Role In Ovarian Differentiation Research Articles

Cloning, identification, and functional analysis of foxl2 gene and its expression after 17β‐estradiol (E2) treatment in Dabry’s sturgeon, Acipenser dabryanus

The study focuses on the critically endangered Dabry’s sturgeon (Acipenser dabryanus), a species on the brink of extinction in the wild. This research investigates the role of the Forkhead box protein L2 (foxl2) in the gonadal development and differentiation of this species. The foxl2 gene, known for its role in various physiological processes, including sexual maturation, is hypothesized to play a significant role in the sex differentiation of Dabry’s sturgeon. This study cloned the full-length cds sequence of the foxl2 gene and analyzed its expression across various tissues, focusing on its response to estradiol treatment. Our findings indicate that foxl2 is predominantly expressed in ovaries and shows a dose-dependent response to estradiol, suggesting its potential role in ovarian differentiation. This research underscores the importance of foxl2 in understanding reproductive biology and offers a foundation for future conservation strategies.

First Data on Sexual Dimorphic Growth of Cultured Pacific Bluefin Tuna, Thunnus orientalis (Temminck et Schlegel), and Its Sex Manipulation by Treatment with an Aromatase Inhibitor

Tuna (genus Thunnus), particularly Pacific bluefin tuna (T. orientalis; PBT), are commercially important fish in the aquaculture industry worldwide. The objective of this study was to investigate sexual dimorphism in the growth performance of aquaculture-produced PBT and develop techniques for its sex manipulation, for the first time in tuna. A comparison of the body size between sexes revealed that male-cultured PBTs were larger than females at harvest. We also confirmed that cyp19a1a (encoding a gonadal aromatase) expression increased specifically in the genotypic female PBT gonads during sex differentiation. This suggested that aromatase plays an important role in ovarian differentiation and that the suppression of aromatase activity may effectively induce masculinization in genotypic females. Therefore, we administered letrozole—an aromatase inhibitor (AI)—into sexually undifferentiated PBT through the oral route. AI administration resulted in a 100% sex reversal of genotypic females into phenotypic males at the molecular level. Our results provide the basis for future studies on the establishment of mono-sex male production technology in PBT, which would help improve the productivity of closed-cycle PBT aquaculture. Furthermore, this study offers important insights into the understanding of the sex-wise growth of tuna species in aquacultural settings, and developing sex manipulation techniques.

Pengaruh Suhu Inkubasi Terhadap Profil Ekspresi Gen Determinasi Seks Tukik Lepidochelys olivacea

Sex determination in turtles is not only based on genotype, but also depends on the incubation temperature. In addition, sex differentiation takes place during the thermosensitive period (TSP). This review article aims to examine the effect of incubation temperature on the expression profile of the sex determination gene for tortoises. Fertile eggs are incubated at two temperatures, namely the feminine temperature (30-33 ° C), and the masculine temperature (26-27 ° C). The results of bioreproduction and phenotype measurements of hatchlings, namely, embryo growth, incubation time, morphometrics, and locomotor performance of hatchlings were analyzed by regression analysis and Student's t test. The results of analysis from several literatures showed that the expression of aromatase and Rspond 1 genes (which play a role in ovarian differentiation) after TSP from incubated embryos at a higher feminine temperature and different from the masculine temperature. Regarding bioreproduction, the incubation period at the feminine temperature is shorter than the masculine temperature. Likewise embryos grow more rapidly at feminine temperatures. Incubation at feminine temperature had a significant effect on the phenotype of hatchlings, namely carapace width, plastron length and width, long flipper and hind arms, long neck, and flipper swing frequency.

