Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Metalloproteases in gonad formation and ovulation.

Changes in expression or activation of various metalloproteases including matrix metalloproteases (Mmp), a disintegrin and metalloprotease (Adam) and a disintegrin and metalloprotease with thrombospondin motif (Adamts), and their endogenous inhibitors (tissue inhibitors of metalloproteases, Timp), have been shown to be critical for ovulation in various species from studies in past decades. Some of these metalloproteases such as Adamts1, Adamts9, Mmp2, and Mmp9 have also been shown to be regulated by luteinizing hormone (LH) and/or progestin, which are essential triggers for ovulation in all vertebrate species. Most of these metalloproteases also express broadly in various tissues and cells including germ cells and somatic gonad cells. Thus, metalloproteases likely play roles in gonad formation processes comprising primordial germ cell (PGC) migration, development of germ and somatic cells, and sex determination. However, our knowledge on the functions and mechanisms of metalloproteases in these processes in vertebrates is still lacking. This review will summarize our current knowledge on the metalloproteases in ovulation and gonad formation with emphasis on PGC migration and germ cell development.

Oestrogen Activates the MAP3K1 Cascade and ß-Catenin to Promote Granulosa-like Cell Fate in a Human Testis-Derived Cell Line.

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate ß-catenin-a factor essential for ovarian development. We show that oestrogen can activate ß-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to ß-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.

Gene expression profiles of bovine genital ridges during sex determination and early differentiation of the gonads†.

Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.

Matrix metalloproteinase-dependent regulation of extracellular matrix shapes the structure of sexually differentiating mouse gonads.

The extracellular matrix (ECM) proteins play an important role in the establishment of the sex-dependent structure of developing gonads. The matrix metalloproteinases (MMPs) are the major players in the regulation of ECM. Our hypothesis was that the MMPs-dependent regulation of EMC is crucial for the establishment of the correct, either testis or ovary, structure of developing gonad. We cultured developing mouse gonads in vitro in the presence of the MMPs inhibitors (α-2-macroglobulin, leupeptin, phosphoramidon) or the MMPs activator, APMA (4-aminophenylmercuric acetate). These inhibitors and activator inhibit/activate, to a different degree, matrix metalloproteinases, but the exact mechanism of inhibition/activation remains unknown. We found that the MMP inhibitors increased accumulation of ECM in the developing gonads. The α-2-macroglobulin had the weakest, and the phosphoramidon the strongest effect on the ECM and the structure of the gonads. The α-2-macroglobulin caused a slight increase of ECM and did not disrupt the gonad structure. Leupeptin led to the strong accumulation of ECM, resulted in the formation of the structures resembling testis cords in both testes and ovaries, and caused increase of apoptosis and complete loss of germ cells. Phosphoramidon caused the strongest accumulation of ECM, which separated individual cells and completely prevented intercellular adhesion both in the testes and in the ovaries. As a result of aberrant morphology, the sex of the phosphoramidon-treated gonads was morphologically unrecognizable. The APMA - the activator of MMP caused ECM loss, which led to the loss of cell adhesion, cell dispersion and an aberrant morphology of the gonads. These results indicate that the ECM accumulation is MMPs-dependent and that the correct amount and distribution of ECM during gonad development plays a key role in the formation of the gonad structure.

Effect of perfluorooctanesulfonate exposure on steroid hormone levels and steroidogenic enzyme activities in juvenile Silurana tropicalis.

The impact of the perfluoro-chemical, perfluorooctanesulfonate (PFOS), on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined because of its ubiquity in the environment, bioaccumulative nature and potential to disturb endocrine activity. A partial life cycle study exposing S. tropicalis to varying concentrations of PFOS 0.06, 0.13, 0.25, 0.50 and 1.0 mg PFOS/L [nominal]) was conducted. Gonad and plasma samples were collected from juvenile control specimens and organisms exposed to PFOS from early embryo through 150 days post-metamorphosis. Gonad CYP17, aromatase and 5α-reductase activities were measured. Plasma estradiol, testosterone, dihydrotestosterone (DHT) and gonadal testosterone were measured in both males and females. Increased plasma DHT and gonadal testosterone were found in PFOS-treated juvenile male S. tropicalis compared to controls. Decreased plasma estradiol, but not testosterone, was detected in PFOS-treated female S. tropicalis compared to controls. Plasma DHT was not detected and an increase in gonadal testosterone was detected in PFOS-treated female frogs. Female S. tropicalis exposed to PFOS exhibited a concentration-related decrease in the mean aromatase activity, but not 5α-reductase. PFOS exposure in male frogs induced a concentration-related increase in 5α-reductase activity, but did not alter aromatase activity compared to control frogs. A concentration-related increase in CYP 17,20-lyase activity, but not 17-hydroxylase activity, was found in both female and male S. tropicalis exposed to PFOS.

