PRL Cells Research Articles

Immunohistochemical study of p53 protein in human and animal pituitary tumors.

In many human cancers, p53 gene mutations are frequently occurring genetic abnormalities, which may be detected by immunohistochemical staining for p53 protein. In the present study, p53 immunoreactivity was investigated in formalin-fixed, paraffin-embedded tissues from human and animal pituitary tumors, using the avidin-biotin-peroxidase complex technique. No p53 was detected in 3 nontumorous human adenohypophyses or in 40 human pituitary tumors including 5 GH cell adenomas, 10 PRL cell adenomas, 2 mixed GH cell-PRL cell adenomas, 2 acidophil stem cell adenomas, 8 ACTH cell adenomas, 1 TSH cell adenoma, 1 FSH/LH cell adenoma, 5 null cell adenomas, 5 oncocytomas, and 1 plurihormonal adenoma. Twenty nontumorous and hyperplastic pituitaries of hGRH transgenic mice and 8 tumors in these transgenic animals were immunonegative for p53. All pituitary tumors found in AVP/SV40 transgenic mice contained p53 immunoreactivity in the nuclei, while the nontumorous adenohypophysis of one such transgenic mouse was negative. It can be concluded that p53 mutations are apparently not involved in the pathogenesis of human pituitary adenomas or of the pituitary tumors which develop in hGRH transgenic mice. However, pituitary tumors in AVP/ SV40 transgenic mice are accompanied by p53 expression.

Developmental assessment of hypothalamic tuberoinfundibular dopamine in prolactin-deficient dwarf mice [published erratum appears in Endocrinology 1993 Jul;133(1):327

Development of the hypophysiotropic hypothalamus in PRL-deficient Ames dwarf (df/df) mice was examined for steady state dopamine (DA) by visualization using formaldehyde-induced catecholamine histofluorescence and by quantification using catecholamine HPLC at selected postnatal ages (7, 14, 21, 30, and 90 days). Phenotypically normal (DF/?) littermate mice were compared with dwarfs by both methods at each age. The studies were designed to investigate whether the known deficiency in hypothalamic tuberoinfundibular DA in adult dwarfs is present neonatally or develops over the postnatal period. The anterior pituitary of each mouse was processed for GH and PRL immunocytochemistry. At 7 days of age, GH immunostaining was robust, and scattered PRL-positive cells were noted in DF/? pituitary. Homogeneously distributed PRL cells increased in number through 30 days of age in normal mice. Neither GH nor PRL immunoreactivity was present in df/df mice at any age. At 7, 14, and 21 days of age, hypothalamic DA tuberoinfundibular histofluorescence was comparable in df/df and DF/? mice. At 90 days of age, tuberoinfundibular histofluorescence in normal mice remained intense, but was virtually undetectable in dwarfs. The developmental change affected only tuberoinfundibular neurons, since DA histofluorescence in nonhypophysiotropic areas, such as substantia nigra (SN), was qualitatively comparable for df/df and DF/? for all ages examined. Norepinephrine (NE) fluorescence in hypothalamus was also comparable for df/df and DF/?. Catecholamine HPLC provided quantitative confirmation of histofluorescence observations. DA and NE levels in both hypothalamus and ventral midbrain, including SN, increased during development in both df/df and DF/? brains. NE levels were not different between dwarf and normal animals at any age in either medial basal hypothalamus (MBH) or SN. The DA concentration in SN was not different between df/df and DF/? at any age examined. MBH DA was comparable in df/df and DF/? mice at 7, 14, and 21 days of age; at 30 and 90 days, MBH DA was markedly lower (P < 0.001) in dwarf than in normal mice. Although MBH DA in dwarfs was comparable to that in normal mice at 21 days, the increase in dwarfs between 14 and 21 days was not statistically significant. Thus, the hypothalamic DA deficit that exists in adult dwarf mice is not present neonatally and represents a failure to increase DA compared with normal mice after 14 days of age. The failure of continued development of hypophysiotropic tuberoinfundibular DA neurons in dwarf mice is correlated chronologically with absent pituitary PRL production.

