Electrophysiological Differences Between fshb- and lhb-Expressing Gonadotropes in Primary Culture

Abstract

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²⁺ responses were similar. Expression of Ca²⁺-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²⁺ influx.

Gnrh1-induced responses are indirect in female medaka Fsh cells

“…To our surprise, we did not co-localize any gnrhr with fshb . This contradicts previous reports of gnrhr expression in fshb cells from Atlantic cod (19, 54) and tilapia (28). This disparity may be due to species or sex differences, or to differences in methodology.…”

“…Activation of GnRH receptors on gonadotrope cells elicit increased cytosolic Ca 2+ levels ([Ca 2+ ] i ) with a subsequent alteration in membrane potential and hormone release by exocytosis. A similar response to Gnrh has been shown also in teleost fish (19–25), although a Gnrh pulsatile or frequency dependent release of gonadotropins has not been demonstrated.…”

“…This disparity may be due to species or sex differences, or to differences in methodology. The work in Atlantic cod was performed on primary dissociated pituitary cells pooled from both sexes and conducted at 2 to 7 days after plating (19, 54). This difference in timing may be important, as a previous study found that gnrhr expression increased in pituitary cell culture over time (55), suggesting that either gnrhr expression increases in cells already expressing the receptor or that new cells start to express gnrhr .…”

Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion

“…These channels are activated by elevation in cytosolic Ca 2+ and play a critical role in control of firing properties of excitable cells (35), including pituitary cells (36). The expression of SK channels is well documented in fish (37), rat (38, 39), mouse (40), and ovine gonadotrophs (17), and the level of their expression is dependent on estradiol (20). Whole-cell current recordings confirmed the presence of BK current in several pituitary cell types but not in gonadotrophs (16).…”

Gonadotropes in medaka grow long extensions with varicosity-like swellings, projecting towards each other and blood vessels

“…In transgenic medaka (Oryzias latipes) that express green fluorescent protein (Gfp) under the control of the endogenous lhb promotor (tg(lhb:hrGfpII); see Hildahl et al, 2012), Lh-cells form long extensions in primary culture , and these differ from those previously described in gonadotrope cells from teleosts (Golan et al, 2016b;Hodne et al, 2013) and mammals (Alim et al, 2012;Childs, 1985;Navratil et al, 2007). This observation prompted us to investigate these extensions in more detail.…”