Tilapia, a good model for studying reproductive endocrinology.

The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2, cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1, cyp11a1, cyp17a2, cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a/esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy, dmrt1, gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish.

Genome-wide analysis of two different regions of brain reveals the molecular changes of fertility related genes in rln3a-/- mutants in male Nile tilapia (Oreochromis niloticus).

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.

Effects of juicing methods on the bioactive compounds and flavor quality of 'Black-seed' pomegranate from three producing areas.

BACKGROUND: Color, nutrients and flavor are the key characteristics of pomegranate juice, but they are susceptible to processing methods and raw materials. In this study, the effects of aril juicing and whole fruit juicing methods on the composition of 'Black-seed' pomegranate juice from three producing areas were studied, including physicochemical parameters, color attributes, organic acids, sugars, phenolic compounds, and volatile compounds. RESULTS: The whole fruit juicing method resulted in higher juice yields of pomegranate fruit with 69.01-72.59%, hue angle values were 5.95-6.45°, and the juice remained red. The highest level of citric acid (21.21 g L-1 ), total acids (24.78 g L-1 ), and total anthocyanin content (435.59 mg L-1 ) were found in whole fruit juice, and seven tannins were detected. The most abundant volatile compounds were (Z)-3-hexen-1-ol and 1-hexanol in all juice samples, with alcohol content increased and aldehydes content decreased by whole fruit juicing. Principal component analysis revealed that the 24 indexes (variable important in projection >1) clearly distinguished juice samples obtained by two juicing methods, with ellagic acid hexoside, (E)-2-heptenal, (+)-catechin, and octanoic acid having the best discriminatory potential. CONCLUSION: Overall, the effects of juicing method on 'Black-seed' pomegranate juice were greater than those of raw-material-producing areas. These results confirmed the potential for using the whole 'Black-seed' pomegranate for processing, and also provided a theoretical basis for the healthy product development and utilization of dark-color pomegranate varieties. © 2023 Society of Chemical Industry.

Secrets behind Protein Sequences: Unveiling the Potential Reasons for Varying Allergenicity Caused by Caseins from Cows, Goats, Camels, and Mares Based on Bioinformatics Analyses.

This study systematically investigated the differences in allergenicity of casein in cow milk (CM), goat milk (GM), camel milk (CAM), and mare milk (MM) from protein structures using bioinformatics. Primary structure sequence analysis reveals high sequence similarity between the α-casein of CM and GM, while all allergenic subtypes are likely to have good hydrophilicity and thermal stability. By analyzing linear B-cell epitope, T-cell epitope, and allergenic peptides, the strongest casein allergenicity is observed for CM, followed by GM, and the casein of MM has the weakest allergenicity. Meanwhile, 7, 9, and 16 similar or identical amino acid fragments in linear B-cell epitopes, T-cell epitopes, and allergenic peptides, respectively, were observed in different milks. Among these, the same T-cell epitope FLGAEVQNQ was shared by κ-CN in all four different species' milk. Epitope results may provide targets of allergenic fragments for reducing milk allergenicity through physical or/and chemical methods. This study explained the underlying secrets for the high allergenicity of CM to some extent from the perspective of casein and provided new insights for the dairy industry to reduce milk allergy. Furthermore, it provides a new idea and method for comparing the allergenicity of homologous proteins from different species.

Characterization of the flavor, sensory quality and in vitro bioaccessibility in cloudy pomegranate juice treated by high pressure and thermal processing.

