The lactogenic response of mouse mammary explants to mouse prolactin (mPRL) and mouse growth hormone (mGH) was studied. Mouse mammary explants were cultured, in a defined medium, in the presence of varying concentrations of either mPRL or mGH and the rate of 3H amino acid incorporation into casein was measured. The lactogenic activity of mPRL was compared with that of ovine prolactin (oPRL) and found to be directly comparable. MGH, when compared to oPRL, was found to be less active in stimulating casein synthesis.

The activities of insulin-like growth factors (IGFs) in bone are modulated by a family of binding proteins (IGFBPs) whose physiological roles remain poorly understood. We have previously shown that targeted overexpression of IGF-I in osteoblasts of transgenic (OC-IGF-I) mice stimulates bone formation. In this model, bone formation is markedly but transiently increased in an age-dependent manner, raising the possibility that IGF-I may be influencing IGFBPs to in turn modulate its paracrine actions within bone. We sought to characterize the IGFBPs in normal mouse bone during development and to determine whether osteoblast-targeted overexpression of IGF-I influenced bone IGFBP abundance in vivo. Femoral bone IGFBP content was assessed in control nontransgenic and OC-IGF-I mice by I125-IGF-I ligand and immunoblotting. Bone IGFBP-5 and IGF-I mRNA abundance was determined using real-time reverse transcription (RT)-PCR. Ligand blot of bone extract showed a 30-kDa band, identified as IGFBP-5 by immunoblot, predominated. The abundance of IGFBP-5 declined with age in both control and transgenic bone. Ligand and immunoblot analysis revealed a 5-fold increase in IGFBP-5 protein levels at 3 weeks in transgenic bone (P<0.0001). The elevated IGFBP-5 protein levels were associated with a similar increase in IGF-I mRNA abundance (4-fold, P<0.01) and a significant increase in IGFBP-5 mRNA abundance (1.5-fold). Despite the age-related decline at 6 weeks, IGFBP-5 remained significantly (P<0.01) more abundant in transgenic bone compared to controls. In contrast, bone IGFBP-4 abundance was relatively unchanged by either age or IGF-I overexpression. These studies demonstrate a distinctive developmental pattern of IGFBP-5 content in mouse bone and show that osteoblast-derived IGF-I determines skeletal IGFBP-5 abundance, at least in part by inducing its synthesis. In that IGFBP-5 is thought to stimulate bone formation, directly or via IGF-I action, such changes in bone IGFBP-5 may be important to ensure robust bone acquisition in the early postnatal period.

Objective: To test the involvement of nitric oxide in murine ovarian follicular cysts.Design: Controlled animal study.Setting: Academic research environment.Animal(s): Immature female B6D2F1 mice at 23+/-2 days old.Intervention(s): Ovarian cysts were induced by implanting miniosmotic pumps that delivered and maintained constant levels of hCG. Nitric oxide studies included the delivery of nitric oxide synthase (NOS) inhibitors, N-G-nitro-L-arginine methyl ester (L-NAME), or N-G-nitro-D-arginine methyl ester, by the same method.Main Outcome Measure(s): Ovulation assays measured cumulus oocyte complexes and blood follicle barrier (BFB) function.Result(s): Chronic treatment with hCG induced enlarged ovaries containing multiple follicular cysts, which were approximately double the size of follicles in sham-operated mice. These cysts enclosed few, if any granulosa cells, secreted high levels of testosterone, and had impaired ovarian BFB function. Inhibition of NOS by L-NAME during ovarian cyst formation reduced the size of follicular cysts, sustained normal testosterone levels, and maintained hormonal BFB reactivity in cystic follicles.Conclusion(s): Nitric oxide was found to be involved in the formation of hCG-induced murine follicular cysts and complications associated with these cysts were ameliorated by the NOS inhibitor L-NAME.

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted an uterine homogenates using [I-125]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the est rous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGG-1 action, and that IGF-IR is constitutively expressed in the mouse uterus. (C) 1999 Wiley-Liss, Inc.

PRL is synthesized and released by several extrapituitary tissues, including decidualized endometrial stromal cells. As interleukin-a (IL-2) stimulates the synthesis and release of pituitary PRL, and decidual stromal cells have receptors for IL-2, we examined whether IL-2 also regulates the release of decidual PRL. Exposure of primary cultures of human decidual cells (10(6) cells/well) from term pregnancies to IL-2 (50 ng/mL) inhibited PRL release beginning 48 h after exposure. The inhibition by IL-2 was dose dependent, and the maximal inhibition of PRL release after 5 days of exposure to IL-2 was 71.0 +/- 0.9% (mean +/- SE). IL-2, however, had no effect on decidual cell viability. The inhibitory effect of IL-2 on PRL release was secondary to inhibition of PRL synthesis. Decidualized human endometrial stromal cells transfected with 3 kb of the extrapituitary PRL (exon 1a) promoter coupled to a luciferase expression vector responded to IL-2 (10 ng/mL) with a significant decrease in luciferase activity. These findings strongly suggest that IL-2 inhibits the synthesis and release of decidual PRL and provide further support for a critical role of cytokines in the regulation of decidual PRL gene expression.

