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

Abstract

___________________________________________________________________________Abbreviations: ACTH, Adrenocorticotropic hormone (mammals); DiI, lipophilic carbocyanine dye; FSH/Fsh, Follocle-stimulating hormone protein (mammals/fish); GFP, green fluorescent protein; GnRH/Gnrh, Gonadotropin releasing hormone protein (mammals/fish); IQR, interquartile range; LH/Lh, Luteinizing hormone protein (mammals/fish); Lhβ/Lhβ, Luteinizing hormone β subunit protein (mammals/fish); Lhb/lhb, Luteinizing hormone β subunit gene (mammals/fish); Tamra, 5/6-carboxy-tetramethyl-rhodamine succinimidyl ester AbstractAccumulating evidence in the scientific literature indicates that some pituitary cell types are organized in complex networks. Previous observations have indicated that this may also be the case in medaka (Oryzias latipes), where long cellular extensions with varicosity-like swellings are formed by luteinizing hormone (Lh)-producing gonadotropes expressing green fluorescent protein. In this study, immunofluorescence of intact pituitaries reveal that Lh beta polypeptides are mainly located in the varicosity-like swellings and at the extremity of the extensions. Some extensions approach nearby Lh-producing cells, and other extensions are in close contact with blood vessels. To investigate whether these extensions may contribute to network formation, we followed their development using confocal and fluorescent microscopy on primary cultures.During the first two days in culture, the extensions initiated the formation of homotypic cellular networks and clustering. The extensions were classified as either major or minor. Major extensions were several cell diameters long, dependent on microtubules, and displaying varicosity-like swellings at regular intervals. Minor extensions typically protruded from the major, were significantly shorter and thinner, and dependent on actin. The swellings were dependent on both microtubules and actin. Flash photolysis of caged Ca 2+ showed that the signal was propagated along the major extensions, intensifying in each swelling, indicating a continuous structure. However, the Ca 2+ signal did not transfer to the next cell in the network, but was transferred between cells merged at their somas. In summary, Lh-producing gonadotropes in medaka display a complex cellular structure of extensions, possibly linked to communication with blood vessels and/or other gonadotrope cells.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion

“…We found that Lh and Fsh cells are similar in morphology. They have similar volume in juveniles and in adults and show, both in vivo and in vitro, extensions allowing networking as previously shown for Lh (Grønlien et al 2019). Here, we show that Lh and Fsh cells show similar behaviour as they connect and cluster in cell culture using these extensions.…”

“…In medaka, Lh cells have been found to participate in the plasticity of the pituitary during puberty through hypertrophy and estrogen-sensitive hyperplasia during puberty (Fontaine et al 2019). Lh cells have also been shown to make neuron-like projections allowing homotypic networks (Grønlien et al 2019), to express gnrh receptors (gnrhr), and to respond to gnrh stimuli by increasing their action potential frequency and intracellular calcium concentration (Strandabo et al 2013. However, very little is known about Fsh cells and if and how they contribute to pituitary plasticity during puberty.…”

Functional Pituitary Networks in Vertebrates

“…Long extensions have been described in fish gonadotropes both in situ and in culture ( 32 , 95 , 102 , 103 ). It is difficult to postulate as to the function of these processes but two plausible options are that these appendages are used for cell-cell communication or as a bridge between the cells and the vasculature.…”

“…It is difficult to postulate as to the function of these processes but two plausible options are that these appendages are used for cell-cell communication or as a bridge between the cells and the vasculature. One line of evidence from cultured medaka cells ( 102 ) suggests that these processes do not relay signals between cells but are instead used for cell motility which serve to bring the cells together thus promoting cell-cell contacts and the formation of a network. It is hard to evaluate whether the processes serve the same role in vivo as in the adult medaka pituitary the cells are highly clustered yet the projections are still observed ( 102 ).…”

“…One line of evidence from cultured medaka cells ( 102 ) suggests that these processes do not relay signals between cells but are instead used for cell motility which serve to bring the cells together thus promoting cell-cell contacts and the formation of a network. It is hard to evaluate whether the processes serve the same role in vivo as in the adult medaka pituitary the cells are highly clustered yet the projections are still observed ( 102 ). Findings from the zebrafish model suggest that these processes are more likely to operate as extensions of the cells toward the vasculature than for intercellular communication.…”