Phylogenomic and expression analysis of Colossoma macropomum cyp19a1a and cyp19a1b and their non-classical role in tambaqui sex differentiation

The genes coding for Cytochrome P450 aromatase (cyp19a1a and cyp19a1b) and estrogen (E2) receptors (esr1, esr2a and esr2b) play a conserved role in ovarian differentiation and development among teleosts. Classically, the “gonad form” of aromatase, coded by the cyp19a1a, is responsible for the ovarian differentiation in genetic females via ligation and activation of the Esr, which mediates the endocrine and exocrine signaling to allow or block the establishment of the feminine phenotype. However, in neotropical species, studies on the molecular and endocrine processes involved in gonad differentiation as well as on the effects of sex modulators are recent and scarce. In this study, we combined in silico analysis, real-time quantitative PCR (qPCR) assay and quantification of E2 plasma levels of differentiating tambaqui (Colossoma macropomum) to unveil the roles of the paralogs cypa19a1a and cyp19a1b during sex differentiation. Although the synteny of each gene is very conserved among characids, the genomic environment displays striking differences in comparison to model teleost species, with many rearrangements in cyp19a1a and cyp19a1b adjacencies and transposable element traces in both regulatory regions. The high dissimilarity (DI) of SF-1 binding motifs in cyp19a1a (DI = 10.06 to 14.90 %) and cyp19a1b (DI = 8.41 to 13.50 %) regulatory region, respectively, may reflect in an alternative pathway in tambaqui. Indeed, while low transcription of cyp19a1a was detected prior to sex differentiation, the expression of cyp19a1b and esr2a presented a large variation at this phase, which could be associated with sex-specific differential expression. Histological analysis revealed that anti-estradiol treatments did not affect gonadal sex ratios, although Fadrozole (50 mg kg−1 of food) reduced E2 plasma levels (p < 0,005) as well cyp19a1a transcription; and tamoxifen (200 mg kg−1 of food) down regulated both cyp19a1a and cyp19a1b but did not influence E2 levels. Altogether, our results bring into light new insights about the evolutionary fate of cyp19a1 paralogs in neotropical fish, which may have generated uncommon roles for the gonadal and brain forms of cyp19a1 genes and the unexpected lack of effect of endocrine disruptors on tambaqui sexual differentiation.

Prognostic Value of the Grade and Extent of Coronary Microvascular Dysfunction in Takotsubo Syndrome

The genes coding for Cytochrome P450 aromatase (cyp19a1a and cyp19a1b) and estrogen (E2) receptors (esr1, esr2a and esr2b) play a conserved role in ovarian differentiation and development among teleosts. Classically, the “gonad form” of aromatase, coded by the cyp19a1a, is responsible for the ovarian differentiation in genetic females via ligation and activation of the Esr, which mediates the endocrine and exocrine signaling to allow or block the establishment of the feminine phenotype. However, in neotropical species, studies on the molecular and endocrine processes involved in gonad differentiation as well as on the effects of sex modulators are recent and scarce. In this study, we combined in silico analysis, real-time quantitative PCR (qPCR) assay and quantification of E2 plasma levels of differentiating tambaqui (Colossoma macropomum) to unveil the roles of the paralogs cypa19a1a and cyp19a1b during sex differentiation. Although the synteny of each gene is very conserved among characids, the genomic environment displays striking differences in comparison to model teleost species, with many rearrangements in cyp19a1a and cyp19a1b adjacencies and transposable element traces in both regulatory regions. The high dissimilarity (DI) of SF-1 binding motifs in cyp19a1a (DI = 10.06 to 14.90 %) and cyp19a1b (DI = 8.41 to 13.50 %) regulatory region, respectively, may reflect in an alternative pathway in tambaqui. Indeed, while low transcription of cyp19a1a was detected prior to sex differentiation, the expression of cyp19a1b and esr2a presented a large variation at this phase, which could be associated with sex-specific differential expression. Histological analysis revealed that anti-estradiol treatments did not affect gonadal sex ratios, although Fadrozole (50 mg kg−1 of food) reduced E2 plasma levels (p < 0,005) as well cyp19a1a transcription; and tamoxifen (200 mg kg−1 of food) down regulated both cyp19a1a and cyp19a1b but did not influence E2 levels. Altogether, our results bring into light new insights about the evolutionary fate of cyp19a1 paralogs in neotropical fish, which may have generated uncommon roles for the gonadal and brain forms of cyp19a1 genes and the unexpected lack of effect of endocrine disruptors on tambaqui sexual differentiation.