Evaluation of the developmental toxicity of perfluorooctanesulfonate in the Anuran, Silurana tropicalis.

A 150-day post-metamorphosis (dpm) partial lifecycle study exposing Silurana tropicalis to <0.03 (control), 0.06, 0.13 0.25, 0.5 and 1.0 mg/L perfluorooctanesulfonate (PFOS) was conducted. A subset of specimens from the control and each treatment were evaluated at metamorphic completion. A significant increase in the median metamorphosis time was observed in the 1.0 mg/L PFOS treatment relative to the control. A modest increase in the occurrence, but not severity, of mild follicular hypertrophy was found in thyroid glands from organisms exposed to the 0.62 and 1.1 mg/L PFOS treatments. At 150 dpm, a concentration-dependent increase in whole body PFOS residues was measured ranging from 29.6 to 163.5 mg/kg in the 0.05 and 1.1 mg/L PFOS treatments. Decreased body weight and snout-vent length were noted in specimens exposed to 1.1 mg PFOS/L at the completion of metamorphosis. Body weight was reduced in the 1.1 mg/L PFOS concentration; however, snout-vent length was not affected by PFOS exposure at 150 dpm. An increased proportion of phenotypic males were noted in the 0.62 and 1.1 mg/L PFOS treatments. Abnormal ovary development characterized by size asymmetry, necrosis and formation of excessive fibrous connective tissue was identified in females exposed to 0.29 and 1.1 mg PFOS/L. Asymmetrically misshaped testes were found at 1.1 mg/L PFOS. Results suggested that PFOS is capable of interfering with S. tropicalis growth before metamorphic completion and growth and gonad development during juvenile development.

Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development.

BACKGROUND: Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. RESULTS: At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). CONCLUSION: Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish.

Human foetal ovary shares meiotic preventing factors with the developing testis.

STUDY QUESTION: How can pre-meiotic germ cells persist in the human foetal ovary? SUMMARY ANSWER: Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. WHAT IS KNOWN ALREADY: The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. STUDY DESIGN SIZE, DURATION: In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. PARTICIPANTS/MATERIALS SETTING, METHODS: Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. MAIN RESULTS AND THE ROLE OF CHANCE: Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. WIDER IMPLICATIONS OF THE FINDINGS: We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

Expression of dmrt1 and vtg genes during gonad formation, differentiation and early maturation in cultured Russian sturgeon Acipenser gueldenstaedtii.

Expression of the dmrt1 and vtg genes was described using the real-time PCR (rt-PCR) method from 25 to 1600 days post-hatch (dph) in cultured Russian sturgeon Acipenser gueldenstaedtii. The level of dmrt1 transcription in gonads in subsequent studied periods increased exponentially while vtg expression increased in gonads and livers of A. gueldenstaedtii examined, but in later stages of development. Both dmrt1 and vtg genes showed elevated expression in intersex individuals probably caused by dietary exposure to phyto-oestrogens.

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues.

African clawed frog Xenopus sp. is used extensively for developmental biology and toxicology research. Amid concerns of environmental pollutants disrupting endocrine systems and causing altered reproductive development in wildlife, eco-toxicology research has led to a focus on linking molecular initiating events to population-level effects. As such, efforts to better understand reproductive development at the molecular level in these model species are warranted. To that end, transcriptomes were characterized in differentiating Xenopus tropicalis gonad tissues at Nieuwkoop and Faber (NF) stage 58 (pro-metamorphosis), NF66 (completion of metamorphosis), 1week post-metamorphosis (1WPM), and 2weeks post-metamorphosis (2WPM). Differential expression analysis between tissue types at each developmental stage revealed a substantial divergence of ovary and testis transcriptomes starting between NF58 and NF66; transcriptomes continued to diverge through 2WPM. Generally, testis-enriched transcripts were expressed at relatively constant levels, while ovary-enriched transcripts were up-regulated within this developmental period. Functional analyses of differentially expressed transcripts allowed linkages to be made between their putative human orthologues and specific cellular processes associated with differentiating gonad tissues. In ovary tissue, genetic programs direct germ cells through meiosis to the diplotene stage when maternal mRNAs are transcribed and trafficked to oocytes for translation following fertilization. In the testis, gene expression is consistent with connective tissue development, tubule formation, and germ cell support (Leydig and Sertoli cells). This dataset exhibited remarkable consistency with transcript profiles previously described in gonad tissues across species, and emphasizes the universal importance of certain transcripts for germ cell development and preparation of these tissues for reproduction.

