Transcript trends of leptin system and selected fertility markers in post-spawning zebrafish ovary

Leptin is an adipocytokine produced by various organs, with an evident role on reproduction in mammals. In most teleosts, including zebrafish (Danio rerio), leptin has two paralogues, leptin-a (lepa) and leptin-b (lepb), and a single cognate receptor, leptin receptor (lepr). In these taxa, the leptin system may influence puberty and later sexual maturation stages, but its role in regulating female zebrafish reproduction remains poorly understood. In wild-type zebrafish (WT), the ovary lepb displays peak expression levels over the other organs, while the expression of lepa and lepr is minimal. Subfertility — lower eggs per spawning and fertilisation rate, partial anovulation, slower oocyte maturation, and increased atresia — was shown in female lepr -/- knockdown (loss of function) strains, while various genes related to steroidogenesis, oocyte maturation, ovulation, and atresia were differentially expressed compared to WT [1; 2]. Our study aims to explore the trends and localization of leptin system transcripts in WT ovaries to better characterise their role in late stages of female reproduction. Six-month old female AB strain WT were synchronised (purging by natural mating) and selected when laid > 50 eggs. Latter individuals mated again after 7 days, and were re-selected for the quality and quantity of spawned eggs. Five to 8 individuals were anaesthetized and euthanized at 0, 2, 4, 6, 8, 10, 12, and 14 days post spawning (dps). The full excised left ovary was collected for RNA extraction and the rest of the animal was FFPE for histology. RT-qPCR was conducted for lepa, lepb, lepr, and for selected transcripts related to steroidogenesis (cyp19a1a, star, hsd3b1), oocyte maturation (pgr, pgrmc2) or ovulation (cpla2) previously found to be up- or down-regulated in female lepr -/- [1]. ef1a and rpl13a were used as housekeeping genes. Animal experiments followed Swedish Ethical Committee guidelines and approval in Uppsala. Spearman's rho correlation analysis was performed to assess the relationship between different targets across all time-points collectively, as well as parametric to non-parametric tests to compare single genes at different time points post-spawning. The leptin system 2^-(ΔCt) is one to two orders of magnitude smaller compared to other targets. lepr is significantly and positively correlated with lepb (0.467, p: 0.001), cpla2 (0.466, p: 0.001), and pgr (0.409, p: 0.005). Overall star, hsd3b1, pgr, pgrmc2, and cpla2 show a significant positive correlation between them and against the GSI (gonado-somatic index), while cyp19a1a has a different correlation trend being negatively correlated with the GSI (#0.475, p < 0.001). In a less-than-significant manner, lepa shows its peak at 0 dps, lepb tends to be higher at 12 dps compared to the other time points, while lepr fluctuates peaking at 0 and 6 dps. hsd3b1, pgr, pgrmc2, and cpla have an increasing trend and are significantly upregulated at 8-12 dps, while cyp19a1a has a significant peak at 0 dps and, successively, drops sharply. star remains stable over time. In conclusion, while the leptin system appears crucial in zebrafish reproduction, its dynamic temporal trends post-spawning revealed in this study need collateral investigation through different methods. ISH, histological characterization of the follicular population, and further statistical analysis are ongoing.