No abstract

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control reproduction in vertebrates. Using a transgenic line of medaka, in which green fluorescent protein expression is controlled by the endogenouslhbpromotor, we studied development and plasticity of Lh cells, comparing juveniles and adults of both genders. Confocal imaging and 3D reconstruction revealed hypertrophy and hyperplasia of Lh cells in both genders from juvenile to adult stages. We show that Lh cell hyperplasia may be caused by recruitment of existing pituitary cells that start to producelhb, as evidenced by time lapse recordings of primary pituitary cell cultures, and/or through Lh cell proliferation, demonstrated through a combination of 5-bromo-2′-deoxyuridine incubation experiments and proliferating cell nuclear antigen staining. Proliferating Lh cells do not belong to the classical type of multipotent stem cells, as they do not stain with anti-sox2. Estradiol exposurein vivoincreased pituitary cell proliferation, particularly Lh cells, whereas pituitarylhbandgpaexpression levels decreased. RNA-seq andin situhybridization showed that Lh cells express two estrogen receptors,esr1andesr2b, and the aromatase genecyp19a1b, suggesting a direct effect of estradiol, and possibly androgens, on Lh cell proliferation. In conclusion, our study reveals a high degree of plasticity in the medaka Lh cell population, resulting from a combination of recruitment and cell proliferation.

Reproductive function in vertebrates is stimulated by GnRH that controls the synthesis and release of the two pituitary gonadotropins, FSH and LH. FSH and LH, which regulate different stages of gonadal development, are produced by two different cell types in the fish pituitary. This is in contrast to the situation in mammals and birds, and it enables investigation of their differential regulation. In the present study, we used fluorescence in situ hybridization to show that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This result was confirmed by patch-clamp recordings and by cytosolic Ca2+ measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, responded to Gnrh1 by biphasic alteration in action-potential frequencies and cytosolic Ca2+ levels. In contrast, both Fsh and Lh cells are able to respond to Gnrh1 in brain-pituitary tissue slices both electrically and by elevating the cytosolic Ca2+ levels. Using Ca2+ uncaging in combination with patch-clamp recordings and cytosolic Ca2+ measurements, we show that Fsh and Lh cells form homotypic and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka are indirect and probably mediated via Lh cells.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhβ, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.