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.

De Novo Assembly, Characterization and Comparative Transcriptome Analysis of the Gonads of Jade Perch (Scortum barcoo).

Due to the high meat yield and rich nutritional content, jade perch (Scortum barcoo) has become an important commercial aquaculture species in China. Jade perch has a slow growth rate, taking 3-4 years to reach sexual maturity, and has almost no difference in body size between males and females. However, the study of its gonad development and reproduction regulation is still blank, which limited the yield increase. Herein, the gonad transcriptomes of juvenile males and females of S. barcoo were identified for the first time. A total of 107,060 unigenes were successfully annotated. By comparing male and female gonad transcriptomes, a total of 23,849 differentially expressed genes (DEGs) were identified, of which 9517 were downregulated, and 14,332 were upregulated in the testis. In addition, a large number of DEGs involved in sex differentiation, gonadal development and differentiation and gametogenesis were identified, and the differential expression patterns of some genes were further verified using real-time fluorescence quantitative PCR. The results of this study will provide a valuable resource for further studies on sex determination and gonadal development of S. barcoo.

De novo assembly, characterization and comparative transcriptome analysis of gonads reveals sex-biased genes in Coreoperca whiteheadi.

The wild Coreoperca whiteheadi is considered as the primordial species in sinipercine fish, which has valuable genetic information. Unfortunately, C. whiteheadi was listed as a near-threatened species because of the environmental pollution, over-exploitation and species invasion. Therefore, more genetic information is needed to have a better understanding of gonadal development in C. whiteheadi. Here, the first gonadal transcriptomes analysis of C. whiteheadi was conducted and 277.14 million clean reads were generated. A total of 96,753 unigenes were successfully annotated. By comparing ovary and testis transcriptomes, a total of 21,741 differentially expressed genes (DEGs) were identified, of which 12,057 were upregulated and 9684 were downregulated in testes. Among them, we also identified about 53 differentially expressed sex-biased genes. Subsequently, the expression of twenty-four DEGs were confirmed by real-time fluorescence quantitative PCR. Furthermore, the histological analysis was conducted on ovaries and testes of one-year-old C. whiteheadi. Our results provided basic support for further studies on the function of sex-biased genes and the molecular mechanism of sex determination and reproduction in C. whiteheadi.

Molecular characterization of Smtdc-1 and Smddc-1 discloses roles as male-competence factors for the sexual maturation of Schistosoma mansoni females.

Introduction: Schistosomes are the only mammalian flatworms that have evolved separate sexes. A key question of schistosome research is the male-dependent sexual maturation of the female since a constant pairing contact with a male is required for the onset of gonad development in the female. Although this phenomenon is long known, only recently a first peptide-based pheromone of males was identified that contributes to the control of female sexual development. Beyond this, our understanding of the molecular principles inducing the substantial developmental changes in a paired female is still rudimentary. Objectives: Previous transcriptomic studies have consistently pointed to neuronal genes being differentially expressed and upregulated in paired males. These genes included Smp_135230 and Smp_171580, both annotated as aromatic-L-amino-acid decarboxylases (DOPA decarboxylases). Here, we characterized both genes and investigated their roles in male-female interaction of S. mansoni. Methodologies/findings: Sequence analyses indicated that Smp_135230 represents an L-tyrosine decarboxylase (Smtdc-1), whereas Smp_171580 represents a DOPA decarboxylase (Smddc-1). By qRT-PCR, we confirmed the male-specific and pairing-dependent expression of both genes with a significant bias toward paired males. RNA-interference experiments showed a strong influence of each gene on gonad differentiation in paired females, which was enhanced by double knockdown. Accordingly, egg production was significantly reduced. By confocal laser scanning microscopy, a failure of oocyte maturation was found in paired knockdown females. Whole-mount in situ hybridization patterns exhibited the tissue-specific occurrence of both genes in particular cells at the ventral surface of the male, the gynecophoral canal, which represents the physical interface of both genders. These cells probably belong to the predicted neuronal cluster 2 of S. mansoni. Conclusion: Our results suggest that Smtdc-1 and Smddc-2 are male-competence factors that are expressed in neuronal cells at the contact zone between the genders as a response of pairing to subsequently control processes of female sexual maturation.

Comparative transcriptome analysis of heat-induced domesticated zebrafish during gonadal differentiation.