Fadrozole-Mediated Sex Reversal Induces PAX2+Undifferentiated Supporting Cells in Female Chicken Gonads

During early embryogenesis, the undifferentiated gonad is bipotential and subsequently commits to an ovarian or testicular fate. In birds, double dose of the Z-linked gene DMRT1 is required for testicular differentiation in male embryos (genetically ZZ). In female birds, estrogen plays a key role in ovarian differentiation. 17β-estradiol (E2) induces gonadal feminization when applied to male embryos (ZZ). Conversely, inhibition of estrogen synthesis with the drug fadrozole (FAD) results in testicular development in genetically female embryos (ZW). However, activation of male markers in sex-reversed ZW embryos is typically delayed, raising the possibility that FAD-treated embryos may transition through an undifferentiated state before masculinization. Recently, PAX2 was identified as a marker of undifferentiated supporting cells in the chicken embryo, being downregulated in both sexes at the onset of gonadal sex determination. To investigate the supporting cell differentiation process in estrogen-mediated sex reversal, we injected 1 mg of fadrozole in 100µl of PBS or vehicle into embryonic day 3.5 (E3.5) chicken eggs. Eggs were incubated until E9.5, genotypically sexed (ZZ or ZW) and processed for qRT-PCR and immunofluorescence. Quantitative RT-PCR confirmed that sex reversal had occurred in FAD-treated females, showing a reduction of pre-granulosa cell markers aromatase (P<0.005) and FOXL2 (P<0.05), compared to the control. Interestingly, PAX2 mRNA expression was up-regulated (P<0.05) in sex-reversed females, suggesting an increase in undifferentiated supporting cells (n=6). To confirm this observation, immunofluorescence was used to detect aromatase, SOX9 (male marker) and PAX2. In FAD-treated females, both SOX9+ (male) and aromatase+ (female) cells co-existed in the same gonad, but in separated defined regions. Aromatase positive cells were located in the most apical region of the gonad whereas SOX9 positive cells were detected in the basal region. We detected an increase in PAX2 positive cells in the gonadal medulla between the SOX9 and aromatase positive supporting cells. No SOX9 or PAX2 positive cells were detected in control female gonads (n=3). For feminization experiments 100µl of a 1mg/ml solution of E2 or vehicle (Oil) was injected into E3.5 chicken eggs. No significant increase in PAX2 was detected by qRT-PCR (p>0.05, n=6) and no PAX2 positive cells were detected in E2 treated gonads at E9.5. These results suggest that in fadrozole-mediated masculinization (but not in estrogen-induced feminization) there is an increase in undifferentiated supporting cells. The absence of both estrogens (feminizing) and elevated DMRT1 (masculinizing) could explain why the supporting cells remain in an undifferentiated state in ZW (genetically female) embryos. Further research is required to evaluate the fate of these undifferentiated cells in gonadal sex differentiation.

Common carp pentraxin gene: Evidence for its role in ovarian differentiation and growth

