In vitro modifications in the proliferation

Abstract

In order to establish the correlation between in vitro proliferation rate and morphometric variations of prolactin immunoreactive cells, a morphometric study was carried out in rat pituitary monolayer cultures by means of the double immunocytochemical staining methods employing mouse monoclonal antiproliferative cell nuclear antigen (PCNA) and rabbit anti-prolactin (PRL) as primary antibodies. PCNA was found to be an adequeate marker for proliferation in pituitary monolayer cultures. 48.35+2.78% of the cells present in the culture were in active cell cycle after 3 days of incubation and a similar proportion, 54.93&2.83% was found after 7 days. On the 3rd day, PRL imrnunopositive cells accounted for 15.16*0.21% of the total cellular content in the dishes and 8.68*0.12% of the PCNA immunoreactive cells were also PRL immunopositive cells and, 60.95112.65% of PRL cells stained for PRL and PCNA. On the 7th day, an increase to 32.18f 0.60% of PRL cells was found; the PCNA and PRL cells accounted for 60.32*2.34% of the total PRL cells, and 19.88I1.09% of the PCNA reactive cells stained for PRL. Additionally, the morphometric analysis performed after 3 or 7 days of incubation showed that, while the size of PRL cells remained unmodified, the nuclear area had increased on the 7th day in relation to the 3rd day (pc0.01). These results suggest: 1) PCNA is a valid proliferative marker for pituitary cells in cultures; 2) a very high percentage of the PRL cells was in early proliferation; 3) on the 7th day of incubation, the proliferative rate of PRL cells was very similar to that observed on the 3rd day, suggesting a maintained proliferation for PRL cells at early incubation phases; and 4) the cellular activity, expressed as variations in the nuclear size, was higher on the 7th day than on the 3rd day; in addition, the numerical density of PRL cells increased.