypothalamic-pituitary-thyroid (HPT) axes, as well as the levels of hormone 3,5,3′- triiodothyronine (T3) and thyroxine (T4), were significantly decreased at all time points. A significant decrease in the ratio of testosterone and estradiol (T/E2) were detected at 28 and 42 dpf in MC-LR group along with changes in genes related to the hypothalamic-pituitary–gonadal (HPG) axis. The result of sex ratio showed that the percentage of females was up to 61.84%, indicating a estrogenic effect induced by MC-LR. The significant changes on hormone levels and gene transcripts occurred mainly in the stage of sex differentiation. The correlation analysis further suggested that key cross-talks among three endocrine axes may be the growth hormone releasing hormone (GHRH), transthyretin (TTR) and gonadotropin releasing hormone (GnRH) signaling molecules. Overall, our findings provide a new insight for understanding the mechanisms by which MC-LR affects fish growth and reproduction during gonadal development.

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.

Transcriptome profiling of the gonad during sex differentiation in hermaphroditic brown-marbled grouper (Epinephelus fuscoguttatus)

Sex differentiation is an intricate process regulated by a complicatedly organized network especially in hermaphroditic fish. The brown-marbled grouper (Epinephelus fuscoguttatus) is a protogynous hermaphroditic fish, however, the regulatory mechanism of its sex differentiation is still unclear. In present study, transcriptome sequencing of the gonads at 117 dph and 259 dph was performed to explore the molecular mechanism underlying sex differentiation. A total of 37,434,906,973 clean reads were generated from six cDNA libraries. QPCR results of 12 genes related to sex differentiation indicated the reliability of transcriptome data. RNA-seq data showed that the genes in retinoic acid signal pathway participate in the early stage of sex differentiation. When the oocyte was observed in histology, the germ cell had finished the preparation of first meiosis with high expression of many meiosis-related genes, including sycp1, sycp3, mlh3, crem, spo11, msh5, and rec8. The germ cells in 259 dph gonad were classified into six stages by their histological morphology. Besides, male-related genes, gsdf, sox9, dmrt1, and dmrt3, might play roles in sex differentiation. Our study, for the first time, investigated the transcriptome from brown-marbled grouper, and identified functional genes, GO terms, and KEGG pathways involved in sex differentiation. These data will contribute to future studies on the molecular mechanism of sex differentiation in teleost.

The role of phytohormones and their related miRNAs in sex differentiation of Xanthoceras sorbifolium Bunge

• Determined the influence of IAA, GA, ABA, ZT on sex differentiation of Xanthoceras sorbifolium . • Identified 20 significantly differently expressed miRNAs (83 miRNA/target pairs) being related to phytohormone synthesis and signal transduction during sex differentiation. Xanthoceras sorbifolium Bunge is an important woody oilseed tree in north China. However, its yield is severely restricted by a low ratio of female to male flowers, and little is known about the physiological and molecular mechanisms underlying the sex differentiation of flowers. Therefore, carrying out the research in its sex differentiation regulation mechanism is the basis of increasing seed yield. Here, we investigated the cytology of sex differentiation, the content and their ratios of indole-3-acetic acid (IAA), gibberellins (GA 3 ), zeatin (ZT), abscisic acid (ABA), the effects of plant growth regulators on flower development, and the expression profiles of phytohormone-related miRNAs. The results showed that both functional female and male flowers were bisexual at the early stage of flower bud differentiation, while the development of the pistils of male flowers and the stamens of female flowers was arrested at later stage of sex differentiation. Furthermore, IAA, ABA and ZT were shown to be beneficial to pistil development in X. sorbifolium . GA 3 was the key phytohormone in pistil abortion. High level of ABA/ZT was beneficial for microspore mother cell meiosis. ZT/IAA was probably related to pistil abortion. High level of ZT/GA 3 contributed to ovule development. Spraying 6-BA was more effective than GA 3 in increasing female-to-male ratio regardless of CK. In addition, several miRNAs played key roles in sex differentiation, especially miR393, miR396, and miR399. They participated in the regulation of pollen maturation and multiple phytohormone pathways. The present study comprehensively identified the roles of phytohormones and their related miRNAs during sex differentiation in X. sorbifolium , which will be a valuable resource for sex regulation using genetic and physiological methods in the future.

