Electron‐microscopic cytochemical studies on the secretory process in rat prolactin cells in primary culture

Abstract

Three aspects of the secretory process in male rat prolactin (PRL) cells grown in primary cultures for 7--14 days have been investigated by cytochemical methods. The subcellular localization of prolactin has been studied using preembedding or postembedding immunocytochemical methods after various fixatives. With the postembedding method, PRL is localized essentially in secretory granules. The maximum intensity of staining is obtained with PAF fixative. With the preembedding method, subcellular localization of the staining varies depending on the fixative. After PAF-fixation, positive staining is observed on secretory granules, ground cytoplasm, the outer face of some RER cisternae and, in a few cells, on the innermost Golgi cisternae, as well as on masses of condensing secretory material. After Ohtsuki's hypotonic fixative followed by saponin permeabilization, PRL is visualized within the totality of RER cisternae, including the perinuclear cisternae and the peripheral saccules on the cis-Golgi face. Secretory granules are unstained. Membrane traffic was investigated using the Con A-HRP indirect method as a tracer of surface saccharides. Plasma membrane, coated with Con A-HRP at 4 degrees C, is slowly internalized at 37 degrees C. This involves both randomly distributed invaginations and capping. The final step of endocytosis (1--2 hours) is located in the Golgi zone, where very few smooth membranes are stained. In contrast, a conspicuous deposit is found around the dense content of secretory granules. This suggests a recycling of internalized membrane and a transfer of Con A-HRP from the inner face of smooth cisternae to the secretory material. The internalization of Con A-HRP-coated membrane leads to an inhibition of PRL release starting after 30 minutes. This is accompanied by a marked increase of acid phosphatase activity, mostly around forming and mature secretory granules.

Axons Containing a Prolactin-Like Peptide Project into the Perivascular Layer of the Median Eminence: An Immunocytochemical Light and Electron Microscope Study in Adult and Infant Rats

“…a gift from NIAMDD). As previously reported, these antisera do not cross-react with growth hormone or with any other pituitary hor mone [26]. The specificity of the antisera for hypophysial prolactin was further assessed by immunoblotting for rat anterior pituitary homogenates ( fig.…”

Ultrastructural Characterization of Prolactin-Like Immunoreactivity in Rat Medial Basal Hypothalamus

“…With respect to ultrastructural-immunocytochemicai tech nique, the present results can be compared with those from rat pituitary prolactin cells in culture [20], There, also, specificity of immunoreactivity was confirmed with preadsorption controls, and the preembedding method was used successfully. Inter estingly, the intensely stained granules, about 200 nm in diameter, in that study matches quite well with the large immunoreactive granules in neurons, found in the present work.…”

Proteolytic Processing of Sulfated Secretogranin II in the trans-Golgi Network of GH3B6 Prolactin Cells

“…The acidification of pH may thus be responsible for both the aggregation and the cleavage of SgII in the TGN. Ultrastructural studies have evidenced the aggregation of proteins in specific regions of the more distal part of the TGN in endocrine cells (48), including prolactin cells (49,50). The TGN is a very dynamic structure whose size and shape vary greatly according to the cell type and activity (51,52).…”