BACKGROUND: Recently, cloudy pomegranate juice (PJ) has become popular due to its rich phenolic and health-promoting effects. The aim of the present work was to evaluate the application of high hydrostatic pressure processing (HPP), pasteurization (PT) and high-temperature short-time sterilization (HTST) on physicochemical properties (color, flow behavior, turbidity, sugars, organic acids, aroma and sensory evaluation) and in vitro bioaccessibility of total phenolics content (TPC), total flavonoids content (TFC) and phenolics of cloudy PJ. RESULTS: Compared to HPP, thermal sterilization significantly increased the brightness (L*), redness (a*), total color difference (ΔE) and turbidity, and decreased the TPC and TFC. HPP maintained the volatile profile of cloudy PJ better, while thermal sterilization significantly changed the profile by decreasing alcohols 23.8-32.7% and increasing acids by 33.6%-182.8%. The bioaccessibility of flavonoids, phenolic acids and tannins in the control cloudy PJ after in vitro oral-gastric-intestinal digestion were 1.5%, 4.9%, and 9.0%, respectively, which were not significantly changed by different treatments. CONCLUSION: These results contributed to promoting the color quality and health benefits of cloudy PJ rich in phenolics by optimizing the processing conditions in the food industry. © 2022 Society of Chemical Industry.

Desert hedgehog mediates the proliferation of medaka spermatogonia through Smoothened signaling.

Desert hedgehog (DHH) signaling has been reported to be involved in spermatogenesis and the self-renewal of spermatogonial stem cells (SSCs). However, the role of DHH in proliferation of spermatogonia including SSCs remains to be elucidated. Here, we report that Dhh from medaka (Oryizas latipes) (named as OlDhh) could directly mediate the proliferation of spermatogonia via Smoothened (Smo) signaling. Oldhh is 1362 bp in length and encodes 453 amino acid (aa) residues with more than 50% identity with the homologs in other species. It has expression predominantly restricted to testis. The soluble and tag-free 176-aa mature OlDhh (named as mOlDhh) were successfully obtained by fusing with the N-terminal tag of cleavable 6-histidine and small ubiquitin-related modifier and then removing the tag. Notably, mOlDhh significantly promoted the proliferation of SG3 (a spermatogonial stem cell line from medaka testis) in a dose-dependent manner and spermatogonia in testicular organ culture. Furthermore, the proliferation of SG3 in the presence of mOlDhh could be inhibited by Smo antagonist (cyclopamine) resulting in apoptosis. Additionally, mOlDhh significantly upregulated the expression of smo as well as the pluripotent-related genes bcl6b and sall4. These data suggest that Smo is an indispensable downstream component in the Dhh signaling pathway. In conclusion, our findings unambiguously demonstrate that Dhh could directly mediate the proliferation of spermatogonia through Smo signaling. This study provides new knowledge about the proliferation regulation of spermatogonia.

The Stability of Intact Parathyroid Hormone (PTH) in Different Types of Blood Collection Tubes.

BACKGROUND: Over past decades, the instability of parathyroid hormone (PTH) causes great interference for the clinical laboratory. Contradictory results were reported in many reports about storage conditions and suitable blood collection tubes to ensure PTH stability in the pretreatment phase. METHODS: This study recruited 30 participants including 10 healthy persons, 10 hemodialysis, and 10 hyperparathyroidism patients. Five types of blood collection tubes (EDTA-K3 tube, coagulant tube, heparin anticoagulant tube, gel separating tube, and plain tube) were included to determine whether they were suitable as blood-collecting vessels. The time points and conditions for testing samples included less than 2 hours, 4 hours, and 8 hours at room temperature, and, in parallel, 24 hours, 48 hours, and 72 hours in refrigeration. Two different judgement criteria were used to compare the stability of PTH in different blood vessels. RESULTS: Purely statistical analysis showed that 4 types of blood collection tubes could not perform the same storage ability as EDTA-K3 tube at "T0" time point. Plain tube had the largest drop among all types of blood collection tubes. Compared by pairwise t-test, EDTA-K3 tube could maintain intact PTH for 8 hours (p = 0.998) at room temperature and 24 hours (p = 0.053) in refrigeration. When comparing the total change limit (TCL = 18.8%), at room temperature, EDTA-K3 tube (7.0%), heparin tube (12.7%), coagulant tube (16.2%), and plain tube (17.6%) could maintain intact PTH for 8 hours, and GST can preserve PTH for 4 hours (18.2%). In refrigeration, EDTA-K3 tube could maintain PTH for 72 hours (7.5%) and heparin tube could maintain 24 hours (18.4%). The other three blood collection tubes could not preserve PTH in refrigeration (GST = 22.1%, coagulant tube = 20.3%, plain tube = 20.8%). CONCLUSIONS: PTH seems more stable in the EDTA-K3 tube than any other blood collection tubes and is followed next by the heparin anticoagulant tube. Plain tube and GST have faster degradation than other tubes and are not suggested to preserve intact PTH.

