Activation of Novel Secretoneurin Circuits in Ovulating Zebrafish
Creators: name: Spadacini, Victoria
Publisher: name: Université d'Ottawa | University of Ottawa
Year: 2024
Subjects: reproduction
endocrinology
ovulation
zebrafish
hormone
pituitary

description: The hypothalamus-pituitary-gonad (HPG) axis is central to vertebrate reproduction. Hypothalamic neurons release the gonadotropin-releasing hormone (Gnrh) which stimulates the synthesis and release of the gonadotropins hormone (Lh) and follicle stimulating hormone (Fsh) in the anterior pituitary. Gonadotropins trigger and regulate essential reproductive events including spermatogenesis, gametogenesis, oocyte maturation, ovulation, and modulates sexual behaviours. Gnrh is considered the initiator of reproduction because it stimulates the Lh surge that in turn triggers ovulation. However, the novel neuropeptide secretoneurin (SN) can stimulate Lh release independent of Gnrh and mutations of the two SN precursor genes (scg2a and scg2b) result in significant reproductive deficits in zebrafish, most notably in ovulation and courtship behaviours. Thus far, there have been no demonstrations that SN-producing neurons are specifically activated prior to or during ovulation; this critical step is the focus of this thesis project. We tested the hypothesis that SNa is involved in the ovulatory process in female zebrafish because neurons are activated prior to ovulation. The main objective of this project was to determine when during the ovulatory cycle SNa neurons are being activated. We first validated a mouse anti-phospho-ERK1/2 primary antibody not yet tested in zebrafish and verified phospho-ERK as an effective biomarker for neuron activation through blocking immunoreaction with a phospho-peptide of the targeted ERK epitope and glutamatergic stimulation of key neuroendocrine regions. Using this antibody, activation of SNa neurons in the preoptic area (POA) was investigated as well as SNa action on Lh cells in the pituitary following SNa intraperitoneal injection. In the preoptic area, magnocellular neurons immunoreactive for SNa were progressively activated within the periovulatory window, with activation levels peaking at the time of presumptive ovulation (8:30h). At the time of the Lh surge (5:00h), 9.9% of SNa positive cells were active in the POA, increasing to 21% at ovulation. Intraperitoneal injection of a low (1 nmol/g) and high (2.5 nmol/g) dose of SNa in mature females isolated from males stimulated ovulation to a level equivalent to those injected with human chorionic gonadotropin (hCG). The low and high dose stimulated ovulation in 58% (7/12) and 75% (9/12) females respectively, compared to 83% (11/12) in the hCG group. Additionally, injections of both doses resulted in activation of Lh pituitary cells, with a 5.5-fold increase observed in ovulated fish injected with high dose SNa compared to unovulated saline injected controls. These results demonstrate that SNa neuronal networks are active prior to ovulation and are likely modulating Lh cell activity in the pituitary. These data support the hypothesis that the Scg2/SN system plays a stimulatory role as a reproductive hormone in zebrafish.