Leptin and Its Signaling Are Not Involved in Zebrafish Puberty Onset.

Leptin is a peptide hormone secreted from the adipose tissues and its signaling plays a central role in metabolic regulation of growth, especially on fat mass. In addition, leptin is also involved in regulating reproduction in mammals. In teleosts, there are two leptin ligands (lepa and lepb) and one cognate leptin receptor (lepr); however, their functions are still elusive. In this study, we created null-function mutants for lepa, lepb and lepr in zebrafish using CRISPR/Cas9 method and analyzed their phenotypes with emphasis on puberty onset, one major function widely reported for leptin in mammals. We demonstrated that the loss of leptin ligands or their receptor resulted in no obesity from prepubertal stage to adulthood. We then focused on leptin involvement in controlling puberty onset. We first confirmed the somatic threshold for puberty onset in females and proposed a criterion and somatic threshold for male puberty onset. We examined gonadal development and sex maturation in different genotypic combinations including single mutants (lepa-/-, lepb-/- and lepr-/-), double mutants (lepa-/-;lepb-/-) and triple mutants (lepa-/-;lepb-/-;lepr-/-). Our results showed that once the fish reached the thresholds, the siblings of all genotypes displayed comparable gonadal development in both sexes without obvious signs of changed puberty onset. In conclusion, this comprehensive genetic study on the lep-lepr system demonstrated that in contrast to its counterpart in mammals, leptin system plays little role in controlling growth and reproduction especially puberty onset in zebrafish.

The impact and cost-effectiveness of 9-valent human papillomavirus vaccine in adolescent females in Hong Kong.

INTRODUCTION: In Hong Kong (HK), a single-cohort vaccination program for 10-12-year-old girls with the 9-valent human papillomavirus (HPV) vaccine (9vHPV; types 6/11/16/18/31/33/45/52/58) has been launched. This study assessed the public health impact and cost-effectiveness of implementing routine 9vHPV vaccination (12-year-olds) with or without catch-up 9vHPV vaccination (13-18-year-olds) in HK. METHODS: The health impact and costs of implementing routine 9vHPV vaccination with or without catch-up vaccination over a 100-year time horizon were evaluated using a validated HPV-type transmission dynamic model adapted to the HK population; analyses were performed from a healthcare payer perspective. Routine vaccination (12-year-old girls) and catch-up vaccination (13-18 years) assumed vaccine coverage rates of 70% (base case) and 30%, respectively. The model also assumed herd immunity, lifelong vaccine protection, a discount rate of 3%, and a cost per dose of HK dollars (HKD) 858 [United States dollars (USD) 110] and HKD 1390 (USD 179) for the 2-valent HPV (2vHPV) and 9vHPV vaccines, respectively. HPV disease-related incidence and the incremental cost-effectiveness ratio (ICER) per quality-adjusted-life-year (QALY) were estimated. Cost-effectiveness was determined at a ceiling threshold of HK dollars (HKD) 382,046 (USD 49,142) or 1.0 times the gross domestic product per capita of HK. RESULTS: Compared with routine 9vHPV alone, routine plus catch-up 9vHPV is projected to reduce cervical cancer incidence by 3.4%. Routine plus catch-up 9vHPV will also reduce genital warts incident cases for males/females by 2.6%/5.4%. The incremental cost-effectiveness ratios were HKD 29,911 (USD 3847)/quality-adjusted life-year (QALY) for routine plus catch-up 9vHPV versus routine 9vHPV alone and HKD 25,524 (USD 3283)/QALY for routine 9vHPV alone versus screening only. Sensitivity analyses indicated that routine plus catch-up 9vHPV compared with routine 9vHPV alone remained cost-effective at coverage rates of 30% and 90%. CONCLUSIONS: This analysis predicts that the current HK vaccination strategy can be considered cost-effective and will provide maximum health benefit. These results support addition of the routine 9vHPV vaccine with or without catch-up 9vHPV vaccination to the regional vaccination program in HK.

Identification and characterization of a specific 13-miRNA expression signature during follicle activation in the zebrafish ovary.

Ovarian folliculogenesis is always of great interest in reproductive biology. However, the molecular mechanisms that control follicle development, particularly the early phase of follicle activation or recruitment, still remain poorly understood. In an attempt to decipher the gene networks and signaling pathways involved in such transition, we conducted a transcriptomic analysis (RNA-seq) on zebrafish primary growth (PG, stage I; inactive) and previtellogenic (PV, stage II; activated) follicles. A total of 118 unique microRNAs (miRNAs) (11 downregulated and 83 upregulated during PG/PV transition) and 56711 unique messenger RNAs (mRNAs) (1839 downregulated and 7243 upregulated during PG/PV transition) were identified. Real-time quantitative polymerase chain reaction analysis confirmed differential expression of 46 miRNAs from 66 candidates (66.67%). Among which, we chose to focus on 13 miRNAs (let-7a, -7b, -7c-5p, -7d-5p, -7h, -7i; miR-21, -23a-3p, -27c-3p, -107a-3p, -125b-5p, -145-3p, and -202-5p) that exhibited significant differential expression between PG and PV follicles (P ≤ 0.045*). With this 13-miRNA expression signature alone, PG follicles can be well differentiated from PV follicles by hierarchical clustering, suggesting their functional relevance during PG-to-PV transition. By overlaying predicted target genes and the differentially expressed mRNAs revealed by the RNA-seq analysis, especially those showing reciprocal miRNA-mRNA expression patterns, we shortlisted a panel of miRNA downstream targets for luciferase reporter validation. The reporter assay confirmed the interactions of let-7i:: atg4a (P = 0.01*), miR-202-5p::c23h20orf24 (P = 0.0004***), and miR-144-5p::ybx1 (P = 0.003**), implicating these potential miRNA-mRNA gene pairs in follicle activation during folliculogenesis. Our transcriptomic data analyses suggest that miRNA-mediated post-transcriptional control may represent an important mechanism underlying follicle activation.