Comparative Transcriptome Analysis Reveals Sex-Biased Expression of Hormone-Related Genes at an Early Stage of Sex Differentiation in Red Bayberry (Morella rubra)

The molecular mechanism of sex development and differentiation in the economically important dioecious fruit tree, red bayberry (Morella rubra), was revealed using next-generation transcriptome sequencing (NGS), and comparative analyses were used to identify differentially expressed genes (DEGs) in female and male flower buds. A total of 7029 of these DEGs were identified at two early development stages. KEGG pathway enrichment analysis revealed that plant hormone signal transduction was significantly overrepresented, and 91 genes related to hormones were identified. An analysis of 7029 DEGs revealed 161 hormone-related genes, with the 42 related to auxin and 26 related to ethylene being the most highly represented. A total of 62 genes were significantly up-regulated in females and 29 were in males, with 18 of them specifically expressed in females and 10 in males. A total of 415 transcription factors were identified, with 129 genes up-regulated in females and 53 in males. Moreover, 38 had female-specific expression and 18 had male-specific expression. Using weighted gene co-expression network analysis (WGCNA), two modules were found to be associated with sexual type. In the module coded light-green, there were five genes related to hormones, one to flower development and ten transcription factors with four genes specifically expressed in the males and four in females. The hub gene in the light-green module is MR0TCONS_00017483.1 (ACO), which is involved in ethylene biosynthesis and had male-specific expression. Among the transcription factors, three of the four male-specific expressed genes involved in flavonoid biosynthesis, including the MYB gene MR1TCONS_00020658.1 and two BHLH genes, MR6G001563.1 and MR8G020751.1, played important roles in male floral differentiation. In the dark-cyan module, six hormone-related genes, five transcription factors and three flower development genes were identified with the hub gene MR1G019545.1 (ETR1), which participates in the ethylene signaling pathway, and MR4G023618.1, which encodes the C3H zinc finger transcription factor. These results indicate that ethylene is the key hormone that interacts with other hormones and transcription factors to regulate sex differentiation in the red bayberry, which also provides new insights into the mechanism of sex determination and differentiation in the red bayberry.

Sex biased expression of hormone related genes at early stage of sex differentiation in papaya flowers

Sex types of papaya are controlled by a pair of nascent sex chromosomes, but molecular genetic mechanisms of sex determination and sex differentiation in papaya are still unclear. We performed comparative analysis of transcriptomic profiles of male and female floral buds at the early development stage before the initiation of reproductive organ primordia at which there is no morphological difference between male and female flowers. A total of 1734 differentially expressed genes (DEGs) were identified, of which 923 showed female-biased expression and 811 showed male-biased expression. Functional annotation revealed that genes related to plant hormone biosynthesis and signaling pathways, especially in abscisic acid and auxin pathways, were overrepresented in the DEGs. Transcription factor binding motifs, such as MYB2, GAMYB, and AP2/EREBP, were enriched in the promoters of the hormone-related DEGs, and transcription factors with those motifs also exhibited differential expression between sex types. Among these DEGs, we also identified 11 genes in the non-recombining region of the papaya sex chromosomes and 9 genes involved in stamen and carpel development. Our results suggested that sex differentiation in papaya may be regulated by multiple layers of regulation and coordination and involved transcriptional, epigenetic, and phytohormone regulation. Hormones, especially ABA and auxin, transcription factors, and genes in the non-recombination region of the sex chromosome could be involved in this process. Our findings may facilitate the elucidation of signal transduction and gene interaction in sex differentiation of unisexual flowers in papaya.

