Effects of Estrogen on Prolactin (PRL) Incorporation by Lutein and Milk Secretory Cells and on Pituitary PRL Secretion in the Postpartum Rat: Correlations with Target Cell Responsiveness to PRL

The relationships between cell function and the incorporation of endogenous prolactin (PRL) by milk secretory cells (MSC) and lutein cells (LC) were examined in lactating rats which had been given graded estradiol treatment designed to dissociate progressively the activities of these two PRL target cell populations, decreasing MSC function while maintaining or augmenting LC function. MSC function was assessed by monitoring pup growth and by histological study of mammary tissue. LC function was assessed by radioimmunoassay (RIA) of circulating progesterone (P) and 20a-hydroxyprogesterone (2Ooi) and by histological evaluation of ovaries. PRL availability was assessed by RIA of serum PRL and by morphological evaluation of pituitary PRL cells. Intra cellular distributions of endogenous PRL in both target tissues were examined immunohisto chemically. The availability of PRL binding sites was also examined in MSC from rats given the highest dose of estrogen. As estrogen dosage increased, PRL secretion was maintained, or increased. Despite this, there Was progressive lactational failure, accompanied by a graded failure of PRL incorporation into MSC. These cells appeared to lose, successively, their abilities to translocate PRL to nuclei, to transport it from basal to apical regions of the cell and ultimately, with the highest dose of estro gen, to internalize or even recognize PRL, as evidenced by the absence of not only PRL, but even PRL-binding sites. In contrast, as estrogen dosage increased, titers of P and P/20o ratios remained high and LC showed progressive morphological signs of not only increased activity (hypertrophy, nucleolar enlargement) but augmented incorporation of PRL. Thus, in both MSC and LC, estrogen produced a spectrum of variations in responsiveness to PRL; this spectrum was correlated with a series of effects on PRL incorporation by these cells. These estrogenic modulations of PRL action might, therefore, involve regulating the access of this protein hormone to various intracellular control points. 394 NOLIN AND BOGDANOVE MATERIALS AND METHODS Two experiments were performed. In the first, 6 rats (CD strain, Charles River Breeding Laboratories, Inc.), each nursing her own litter of 9â€”li pups, were given estrogen by implantation of open-ended 5 mm sections of polyethylene tubing (innramedic PE5O, Clay Adams Co., 0.584 mm i.d. ; 0.965 mm o.d.) packed with crystalline estradiol 3,17-dipropionate (ED Lot no. 179â€”812, Dextran Chemicals, Inc.). A 23 gauge stylus was used to pack the tubing. Each rat was lightly anesthetized with ether on Day 4 of lactation and a short, shallow-beveled 17 gauge needle, fitted with a stylus, was used to inject 1 implant into each of 3 mammary glands. The rats were not handled again until Day 14, when they were decapitated and their ovaries, pituitaries and mammary glands were col lecned, fixed in Bouin's fluid and processed for im munohistochemical study, as described below. The second study was done to examine the possi ble dose dependency of the estrogen effects observed in the first study. Thirty pregnant rats of the same (CD) strain were used. Beginning â€œ? vi week prior no delivery, the rats were caged individually in 23 X 20 X 14 cm clear plastic boxes with wire tops in a room with lights on 14 h/day (0500â€”1900 h) and an ambi cnn temperature of 23 Â±1Â°C.Food and water were continuously available. All rats delivered on the same day (Day 1 of lactation). Immediately after delivery, pups were counted. Six rats that delivered less than 8 pups each were discarded. Some of their pups, as well as those in excess of 10/litter from the other rats, were used where necessary to equalize all litters no 10 pups. Twenty-four families were then divided into 4 groups of 6 families each. At â€œ? â€œ1000 h of Day 4, each mother and each litter were weighed. Mothers were non weighed again until Day 15. Litters were weighed on alternate mornings and on the morning of Day 15. Estradiol benzoate (EB, Progynon benzoane, 6CX2P96706, Corp.) was given to 18 of the mothers at â€œ? â€ v1700 h on Days 4â€”14(11 days). Six mothers were given each dose of EB (1.0, 4.0 or 16.0 Mg)by s.c. injection in 200 p1 sesame oil (U.S.P. 0-154, Fisher Scientific Co.). The remaining 6 mothers, 3 injected with oil and 3 untreated, served as controls Since the 2 control groups did non differ, they are considered as 1 group under Results. Between 1600â€”1800 on Day 15, mothers were rapidly decapitated. Trunk blood was collected and refrigerated until sera could be separated an â€œ? v1830 h. Terminal body weights were obtained by adding the weights of the trunk, head and extravasated blood. Pituinaries, mammary glands and ovaries were removed and placed in Bouin's fluid. Pups were killed by cervical dislocation. Sera collected from 2 adult male rats, killed in the same way, served as controls for the lack of stress-induced PRL release (Nolin en al., 1977). Sera were divided into 2 aliquots/sample and frozen immediately. One sen of aliquons was stored an â€”? 40Â°C until thawed for RIA of PRL, after which any residual serum was refrozen and stored further at â€”? 40Â°C. Through error, the other set was kept an â€”? 5Â°C until, 15 months later, it was assayed for progesterone (P) and 2Os-hydroxyprogesnerone (20o), together with some of the samples which had been stored an â€”? 40Â°C, using a method and antiserum described previously (Beatnie en al., 1977; Resko et al., 1974). Serum PRL radioimmunoassay was done with the NIAMDD antiserum to rat PRL (S-i), as previously reported (Nolin en al., 1977). Duncan's multiple comparison test was used for statistical evaluation of hormone concentration differences. After fixation, pituitaries, mammary glands and ovaries were dehydrated, embedded in paraffin, sectioned serially at 5 pm and stained for endogenous PRL by the immunohistochemical method described in detail previously (Nolin and Witorsch, 1976). Reagents used in the present study were: goat anti serum to monkey 7-globulin (G61-1, Antibodies, Inc.), rabbit antiserum no ran PRL (APRL: NIAMDD anti-rat PRL 53) and goat antiserum to rabbit -y globulin (AR'@C;65-159-1, Miles Laboratories). AR'yG was conjugated no peroxidase by the method of Nakane and Kawaoi (1974). Diaminobenzidine:H2 02 was used as the chromogenic substrate. Fully absorbed APRL (ABS APRL), used in the primary test for PRL specificity of the staining reac tion, was prepared by mixing 1 ml APRL (1:50) with 8 @gNIAMDD rat PRL 1-2. Material stained via APRL binding was identified as PRL only if it was tonally unstained in adjacent serial sections when ABS-APRL was used in place of APRL in an otherwise identical reaction sequence. Pituitaries were subjected to the same protocol as target tissues, providing an adjunctive nest of method specificity (see Nolin 1978a, footnote 2). Mammary glands from the ED implant group were also subjected to immunohisnochemical testing for the presence of PRL binding sites which could bind exogenous PRL (Nolin and Witorsch, 1976). For this, sections were preincubaned with exogenous PRL (NIAMDD ran PRL 1-2, in amounts ranging from 15â€”1500 pg/section) prior to application of APRL in the PRL-staining sequence. Maternal Weights RESULTS Data were non obtained in the first experi ment (rats with ED implants). In the second, mean maternal weight on Day 4 was 288 Â±9 g. On Day 15, the mean weights for controls and the 1, 4 and 16 pg/day EB treated rats were 310 Â± 11, 294Â± 9, 304 Â±7 and 292 Â±8 g, respectively. The differences among the 4 groups were not statistically significant.