PRL Cells Research Articles

Enumeration of lactotropes and somatotropes among male and female pituitary cells in culture: evidence in favor of a mammosomatotrope subpopulation in the rat.

The hemolytic plaque assay technique can be used to detect specific hormone release from single pituitary cells. Using antisera raised against murine GH or rat PRL, we have enumerated the active lactotropes and somatotropes from male and female rat pituitary glands. These studies reveal sex-related differences in the number of cells exporting GH and PRL among anterior pituitary cells in culture. In the presence of human GH-releasing factor (hGRF), the mean percentage of GH cells was 53% in males and 30% in females (P less than 0.005). The mean percentage of PRL cells was 15% in males and 39% in females (P less than 0.008). These values were not significantly altered when hGRF was omitted. The sum of GH and PRL cells identified in separate plaque assays significantly exceeds the number obtained when GH and PRL cells were determined concurrently with a simultaneous plaque assay for both hormones. This difference is dependent on the presence of hGRF, since there was no difference when hGRF was omitted. These data identify the mammosomatotrope in numbers lower than previous reports. By this approach, the mammosomatotrope subpopulation numbers about 5% of all cells in culture. In summary, we demonstrate a sex-related difference in the number of cells exporting GH or PRL among pituitary cells in culture. This difference corresponds with and may underly sex-related differences in the responsiveness of GH and PRL secretion from the pituitary gland. Furthermore, a minor subpopulation of normal pituitary cells appears capable of simultaneous secretion of both GH and PRL.

Immunocytochemical evidence for the ability of the human pharyngeal hypophysis to respond to change in endocrine feedback

Two pharyngeal hypophyses from patients with endocrine disorder were examined light microscopically and immunocytochemically. The pharyngeal hypophysis from a patient with primary hypothyroidism was hypertrophic, with TSH cell hyperplasia; while that from a patient treated with metoclopramide, a dopamine-receptor-blocking drug, showed PRL cell hyperplasia. These findings strongly suggest that under certain circumstances the pharyngeal hypophysis is able to respond with specific changes to variations in the endocrine feedback.

Fine-structural heterogeneity and morphologic changes in rat pituitary prolactin cells after estrogen and testosterone treatment.

This study was conducted to determine the functional and/or developmental relationships among three heterogeneous types of prolactin cells (I, II and III) in rats. Rats were injected subcutaneously daily with estradiol or testosterone propionate on days 10-20 after birth. Estradiol increased the proportion of cell types II and III, increased serum PRL levels 12-fold in males and 15-fold in females, and increased pituitary levels of prolactin 12-fold in males and 5-fold in females. Testosterone mainly increased the proportion of the Type-II cells, decreased serum levels of prolactin in males only, and did not change pituitary levels of prolactin. In a second experiment, treatment of rats with nafoxidine for five days after E2 treatment (days 10-20 after birth) increased the proportion of Type-I cells and decreased the proportion of Type-III cells and decreased serum and pituitary levels of prolactin by 50% in females and by 15 and 45% in males. In a third experiment utilizing adult male rats, estradiol and testosterone were found to modulate the relative ratios of the different types of PRL cells as they did in immature animals. The data taken as a whole suggest the possibility of an estrogen-stimulated conversion of one cell type to another, which may be a reflection of prolactin secretory activity.

Anti-oestrogenic effects of tamoxifen on mammary gland and hypophysis in female rats.

