Secretion of 23 kDa and glycosylated prolactin by rat pituitary cell culture in serum-free media: a comparative morphological, cyto- and immunochemical study

Abstract

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 keratinocyteserum-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 serumfree 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.

Routing, Processing and Export of Rat Pituitary Prolactin: Identification of a 36 kDa Disulphide-bridged Oligomeric Preprolactin

“…If glycosylation of rPRL should be a partly ER event, by remaining longer in the ER one might expect a glycosylation pattern different from the basal one (control pituitary cells). This is not the case, which suggests that the formation of glycosylated rPRL is a pure posttranslational Golgi event and reinforces the evidence we accumulated in the last decade of 26 kDa rPRL being an O-linked glycoform (Bollengier et al, 1995).…”

“…Cell culture: anterior pituitaries were processed as formerly described , and the obtained pituitary cells were then grown as recently reported (Bollengier et al, 1995(Bollengier et al, , 1996. Briefly resumed, cells were cultured for 48 h in total culture medium (DMEM supplemented with 10% FCS, TES and antibiotics), then depleted of this culture medium, and further cultured in Keratinocyte-SFM supplemented with NaSe, transferrin, insulin, T 3 , albumin, CaCl 2 and antibiotics.…”

“…Vertical SDS-PAGE, transfer electrophoresis and PRL specific immunoblotting was as formerly described (Bollengier et al, 1995(Bollengier et al, , 1996. Briefly resumed, vertical SDS-PAGE was carried out in 10-20% gradient gels; the sample buffer was Tris (0.01 M), EDTA (0.001 M), 1% SDS, pH 8.0 and the gel buffer Tris (0.33 M), pH 8.6.…”

“…Previously (Bollengier et al, 1995) we gained a first insight in the basal secretion of rat prolactin molecular size (Mr) isoforms, the major findings being that monomeric 23 kDa rat prolactin (rPRL) is the dominant secretory product of rat pituitary lactotroph cells and 26 kDa rPRL the preferential secreted glycoform. Recently (Bollengier et al, 1996) we investigated the change of both the molecular size heterogeneity and the relative distribution of rPRL variants, synthesized and secreted by rat pituitary cells under different physio-logical stimuli.…”

“…The statistical method used to compare the quantitative parameters was the non-parametric U-test of Wilcoxon (Mann-Whitney) (Bollengier et al, 1995).…”