Nile Tilapia: A Model for Studying Teleost Color Patterns.

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation. Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns. We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish. We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways. Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations. None of F1 heterozygotes had phenotype. By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes. Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia. Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates.

Role of sex steroids in fish sex determination and differentiation as revealed by gene editing.

The involvement of sex steroids in sex determination and differentiation is relatively conserved among non-mammalian vertebrates, especially in fish. Thanks to the advances in genome sequencing and genome editing, significant progresses have been made in the understanding of steroidogenic pathway and hormonal regulation of sex determination and differentiation in fish. It seems that loss of function study of single gene challenges the traditional views that estrogen is required for ovarian differentiation and androgen is needed for testicular development, but it is not so in essence. Steroidogenic enzymes can be classified into two categories based on expression and enzyme activities in fish. One type, encoded by star2, cyp17a1 and cyp19a1a, is involved in estrogen production and exclusively expressed in the gonads. Mutation of these genes results in the up-regulation of male pathway genes and sex reversal from genetic female to male. The other type, encoded by the duplicated paralogs of the above genes, including star1, cyp11a1, cyp17a2 and cyp19a1b, as well as cyp11c1 gene, is dominantly expressed both in gonads and extra-gonadal tissues. Mutation of these genes alters the steroids (androgen, DHP and cortisol) production and spermatogenesis, fertility, secondary sexual characteristics and sexual behavior, but usually does not affect the sex differentiation. For the estrogen receptors (esr1, esr2a and esr2b), single mutation failed to, but double and triple mutation leads to sex reversal from female to male, indicating that at least Esr2a and Esr2b are required to mediate the role of estrogen in sex determination proved by gene editing experiments. Taken together, results from gene editing enrich our understanding of steroid synthesis pathways and further confirm the critical role of estrogen in female sex determination by antagonizing the male pathway in fish.

Clostridium butyricum Supernatant Regulates the Expression of RORγt in HCT-116 Cells by Inhibiting the TLR2/MyD88/NF-κB Signaling Pathway.

In this study, we treated HCT-116 cells with Clostridium butyricum (C. butyricum) supernatant and observed its effects on the TLR2/MyD88/NF-κB signaling pathway and RORγt, to further explore the possible immune regulatory mechanism of C. butyricum. Our results showed that C. butyricum supernatant downregulated the mRNA and protein levels of TLR2, MyD88, NF-κBp65, and RORγt in HCT-116 cells and the protein levels of phospho-NF-κBp65. Partial blockage of TLR2 by CD282 weakened the inhibitory effects of C. butyricum supernatant on the above pathway components. Those component levels were still inhibited by C. butyricum supernatant after Pam3CSK4 activation of TLR2. In summary, C. butyricum supernatant can inhibit the TLR2/MyD88/NF-κB signaling pathway and the expression of RORγt in HCT-116 cells. These effects are at least partly achieved through inhibition of TLR2.

Identification, Expression and Evolution of Short-Chain Dehydrogenases/Reductases in Nile Tilapia (Oreochromis niloticus).

The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.

FAM60A, increased by Helicobacter pylori, promotes proliferation and suppresses apoptosis of gastric cancer cells by targeting the PI3K/AKT pathway.

