Secretogranin-2 Complete Gene Knockouts in Zebrafish Reveal Their Stimulatory Roles in Female Reproduction

Abstract

Secretogranin-2 (Scg2) is a precursor protein in secretory vesicles of neuroendocrine neurons and pituitary hormone-secreting cells. Enzyme processing of Scg2 yields the bioactive peptide secretoneurin (SN) and others. Evidence links SN to reproductive control, enhancing gonadotroph function in several species. We employed CRISPR-Cas9 to excise both the scg2a and scg2b genes and generate single and double knockout (DKO) zebrafish. To confirm the success of gene deletion, the knockout lines were rigorously validated through sequencing, peptide mass spectrometry and immunohistochemistry. The scg2a-/- and scg2b-/- lines were crossed to establish a DKO model to investigate the role of the scg2 genes in zebrafish reproduction. Morphometric assessments indicated that scg2b-/- and DKO females had increased weight but shorter body lengths. Behavioural analysis showed reduced quiver behaviour in male scg2a-/- and scg2b-/- fish, while significant delays in oviposition were observed for females in the scg2b-/- and DKO groups. Lower spawning rates were recorded for within-line crosses of scg2a-/-, scg2b-/- single KO, and DKO fish (28%, 24%, and 16.6%) compared to WT (64.5%), and all KO groups exhibited low fecundity. The unspawned DKO females could not spawn with WT males, evidencing female-specific spawning defects. Ovarian histology revealed a reduction in mature oocytes and increased atresia in the DKO ovary. The injection of synthetic SNa, SNb, or human chorionic gonadotropin did not restore spawning, highlighting severe reproductive dysfunction in DKO females. Molecular analysis using digital PCR for genes encoding neuropeptides and gonadotropins indicated that, at pre-ovulation, DKO females exhibited decreased gnrh3 and increased avp levels in the hypothalamus, along with upregulated pituitary lhb and fshb, and downregulated gnrhr2. Expression of oxt was significantly reduced in both telencephalon and hypothalamus during ovulation, whereas avp was highly expressed only in the hypothalamus. Additionally, during ovulation, gnrhr2 and cga were downregulated in the pituitary, while lhb and fshb levels remained elevated. Post-ovulation, oxt in the telencephalon was consistently downregulated, and levels of hypothalamic gnrh2, avp, and pituitary cga were also reduced compared to WT. Ovarian lhcgr and star1 expression showed significant reduction throughout the ovulation cycle, indicating impaired hormonal signalling and disrupted reproductive regulation in the HPG axis. The shape of the pituitary in DKO fish was altered, having an abnormal elongated shape compared to the teardrop-like oval shape in the WT pituitary. Single-pituitary proteomics identified 3674 proteins and revealed 51 significantly altered proteins between DKO and WT, indicating a major shift in pituitary function. These results demonstrate that scg2a and scg2b are crucial for reproduction in zebrafish, impacting spawning, oviposition timing, fecundity, oocyte maturation, and the regulation of reproductive hormones in the HPG axis. The secretograninergic system is, therefore, important for the regulation of fertility.