Pentraxins (PTX), belong to an evolutionarily conserved family, containing a PTX protein domain, having role in acute immunological responses and fertility in higher vertebrates. However, information regarding the action of ptx on reproduction is extremely limited in fish. To study this, ptx cDNA was cloned for downstream analysis. Tissue distribution and ontogeny expression analysis indicated the prevalence of ptx in ovary. Varied phase-wise expression during carp ovarian cycle and elevated ptx expression after human chorionic gonadotropin induction, in vitro and in vivo, indicated probable regulation of gonadotropin. In situ hybridization and immunohistochemistry revealed the presence of ptx transcript and protein in the follicular layer of stage-III/IV oocytes indicating a role in ovarian growth. To assess the functional significance of ptx, transient silencing was performed using follicular primary cell culture, in vitro and in common carp, in vivo, through ovary-targeted injection of PEI-siRNA. Transient silencing of ptx-siRNA reduced the expression of various genes/factors related to oogenesis such as transcription factors, several steroidogenic enzymes, and esrs genes. These alterations in expression suggested a plausible role for ptx in ovarian steroidogenesis either, directly or indirectly, which is evident from the changes in the serum estradiol-17β (E2) and 17α,20β-dihydroxyprogesterone levels. Furthermore, downregulation of aromatase activity was also noticed after transient silencing. Increased ptx expression after E2 induced sex reversal to juvenile carp showed the correlative role of ptx during ovarian differentiation and development. Taken together, these findings suggest that ptx exerts an important role during ovarian growth, maturation and/or recrudescence of common carp.

Characterization and expression profiles of cyp19a1a in the schizothoracine fish Schizothorax prenanti

Aromatase plays a central role in ovarian differentiation and development in teleosts. In the present study, we identified a cyp19a1a homologue from the ovary of Schizothorax prenanti and analysed its expression at both the mRNA and protein levels. Cyp19a1a of S. prenanti showed high homology with that of other teleosts, especially S. kozlovi. The ovary and testis were the main sites of cyp19a1a expression in S. prenanti, and cyp19a1a transcript levels peaked in the mid-vitellogenic (MVG)-stage ovary and the mid-spermatogenic (MS)-stage testis. Signals of Cyp19a1a immunopositivity were detected in the spermatocytes and follicular cells of cortical alveolar-stage and MVG oocytes but not in spermatogonia or spermatids. Taken together, these findings indicate that Cyp19a1a may play an important role in oocyte vitellogenesis as well as spermatocyte development in S. prenanti.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17β-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty

Although estrogens have been generally considered to play a critical role in ovarian differentiation in non-mammalian vertebrates, the specific functions of estrogens during ovarian differentiation remain unclear. We isolated two mutants with premature stops in the ovarian aromatase (cyp19a1) gene from an N-ethyl-N-nitrosourea-based gene-driven mutagenesis library of the medaka, Oryzias latipes. In XX mutants, gonads first differentiated into normal ovaries containing many ovarian follicles that failed to accumulate yolk. Subsequently, ovarian tissues underwent extensive degeneration, followed by the appearance of testicular tissues on the dorsal side of ovaries. In the newly formed testicular tissue, strong expression of gsdf was detected in sox9a2-positive somatic cells surrounding germline stem cells suggesting that gsdf plays an important role in testicular differentiation during estrogen-depleted female-to-male sex reversal. We conclude that endogenous estrogens synthesized after fertilization are not essential for early ovarian differentiation but are critical for the maintenance of adult ovaries.

Functional characterization of &amp;lt;italic&amp;gt;Cyp19a1&amp;lt;/italic&amp;gt; in female sexual differentiation in &amp;lt;italic&amp;gt;Pelodiscus sinensis&amp;lt;/italic&amp;gt;

Aromatase is the key enzyme for estrogen synthesis, and functions as a key regulator in early female sexual differentiation in non-mammalian vertebrates. In this study, the expression pattern of Cyp19a1 was examined in tissues and its functional role in ovarian differentiation was investigated through lentivirus-mediated RNA interference and over-expression systems. Results showed that Cyp19a1 was robustly expressed in adult ovary, and was not or weakly detected in testis, heart, liver, spleen, lung, kidney, muscle and intestine. Moreover, Cyp19a1 exhibited female sexual dimorphic expression pattern in embryonic gonads early at stage 18, preceding the onset time of gonadal differentiation (stage 19). Loss-of-function analysis revealed that ZW embryos with AI treatment or Cyp19a1 knockdown exhibited female to male sex reversal, characterized by obviously masculinized gonads with well-organized testis cord structure in the medulla and a degenerated cortex, an upregulation of testicular marker Sox9 and Amh , and a decline in ovarian marker Foxl2 . Gain-of-function analysis showed that ZZ gonads overexpressing Cyp19a1 were feminized, determined by a thickened outer cortex with a number of germ cells and a vacuolated medulla, as well as upregulation of Foxl2 and downregulation of Sox9 and Amh . In summary, Cyp19a1 is both necessary and sufficient to initiate ovarian development, thereby functioning as a key regulator of female sexual differentiation. This study provides a foundation for understanding the mechanism of sex determination in Pelodiscus sinensis.