Adult ovariectomized rats were treated for 14 days with oestradiol benzoate (E2B) 15 micrograms/kg/d and oestradiol benzoate 15 micrograms/kg/d + progesterone (PRO) 15 mg/kg/d for induction of mammary gland parenchymal stimulation. Histological examination and whole mount preparation demonstrated that ductal growth in the mammary gland after E2B treatment was completely antagonized by tamoxifen (TAM) 0.5 mg/kg/d. Parallel DNA concentrations in the mammary gland were decreased to control levels by TAM 0.5, 5 and 15 mg/kg/d. E2B-induced hyperprolactinaemia in the forenoon (basal secretion was equally reduced by TAM 0.5, 5 and 15 mg/kg/d). In the afternoon, when prolactin (Prl) secretion is at its maximum, TAM 0.5 mg/kg/d turned out to be ineffective to abolish Prl surge, but TAM 5 and 15 mg/kg/d reduced serum Prl concentrations in a dose-related manner. Immunoperoxidase staining of Prl cells in the pars distalis of the hypophysis indicated that adaptive hypertrophy and signs of hypersecretion after E2B were abolished by TAM 5 mg/kg/d. Luteotrophic cells clearly showed cellular atrophy, regression and secretory inactivity. Maximal tubulo-alveolar mammary parenchymal stimulation in rats treated with E2B-PRO was slightly inhibited by TAM 0.5 mg/kg/d. Histology showed a small disseminated parenchymal islet. DNA concentrations only were partially decreased by the anti-oestrogen though serum Prl concentrations were found to be completely decreased to control levels. Secretory activity of Prl cells was reduced by TAM 0.5 mg/kg/d. In E2B-PRO treated rats lisuride had poor inhibitory activity on Prl levels and none on DNA concentrations in the mammary gland. Combined treatment with TAM and lisuride significantly decreased DNA concentration in the mammary gland compared to animals which received E2B-PRO. Also Prl levels were at a minimum. Histology performed on the mammary gland showed only slight tubulo- but no tubulo-alveolar activation. Luteotrophic cells in the pituitary gland stained by the immunoperoxidase technique appeared regressive, shrunken and atrophied.

Study of growth hormone in spontaneous dwarf rat

In 1977, in the Research Laboratories, Morishita Pharmaceutical Co. Ltd., we were fortunate to discover that small mutant rats named Spontaneous Dwarf Rat (gene symbol dr), inherited an autosomal recessive. In previous papers, we reported that these rats might be characterized by a defect of growth hormone (GH). In this paper, we carried out the studies of the secretory cells and GH deficiency in the pituitary glands of +/dr and dr/dr rats using immunohistochemistry, electron microscopy and acrylamide gel electrophoresis. ACTH, PRL and GH secretory cells in the pituitary gland respectively identified with antiserum to porcine ACTH, rat PRL and ovine GH by indirect method of immunohistochemistry. In the pituitary of dr/dr rats, ACTH and PRL secretory cells were confirmed, but none of the cells were stained with ovine GH antiserum. Using electron microscopy, GH secretory cells could not be identified precisely although the other cells were identified in the pituitary gland of dr/dr rats. With the analysis of pituitary homogenate by acrylamide gel electrophoresis, PRL band appeared in dr/dr rats but GH did not. These results obviously indicated that the inhibition of body weight in dr/dr rats was caused by the absence of GH in the anterior pituitary. Therefore, the Spontaneous Dwarf Rat may be a useful animal model for human dwarfism caused by isolated GH deficiency.

The role of estrogen in the differentiation of prolactin producing cells

The capacity to synthesize PRL and GH was studied in normal mice and in Snell and Ames dwarfs. In normal mice GH synthesis turned on dramatically between day 16-17 of gestation whereas PRL was not detectable throughout gestation or the first week of life, was barely detectable in 8-day-old mice and was clearly demonstrable by 12 days of age. However, transplantation of pituitaries from newborn mice into adult female hosts resulted in substantial PRL synthesis. Treatment of mice with DES at various ages showed that neonates failed to respond to the hormone; in older pups, an age-dependent increase in the magnitude of PRL synthesis was observed. There was a direct correlation between nuclear estrogen receptor levels and PRL cell function. Snell and Ames dwarf mice failed to synthesize PRL or GH at any stage of development, nor could we detect immunoreactive peptides which might represent mutant forms of the hormones. Our findings suggest that: (1) GH gene expression precedes that of PRL by about 2 weeks; (2) the development of PRL cell function is dependent on estrogen; (3) the capacity to respond to estrogen is present before the endogenous initiation of PRL synthesis and is limited by the availability of estrogen receptor; and (4) in Snell and Ames mutants, both types of alleles result in failure of the pituitary to initiate PRL and GH synthesis.

Immunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin

An antiserum raised to highly purified chum salmon ( Ocorhynchus keta) prolactin (sPRL) was used to identify prolactin-producing cells in the adenohypophysis of 15 species of teleosts by the immunocytochemical peroxidase-antiperoxidase method. In the chum salmon, the only pituitary cells that reacted with sPRL antibody were the PRL cells organized as follicular structures in the rostral pars distalis. When the antiserum was absorbed with sPRL, on the other hand, no immunoreactive cell was observed in the pituitary, indicating the specificity of the antiserum. Furthermore, the antibody to sPRL reacted only with PRL cells in the pituitaries of three species of salmonids, a plecoglossid, eel, carp, goldfish, killifish, tilapia, and five species of marine fishes, thus showing no species specificity of the antibody among the teleosts tested. The PRL cells of the eel decreased in number and also in immunoreactivity after adaptation to seawater for 1 month. On the other hand, highly immunoreactive PRL cells were observed in the pituitaries of marine fishes, although the cells were much fewer in number than in eels and in other fishes in fresh water.

Dopamine receptors of the monkey anterior pituitary in various endocrine states.

The anterior pituitary of adult Macaca fascicularis monkeys was removed when they were killed and the potent dopamine (DA) antagonist [3H]spiperone was used to label DA receptors. Saturation isotherms of [3H]piperone binding indicated a dissociation constant of 0.09 nM and maximal sites of 170 fmol/mg assay protein (n = 2). Competition studies validated the hypothesis that [3H]spiperone labeled DA receptors; the order of potency of 12 compounds was appropriate for a dopaminergic interaction and was similar to the order found in the anterior pituitary of other mammals. A concentration of [3H]spiperone that would label 90% of the specific binding sites (0.76 nM) was used to estimate the total DA receptor activity in the anterior pituitary of individual monkeys. [3H]spiperone binding in pregnant monkeys was greater than the binding in ovariectomized and lactating females or intact males (P less than 0.05 or 0.01). The postpartum lactating group contained more [3H]spiperone binding activity than the cycling group (P less than 0.05). Determination of the serum concentrations of PRL, estradiol, testosterone, progesterone, and dehydroepiandrosterone confirmed the predicted hormonal status of the individual monkeys at the time of sacrifice. Because DA is a PRL inhibitory hormone, it was of interest that the serum PRL concentration was positively correlated with the degree of [3H]spiperone binding (r2 = 0.74). This observation, combined with the known hypertrophy and/or hyperplasia of the mammotroph during pregnancy, supports the hypothesis that the DA receptor is associated with the PRL cell of the anterior pituitary.

Dopamine internalization by and intracellular distribution within prolactin cells and somatotrophs of the rat anterior pituitary as determined by quantitative electron microscopic autoradiography.

The internalization and intracellular distribution of [3H]dopamine in PRL cells and somatotrophs of the female rat anterior pituitary were investigated. Dissociated pituitary cells were incubated with [3H]dopamine for 5 min and subsequently chased in medium lacking radioactive dopamine for various time periods up to 3 h. The localization of [3H]dopamine was monitored by high resolution quantitative electron microscopic autoradiography. Results obtained by grain density analysis revealed that dopamine is internalized by these cells and sequestered in a multiplicity of intracellular membranous organelles. These included the lysosomes and those organelles involved in the secretory pathway, chief among which were the Golgi cisternae and the immature (type I and II) and mature (type III and IV) secretory granules. The highest grain density values throughout the entire chase period were recorded for the lysosomal compartment. This is consistent with the finding that approximately 70% of the internalized dopamine was lost from the PRL cells within the time frame of the chase period, most likely due to its metabolism or release. Although the overall grain density of the nucleus was low at all chase periods, analysis of the grain density distribution of the various nuclear profiles of the post-pulse time point revealed that nuclear labeling was not uniform, suggesting that dopamine is taken up by the nucleus and localized in discrete areas of this compartment. The internalization event itself was found to be a receptor-dependent one since the simultaneous incubation of pituitary cells with [3H] dopamine and physiological concentrations (10-5 M and 10-7 M) of its unlabeled analog resulted in a 50–60% decrease in the uptake of [3H]dopamine by PRL cells, as indicated by grain density analysis. Somatotrophs showed a similar pattern of dopamine internalization and sequestration with the exception that labeling of the Golgi cisternae could not be established with certainty by the analytical methods employed. The data obtained provide a firm morphological basis for the intracellular distribution of dopamine in the two numerically most frequent (PRL cells and somatotrophs) cell types of the rat anterior pituitary gland.