Helicobacter pylori (H. pylori) infection can promote the development of gastric cancer (GC); however, the underlying mechanism is not clear. FAM60A has been found showing high levels in some cancer cells, including lung cancer (A549), and pancreatic cancer (Capan-2) cell lines. Data in oncomine showed that FAM60A overexpression was an critical prognostic factor in GC. In this study, we showed that knockdown of FAM60A could revert the increase of proliferation and the decrease of apoptosis caused by H.pylori infection in HGC-27 and AGS cells. Conversely, FAM60A upregulation promoted proliferation and inhibited apoptosis in HGC-27 and AGS cells. We also found that the PI3K/AKT pathway inhibitor LY294002 could revert the changes caused by FAM60A upregulation in HGC-27 and AGS cells. Thus, our study provides evidence that FAM60A act as a carcinogen and suggests that H. pylori-induced upregulation of FAM60A may contribute to the development of gastric cancer.

Rapamycin preserves the primordial follicle pool during cisplatin treatment in vitro and in vivo.

Rapamycin has been proven to effectively inhibit the activation of primordial follicles while cisplatin-induced the loss of primordial follicles due to the over-activation of the primordial follicle stockpile. Whether rapamycin could inhibit the loss of primordial follicles induced by cisplatin is still unknown. The ovaries of neonatal Sprague Dawley rats were cultured in vitro in different doses of rapamycin (0.08, 0.16, and 0.32 µg/ml) and cisplatin (0.1, 0.4, and 0.8 µg/ml). The immature BALB/c mice were administered cisplatin with or without rapamycin by intraperitoneal injection. Ovaries were collected to analyze the histomorphology, the messenger RNA (mRNA) expression of anti-Mullerian hormone (AMH), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) and the expression of key proteins of mammalian target of rapamycin (mTOR) pathway. Growing follicle counts of ovaries cultured in vitro in the R0.16 and R0.32 groups were decreased and the ratio of growing to primordial follicles was also decreased in a dose-dependent manner. In the C0.8 group, growing follicles were decreased compared with the other groups while the ratio was substantially increased in the C0.4 and C0.8 group. Co-treatment attenuated primordial follicle loss and reduced the upregulated ratio induced by cisplatin. Ovarian follicle dynamics in vivo was consistent with the in vitro results. Primordial follicles counts were statistically increased and the ratio was reduced in the rapamycin group compared with the control group. Primordial follicle counts were dramatically reduced in the cisplatin group whereas co-treatment with rapamycin slightly recovered its counts. There was no obvious difference in the number of growing follicles between the cisplatin group and other groups. The ratio was significantly increased in cisplatin-treated mice whereas decreased in the co-treatment group. The apoptosis rate of antral follicles in cisplatin-treated mice was higher than the other groups while the apoptosis rate was decreased in the co-treatment group in vivo. Compared with the control and rapamycin group, the mRNA expression of AMH, GDF9, and BMP15 were downregulated in the cisplatin group. The co-treatment group recovered the mRNA expression of BMP15. In addition, the expression of key protein of mTOR pathway rpS6 and its phosphorylated forms were increased in the cisplatin-treated group while co-treatment decreased their expression. Rapamycin attenuated the loss of primordial follicles induced by cisplatin through the inhibitory effect of rapamycin on the mTOR pathway. These results suggest that rapamycin may be an effective drug for the protection of ovarian function during chemotherapy.

Cloning and characterization of wnt4a gene in a natural triploid teleost, Qi river crucian carp (Carassius auratus).

