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.

Fish skin is mainly composed of the epidermis, dermis, and its derivative scales. There is a wide diversity in scale number in fishes, but the diversity of skin structure lacks systematic histological comparison. This research aimed to improve our understanding of the functional relationship between the scale number and the skin structure in freshwater fishes and to determine which ecological factors affect the scale number and skin structure. First, we presented a method to quantify skin structure in fish and histologically quantified the skin structure of 54 freshwater fishes. Second, we collected the scale number and habitat information of 509 Cyprinidae fishes in China and explored which ecological factors were related to their scale number. Third, common carp and scaleless carp were used as models to study the effects of scale loss on swimming. We found a strong negative correlation between scale thickness and scale number. The main factor affecting the skin structure of fishes was the species’ water column position, and the skin of benthic fishes was the most well-developed (thicker skin layers (dermis, epidermis) or more/larger goblet cells and club cells). The scale number was related to two factors, namely, temperature and water column position, and cold, benthic and pelagic adaptation may have contributed to increased scale numbers. Only in benthic fishes, the more well-developed their skin, the more scales. In common carp, scale loss did not affect its swimming performance. In summary, we suggest that there is a rich diversity of skin structure in freshwater fishes, and the scales of fish with well-developed skin tend to degenerate (greater number/smaller size/thinner, or even disappear), but the skin of fish with degenerated scales is not necessarily well developed.

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

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 interactions 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 by using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wide-types. The steroid hormone levels (testosterone, 11-ketotestosterone and estrogen) 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 key 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 key 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.