Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish

The regularity of Piwi-interacting RNA (piRNA) biogenesis is crucial to germline development. Functioning as Piwi-interacting proteins, Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and the piRNA pathway. In this study, zebrafish tdrd12 was identified, and the maternal and germ cell-specific expression patterns of zebrafish tdrd12 were observed. Utilizing TALEN (transcription activator-like effector nuclease) techniques, two independent tdrd12 mutant zebrafish lines were generated. Although no defects were found during the generation of the primordial germ cells (PGCs) in the tdrd12-null fish progenies obtained from the heterozygous tdrd12 mutant parents, all Tdrd12-deficient fish developed into infertile males. The reduced numbers and eventually loss of the germ cells by 35 days post fertilization (dpf) led to masculinization and infertility of the Tdrd12-deficient fish. Meiosis defects of the germ cells in the tdrd12 mutants during the gonad-transitioning period were observed, revealing the indispensable functions of Tdrd12 in gametogenesis. Our studies demonstrated that zebrafish Tdrd12 is essential for germ cell development and maintenance

High Photocurrent Density and Continuous Electron Emission Characterization of a Multi-Alkali Antimonide Photocathode

High photocurrent density cathodes that enable small cross-section electron beams are required for high-power terahertz vacuum devices. Multi-alkali antimonide photocathodes may be well suited for generating sub-mm electron beam sources. This paper involves the repeatability, stability, uniformity, and linearity experiments of the multi-alkali antimonide photocathodes electron emission operations under a continuous-wave 450 nm laser with a bias voltage of 5000 V. The effect of heat, electric contact, and cathode surface roughness to emission characterizations is analyzed. The methods to maintain the high-current-density emission and avoid the fatigue of the photocathode are verified. The emission can be repeated with increased optical power. The stable photocurrent density of near 1 A/cm2 and maximum current density of near 1.43 A/cm2 is recorded. The continuous photocurrent density is significantly improved compared to the current density reported in traditional applications. It is found that the current curves measuring at different areas of the photocathode differ greatly after the laser power of 800 mW. The increase in current for some areas may be attributed to the conductive current caused by built-in electric fields between the emission rough area and its adjacent areas

Sufficient numbers of early germ cells are essential for female sex development in zebrafish.

The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd) gene was identified. Utilizing the metrodinazole (MTZ)/ bacterial nitroreductase (NTR) system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP(-3'UTR)) and Tg (dnd:NTR-EGFP(+3'UTR)) zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies

Zebrafish gonad mutant models reveal neuroendocrine mechanisms of brain sexual dimorphism and male mating behaviors of different brain regions

Abstract Background Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis enzyme cyp17a1-deficient zebrafish to investigate the differences and interplays in the brain–gonad–behavior axis, and the molecular control of brain dimorphism and male mating behaviors. Methods Tdrd12 +/−; cyp17a1 +/− double heterozygous parents were crossed to obtain tdrd12 −/− ; cyp17a1 +/+ (tdrd12 KO), tdrd12 +/+; cyp17a1 −/− (cyp17a1 KO), and tdrd12 −/− ; cyp17a1 −/− (double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid hormone levels (testosterone, 11-ketotestosterone and 17β-estradiol) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis. Results Qualitative analysis of mating behaviors demonstrated that tdrd12 −/− fish behaved in the same manner as wild-type males to trigger oviposition behavior, while cyp17a1 −/− and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain–gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile of tdrd12 −/− fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles of cyp17a1 −/− and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish. Conclusions Our results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain–gonad–behavior axis. Moreover, this study revealed the crucial candidate genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish. Graphical Abstrac

Gonadal expression of the <i>dnd^+3’UTR</i>:NTR-EGFP and <i>dnd^-3’UTR</i>:NTR-EGFP transgene in adult zebrafish.

<p>Anatomical views of the gonadal expression of the NTR-EGFP transgene are presented in A-D; Views of NTR-EGFP trangene expression and histological features with HE staining in the cyosectioned gonadal tissue are presented in E-H and I-L; A and B) Expression of the transgene in zebrafish ovary (A) and testis (B) in <i>dnd^+3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; C and D) Expression of the transgene in zebrafish ovary (C) and testis (D) in <i>dnd^-3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; E and F) NTR-EGFP transgene expression in the cryosections of the ovary (E) and testis (F) of the F1 progenies of <i>dnd^+3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; G and H) NTR-EGFP transgene expression in the cryosections of the ovary (G) and testis (H) of the F1 progenies of <i>dnd^-3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; I-J) histological features with HE staining for the I-J) shows the HE staining for the cryosections of the ovary (I) and testis (J) of the F1 progenies of <i>dnd^+3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; K-L) shows the HE staining for the cryosections of the ovary (K) and testis (L) of the F1 progenies of <i>dnd^-3’UTR</i>:NTR-EGFP transgenic fish at 100 dpf; Fluorescence was observed in the oocytes at all stages (E, G, I, K) and in spermatocytes(sc) and sperm(sp) (F, H, J, L).</p