Effect of luteinizing hormone-releasing hormone analogs containing cytotoxic radicals on the function of rat pituitary cells: tests in a long term superfusion system.

A novel class of targeted chemotherapeutic agents based on cytotoxic compounds linked to LHRH analogs was tested in a long term superfusion system in order to determine their effects on different types of rat pituitary cells. The compounds investigated included two cytotoxic LHRH agonists, T-98 ([D-Lys6]LHRH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)) and T-107 ([D-Lys6]LHRH linked to doxorubicin (DOX) through glutaric acid spacer), and two cytotoxic LHRH antagonists T-121 and T-144, both containing two residues of G-HMAQ. The analogs were infused for 24 h at pharmacological (micromolar) concentrations. The secretions of LH, GH, and PRL cells after a short (2-h) and a long (20-h) recovery period following the treatment were compared to those before the analog infusion. All four cytotoxic LHRH analogs selectively decreased the stimulated LH response, while basal secretions of LH, GH, and PRL as well as the stimulated release of GH and PRL were not influenced in spite of the high doses applied and the long duration of exposure. The inhibitory effects of cytotoxic agonists and antagonists were significantly greater than those of their parent peptides. Equimolar concentrations of cytotoxic compounds alone (DOX or G-HMAQ), however, caused functional damage to all types of cells tested. To evaluate the mechanisms of the observed inhibitory actions, we compared the amount of LH released in response to 1) a specific stimulus to receptors (3 nM LHRH), 2) membrane depolarization with 100 MM KCl, not involving the receptor, and 3) the extraction of cells with 0.01 N HCl at the end of experiment. Cytotoxic LHRH agonists and their carriers caused depletion of LH pools, while cytotoxic LHRH antagonists or their parent peptides decreased the available binding sites for LHRH. The inhibitory effect of DOX on stimulated secretion of LH, GH, and PRL in our system could be due to an action on cell membranes. Our work indicates that in the pituitary cell superfusion system, targeted cytotoxic conjugates, based on LHRH analogs, selectively affect LH cells, in contrast to unconjugated cytotoxic compounds, which also damage GH and PRL cells.

Effect of luteinizing hormone-releasing hormone analogs containing cytotoxic radicals on the function of rat pituitary cells: tests in a long term superfusion system

A novel class of targeted chemotherapeutic agents based on cytotoxic compounds linked to LHRH analogs was tested in a long term superfusion system in order to determine their effects on different types of rat pituitary cells. The compounds investigated included two cytotoxic LHRH agonists, T-98 ([D-Lys6]LHRH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)) and T-107 ([D-Lys6]LHRH linked to doxorubicin (DOX) through glutaric acid spacer), and two cytotoxic LHRH antagonists T-121 and T-144, both containing two residues of G-HMAQ. The analogs were infused for 24 h at pharmacological (micromolar) concentrations. The secretions of LH, GH, and PRL cells after a short (2-h) and a long (20-h) recovery period following the treatment were compared to those before the analog infusion. All four cytotoxic LHRH analogs selectively decreased the stimulated LH response, while basal secretions of LH, GH, and PRL as well as the stimulated release of GH and PRL were not influenced in spite of the high doses applied and the long duration of exposure. The inhibitory effects of cytotoxic agonists and antagonists were significantly greater than those of their parent peptides. Equimolar concentrations of cytotoxic compounds alone (DOX or G-HMAQ), however, caused functional damage to all types of cells tested. To evaluate the mechanisms of the observed inhibitory actions, we compared the amount of LH released in response to 1) a specific stimulus to receptors (3 nM LHRH), 2) membrane depolarization with 100 MM KCl, not involving the receptor, and 3) the extraction of cells with 0.01 N HCl at the end of experiment. Cytotoxic LHRH agonists and their carriers caused depletion of LH pools, while cytotoxic LHRH antagonists or their parent peptides decreased the available binding sites for LHRH. The inhibitory effect of DOX on stimulated secretion of LH, GH, and PRL in our system could be due to an action on cell membranes. Our work indicates that in the pituitary cell superfusion system, targeted cytotoxic conjugates, based on LHRH analogs, selectively affect LH cells, in contrast to unconjugated cytotoxic compounds, which also damage GH and PRL cells.

