Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

Abstract

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the <i>scg2a</i> and <i>scg2b</i> genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (<i>n</i> = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (<i>P</i> = 0.006), 44% (<i>P</i> = 0.0169), and 6% (<i>P</i> < 0.0001) for <i>scg2a</i>^<i>-/-</i> , <i>scg2b</i>^<i>-/-</i> , and <i>scg2a</i>^<i>-/-</i><i>;scg2b</i>^<i>-/-</i> mutants, respectively. Comprehensive video analysis indicates that <i>scg2a</i>^<i>-/-</i><i>;scg2b</i>^<i>-/-</i> mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected <i>scg2a</i>^<i>-/-</i><i>;scg2b</i>^<i>-/-</i> double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (<i>P</i> = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in <i>scg2a</i>^<i>-/-</i><i>;scg2b</i>^<i>-/-</i> fish compared to WT injected with secretoneurin-a (<i>P</i> < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in <i>scg2a</i> and <i>scg2b</i> mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.