RPL39L is an example of a recently evolved ribosomal protein paralog that shows highly specific tissue expression patterns and is upregulated in ESCs and HCC tumors.

Ribosomal proteins (RPs) have been shown to be able to impart selectivity on the translating ribosome implicating them in gene expression control. Many ribosomal proteins are highly conserved and recently a number of ribosomal protein paralogs have been described in mammals. We examined the expression pattern of RPs in differentiating mouse Embryonic Stem Cells (ESCs), paying particular attention to the RP paralogs. We find the RP paralog Rpl39l is highly expressed in ESC and its expression strongly correlates with hepatocellular carcinoma tumor (HCC) samples with high tumor grading and alpha-fetoprotein level giving it diagnostic potential. We further screen the expression pattern of all RPs and their paralogs across 22 different tissues. We find that the more recently evolved RP paralogs show a much greater level of tissue-specific expression. We propose that these RP paralogs evolved more recently to provide a greater level of gene expression control to higher eukaryotes.

Loss of Growth Hormone Gene (gh1) in Zebrafish Arrests Folliculogenesis in Females and Delays Spermatogenesis in Males.

As a master hormone controlling growth and metabolism, GH is also known to regulate reproduction. Studies in mammals have shown that mutations in GH or its receptor (GHR) not only result in retardation in body growth but also reproductive dysfunctions in both sexes. However, the roles of GH in reproduction of other vertebrates are poorly defined. In this study, we created two zebrafish GH (gh1) mutant lines using CRISPR/Cas9. The mutant developed normally up to 14 days postfertilization (dpf); however, a high rate of mortality was observed afterward in both lines, and only a small number of mutant fish could survive to adult stage. The body growth of the mutants was significantly retarded in both sexes in a gene dose-dependent manner compared with their wild-type siblings. A severe dysfunction of gonadal development was observed in survived mutant females, with ovarian folliculogenesis being arrested completely at primary growth stage until 100 dpf. Interestingly, the folliculogenesis in the mutant resumed after months of delay with a certain number of follicles entering vitellogenic growth. As for male reproduction, although the spermatogenesis in mutant males seemed normal in adults, the GH-insufficient heterozygote showed an obvious delay of spermatogenesis (puberty onset) at early developmental stages. The adult mutant males could not breed with wild-type females through natural spawning; however, the sperm isolated from the mutant testes could fertilize eggs through artificial fertilization. This study provides further genetic evidence for the dependence of puberty onset on somatic growth, but not age, in fish.

Roles of Figla/figla in Juvenile Ovary Development and Follicle Formation During Zebrafish Gonadogenesis.

Sex determination and differentiation are complex processes. As a juvenile hermaphrodite or undifferentiated gonochorist, zebrafish undergo a special juvenile ovarian phase during sex differentiation, making it an excellent model for studying early oogenesis and folliculogenesis. We provide lines of evidence at morphological, molecular, and genetic levels for roles of factor in the germline α (Figla), an oocyte-specific transcription factor, in early zebrafish gonadogenesis. As in mammals, Figla/figla was also expressed in the gonads and its expression in the ovary was also restricted to early oocytes. Disruption of figla gene by CRISPR/Cas9 led to an all-male phenotype in the mutant. Detailed analysis of early gonadal development showed that the germ cells in the mutant were clustered in cysts and underwent meiosis, forming oocytes at prefollicular chromatin nucleolar (CN) stage (stage IA). However, the subsequent transition from cystic CN oocytes to individual follicular perinucleolar oocytes (stage IB) was blocked, resulting in an all-male phenotype in the mutant. The phenotype of figla mutant could not be rescued by estrogen treatment, in contrast to cyp19a1a mutant, and introduction of tp53 mutation also had no effect, unlike in fancd1 and fancl mutants. Transcriptome analysis revealed that many biological processes and pathways related to germ cell development, especially oogenesis, were upregulated in the presence of Figla and that the regulation of figla expression may involve heat shock proteins. Our results strongly suggest important roles for Figla in juvenile ovary development, especially the formation of individual follicles from cystic oocytes.

Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

Clonal relationship of tumor nodules in hepatocellular carcinoma: a hierarchical clustering analysis of comparative genomic hybridization data.

Information on the clonal relationship between tumor nodules in patients diagnosed with hepatocellular carcinoma (HCC) holds prognostic significance in the prediction of recurrence and postoperative treatment. Here, we investigated the clonal relationships in 11 focal nodules from 5 patients with HCC by comparative genomic hybridization (CGH) and spectral karyotyping. CGH analysis indicated tumor nodules in 5 (100%) of 5 cases to share similar patterns of genomic imbalances, suggesting nodules to be clonally related. This clonal relationship was further substantiated in 2 cases, where spectral karyotyping analysis indicated identical structural rearrangements between focal tumors. Few studies have attempted to differentiate multicentric growth from intrahepatic dissemination in HCC using CGH analysis; however, data interpretation remained subjective. In this study, we have extended our data to include published CGH findings on 57 nodules derived from 19 HCC cases. Simulation of CGH findings by unsupervised 2-way hierarchical clustering indicated tumor nodules in 20 (83.3%) of 24 cases to cluster concordantly, signifying a high incidence of clonal similarity. In sum, bioinformatic analysis of genomic profiles may represent a reliable research tool in analyzing clonal relationships among tumor nodules.