WNT4 (wingless-type MMTV integration site family, member 4) plays a key role in the ovarian differentiation and development in mammals. However, the possible roles of Wnt4 during gonadal differentiation and development need further clarification in teleosts. In this study, we cloned and characterized the full-length cDNA of Qi river crucian carp (Carassius auratus) wnt4a gene (CA-wnt4a). The cDNA of CA-wnt4a is 2337 bp, including the ORF of 1059 bp, encoding a putative protein with a transmembrane domain and a WNT family domain. Sequence and phylogenetic analyses revealed that the CA-Wnt4a identified is a genuine Wnt4a. Tissue distribution analysis showed that CA-wnt4a is expressed in all the tissues examined, including ovary. CA-wnt4a undergoes a stepwise increase in the embryonic stages, suggesting that CA-wnt4a might be involved in the early developmental stage. Ontogenic analysis demonstrated that CA-wnt4a expression is upregulated in the ovaries at 30-50 days after hatching (dah), the critical period of sex determination/differentiation in Qi river crucian carp. From 90 dah, the expression of CA-wnt4a was gradually downregulated in the developing ovaries. Immunohistochemistry demonstrated that CA-Wnt4a was expressed in the somatic and germ cells of the ovary by 30 dah, thereafter, positive signals of Wnt4a were detected in the somatic cells, oogonia and primary growth oocytes from 60 dah. In the sex-reversed testis induced by letrozole treatment, the expression level of CA-wnt4a was significantly downregulated. When CA-wnt4a expression was inhibited by injection of FH535 (an inhibitor of canonical Wnt/ß-catenin signal pathway) in the ovaries, levels of cyp19a1a, foxl2 mRNA were significantly downregulated, while sox9b and cyp11c1 were upregulated, which suggested that together with Foxl2-leading estrogen pathway, CA-wnt4a signaling pathway might be involved in ovarian differentiation and repression of the male pathway gene expression in Qi river crucian carp.

Comparative transcriptome profiling and characterization of gene expression for ovarian differentiation under RU486 treatment.

17α, 20ß-dihydroxypregn-4-en-3-one (17α, 20ß-DP, DHP), a teleost specific biologically active progestin, has been proved to play a critical role in oocytes maturation, ovulation and spermiation. RU486 (Mifepristone, an antagonist of progestin receptor) has been applied in contraceptives, abortion and hormone therapy in clinical medicine. To get further insights into the molecular mechanisms of nuclear progestin receptor (Pgr) activated ovarian differentiation and maintenance, we conducted comparative gonadal transcriptome analysis, and investigated histological and transcriptional differences using 4 months after hatching (mah) RU486-treated XX and control XX/XY Nile tilapia (Oreochromis niloticus). DESeq analysis identified 7148 DEGs (differentially expressed genes) between RU486-treated and control XX gonads, while merely 442 DEGs were screened between the gonads of RU486-treated XX and control XY fish highlighting that RU486 treatment set forwards masculinity in XX fish. Comprehensive analysis of gene hierarchical clustering revealed that RU486 treatment in XX fish resulted in robust changes of gene expression profiles. In comparison with XX group, female-dominant genes were significantly repressed in RU486 treated XX fish gonads. Moreover, most parts of down-regulated genes in wild type female were evidently up-regulated genes in RU486-treated XX fish gonads. Comparing with control XY group, the majority of male-dominant genes represent a high level of expression. However, RU486-treatment led to an up-regulation of a cluster genes specifically which showed relative lower expression in both control XX and XY group. RU486-treatment mediated global changes of gene expression profiles in steroidogenesis, germ cell differentiation and follicular cell trans-differentiation were verified by quantitative PCR. Both morphological and immunohistochemistry results further proved that RU486 treatment initiates testicular-like gonads development in XX fish via simultaneously enhancing the male responsive genes and suppressing the female-dominant genes. Moreover, RU486 treatment caused significant decline of fshr, lhr and increase of ars. Taken together, our data confirms blocking of DHP physiology by RU486 treatment induces masculinization in XX gonad preferably via repressing of gonadotropin physiology, germ cell differentiation and promoting follicular trans-differentiation in teleosts.

A Tandem Duplicate of Anti-Müllerian Hormone with a Missense SNP on the Y Chromosome Is Essential for Male Sex Determination in Nile Tilapia, Oreochromis niloticus.

Variation in the TGF-ß signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that amhy, a Y-specific duplicate of the anti-Müllerian hormone (amh) gene, induces male sex determination in Nile tilapia. amhy is a tandem duplicate located immediately downstream of amhΔ-y on the Y chromosome. The coding sequence of amhy was identical to the X-linked amh (amh) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region. amhy lacks 5608 bp of promoter sequence that is found in the X-linked amh homolog. The amhΔ-y contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-ß binding domain. Both amhy and amhΔ-y expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of amhy in XY fish resulted in male to female sex reversal, while mutation of amhΔ-y alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the amhy/amhΔ-y haplotype or a vector containing amhy ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the anti-Müllerian hormone receptor type II (amhrII) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated amhy with a missense SNP is the candidate sex determining gene and amhy/amhrII signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-ß signaling pathway in fish sex determination.

