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Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Toll-like receptor (TLR) signaling is conserved between fish and mammals, except for TLR4, which is absent in most fish. In the present study, we aimed to evaluate whether TLR4 is expressed in Schizothorax prenanti ( SpTLR4 ). The SpTLR2 and SpTLR4 were cloned and identified, and their tissue distribution was examined. The cDNA encoding SpTLR4 and SpTLR2 complete coding sequences (CDS) were identified and cloned. Additionally, we examined the expression levels of seven SpTLRs ( SpTLR2 , 3 , 4 , 18 , 22-1 , 22-2 , and 22-3 ), as well as SpMyD88 and SpIRF3 in the liver, head kidney, hindgut, and spleen of S. prenanti , after intraperitoneal injection of polyinosinic-polycytidylic acid (poly (I:C)). The Sp TLR2 and Sp TLR4 shared amino acid sequence identity of 42.15-96.21% and 36.21-93.58%, respectively, with sequences from other vertebrates. SpTLR2 and SpTLR4 were expressed in all S. prenanti tissues examined, particularly in immune-related tissues. Poly (I:C) significantly upregulated most of the genes evaluated in the four immune organs compared with the PBS-control ( p < 0.05); expression of these different genes was tissue-specific. Our findings demonstrate that TLR2 and TLR4 are expressed in S. prenanti and that poly (I:C) affects the expression of nine TLR-related genes, which are potentially involved in S. prenanti antiviral immunity or mediating pathological processes with differential kinetics. This will contribute to a better understanding of the roles of these TLR-related genes in antiviral immunity.

Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94−100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.

We identified and cloned cDNA encoding the heat shock protein (Hsp) 27 gene from Schizothorax prenanti (SpHsp27), and compared its expression with that of SpHsp60, SpHsp70, and SpHsp90 in the liver, head kidney, hindgut, and spleen of S. prenanti that were injected with polyinosinic-polycytidylic acid [Poly (I:C)]. The SpHsp27 partial cDNA (sequence length, 653 bp; estimated molecular mass, 5.31 kDa; theoretical isoelectric point, 5.09) contained an open reading frame of 636 bp and a gene encoding 211 amino acids. The SpHsp27 amino acid sequence shared 61.0−92.89% identity with Hsp27 sequences from other vertebrates and SpHsp27 was expressed in seven S. prenanti tissues. Poly (I:C) significantly upregulated most SpHsps genes in the tissues at 12 or 24 h (p < 0.05) compared with control fish that were injected with phosphate-buffered saline. However, the intensity of responses of the four SpHsps was organ-specifically increased. The expression of SpHsp27 was increased 163-fold in the head kidney and 26.6-fold SpHsp27 in the liver at 24 h after Poly (I:C) injection. In contrast, SpHsp60 was increased 0.97−1.46-fold in four tissues and SpHsp90 was increased 1.21- and 1.16-fold in the liver and spleen at 12 h after Poly (I:C) injection. Our findings indicated that Poly (I:C) induced SpHsp27, SpHsp60, SpHsp70, and SpHsp90 expression and these organ-specific SpHsps are potentially involved in S. prenanti antiviral immunity or mediate pathological process.

Minimally invasive sampling was used to determine the sex of Chinese giant salamanders (Andrias davidianus). Urine samples (n = 25) were collected from 6 adults in the breeding season and from 19 individuals (7 adults and 12 juveniles) in the non-breeding season. The hormone testosterone (T) and estrone-3-glucuronide (E1G) in urine were collected from Chinese giant salamanders (CGSs), and the hormone extracts were analyzed by enzyme immunoassays (EIA). The data demonstrated that the urine T concentration of the male CGSs was significantly higher than that of the females during the breeding season (p < 0.05) and even more pronounced during the non-breeding season (p < 0.01). The urine E1G concentration of the males was less pronounced than that of the females during the breeding season (p < 0.01) and significantly lower during the non-breeding season (p < 0.05). The urine T/E1G values of all the male salamanders were significantly higher than those of the females (p < 0.01) during both the breeding season and the non-breeding season. An interesting pattern was found in this study: the value of urine log10(T/E1G) of the male CGSs was higher than 1, whereas the value for the females was lower than 1, during both the breeding and non-breeding seasons, and in the adult and sub-adult age groups of CGSs. There were 25 salamanders in this study and the accuracy rate reached 100% by using a log10(T/E1G) value of 1. The results of the log10(T/E1G) value provide new insight into the future development of the sex identification of CGSs and also lay the foundation for accurate sex identification in the preparation for artificial release. This is the first study to show that the T/E1G ratio in urinary hormones is reliable for the sex identification of CGSs. Additionally, urinary hormone T/E1G measures are promising sex identification tools for amphibian or monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.

N-3 highly unsaturated fatty acids (n-3 HUFA) have been shown to inhibit body fat accumulation in animals. To clarify the mechanism of this fat-lowering effect of n-3 HUFA in grass carp (Ctenopharyngodon idellus), two experiments were conducted. In experiment 1, isolated grass carp mature adipocytes were incubated with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at different concentrations for 6 h. The release of glycerol to the medium was detected, and the expression of the lipolysis-related genes was analyzed. In experiment 2, a 95-day feeding trial was conducted with two diets formulated with either lard oil (as control) or fish oil (supplying n-3 HUFA as treatment) as the main lipid source. The glycerol and free fatty acid (FFA) released from the isolated adipocytes of both groups were detected after the feeding period. The expression of select lipolysis-related genes in adipose tissue was also analyzed. The results from experiment 1 showed that the release of glycerol was significantly increased by DHA and EPA (P < 0.05). Moreover, the expression of lipolysis-related genes, such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), tumor necrosis factor α (TNFα) and leptin, was also significantly elevated in the treatment group (P < 0.05). Experiment 2 demonstrated that glycerol and FFA release from the isolated adipocytes were significantly higher in the treatment group compared to the control group (P < 0.05). The expression level of ATGL, HSL, TNFα and leptin in the treatment group was significantly higher than in the control group (P < 0.05). The present results provide novel evidence that n-3 HUFAs could regulate grass carp adipocyte lipolysis in vitro or in vivo, and the effect might be in part associated with their influence on the expression of lipolysis-related genes and lipolysis-related adipokines genes.