Identification and expression analysis of two Wnt4 genes in the spotted scat (Scatophagus argus)

Background: WNT4 is a protein that plays a crucial role in ovarian differentiation and development in mammals, with a relatively well understood function in mammalian gonadal differentiation. The role of WNT4 in teleost fish; however, remains unclear. In the present study, cDNAs of Wnt4a and Wnt4b were cloned and characterized in the spotted scat. The expression patterns of two Wnt4 genes in the gonads at different stages of development and in fish after treatment with 17α-methyltestosterone (MT) were investigated. Results: The tissue distribution showed that Wnt4a was expressed in various tissues, including the gonads, gills, spleen, brain, and fin. Interestingly, Wnt4b not only was expressed in the gills, brain, and spleen, but also was obviously expressed in the ovary. During gonad development, Wnt4a was highly expressed in the testis at stage I and Wnt4b was mainly expressed in the ovary at stages II-III. After MT treatment, the mRNA expression of Wnt4a increased significantly up to 40 d, and the transcript level of Wnt4b decreased at 20 d. Conclusions: These results suggest that Wnt4a may be involved in gonad development and plays a role in the process of spermatogonial proliferation. Our results also demonstrate that Wnt4b is not only expressed in the nervous system, but also in the ovary and it may be involved in ovary development of the spotted scat. Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-fareast-language:EN-US;}

EXPRESSION PROFILE OF SEX DETERMINATION GENE, BIOREPRODUCTION, PHENOTYPE, AND LOCOMOTORY PERFORMANCES OF OLIVE RIDLEY, Lepidochelys olivacea INDUCED BY DIFFERENT INCUBATION TEMPERATURE

&lt;p&gt;&lt;em&gt;S&lt;/em&gt;&lt;em&gt;ex determination &lt;/em&gt;&lt;em&gt;in turtle species &lt;/em&gt;&lt;em&gt;is not only based on genotype, but also rely on the incubation temperature. In addition, sexual differentiation takes place during the thermo-sensitive period (TSP). This study was conducted to determine the effects of incubation temperature on &lt;/em&gt;&lt;em&gt;sex &lt;/em&gt;&lt;em&gt;expression profile of determination gene, bioreproduction, &lt;/em&gt;&lt;em&gt; phenotype&lt;/em&gt;&lt;em&gt;, and locomotory performances of olive ridley turtle hatchlings. Fertile eggs incubated at two temperatures, namely feminine temperature (30-33°C), and masculine temperature (26-27°C). Value of cycle threshold (C&lt;sub&gt;T&lt;/sub&gt;) measured during TSP, i.e 23-25 embryonic development stage, and after TSP, i.e 26-27 embryonic development stage using real time PCR techniques. Comparison of gene expression at both incubation temperatures were analyzed by ANOVA, and Student’s t test. Hatchling bioreproduction and phenotype measurement consist of the incubation period, embryo growth, morphometrics, and locomotori performances hatchlings were analyzed with regression analysis and Student’s t test. The results showed expression of both aromatase and Rspond 1 genes (which plays a role in ovarian differentiation) after the TSP that incubated at feminine temperature higher and different with masculine temperature. In conjunction with the bioreproduction and phenotype, the incubation period of feminine temperature shorter than that of masculine. Likewise, growth of the embryo of feminine temperature was faster than that of masculine. Incubation at feminine temperature significantly affect to carapace width, length and width of the plastron, long flippers and rear arms, long neck, and the frequency of the swing flippers.&lt;/em&gt;&lt;em&gt; &lt;/em&gt;&lt;/p&gt; &lt;strong&gt;&lt;em&gt;Keywords: &lt;/em&gt;&lt;/strong&gt;&lt;em&gt;thermo-sensitive period (TSP), gene expression, phenotype, Lepidochelys olivacea&lt;/em&gt;