Human prolactin-producing adenomas and bromocriptine: a histological, immunocytochemical, ultrastructural, and morphometric study.

Bromocriptine inhibits PRL secretion and causes a size reduction of PRL-producing adenomas. To clarify its antitumor effect, PRL cell adenomas were studied by histology, immunocytochemistry, transmission electron microscopy, and light and electron microscopic morphometry. All patients had macroadenomas (diameter, >10 mm) and serum PRL levels from 420–9800 ng/ml. Patients 1 and 2 received no medication. Four patients were treated with bromocriptine (7.5 mg/day for 4–weeks); patients 3 and 4 continued therapy up until the operation, whereas patients 5 and 6 discontinued bromocriptine 7 and 14 days, respectively, before tumor removal. All four bromocriptine- treated patients demonstrated a reduction in serum PRL levels and tumor size on computed tomography. Patients 5 and 6 showed reexpansion of their tumors after drug withdrawal. By light microscopy, all six tumors represented chromophobe adenomas and were positive for PRL by the immunoperoxidase technique. The intensity of immunostaining and the number of ...

Immunocytochemical and ultrastructural correlates of the pineal inhibition of prolactin cell activity in blind-anosmic female rats.

The effects of the pineal gland on the light microscopic-immunocytochemical and ultrastructural appearance of pituitary mammotrophs were studied in female rats eight weeks after prepubertal blinding and olfactory bulbectomy. Blinding and anosmia resulted in a marked decrease in the size of the pars distalis concomitant with a reduction in the apparent number and size of PRL cells as compared with intact animals. Ultrastructurally, these cells appeared much less active than those of intact rats. The small and angular-shaped mammotrophs of blind-anosmic rats characteristically exhibited scant arrays of rough endoplasmic reticulum, small Golgi complexes with few immature secretory granules, few mature secretory granules and rare exocytosis patterns. Pinealectomy tended to reverse the effects of blinding and anosmia on pars distalis size and PRL cell size, apparent number and ultrastructure. In fact, the mammotrophs of blind-anosmic-pinealectomized rats were quite similar in ultrastructural appearance to those of intact rats. From these data we conclude that the pineal causes mammotroph hypotrophy and hypoplasia in blind-anosmic female rats.

Cytological study on the anterior pituitary of senile untreated beagle bitches with spontaneous mammary tumours.

Pituitaries were obtained from senile untreated Beagle bitches of comparable age (7-9 years) and genital status. The animals were divided into three groups; one was normal (without mammary lesions), one had benign tumours and one had mammary adenocarcinomas. PRL-, STH-, ACTH- and gonadotrophin-producing cells are studied and counted in serial paraffin sections stained with histochemical techniques. The animals with mammary malignancy displayed a marked increase in the relative number of PRL and ACTH cells with morphological signs of higher secretory activity in most cells, compared with that in normal bitches or bitches with benign tumours. STH cells in bitches with adenocarcinomas were reduced in number; however the secretory activity in these animals was the same as that observed in the normal bitches. In the animals with benign mammary tumours, STH cells showed morphological indication of higher secretory activity than in the other groups. PRL and ACTH cells were slightly increased in number and had slightly higher activity than that in normal bitches. These findings may suggest a role for hypophyseal hormones in mammary neoplasias.

Pineal gland inhibition of prolactin cell activity is independent of gonadal regression.