Prolactin receptor expression by lymphoid tissues in normal and immunized rats

Prolactin receptor (PRLr) expression and distribution in thymus, spleen, bone marrow, lymph nodes, and peripheral blood lymphocytes from young adult Lewis rats are analyzed using single-color flow cytometry and a well-characterized monoclonal antibody directed against the rat liver PRLr. The in vivo effects of regional immunization on PRLr expression are also examined. PRLr is found to be widely distributed among cells of the immune system and demonstrates lymphoid tissue-specific patterns of expression. Footpad immunization caused the rapid, but transient, induction of PRLr expression in the draining lymph node, with only modest effects on PRLr expression in other distant lymphoid tissues. These studies indicate that PRL may be capable of direct interaction with the immune system through differential expression of the PRL cell surface receptor on select lymphoid target cell populations.

Immunocytochemical and Ultrastructural Study of Rana dalmatina PRL and GH Pituitary Cells during Larval Development

Single and double simultaneous indirect immunogold methods and ultrathin sagittal sections of Rana dalmatina tadpole heads were used to follow cytodifferentiation of PRL and GH cells in the adenohypophysial primordium from developmental stage 26 to the end of metamorphosis. Rabbit anti-frog ( Rana catesbeiana) PRL and rabbit anti-frog GH were employed in the single method, and rabbit anti-frog PRL and monkey anti-rat GH were used in the double simultaneous technique as primary antisera. At stage 27, a couple of cells with few small secretory granules immunoreactive with anti-frog GH are first identifiable. At stage 28, some cells with few membrane-bound secretory granules, 70-90 nm in diameter, and immunoreactive with anti-frog PRL and other cells with few secretory granules, 80-110 nm in diameter, and immunoreactive with anti-frog GH are present. As development progresses, differentiation in both cell types is shown by an increase in the cytoplasmic volume and in the number, size, and immunoreactivity of secretory granules. Neither comparison of adjacent ultrathin sections stained with the single labeling technique nor results of double simultaneous labeling showed the coexistence of PRL and GH within the same cell.

Influence of exogenously administered oxytocin on prolactin-producing cells in adult male rats as revealed by immuno-electron microscopy.

Prolactin-producing cells (PRL cells) identified by immuno-electron microscopy were studied in male rats with chronic intraperitoneal injection of synthetic oxytocin (OT). The PRL cells are usually classified into three types: the immature type containing round secretory granules about 100 nm in diameter with poorly developed cell organelles; the intermediate type containing medium-sized (150-250 nm in diameter) secretory granules with moderately developed cell organelles; and mature type containing large pleomorphic secretory granules ranging from 300 to 700 nm in diameter with well-developed cell organelles. In male rats, the intermediate type comprises typical PRL cells that constitute about 50% of all immunoreactive PRL cells. Chronic intraperitoneal OT administration to rats caused morphologically hypertrophic and hyperfunctioning PRL cells which are identified as mature type PRL cells. The frequency of occurrence of the mature type PRL cells increased after treatment. The contents of pituitary and serum PRL as measured by radioimmunoassay essentially paralleled the morphological results. These data indicate that OT may play a physiological role in PRL secretion as a releasing factor, and that OT administration causes the interconversion of the three types of PRL cells, indicating the mature type to be at the most activated state of secretory function.

Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands

Two novel peptides, named PACAP (pituitary adenylate cyclase activating polypeptide) containing 38 (PACAP38) and 27 residues (PACAP27) were recently isolated from ovine hypothalami. In order to investigate the pituitary cell type(s) that bear a receptor for PACAP, PACAP38 was biotinylated and used for cytochemical examination of binding. The cells were also identified by immunocytochemical methods using the antisera against each of the rat anterior pituitary hormones or an antiserum against S-100 protein, a marker for pituitary folliculo-stellate (FS) cells. Biotinylated PACAP38 (biot-PACAP) exhibited adenylate cyclase stimulating activity (ACSA) comparable to PACAP38 in rat pituitary cell cultures, and displaced the bound 125I-PACAP27 to the rat pituitary membrane preparation to the same extent as PACAP38. After 2–4 days of culture, dispersed rat pituitary cells were incubated with varying concentrations of biot-PACAP at room temperature or 4°C. The bound biot-PACAP38 was visualized by avidin-biotin-peroxidase complex (ABC) method with nickel intensification. Biot-PACAP-positive and pituitary hormone or S-100-positive cells were counted. More than 90% of S-100-positive cells bound biot-PACAP38. A considerable number of GH and PRL cells and a lesser number of ACTH cells also bound biot-PACAP38, whereas only a few identified LH, FSH, or TSH cells bound biot-PACAP38. These results suggest that FS cells are a major target cell type for PACAP. A recent study from our laboratory demonstrated that PACAP stimulated the release of interleukin (IL)-6 in rat pituitary cell cultures. FS cells are known to produce IL-6.

Localization of mRNAs for a Pair of Prolactins and Growth Hormone in the Tilapia Pituitary Using in Situ Hybridization with Oligonucleotide Probes

Oligonucleotide probes were synthesized for the mRNAs of a pair of tilapia prolactins (tPRL 177 and tPRL 188) and growth hormone (tGH) based on cDNAs for the hormones of Oreochromis niloticus and amino acid sequences for the hormones of O. mossambicus. The three 45mer probes were labeled with 35S for hybridization studies on pituitary sections of O. mossambicus adapted to fresh water (FW) or seawater (SW). Expression of tPRL mRNA in the rostral pars distalis was clearly evident with either PRL probe in adjacent sections in PRL cells of the rostral pars distalis; mRNAs of both PRLs were colocalized in the same cells. In addition, the tGH probe demonstrated expression of tGH mRNA specifically in GH cells in the proximal pars distalis. The hybridization signals for both PRLs were significantly greater in the rostral pars distalis of FW fish than in that of SW fish, as judged by computer-aided analysis. In addition, grain concentration for both PRLs was significantly greater over centrally located PRL cells of FW fish. In addition, although overall grain concentrations were lower in SW fish, there were significantly more grains over the centrally located PRL cells with the tPRL 177 probe, whereas there was no difference with the tPRL 188 probe. There was no detectable difference in the occurrence of tGH mRNA between FW and SW fish.

Immunocytochemical and Immunogold Localization of Two Prolactin Isoforms in the Same Pituitary Cells and in the Same Granules in the Tilapia ( Oreochromis mossambicus)

The tilapia pituitary secretes two forms of prolactin (tPRL) and a single growth hormone (tGH). The tPRLs share only 69% sequence identity and are designated tPRL 177 and tPRL 188 to indicate the number of amino acid residues in each isoform. Our aim was to develop specific antisera for detection of these three related polypeptides. Ten peptides corresponding to unique epitopes on the tPRLs and two peptides of tGH were synthesized using solid-phase methods, conjugated to carrier proteins, and used as immunogens for antibody production in rabbits. Select antisera for the tPRLs were highly specific, exhibiting only 1% cross-reactivity to the alternate tPRL under noncompetitive ELISA conditions at dilutions used in immunocytochemical analysis. The anti-tGH specifically bound to cells in the proximal pars distalis. Production of both tPRLs by a single cell type was indicated by the binding of both anti-tPRL 177 and anti-tPRL 188 to the same cells in the rostral pars distalis. Ultrastructural analysis of PRL-producing cells stained sequentially using the two different anti-tPRL antibodies labeled with immunogold of two size classes indicated that both tPRLs appear in the same granules. These findings suggest that the biological significance of two forms of PRL in the adult tilapia is not a function of differential regulation of two different classes of PRL cells or differential release of unique secretory granules.

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.

Effects of fasting, medium glucose, and amino acid concentrations on prolactin and growth hormone release, in vitro, from the pituitary of the tilapia Oreochromis mossambicus

Previous investigations have shown that the release of PRL and GH from the tilapia pituitary is directly sensitive to osmotic pressure and a variety of endocrine and neuroendocrine factors. The present studies were aimed at determining whether the spontaneous release of PRL and GH, in vitro, is: (1) sensitive to the nutritional status of the fish, and (2) responsive to variations in the d-glucose and total amino acid content of the incubation medium. In the first series of experiments, male fish (50 to 60 g) were divided into two groups. One group was fed twice daily for 2 weeks while the second received no food. A nearly homogeneous mass of PRL-secreting cells was dissected from the rostral pars distalis (RPD) and incubated for 18 to 20 hr in either hyposmotic (300 mOsmolal) or hyperosmotic (355 mOsmolal) medium. Similarly, a mass of GH-secreting cells was dissected from the proximal pars distalis (PPD) and incubated for 18 to 20 hr in isosmotic (320 mOsmolal) medium. Fasting was found to alter the total amount of PRL and GH in the culture well (tissue + medium) at the end of the incubations, decreasing PRL and increasing GH. Fasting was also found to both reduce spontaneous PRL release in vitro and suppress its stimulation by reduced osmotic pressure ( P < 0.01). By contrast, fasting resulted in a substantial increase in spontaneous GH release from the PPD in vitro ( P < 0.01). In the second series of experiments, GH release was found to increase as the d-glucose concentration of the medium decreased ( P < 0.01), while prolactin release was unresponsive. The spontaneous release of both PRL and GH in vitro was inversely related to the quantity of essential amino acids (Eagle's minimum essential medium; MEM) added to the incubation medium ( P < 0.01). These findings suggest that the activity and regulation of PRL and GH cells in the tilapia pituitary may be sensitive to the nutritional status of the fish. It would also appear that GH cells, but not PRL cells, may respond to changes in circulating d-glucose levels and that both may respond to variations in circulating amino acids. Additional studies will be required to determine whether the latter response is directed by specific amino acids.

Regulation of Prolactin Messenger Ribonucleic Acid Levels by Estradiol and Dihydrotestosterone as Evaluated by in situ Hybridization Performed on Implanted Pituitary Glands and Anterior Pituitary Cells in Culture in the Male Rat

We have recently demonstrated that 17ß-estradiol (E(2) ) administration increases protactin (PRL) mRNA levels in the male rat anterior pituitary gland and that this stimulatory effect is partially inhibited by concomitant administration of dihydrotestosterone. In order to gain more information about the site(s) of action of E(2) and dihydrotestosterone on PRL gene expression, we have studied the effects of these two hormones in pituitaries implanted under the kidney capsule as well as in anterior pituitary cells in culture. In implanted pituitaries, PRL mRNA levels were increased by 90% as compared to values obtained in the stalk-connected pituitaries from the same animals. Administration of E(2) induced a further increase of PRL mRNA levels in implanted pituitaries, while dihydrotestosterone did not produce any change in animals which had been treated or not with E(2) . In anterior pituitary cells in culture, addition of E(2) to the culture medium resulted in a 60% increase of PRL mRNA levels over control values. Supplementation with dihydrotestosterone did not induce any variation in the concentration of PRL mRNA in cells which were treated or not with E(2) . These results indicate that E(2) exerts a direct action on PRL cells at the pituitary level and strongly support the key role of the hypothalamus in the inhibitory effect of androgens on estrogen-induced stimulation of PRL mRNA in the male rat pituitary.

Mammotropes and somatotropes in the adenohypophysis of androgenized female mice: morphological and immunohistochemical studies by light microscopy correlated with routine electron microscopy.

Female mice were divided into androgenized (AF) and control (CF) groups. The AF mice were injected subcutaneously with testosterone propionate (Tp) and the CF mice with sesame oil at 5 days of age. Mammotropes (PRL cells) and somatotropes (GH cells) in the adenohypophyses of these mice when they became adults were studied with immunohistochemistry and morphometry by light microscopy correlated with routine electron microscopy. In CF mice, almost all of the PRL-immunoreactive cells (about 43% of all parenchymal cells) were type I (classical) PRL cells, and almost all of the GH-immunoreactive cells (about 30% of all parenchymal cells) were type I (classical) GH cells. Type II PRL cells accounted for about 0.4% of parenchymal cells, and type II GH cells were about 2.5% of all parenchymal cells. In AF mice, the percentages of PRL and GH cells were not significantly different from those of CF mice. Mammosomatotropes (Ms cells) in both groups were less than 1% of all parenchymal cells. Numbers of all parenchymal, PRL and GH cells, however, were increased significantly in AF mice when compared to those in CF, because the adenohypophysis was increased in volume in AF mice. Type I PRL cells were larger in AF than in CF. The ultrastructure suggested that type I PRL cells may show increased PRL synthesis and secretion in AF mice. Furthermore, AF mice, in which the hypothalamus is masculinized by the neonatal treatment with Tp, retained feminine characteristics in the population and size of PRL cells and GH cells in the adenohypophysis.