Duplication and gene expression patterns of ß-catenin in Nile tilapia.

ß-Catenin, a key transcriptional coactivator of the Wnt pathway, plays an important role in animal embryonic development and organogenesis. In our earlier study, we have reported that two types of ß-catenin (ß-catenin-1 and ß-catenin-2) were ubiquitously expressed in almost all the tissues examined in tilapia. However, the immunolocalization of ß-catenin in those tissues, especially in extra-gonadal tissues, remains unclear. In the present study, we further confirm that these two types of ß-catenin gene exist only in teleosts and are derived from 3R (third round of genome duplication) by phylogenetic and syntenic analyses. Moreover, the transcriptome analysis conducted in this investigation reveals that two ß-catenins exhibited similar expression patterns in seven adult tissues and four key developmental stages of XX and XY gonads. Finally, immunohistochemistry analysis was performed to detect the cell localization of ß-catenin. A positive signal of ß-catenin was observed in various tissues of tilapia, such as the intestine, liver, kidney, spleen, eye, brain, and gonads. The results of our study indicate that tilapia ß-catenin might be involved in the organ development and play some specific functions in biological processes; all these data will provide basic reference for understanding the molecular mechanism of ß-catenin in regulating of teleost organogenesis.

Kinetic modelling of non-enzymatic browning and changes of physio-chemical parameters of peach juice during storage.

Kinetics of non-enzymatic browning and loss of free amino acids during different storage temperature (4, 25, 37 °C) were investigated. Changes of browning degree (A420), color parameters, Vitamin C (Vc), free amino acids and 5-hydroxymethylfurfural (5-HMF) were analyzed to evaluate the non-enzymatic browning reactions, which were significantly affected by storage temperature. The lower temperature (4 °C) decreased the loss of Vc and the generation of 5-HMF, but induce the highest loss of serine. At the end of storage, loss of serine, alanine and aspartic acid were mainly lost. Results showed that zero-order kinetic model (R2 > 0.859), the first-order model (R2 > 0.926) and the combined kinetic model (R2 > 0.916) were the most appropriate to describe the changes of a* and b* values, the degradation of Vc and the changes of A420, L* and 5-HMF during different storage temperatures. These kinetic models can be applied for predicting and minimizing the non-enzymatic browning of fresh peach juice during storage.

Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition.

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.

Evaluation of browning ratio in an image analysis of apple slices at different stages of instant controlled pressure drop-assisted hot-air drying (AD-DIC).

BACKGROUND: Computer vision-based image analysis systems are widely used in food processing to evaluate quality changes. They are able to objectively measure the surface colour of various products since, providing some obvious advantages with their objectivity and quantitative capabilities. In this study, a computer vision-based image analysis system was used to investigate the colour changes of apple slices dried by instant controlled pressure drop-assisted hot air drying (AD-DIC). RESULTS: The CIE L* value and polyphenol oxidase activity in apple slices decreased during the entire drying process, whereas other colour indexes, including CIE a*, b*, ΔE and C* values, increased. The browning ratio calculated by image analysis increased during the drying process, and a sharp increment was observed for the DIC process. The change in 5-hydroxymethylfurfural (5-HMF) and fluorescent compounds (FIC) showed the same trend with browning ratio due to Maillard reaction. Moreover, the concentrations of 5-HMF and FIC both had a good quadratic correlation (R2 > 0.998) with the browning ratio. CONCLUSION: Browning ratio was a reliable indicator of 5-HMF and FIC changes in apple slices during drying. The image analysis system could be used to monitor colour changes, 5-HMF and FIC in dehydrated apple slices during the AD-DIC process. © 2016 Society of Chemical Industry.