BACKGROUND: The influence of environmental factors, especially temperature, on sex ratio is of great significance to elucidate the mechanism of sex determination. However, the molecular mechanisms by which temperature affects sex determination remains unclear, although a few candidate genes have been found to play a role in the process. In this study, we conducted transcriptome analysis of the effects induced by high temperature on zebrafish during gonad differentiation period. RESULTS: Totals of 1171, 1022 and 2921 differentially expressed genes (DEGs) between high temperature and normal temperature were identified at 35, 45 and 60 days post-fertilization (dpf) respectively, revealing that heat shock proteins (HSPs) and DNA methyltransferases (DNMTs) were involved in the heat-exposed sex reversal. The Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway that were enriched in individuals after heat treatment included Fanconi anemia (FA) pathway, cell cycle, oocyte meiosis and homologous recombination. CONCLUSIONS: Our study provides the results of comparative transcriptome analyses between high temperature and normal temperature, and reveals that the molecular mechanism of heat-induced masculinization in zebrafish is strongly related to the expression of HSPs and DNMTs and FA pathway during gonad differentiation.

De Novo Assembly, Characterization and Comparative Transcriptome Analysis of the Mature Gonads in Spinibarbus hollandi.

Spinibarbus hollandi is an important commercial aquaculture species in southeastern China, but with long maturity period and low egg laying amount. However, there has been little study of its gonad development and reproductive regulation, which limits aquaculture production. Here, for the first time, gonadal transcriptomes of male and female S. hollandi were analyzed. A total of 167,152 unigenes were assembled, with only 48,275 annotated successfully. After comparison, a total of 21,903 differentially expressed genes were identified between male and female gonads, of which 16,395 were upregulated and 5508 were downregulated in the testis. In addition, a large number of differentially expressed genes participating in reproduction, gonad formation and differentiation, and gametogenesis were screened out and the differential expression profiles of partial genes were further validated using quantitative real-time PCR. These results will provide basic information for further research on gonad differentiation and development in S. hollandi.

A Simple Method for Inducing Masculinization of Zebrafish Stocks Using 17α-Methyltestosterone.

Severely skewed sex ratios in zebrafish stocks can pose significant hurdles for line propagation and sperm cryopreservation. To overcome female-biased sex ratios in stocks derived from imported sperm samples, the Zebrafish International Resource Center has implemented routine supplementation of larval food with 17α-methyltestosterone to skew gonadal sex differentiation toward masculinization. Resulting stocks averaged 80% males.

Identification of Important Genes Involved in the Sex-Differentiation Mechanism of Oriental River Prawn, Macrobrachium nipponense, During the Gonad Differentiation and Development Period.

Identification of important genes, involved in the gonad differentiation and development, plays essential roles in the establishment of the artificial technique to regulate the process of testis development in M. nipponense. In this study, we aimed to determine the sensitive period of gonad differentiation and development through hematoxylin and eosin (HE) staining. The important genes involved in the gonad differentiation and development of M. nipponense were then identified through transcriptome profiling analysis during the sensitive period of gonad differentiation and development. HE staining analysis revealed that the sensitive period of gonad differentiation and development was from the post-larval developmental stages 5 (PL5) to PL25, which was dramatically faster than was for the other identified aquatic animals. The transcriptome profiling analysis predicted that phagosome, lysosome, oxidative phosphorylation, and glycolysis/gluconeogenesis play essential roles in the mechanism of gonad differentiation and development in M. nipponense. A total of 29 genes were further identified as the candidate genes, involved in the process of gonad differentiation and development in M. nipponense, based on the gene annotation and gene expression pattern. The qPCR analysis of Mn-JHEH, Mn-DHP, Mn-ALY, and Mn-SMA6 during the whole developmental process revealed that all of these four genes showed high expression levels during the sensitive period of gonad differentiation and development in M. nipponense. Mn-JHEH, Mn-DHP, and Mn-ALY showed higher expressions at PL25F than at PL25M, while Mn-SMA6 showed a higher expression at PL25M. The RNA interference (RNAi) analysis was further used to investigate the potential functions of SMA6 in male sexual development of M. nipponense. The RNAi analysis revealed that SMA6 positively regulated the testis development in M. nipponense by affecting the expression of Mn-IAG. This study provided valuable evidences for the establishment of the technique to regulate the process of gonad development in M. nipponense.

Tandem Mass Tag-Based Quantitative Proteomics Analysis of Gonads Reveals New Insight into Sexual Reversal Mechanism in Chinese Soft-Shelled Turtles.

Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis.