Flow cytometric analysis of functional anterior pituitary cells from female rats
Laser-light scatter signals generated from living cells provide useful information with regard to both cell size (forward-angle light scatter) and granularity (ninety-degree or perpendicular light scatter). By measuring angles of light scatter and fluorescence, a fluorescence-activated cell sorter is capable of analysing and sorting cells on the basis of their size, granularity and cell-surface fluorescence. Using an electronically programmable individual cell sorter we were able to analyse single, viable, dispersed anterior pituitary cells of the female rat on the basis of their laser light scatter characteristics. Two distinct populations of differing granularity were defined: 26 +/- 2.2% (mean +/- S.E.M.) were more granular and 74 +/- 3.5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest, whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependent upon the secretory state of the cell. Hypothalamic stimulating factors increased cell-surface labelling, whilst dopamine and somatostatin decreased labelling. These changes compare favourably with published data obtained by immunocytochemistry. Using dual-colour fluorescence cell surface labelling we were unable to define a population of cells secreting both prolactin and growth hormone (mammosomatotrophs).
[Show abstract] [Hide abstract] ABSTRACT: The presence of neuronal nitric oxide synthase (nNOS) in two populations of pituitary cells, gonadotrophs (LH) and folliculostellate (FS) cells, suggests that pituitary nitric oxide (NO) is involved in the control of hormone secretion. We have used single and double immunostaining and quantitative procedures to investigate possible gender-related differences in the nNOS expression pattern in the anterior pituitary lobe and its possible alterations in different endocrine situations. Our results reveal a sexual dimorphism in the pattern of nNOS expression. In males, nNOS is mainly found in FS cells, whereas only a few LH cells express nNOS. Conversely, in females, nNOS is mainly found in LH cells. After gonadectomy, paralleling an increase in LH cell size and serum luteinizing hormone (LH) levels, there is nNOS upregulation in LH cells and nNOS downregulation in FS cells. After testoterone replacement, LH cells become nNOS-immunonegative again. In lactating rats, LH cells overexpress nNOS, but LH cell size and serum LH levels are low. This suggests that, depending on its cellular source, pituitary NO can exert either an inhibitory or a stimulatory effect on hormone secretion. When released from FS cells, NO exerts a paracrine inhibitory effect, and when released from gonadotrophs it exerts an autocrine or paracrine stimulatory effect on LH or prolactin secretion, respectively.0Comments 9Citations
- "With the aim of contributing to a better understanding of the functional meaning of NO in the pituitary gland, and particularly in LH secretion, we have investigated possible gender differences in the pattern of nNOS activity under normal conditions, and the effect of different endocrine manipulations on its expression , by using immunocytochemical and double immunofluorescence techniques. In addition, we used quantitative analysis of the number and size of LH cells (Kovacs and Horvath 1975; Wynick et al. 1990; Catt and Dufau 1991 ) and serum LH levels as functional parameters of endocrine activity. "
- [Show abstract] [Hide abstract] ABSTRACT: As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.0Comments 12Citations
- [Show abstract] [Hide abstract] ABSTRACT: The use of magnetic beads coated with anti-IgG antibodies should allow simultaneous purification and depletion of differing anterior pituitary cell types labelled with anti-hormone antibodies. This technique would be expected to give very similar results to fluorescence-activated cell sorting (FACS). Magnetic bead separation of dispersed, labelled anterior pituitary cells is cheap, easy and quick to perform (time from the end of anti-hormone antibody labelling to completion of purification is approximately 30 min) and the resulting cells are viable for at least 24 hours after purification. While the cell recovery for beads and FACS, 94% (SEM +/- 4.4) vs. 89% (SEM +/- 3.9) and purity of 88% (SEM +/- 2.2) vs. 96.7% (SEM +/- 1.7) for lactotrophs and purity of 87% (SEM +/- 1.9) vs. 98% (SEM +/- 1) for somatotrophs are similar, the results for depletion by the magnetic bead separation method are disappointing, only 30-40% of the labelled lactotrophs or somatotrophs cells bind to the beads and thus only a sub-population of cells may be purified by this method. These results are explicable on the basis of the sensitivity of the two techniques. Pituitary cells co-incubated with two specific anti-prolactin antibodies (one raised in rabbit and one in sheep) demonstrate that removal by Dynal magnetic beads (coated with rabbit IgG antibody) of those prolactin molecules bound to the rabbit anti-prolactin antibody also removed those prolactin molecules bound to a sheep anti-prolactin antibody. In contrast, co-incubating cells with the rabbit anti-prolactin antibody and a sheep anti-growth hormone antibody did not remove growth hormone labelling when the prolactin bound to the beads was removed.(ABSTRACT TRUNCATED AT 250 WORDS)0Comments 13Citations