Systematic identification and expression analysis of the Sox gene family in spotted sea bass (Lateolabrax maculatus)

The Sox gene family encodes a set of transcription factors characterized by a conserved Sry-related high mobility group (HMG)-box domain, which performs a series of essential biological functions in diverse tissues and developmental processes. In this study, the Sox gene family was systematically characterized in spotted sea bass (Lateolabrax maculatus). A total of 26 Sox genes were identified and classified into eight subfamilies, namely, SoxB1, SoxB2, SoxC, SoxD, SoxE, SoxF, SoxH and SoxK. The phylogenetic relationship, exon-intron and domain structure analyses supported their annotation and classification. Comparison of gene copy numbers and chromosome locations among different species indicated that except tandem duplicated paralogs of Sox17/Sox32, duplicated Sox genes in spotted sea bass were generated from teleost-specific whole genome duplication during evolution. In addition, qRT-PCR was performed to detect the expression profiles of Sox genes during development and adulthood. The results showed that the expression of 16 out of 26 Sox genes was induced dramatically at different starting points after the multicellular stage, which is consistent with embryogenesis. At the early stage of sex differentiation, 9 Sox genes exhibited sexually dimorphic expression patterns, among which Sox3, Sox19 and Sox6b showed the most significant ovary-biased expression. Moreover, the distinct expression pattern of Sox genes was observed in different adult tissues. Our results provide a fundamental resource for further investigating the functions of Sox genes in embryonic processes, sex determination and differentiation as well as controlling the homeostasis of adult tissues in spotted sea bass.

Transcriptome Profiling and Analysis of Genes Associated with High Temperature-Induced Masculinization in Sex-Undifferentiated Nile Tilapia Gonad.

Artificially high temperatures during critical thermosensitive periods (TSPs) can induce the sex reversal of Nile tilapia (Oreochromis niloticus) females into pseudomales; Nile tilapia is a GSD + TE (genotypic plus temperature effects) fish species. Previous studies have shown that water temperature affects the expression levels of many genes in the gonad or brain in various teleost species. However, few studies on the effect of temperature at the whole-gonad transcriptomic level in the early stage of sex differentiation have been reported in fish species exhibiting GSD + TE. In this study, RNA-Seq was performed to characterize the transcriptomic profile and identify genes exhibiting temperature- and sex-biased expressions in the Nile tilapia gonad at 21dpf. A total of 42 genes were found to be associated with both high-temperature treatment and sex development, as the expression levels of these genes differed in both FC (female control) vs MC (male control) and FC vs FT (high temperature-treated females in the TSP). Among these genes, the transcriptional alterations of many male sex determination and differentiation genes, such as Dmrt1, Gsdf, and the DNA damage-inducible protein GADD45 alpha, suggested that the male pathway is initiated after high-temperature treatment and that its initiation may play a role in high temperature-induced masculinization in Nile tilapia. The qRT-PCR validation results for thirteen differentially expressed genes showed that the Pearson's correlation of the log10 fold change values between the qPCR and RNA-Seq results was 0.70 (p < 0.001), indicating the accuracy and reliability of the RNA-Seq results. Our study provides insights into how high-temperature treatment induces the sex reversal of Nile tilapia females.

Transcriptomic analyses reveal novel genes with sexually dimorphic expression in Takifugu rubripes brain during gonadal sex differentiation.

Quantification of mRNAs in gonads and other tissues at the early critical development stage of sex differentiation may help to provide a global view of regulatory mechanisms underlying sex differentiation. We have recently reported the transcriptomic profiling of fugu gonad associated with sex differentiation. This study attempted to identify the genes in the brain that are involved in gonadal differentiation and development. In this study, a transcriptomic scan of potential candidate genes involved in sex differentiation was conducted in the brains of fugu larvae at 30 and 40 dah (morphological gonadal sex differentiation had not yet occurred). The dimorphic expression patterns of several candidate genes were verified using quantitative PCR. A total of 28.24Gb of clean reads were obtained and 22,337 genes were identified in the brains of fugu larvae. These included 1008 novel genes that provide abundant data for functional analysis of sex differentiation. 229 genes were identified in the 30 dah larvae that were abundant in the XY brain and 21 that were abundant in the XX brain. In the 40 dah larvae, 325 genes were identified abundant in the XY brain and 174 were identified abundant in the XX brain. This is the first investigation into the transcriptome of the fugu larvae brain at the early sex differentiation stage. The results obtained here will enhance the understanding of molecular mechanisms that underly fugu sex 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.