EXPRESSION PROFILE OF SEX DETERMINATION GENE, BIOREPRODUCTION, PHENOTYPE, AND LOCOMOTORY PERFORMANCES OF OLIVE RIDLEY, Lepidochelys olivacea INDUCED BY DIFFERENT INCUBATION TEMPERATURE

Sex determination in turtle species is not only based on genotype, but also rely on the incubation temperature. In addition, sexual differentiation takes place during the thermo-sensitive period (TSP). This study was conducted to determine the effects of incubation temperature on sex expression profile of determination gene, bioreproduction, phenotype, and locomotory performances of olive ridley turtle hatchlings. Fertile eggs incubated at two temperatures, namely feminine temperature (30-33°C), and masculine temperature (26-27°C). Value of cycle threshold (CT) measured during TSP, i.e 23-25 embryonic development stage, and after TSP, i.e 26-27 embryonic development stage using real time PCR techniques. Comparison of gene expression at both incubation temperatures were analyzed by ANOVA, and Student’s t test. Hatchling bioreproduction and phenotype measurement consist of the incubation period, embryo growth, morphometrics, and locomotori performances hatchlings were analyzed with regression analysis and Student’s t test. The results showed expression of both aromatase and Rspond 1 genes (which plays a role in ovarian differentiation) after the TSP that incubated at feminine temperature higher and different with masculine temperature. In conjunction with the bioreproduction and phenotype, the incubation period of feminine temperature shorter than that of masculine. Likewise, growth of the embryo of feminine temperature was faster than that of masculine. Incubation at feminine temperature significantly affect to carapace width, length and width of the plastron, long flippers and rear arms, long neck, and the frequency of the swing flippers. Keywords: thermo-sensitive period (TSP), gene expression, phenotype, Lepidochelys olivacea

Cloning and expression analysis of Foxl2 during the reproductive cycle in Korean rockfish, Sebastes schlegeli

Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to regulate ovarian aromatase, which plays a crucial role in ovarian differentiation. To address the role of Foxl2 in gonads and brain during gonadal development, we isolated the full-length cDNA of Foxl2 and analyzed its spatiotemporal expression patterns in the viviparous teleost Korean rockfish, Sebastes schlegeli. Tissue distribution pattern revealed that the Foxl2 was detected in the liver, fat, gill, brain, and ovary, but could hardly be found in the testis. Reverse transcriptase PCR suggested that Foxl2 in Korean rockfish may involve in ovary development in the study of expression level during gonads development. It also revealed that the stage of highest expression level for Foxl2 was almost much earlier than cyp19a1a and cyp19a1b during the gonadal development stage in gonads and brain except for cyp19a1a in brain. Furthermore, the expression pattern of Foxl2 as well as aromatases may imply the role of Foxl2 in the up-regulation of aromatases not only in the female fish but also in male.

Characterisation and expression during sex differentiation of Sox19 from the sea bass Dicentrarchus labrax

The Sox family of transcription factors are involved in a variety of developmental processes including sex determination and gonadal differentiation. Sox19 is a particularly interesting member of this family that has been found only in fish, though mammals have a very diverged orthologue that is designated Sox15 and assigned to a different Sox family subgroup. Here we describe the cloning and characterisation of sox19 from the European sea bass (Dicentrarchus labrax), an important aquaculture species in which sex ratios skewed in favour of males are frequently encountered. The sea bass sox19 gene contains a single intron, encodes a protein of 309 amino acids, has multiple transcription start sites and may produce a truncated splice variant. Sox19 mRNA is present in many adult tissues, with the highest expression in the brain and gonads. Interestingly, the gene is strongly upregulated in the differentiation of the ovary but not the testis, suggesting a role in ovarian differentiation.

