FRI273 Generation And In Vitro Characterization Of A Novel Immortalized Ovine Neuronal KNDy Cell Line

Abstract

Abstract Disclosure: R. Toyoizumi: None. C.Y. Markgraf: None. A. Starman: None. E. Rodriguez: None. B.L. Lee: None. R. Bahn-Bales: None. O. Stricklin: None. C. Estill: None. C.E. Roselli: None. P.E. Chappell: None. The multiphenotypic hypothalamic KNDy neurons play a critical role in the neuroendocrine regulation of reproduction, and via their stimulation of GnRH release, are requisite for fertility and pubertal progression. In vivo evidence has accumulated identifying this more caudal population of Kisspeptin neurons in mediating predominantly negative feedback effects of gonadal steroids, and their ability to synthesize and secrete kisspeptin (KP), neurokinin B (NKB/Tac3), and dynorphin (Dyn) implicates them as crucial regulators of GnRH pulse generation. We have previously generated murine immortalized arcuate KNDy neuronal cell lines to explore molecular mechanisms underlying the steroid hormone regulation of these cells, and in the current study, we characterize a novel immortalized ovine neuronal cell line model of KNDy (oKNDy) cells derived from fetal female sheep brain (∼133 days of fetal development). Clonal lines were derived from isolated mediobasal hypothalamic explants using lentiviral infection of neurons with plasmids encoding SV40 large T-antigen. These neurons express ovine kiss1, tac3, and pdyn, as well as steroid receptors esr1, esr2, and pgr. Similar to KNDy neurons in vivo, receptors for Tac3 (tacr3) and Dyn (oprk1) were also found to be expressed in our oKNDy cells. Further, low physiological concentrations (5-50pM) of 17β-estradiol (E2) were found to significantly repress kiss1 and tac3 expression, while having no effect on tacr3. Concentrations of E2 and progesterone (P4) approximating proestrous levels maximally repressed kiss1 and tac3 after 24h, with elevations in pdyn noted under the same conditions. While cells exposed to higher E2 levels exhibited greater kiss1/tac3 repression, P4 exposure alone revealed stimulation of expression with increasing P4. Since these neurons were derived from the short-day seasonally breeding sheep, they can also be used to explore how circannual timing cues derived from the pituitary pars tuberalis may exert gating signals on the reproductive axis as previously modeled in vivo. Consistent with this, we have found high levels of thyroid hormone receptor α (thra) expression in our oKNDy neurons, suggesting they can respond to locally-synthesized T3. We are continuing to characterize these oKNDy neuronal cells in culture in order to provide insight into GnRH pulse generation and construct molecular models of neuronal responsiveness to steroids and other regulatory inputs. Presentation: Friday, June 16, 2023