Immunohistological analysis in the distribution of cells in the fetal porcine adenohypophysis.

Adenohypophyses of porcine fetuses from 25 to 110 days of gestation were studied by immunohistochemical staining to ascertain the ontogeny of specific cell types and their spatial distribution in the pars distalis. No hormone-containing cells were found before 30 days of gestation. ACTH cells were observed first at 40 days, while GH and LH cells appeared first at 60 days. PRL cells were initially detected at 105 days. ACTH immunoreactive cells were also observed in the pars intermedia at 40 days. Blood capillaries were interposed between cell cords of the pars distalis after 40 days of gestation. ACTH cells were evenly distribution in all areas of the pars distalis except the rostral area (sex zone). GH cells were densely distributed in lateral wings of the pars distalis and immediately anterior to Rathke's lumen. PRL cells resembled GH cells in their distribution pattern, but PRL cells were fewer in number. LH cells were scattered in the sex zone of the pars distalis from 60 to 80 days of gestation. After 90 days, they became scattered throughout the pars distalis but were more numerous in the sex zone than in other areas. The inductive elements of adenohypophysial cells from Rathke's pouch epithelia are discussed. We hypothesize that cell cords of specific areas facing Rathke's lumen may differentiate into specific cell types of the pars distalis during fetal life.

Pituitary Enlargement With Suprasellar Extension in Functional Hyperprolactinemia Due to Lactotroph Hyperplasia

PEILLON, F.; DUPUY, M.; LI, J. Y.; KUJAS, M.; VINCENS, M.; MOWSZOWICZ, I.; DEROME, P. Author Information

Alpha-melanocyte-stimulating hormone is a mammotrophic factor released by neurointermediate lobe cells after estrogen treatment

When neurointermediate lobe (NIL) cells from ovariectomized rats are exposed to 17 beta-estradiol (E2) in vitro, they release a substance that rapidly induces the recruitment of additional PRL cells into the secretory pool. In the present study we tested the hypothesis that this mammotrophic factor is alpha MSH. Cocultures of anterior pituitary and NIL cells were incubated for 18 h, exposed to various treatments for 3 h, and then subjected to a reverse hemolytic plaque assay to quantify the percentage of all pituitary cells that released PRL. Combined exposure to E2 and NIL cells caused a significant increase in the fraction of anterior pituitary cells that released PRL, and the presence of TRH during the reverse hemolytic plaque assay did not affect the magnitude of the response. Treatment with dopamine (which inhibits alpha MSH release) reversed the recruitment of PRL secretors induced by E2 stimulation of NIL cells. Likewise, immunoneutralization with an antiserum directed against alpha MSH abolished the response. Furthermore, alpha MSH alone could substitute for E2 and NIL cells in evoking the recruitment response, whereas none of several other POMC-derived peptides had any consistent effect. Taken together, these results demonstrate that alpha MSH is a mammotrophic factor released by NIL cells in response to E2.

Alpha-melanocyte-stimulating hormone is a mammotrophic factor released by neurointermediate lobe cells after estrogen treatment.

When neurointermediate lobe (NIL) cells from ovariectomized rats are exposed to 17 beta-estradiol (E2) in vitro, they release a substance that rapidly induces the recruitment of additional PRL cells into the secretory pool. In the present study we tested the hypothesis that this mammotrophic factor is alpha MSH. Cocultures of anterior pituitary and NIL cells were incubated for 18 h, exposed to various treatments for 3 h, and then subjected to a reverse hemolytic plaque assay to quantify the percentage of all pituitary cells that released PRL. Combined exposure to E2 and NIL cells caused a significant increase in the fraction of anterior pituitary cells that released PRL, and the presence of TRH during the reverse hemolytic plaque assay did not affect the magnitude of the response. Treatment with dopamine (which inhibits alpha MSH release) reversed the recruitment of PRL secretors induced by E2 stimulation of NIL cells. Likewise, immunoneutralization with an antiserum directed against alpha MSH abolished the response. Furthermore, alpha MSH alone could substitute for E2 and NIL cells in evoking the recruitment response, whereas none of several other POMC-derived peptides had any consistent effect. Taken together, these results demonstrate that alpha MSH is a mammotrophic factor released by NIL cells in response to E2.

