SECRETION OF 23-KDA AND GLYCOSYLATED PROLACTIN BY RAT PITUITARY CELL-CULTURE IN SERUM-FREE MEDIA - A COMPARATIVE MORPHOLOGICAL, CYTOCHEMICAL AND IMMUNOCHEMICAL STUDY

The secretion of 23 kDa prolactin by rat pituitary cells has been thoroughly investigated, but secretion of glycosylated rat prolactin is not currently known. This is mainly due to the lack of an antiserum which is solely specific for glycosylated rat prolactin and therefore we studied the basal secretion of this variant by an indirect method. Rat pituitary cells were cultured in total culture medium and three different serum-free media (DMEM, keratinocyte-serum-free medium, protein-free hybridoma medium) and secretion of 23 kDa and glycosylated rat prolactin was recorded by radioactive techniques and immunoblotting. The pituitary cell quality was monitored by electron microscopy, cell activation-and cell death assessment. In short-range culture (2 days) the pituitary cell quality and behaviour was very good and comparable in total culture medium, DMEM and keratinocyte-serum-free medium, i.e. numerous secretory granules, moderate amount of ER, cristae well in place in the mitochondriae. In medium-range culture (8 days) only cells cultured in total culture medium and DMEM presented a parallel behaviour: migration of cells toward each other, marked degranulation, massive array of ER. The inner membrane of the mitochondria was no longer folded into cristae leaving an unoccupied central space. At day 2 of the culture span secretion of 23 kDa rat prolactin was very comparable in all media used; hereafter, secretion of 23 kDa rat prolactin in total culture medium and DMEM assumed the well known pattern of peaking and slowing down, whereas in the other serum-free media it steadily decreased over the culture span. Pertaining to the important novel point of glycosylated rat prolactin secretion, it was low in comparison to the one of 23 kDa rat prolactin and it assumed a near steady pattern in all media used. 26 kDa rat prolactin was identified as the preferentially secreted glycoform, and the 23 kDa isoform as the major secretory product of rat pituitary lactotroph cells.