Vasoactive intestinal peptide indirectly elicits pituitary LH secretion independent of GnRH in female zebrafish

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhβ mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild type and gnrh3-/- females at 1 hour post-treatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA’s manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and E2 in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

What do zebrafish prefer? Directional and color preferences in maze tasks

Studies regarding the animals innate preferences help elucidate and avoid probable sources of bias and serve as a reference to improve and develop new behavioral tasks. In zebrafish research, the results of innate directional and color preferences are often not replicated between research groups or even inside the same laboratory raising huge concerns on the replicability and reproducibility. Thus, this study aimed to investigate the male and female zebrafish innate directional and color preferences in the plus-maze and T-maze behavioral tasks. As revealed by the percentage of time spent in each zone of the maze, our results showed that males and females zebrafish demonstrated no difference in directional preference in the plus-maze task. Surprisingly, male and female zebrafish showed color preference differences in the plus-maze task; males did not show any color preference, while female zebrafish demonstrated a red preference compared to white, blue, and yellow colors. Moreover, both male and female zebrafish demonstrated a strong black color preference compared to the white color in the T-maze task. Thus, our results demonstrate the importance of innate preference assays involved with the directionality of the apparatus or the application of colors as a screening process conducting behavioral tests (e.g., anxiety, learning and memory assessment, locomotion, and preference) and highlight the need to analyze sex differences.

Social Stress Increases Anxiety-Like Behavior Equally in Male and Female Zebrafish

Zebrafish anxiety-like behavior was assessed in the novel tank test after the formation of dominant-subordinate hierarchies. Ten pairs of animals were subjected to dyadic interactions for 5 days, and compared with control animals. After this period, a clear dominance hierarchy was established across all dyads, irrespective of sex. Social status affected parameters of anxiety-like behavior in the novel tank test, with subordinate males and females displaying more bottom-dwelling, absolute turn angle, and freezing than dominant animals and controls. The results suggest that subordinate male and female zebrafish show higher anxiety-like behavior, which together with previous literature suggests that subordination stress is conserved across vertebrates.

Nuclear progesterone receptor regulates blood coagulation facilitating ovulation in zebrafish

Ovulation is a remodeling process including blood capillary rupture and coagulation. Until now, there is no regulation and functional studies of coagulation factors in ovulation. Here, we report dramatic increases of coagulation factors (f5, f3a) in zebrafish preovulatory follicles. This upregulation was induced by progestin (DHP: 17α, 20β-dihydroxy-4-pregnen-3-one), a native ligand for nuclear progestin receptor (Pgr) that is essential for ovulation in zebrafish; but was abolished in pgr-/-. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin via zebrafish Pgr. Similarly, we found promoter activities of human F5 were significantly stimulated by progesterone (P4) via human PGRB. Moreover, a dramatic increase of erythrocyte numbers in capillaries on ovarian follicles was associated with ovulation. Importantly, heparin, an anticoagulant, inhibited ovulation. Furthermore, reduced fecundity and impaired ovulation were observed in f5+/- female zebrafish. Together, our results provide plausible evidence for an exceptional function of coagulation factors in ovulation.

Long-Term Exposure of Binary Mixture of Cadmium And Mercury Through Alteration of Nrf2 Signalling Pathway Damages The Developed Ovary of Zebrafish

The toxicity of the binary mixture of cadmium (Cd) and mercury (Hg) on the ovary of adult zebrafish was evaluated in the present study. Adult female zebrafish were exposed to cadmium chloride (1 mg/L), mercury chloride (30 µg/L) and a binary mixture of both metals for 21 days. The toxic effects of both metals on the ovary were investigated by evaluating the oxidative stress markers and related gene expression in ovarian tissue along with the histopathological examination. The significantly decreased level of GSH and increased level of MDA in ovarian tissue of adult female zebrafish exposed to Cd + Hg indicated that the exposure of binary mixture of Cd and Hg caused more lipid peroxidation in the ovary. The decreased expression of mRNA of catalase (CAT) and Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) were observed in the ovary of zebrafish exposed to binary mixture. The down regulation of Nrf2 might be responsible for suppression of the antioxidant system which resulted in cellular alterations in the ovary. Upon histological evaluation, a decreased number of full-growth (mature) oocytes along with degenerative changes due to Cd exposure were noticed. While, ovary of zebrafish of Hg-exposed group had shown decreased number of pre- and early-vitellogenic oocytes along with atretic pre-vitellogenic oocytes compared to the control group. The ovary of zebrafish of Cd + Hg-exposed group had shown decreased number of pre-vitellogenic oocytes with marked pathological changes in mature oocytes. Present findings elucidate that simultaneous long-term exposure of Cd and Hg may significantly affect the ovary through alteration of Nrf2 mediated cascade in adult zebrafish.

Effects of Di-Isononyl Phthalate (DiNP) on Follicular Atresia in Zebrafish Ovary

Di-isononyl phthalate (DiNP) is a plasticizer reported to elicit hormone-like activity and disrupt metabolism and reproduction in fish and other vertebrates. In general, phthalates have been used at high concentrations beyond reported environmental levels to assess their adverse effects on fish gonadal physiology. The present study exposed adult female zebrafish to a wide range of DiNP concentrations [0.42 µg L−1 (10−9 M), 4.2 µg L−1 (10−8 M), and 42 µg L−1 (10−7 M)] for 21 days. We evaluated gene expression profiles related to apoptosis, autophagy, and oxidative stress; DNA fragmentation (TUNEL assay: terminal deoxynucleotidyl transferase dUTP nick end labeling) and caspase activity (CAS3) were also examined. Exposure to 0.42 and 4.2 µg L−1 upregulated the genes coding for tnfa and baxa, sod1, prkaa1, respectively. CAS3 immunohistochemistry revealed a higher number of positive vitellogenic oocytes in ovaries exposed to 0.42 µg L−1. Subsequently, we examined the relationship between CAS3 signaling and DNA fragmentation. Accordingly, DNA fragmentation was observed in vitellogenic follicles of fish exposed to 0.42 and 4.2 μg L−1. Our results demonstrate that follicular atresia can occur after exposure to environmental levels of DiNP for 21 days, which may adversely affect the reproductive performance of female zebrafish in a non-monotonic manner.