Stimulation of Prolactin Cell Differentiationin Vitroby a Milk-Borne Peptide*

We have previously reported that the normal expression of PRL-secreting cells in neonatal rats requires a maternal signal specific to the first few days of lactation. These results raised the possibility that a milk-borne factor(s) ingested by the neonate and absorbed into the circulation might induce the ontogenic appearance of PRL cells. The purpose of the present study was to determine whether milk from this period could directly stimulate the differentiation of PRL secretors in culture. Monodispersed anterior pituitary cells from 1-day-old pups were cultured for 6 days with aqueous extracts of milk from early (days 2, 3, and 4) and late (days 15 and 16) lactation and then subjected to reverse hemolytic plaque assays for PRL and GH release. We found that the addition of milk extracts (10 mg/ml) from either early or late lactation stimulated the differentiation of PRL secretors (to 6.1 +/- 1.0% and 2.4 +/- 0.7% of all pituitary cells, respectively; mean +/- SE; n = 3) above that in control cultures without milk (0.2 +/- 0.2%). Thus, early milk was more than twice as effective as late milk in this regard (P less than 0.05). This effect appeared to be specific to PRL cell differentiation, since the relative abundance of GH secretors was not different between cells treated with either early or late milk (29.3 +/- 4.8% and 33.7 +/- 3.9%, respectively). On the other hand, late milk was more than twice as effective as early milk at increasing the capacity of GH secretors to release hormone (P less than 0.05). Preliminary characterization by gel filtration chromatography and proteolytic hydrolysis indicates that the bioactivity that differentiates PRL secretors is a small peptide(s) of 2000-6000 daltons. Taken together, our results demonstrate that a milk-borne peptide(s) is capable of specifically stimulating the differentiation of PRL-secreting cells in vitro, and that this bioactivity is more prevalent in milk from early lactation.

Pituitary Enlargement with Suprasellar Extension in Functional Hyperprolactinemia due to Lactotroph Hyperplasia: A Pseudotumoral Disease

Beside the well characterized PRL-secreting adenomas, a wide spectrum of functional hyperprolactinemic states exists. We describe here five women, 21-38 yr old, all suspected of having a PRL-secreting adenoma because of a pseudotumoral appearance of the pituitary on computerized tomographic (CT) scan or magnetic resonance imaging (MRI). Four had oligomenorrhea with or without galactorrhea, one had amenorrhea with galactorrhea, and two complained of infertility. In the same patient, basal plasma PRL levels were variable on different days, sometimes normal (mean +/- SEM, 11.3 +/- 1.5 micrograms/L), sometimes elevated (49 +/- 7 micrograms/L), but in all cases, a PRL response of large amplitude to TRH (6- to 8-fold increase in the basal value) was observed. Basal plasma levels of estradiol were within luteal phase normal values (0.41 +/- 0.13 pmol/L), while progesterone levels were low (1.92 +/- 0.47 nmol/L). CT scan or MRI showed an intrasellar mass with suprasellar extension, suggesting a tumoral process. However, the signal intensity was homogeneous, and on coronal views, the suprasellar extension was pyramidal and symmetrical, and the pituitary stalk was always in the midline. The five patients were operated on by the transsphenoidal route, but no adenoma was found. Surgical biopsies were taken in four cases, and lactotroph hyperplasia, i.e. enlarged cell cords consisting mainly of PRL cells, was found in three of them. One case displayed a continuum between areas of lactotroph hyperplasia and adenomatous PRL cells. We conclude that functional hyperprolactinemia may mimic on CT scan or MRI a PRL-secreting adenoma.