Corrigendum] Alterations in gene expression during sexual differentiation in androgen receptor knockout mice induced by environmental endocrine disruptors

Following the publication of the above article, an interested reader drew to our attention a number of issues and concerns related to the descriptions of the genes and mouse models in the above paper. After having consulted with the authors, they have acknowledged that their paper did indeed contain a number of errors, and these are listed below in this Corrigendum. First, the authors realized that an incorrect figure was included as Fig.1 in the paper. The correct (and entirely different) version of Fig.1, together with its figure legend, is shown opposite. The following textual errors in the paper should also be noted: i) In the Abstract, lines3‑4 in the left‑hand column (LHC): "androgen receptor (AR)‑/‑, AR+/‑ and AR+/+ male mice" should have been written as "androgen receptor (AR) conditional knockout mice"; line5 in the LHC: "flox‑AR" should have been written as "ARflox/+"; line6 in the LHC: "AR‑Cre" should have been written as "Amhr2‑Cre"; line22 in the LHC: "AR+/‑" should have been written as "ARflox/Y"; line1 in the right‑hand column (RHC): "heterozygous" should have been written as "ARflox/Y"; and line2 in the RHC, "heterozygous" should have been written as "ARflox/Y Cre+". ii) In the Introduction, paragraph 3, lines6‑7 in the LHC: "or female ARKO mice" should have been deleted; paragraph4, line5 in the LHC: "AR‑/‑", "AR+/‑" and "AR+/+" should have been deleted; paragraph4, line11 in the LHC: "AR‑/‑", and "AR+/‑" should have been written as "AR‑/Y", whereas "AR+/+" should have been written as "AR+/Y". iii) In the Materials and methods, paragraph3, the last sentence in the RHC {"The identification of the plasmid containing leydig cells expressing Cre recombinant enzyme and the specificity of the genetically modified (gm) sequence [Amhr2‑Cre (7.1)] are shown in Fig.1} should have been written as "The positive clones were identified by digestion and sequencing." iv) In the Results, the first subheading in the RHC ("...transgenic mouse model (Amhr2‑Cre)" should have been written as "...AR conditional knockout targeting vector"; paragraph1, lines1‑3 in the RHC: The sentence "The AR gene condition knockout plasmid was identified and confirmed by enzyme digestion with BamHI" should have been written as "The AR gene conditional knockout strategy is shown in Fig.1. The gene targeting vector was identified and confirmed by enzyme digestion with BamHI"; paragraph1, line4 in the RHC: "2.7kb" should have been written as "7.8 kb"; Table III, in the title and in the Table heading, "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y"; in the subheading "Comparison between AR‑/‑ male mice and normal mice", "AR‑/‑" should have been written as "AR‑/Y", and "normal" should have been written as "AR+/Y"; and paragraph4, lines 3‑5 in the LHC: "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y". All the authors thank the interested reader for drawing these matters to their attention, and agree to the contents of this Corrigendum. The authors also stress that these errors did not significantly influence either the results or the conclusions of the paper. Furthermore, the authors apologize to the Editor of International Journal of Molecular Medicine and to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 35: 399‑404, 2015; DOI: 10.3892/ijmm.2014.2015].

Sex Determination During Inflorescence Bud Differentiation in Monoecious Pistacia chinensis Bunge

Pistacia chinensis Bunge is widely acknowledged to be dioecious, but rare monoecious individuals have been found. However, the origin of monoecism and the sex differentiation of different sex types remain intriguing questions. Here, sex expressions were explored by identification of sex-associated DNA markers, determination of the sex stability after grafting, and histological characterization of inflorescence bud development using anatomical analysis. The results showed that (1) although polymorphisms among individuals existed, the banding patterns of Polymerase Chain Reaction (PCR) products for different sex types on the same monoecious tree were consistent; (2) the sex expressions of grafted trees were not consistent with those of scions, indicating that monoecism probably did not originate from a stable bud mutation; and (3) both males and females underwent a bisexual period, then the stamen primordia in female buds degenerated into the second round tepals, while the pistil primordia in male buds gradually disappeared. During the sex differentiation phase, female buds were spindle-shaped, while the male buds were full teardrop-shaped, and male buds were bigger than female buds. Taken together, no sex-associated DNA marker was found, sex expressions were unstable after grafting, and the alternative sex organs appeared in the early stage of sex differentiation, suggesting that sex determination occurred during floral development instead of the early vegetative period. These results indicated that the sex expressions may be affected by environmental factors, increasing the understanding of sex determination mechanisms in P. chinensis and other species.

Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes gonad at the early stage of sex differentiation.

Quantifying the expression of mRNAs in the gonads at the critical stage of molecular sex differentiation stage might help to clarify the regulatory network during early sex differentiation and provide new information on the role of sex-related genes in gonadal function. In this study, transcriptomic analysis of sex-related genes expression profiles in fugu gonads at 60 and 90days after hatching (dah) was conducted firstly, and a total of 112,504,991 clean reads, encompassing 28.35Gb of sequences were retrieved. Twenty-three thousand eight hundred ten genes were found to be expressed in juvenile fugu gonads, and we mainly focused on the differentially expressed genes that have the potential to be involved in the gonadal sex differentiation. For 60-dah juveniles, we identified 1014 genes that were upregulated in the ovary and 1570 that were upregulated in the testis. For 90-dah juveniles, we identified 1287 genes that were upregulated in the ovary and 1500 that were upregulated in the testis. The dimorphic expression patterns of 15 genes in gonads at 30 and 40dah were further investigate using qPCR. Cyp11b and star were expressed at higher levels in XY than in XX, while cyp11a1 and cyp19a1a were expressed at higher levels in XX than in XY at 30dah. At 40dah, the levels of gsdf, dmrt1, dmrt3, cyp11c1, star, and hsd3b expression were higher in XY, while the levels of foxl2, cyp19a1a, wnt9b, and foxD4 expression were higher in XX. Sox9, cyp11a1, cyp17a1, cyp17a2, and nr5a2 were expressed at similar levels in XX and XY at 40dah. This is the first report of gonadal transcriptome of fugu at early sex differentiation stage, and our results provide an archive for further study on molecular mechanism underlying sex differentiation in this species.

Effects of exposure to BPF on development and sexual differentiation during early life stages of zebrafish (Danio rerio)

Bisphenol F (BPF) has become a predominant bisphenol contaminant in recent years. It has significant estrogenic properties in both in vivo and in vitro studies. We have previously studied the disrupting mechanisms of BPF on the hypothalamic–pituitary–gonadal axis of adult zebrafish. However, the effects of BPF exposure on development and sexual differentiation of zebrafish embryos/larvae remain unclear. To determine the effects of BPF on the critical stage of sex differentiation in zebrafish, zebrafish embryos/larvae were exposed to 1, 10, 100, and 1000 μg/L BPF from fertilization to 60 days post-fertilization (dpf). Developmental malformations were induced by exposure to BPF from 2 h post-fertilization (hpf), with a LC50 of 10,030 μg/L at 96 hpf and 9391 μg/L at 120 hpf. Long-term exposure during sex differentiation tended to result in a female sex ratio bias. Histological analyses at 60 dpf indicated that the development of ovo-testes and immature ovaries was induced by 100 and 1000 μg/L BPF. Homogenate testosterone levels decreased and 17β-estradiol levels increased in zebrafish in a concentration-dependent manner. BPF exposure suppressed gene expression of double sex, Mab3-related transcription factor 1(dmrt1), fushi tarazu factor 1d (ff1d), sry-box containing gene 9a (sox9a) and anti-Mullerian hormone (amh); induced expression of the forkhead box L2 transcription factor (foxl2), leading to increased expression of aromatase (cyp19a1a), which promoted production of estrogens, and further caused phenotypic feminization of zebrafish. These results suggest that developmental exposure to BPF has adverse effects on sexual differentiation, and the results were useful for a BPF risk assessment.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish

Receptor, c-Kit is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analyzed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50–75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of c-kit was analyzed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression during the pre-spawning phase in male compared to females. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression levels of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes that are involved in germ cell development. Decrease in the levels of 11-ketotestosterone and testosterone in serum were also observed after esiRNA silencing. The findings suggests that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

BackgroundMicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia.ResultsWe identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish.ConclusionsThe sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2636-z) contains supplementary material, which is available to authorized users.

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.