Cloning and differential expression of FOXL2 during ovarian development and recrudescence of the catfish, Clarias gariepinus

FOXL2 is a member of the forkhead/HNF-3-related family of transcription factors which provides tissue-specific gene regulation. It is known to regulate ovarian aromatase, ( cyp19a1a) which plays a crucial role in ovarian differentiation. To understand the role of FOXL2 in gonads and brain during ovarian development and recrudescence, we cloned the full-length cDNA of FOXL2 and analyzed its spatio-temporal expression both at transcript and protein levels in the air-breathing catfish, Clarias gariepinus. Based on its deduced amino acid sequence, an antigenic peptide conjugated with a carrier protein was synthesized which was then used for raising antibody that reacted specifically with FOXL2. Tissue distribution pattern of FOXL2 revealed its presence prominently in ovary and female brain with sexual dimorphism. Highest expression of FOXL2 was observed in ovary and brain during prespawning phase indicating an important role for this correlate in ovarian recrudescence. Human chorionic gonadotropin (hCG) treatment, in vitro and in vivo, induced FOXL2 expression in the ovary during preparatory and prespawning phases. Similar type of enhanced expression was evident in brain after hCG-induction during the prespawning phase. The ontogeny of FOXL2 showed sexual dimorphic expression pattern both in gonads and brain. Based on our previous studies, the expression pattern of FOXL2 was found to be synchronous not only with that of ovarian cyp19a1a but also with brain cyp19a1b. Present study substantiates the role of FOXL2 in the regulation of aromatase in teleosts and also designates FOXL2 as a potential ovary and brain marker during female sex development in catfish.

Molecular cloning of Foxl2 gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow, Gobiocypris rarus

Endocrine-disrupting chemicals (EDCs) can affect normal sexual differentiation in fish. Foxl2, one forkhead transcription factor, plays an important role in ovarian differentiation in the early development of the female gonad in mammals and fish. How EDCs affect Foxl2 expression is little known. In this study, we isolated a Foxl2 cDNA from the ovary of rare minnow Gobiocypris rarus and examined its expression during early development stages and in different adult tissues. Then, we analyzed Foxl2 expression in G. rarus juvenile following 3-day exposure to 17α- ethinylestradiol (EE2), 4-n-nonylphenol (NP), and bisphenol A (BPA). Alignment of known Foxl2 sequences among vertebrates showed high identity in forkhead domain and C-terminal region with other vertebrate proteins. Quantitative RT-PCR analysis showed that Foxl2 expression was linear decrease and cyp19a1a, the downstream target gene of Foxl2, had no correlation with Foxl2 from 18 to 50days post fertilization (dpf). Among different adult tissues, Foxl2 is mainly expressed in ovary, brain, gill, eye, and male spleen. In the 3-day exposure, the juvenile fish to EDCs, 0.1nM EE2, and 1nM BPA significantly up-regulated the expression of Foxl2 gene, while NP had no effect on Foxl2 expression. Altogether, these results provide basic data for further study on how Foxl2 mediates EDCs impact on the sexual differentiation in G. rarus.

Dimorphic Expression of Various Transcription Factor and Steroidogenic Enzyme Genes during Gonadal Ontogeny in the Air-Breathing Catfish, Clarias gariepinus

In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17–20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30–40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30–40 dph when sex-specific genes showed differential expression.

Females Battle to Suppress Their Inner Male

Conventional wisdom holds that the ovary and testis are terminally differentiated organs in adult mammals. However, Uhlenhaut et al. (2009) now report that deletion of a single gene, Foxl2, is sufficient to induce transdifferentiation of ovary into testis in adult mice, suggesting that testicular development is actively repressed throughout the life of females.