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ABSTRACT

Addition of LHRH for 40 h to aggregate cell cultures of 14-day-old rat pituitary significantly decreased the number of [³H]thymidine ([³H]T)-incorporating cells which immunostained for GH protein as well as the number of [³H]T-labelled cells expressing GH mRNA detectable by in situ hybridization with a digoxigenin-labelled riboprobe. The effect at the level of GH protein was seen at a dose of 1 nm LHRH. However, the effect at the GH mRNA level required a higher dose of LHRH (10 nm) or a longer incubation time (64 h). Treatment of the cultures for 40 h with 0·1 nm GH-releasing factor (GRF) provoked a 54% increase in the number of [³H]T-labelled cells containing GH mRNA and a 30% increase in the number of cells immunostained for GH protein. The latter effects of GRF were completely blocked by simultaneous addition of LHRH (1 nm) to the cultures. In the absence of GRF, LHRH (1 nM) also had an inhibitory effect on the total number of cells containing GH mRNA and a comparable effect on the total number of cells stained for GH protein. The present data show that LHRH is capable of inhibiting the GRF-independent as well as the GRF-dependent development of somatotrophs in postnatal rat pituitary in culture.

ABSTRACT

We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [³H]thymidine ([³H]T) into DNA over 16 h. The fraction of [³H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [³H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population.

The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.