In order to determine whether the inhibitory effect of the pineal gland on the PRL cells of blind-anosmic female rats is a result of the attendant gonadal regression observed in these animals, mammotroph activity was compared between intact and ovariectomized rats eight weeks after prepubertal blinding and olfactory bulbectomy. PRL synthesis was evaluated by measuring the amount of 3H-leucine incorporated into PRL by interior pituitaries in vitro. PRL synthesis was reduced by 47% in blind-anosmic rats, an effect which was reversed by pinealectomy. Although ovariectomy itself led to a 64% decrease in PRL synthesis, dual-sensory deprivation in these animals resulted in a further 59% suppression of PRL production. PRL storage, as estimated by measuring total immunoreactive PRL in vitro also appeared to be significantly depressed in blind-anosmic female rats. Once again, ovariectomy had no effect on this reduction. PRL release, as assessed by monitoring serum levels of the hormone, was decreased in blind-anosmic rats. Ovariectomy also caused a reduction in serum PRL levels; however, the combination of blinding and olfactory bulbectomy had no further depressive effect on circulating PRL titers in these animals. Correlated with the decrease in PRL cell activity in both intact and ovariectomized blind-anosmic rats was a pineal-induced decrease in anterior pituitary weight and DNA content in both sets of animals. From these data we conclude that the pineal's inhibition of the PRL cell in blind-anosmic female rats is largely independent of the gonads and, therefore, could not be secondary to gonadal involution.

A possible role for lysosomes in the inhibitory action of dopamine on prolactin release.

The effect of dopamine on lysosomal function in the anterior pituitary gland and on PRL secretion was investigated in vivo and in vitro. When anterior pituitary glands were examined by electron microscopy, it was found that PRL cells in pituitary tissue from rats that had been treated for 8 h with the dopamine precursor, L-dihydroxyphenylalanine (L-dopa), contained more multivesicular bodies (i.e. lysosomes) than did PRL cells in pituitary tissue from vehicle-treated animals. In addition, a large number of the lysosomes in PRL cells of L-dopa-treated rats appeared to have undergone fusion with PRL secretory granules (i.e. crinophagy). When anterior pituitary glands were homogenized and fractionated by continuous sucrose density gradient centrifugation, it was found that most of the activity of the lysosomal enzyme, β-glucuronidase, was present in gradient fractions containing subcellular particles, viz. lysosomes, that had an approximate density of 1.2. Treatment of rats with Ldopa resulted in an increase...

Characterization of seasonal changes in prolactin and growth hormone cells in the hypophyses of white-tailed deer (Odocoileus borealis) by ultrastructural and immunocytochemical techniques.

Prolactin and GH cells were identified in thin sections taken from the adenohypophyses of adult male (6) and female (8) white-tailed deer, collected during all seasons of the year, by locating portions of the same cells in adjacent thick plastic sections immunostained for either PRL or GH. Growth hormone cells were round to ovoid in shape and filled with dense spherical secretory granules which ranged in size from 168 to 682 nm, with a mean diameter of 367 +/- 18 nm (X +/- SD) in the 14 glands studied. No apparent seasonal changes were evident in the ultrastructural appearance of GH cells. Prolactin cells were small and angular in shape during the winter months and contained only a few small secretory granules. By early summer, they wee markedly hypertrophied, round to oval in shape, and densely packed with large spherical secretory granules. The increase in size of PRL secretory granules was most prominent in pregnant females in May, when their mean diameter was approximately double than in midwinter. The ultrastructural appearance of PRL cells in September was similar to that of cells studied in March. The size distribution of PRL secretory granules overlapped that of the GH granules, ranging from 114 to 564 nm, and the mean diameter was 246 +/- 53 nm, calculated from all 14 individual glands. Our observations suggest that the synthesis and secretion of PRL are closely linked to photoperiodic changes in this species, and they demonstrate the necessity of using specific immunocytochemical techniques in the ultrastructural identification of pituitary acidophils, and of specifying the time of year and location (specifies photoperiod) in studies concerning PRL physiology, particularly when dealing with wild animal populations.

Role of estrogen in the dopaminergic control of prolactin secretion.

The effect of estrogen on the dopaminergic control of PRL secretion was investigated. Treatment of ovariectomized rats with estradiol benzoate (25 microgram/kg, sc) daily for 5 days resulted in a marked elevation of the serum PRL concentration. This estrogen-induced increase in serum PRL levels was apparently not the result of a suppressed release of dopamine into hypophysial portal blood, since the mean dopamine concentration in hypophysial portal plasma in estrogen-treated rats was 2.5 times that in vehicle-treated animals. It was found that under in vitro conditions, dopamine was much less effective in inhibiting the release of PRL from pituitary glands of estrogen-treated rats than from glands of vehicle-treated controls. The capacity of PRL cells to internalize dopamine and incorporate it into PRL secretory granules was evaluated in anterior pituitary tissue obtained from estrogen- or vehicle-treated animals. When tissue fragments of the anterior pituitary gland were incubated in the presence of dopamine (10(-5) M) for 30 min at 37 C and then homogenized and fractionated by means of continuous sucrose density gradient centrifugation, it was found that the amount of dopamine associated with PRL granules from anterior lobe tissue of estrogen-treated rats was only 40% of that from the tissue of vehicle-treated controls. These results are supportive of the hypothesis that the ability of estrogen to antagonize the inhibitory action of dopamine on PRL secretion is mediated through an estrogen-induced reduction in the capacity of the PRL cell to incorporate dopamine into PRL secretory granules. (Endocrinology 108: 440, 1981)

Postnatal development, sexual difference and sexual cyclic variation of prolactin cells in rats: special reference to the topographic affinity to gonadotroph.

During the postnatal development of rat pituitary it has been shown that PRL cells appear oval, polygonal or cup shaped in both sexes. The oval cells first appear on Day 10 after birth. Development of the latter two shapes is delayed to Days 20 and 30, respectively. The polygonal shaped cells become predominant with sexual maturation. Some of them surround the gonadotrophs with their elongate, cytoplasmic processes, taking on the appearance of cup cells. Although the PRL cells are more numerous in the female than in the male, the cup shaped cells are inverse. Further it is noted that the immunostainability of the oval and polygonal cells varies during the estrous cycle; the weakest reaction was revealed at estrus and the strongest at dioestrus. Those immunohistochemical findings may suggest that the oval cells are premature, the polygonal ones mature and the cup ones particularly differentiated. The topographic affinity between the cup cells and the gonadotrophs may be a morphological indication of their intimate functional relationship.

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard Anolis carolinensis.

Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its beta-subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine beta-LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.

Rise of Plasma Prolactin and Changes in Fine Structure of the Anterior Hypophysis after Pituitary Stalk Section in Rats*

The effects of pituitary stalk section on the plasma PRL concentration and structure of PRL cells were studied in rats with the use of RIA and electron microscopy. The pituitary stalk was transected in the estrous rat using the parapharyngeal route and blood samples were withdrawn through a cardiac cannula before and after surgery. Plasma PRL concentrations increased significantly 4 min after stalk section. Plasma PRL levels were significantly elevated to one of the highest levels within 8 min after stalk section, and these high levels were maintained until the end of the experiment 2 h after stalk section. No significant changes occurred in sham-operated rats. Ten minutes after stalk section, secretory granules of PRL cells were decreased in number, and most granules were arranged in the peripheral zone of the cytoplasm. The Golgi apparatus and the rough-surfaced endoplasmic reticulum were well developed. Thirty minutes after stalk section, PRL granules were markedly decreased and an abundance of immatur...

Intracellular prolactin in rat corpus luteum and adrenal cortex.

This study was done to examine whether PRL, which appears in both active milk secretory cells (MSC) (1) and milk (1,2) during normal lactation, could also be detected in PRL target cells which do not transfer this hormone into an exocrine secretory product. Ovaries, adrenals, and mammary and pituitary glands were collected from actively nursing rats decapitated on day 15 post-partum. All four tissues were processed for light microscopic immunohistochemical identification of PRL. Immunoreactive PRL was again found in pituitary PRL cells and in MSC. It was also detected within both lutein and adrenal cortical cells (zona fasciculata). In all three target tissues, PRL was present in target cell cytoplasm, but, in MSC and adrenal cells, it was also found occasionally in nuclei. These findings, which seem to indicate that transfer into an exocrine secretory product cannot be the only possible explanation for the appearance of this protein hormone inside its target cells, extend the evidence suggesting that PRL may have intracellular sites of action.