In a previous study, insulin-like growth factor-I (IGF-I) and a potential inhibitory binding protein, IGF binding protein-4 (IGFBP-4), were found to be expressed in a cell-specific and temporally dynamic manner in the mouse uterus during the periimplantation period. The mRNA levels of both IGF-I and IGFBP-4 rapidly increased between Days 5 and 6 of gestation and then declined after the establishment of embryo implantation. In the current study, we conducted in situ hybridization analysis on pregnant mouse uteri and deciduomata-induced mouse uteri to determine whether the presence of an embryo is required for uterine IGF-I and IGFBP-4 mRNA expression. Our data reveal that before implantation, the maternal hormones of pregnancy support IGF-I and IGFBP-4 mRNA expression. Beyond gestational Day 4, however, decidualization of the uterine stroma, either artificially induced or naturally induced by an implanting embryo, is sufficient for maintaining the expression of these two genes. Thus, the presence of specific embryo factors is not required for IGF-I and IGFBP-4 expression in the periimplantation uterus. These studies indicate that the expression of IGF-I and IGFBP-4 mRNAs may be associated with decidualization of uterine stromal cells. The restricted anatomical and temporal expression of IGF-I and IGFBP-4 mRNAs in the periimplantation uterus suggests a physiologic role for IGF-I and IGFBP-4 in the maintenance and expansion of decidualization.

Prolactin (PRL) has been implicated in numerous physiological and developmental processes. The mouse PRL gene was disrupted by homologous recombination. The mutation caused infertility in female mice, but did not prevent female mice from manifesting spontaneous maternal behaviors. PRL-deficient males were fertile and produced offspring with normal Mendelian gender and genotype ratios when they were mated with heterozygous females. Mammary glands of mutant female mice developed a normal ductal tree, but the ducts failed to develop lobular decorations, which is a characteristic of the normal virgin adult mammary gland. The potential effect of PRL gene disruption on antigen-independent primary hematopoiesis was assessed. The results of this analysis indicated that myelopoiesis and primary lymphopoiesis were unaltered in the mutant mice. Consistent with these observations in PRL mutant mice, PRL failed to correct the bone marrow B cell deficiency of Snell dwarf mice. These results argue that PRL does not play any indispensable role in primary lymphocyte development and homeostasis, or in myeloid differentiation. The PRL-/- mouse model provides a new research tool with which to resolve a variety of questions regarding the involvement of both endocrine and paracrine sources of PRL in reproduction, lactogenesis, tumorigenesis and immunoregulation.

Here we describe in detail both the expression of Hoxa-11 in the wild-type mouse uterus and the defects resulting in maternal reproductive failure of Hoxa-11 null female mice. The Hoxa-11 gene is expressed at peak levels in uterine stromal cells during metestrus. Hoxa-11 transcripts were induced beginning on Day 2 of gestation in the stromal cells underlying the uterine epithelium and appeared in the secondary decidual zone between Days 6 and 8 of gestation. At early gestational stages, stromal, decidual, and glandular cell development were deficient in Hoxa-11 null uteri in comparison to wild-type as assessed by histology and immunohistochemical localization of the decidual cell marker epitope, stage-specific embryonic antigen-3 (SSEA-3). Both steroid-induced uterine stromal and glandular cell proliferation as well as oil-induced stromal decidualization after induction of pseudopregnancy were deficient in mutant uteri. Moreover, both Western blotting and immunohistochemistry demonstrated that the burst of glandular leukemia inhibitory factor (LIF) found in normal pregnant uteri at Day 4.5 of gestation was absent in Hoxa-11-deficient uteri. The LIF burst was also not observed in the uteri of bilaterally ovariectomized, hormonally stimulated Hoxa-11 mutants. These results demonstrate that the Hoxa-11 gene is required for normal uterine stromal cell and glandular differentiation during pregnancy, as is the presence of the steroid-induced glandular LIF burst initiating embryo implantation.

Previous studies have suggested a role for insulin-like growth factors (IGFs) in both embryo and trophoblast growth, as well as in uterine differentiation. Included in the IGF family are the IGF binding proteins (IGFBPs). In the current study we have demonstrated, by Western ligand blot of mouse uterine tissue extracts, a dramatic increase in IGFBP-4 at the time of embryo implantation. As an extension of this finding, we used in situ hybridization to examine the ontogeny and anatomical localization of IGFBP-4 mRNA in the mouse uterus during the peri-implantation period. On gestational Day 2, while the embryos are still in the oviduct, no uterine IGFBP-4 mRNA signal was observed. However, on gestational Day 4, when the embryos are free in the uterine lumen, IGFBP-4 mRNA was present in the uterine stroma underlying the luminal epithelium. By gestational Day 6, approximately 24 h after implantation, a IGFBP-4 mRNA signal was intense at each implantation site and extended throughout the decidua. A signal was absent in the uterine tissue between implantation sites. By gestational Day 8, the IGFBP-4 mRNA signal was reduced and confined to the stroma nearest the myometrium. The specific anatomical and temporal nature of the IGFBP-4 mRNA expression suggests a physiologic role for this binding protein in the implantation process.

Glycosylated human prolactin is found in several biological fluids and can only be detected by western immunoblotting. Glycosylated prolactin migrates at 25,000-27,000 molecular weight while prolactin migrates at 23,000. We have discovered that several antihuman prolactin sera crossreact with some light chain components of human immunoglobulins, which also migrate at 25,000 on electrophoretic gels run under reducing conditions. Evidence is provided for prolactin binding to immunoglobulins in biological fluids, which may explain why some polyclonal anti-prolactin sera demonstrate this crossreactivity.

OBJECTIVE:To further characterize the high molecular weight (MW) forms of prolactin (PRL) present in patients with macroprolactinemia.DESIGN:Case reports with laboratory investigations.SETTING:Academic medical centers.PATIENTS:Two patients with macroprolactinemia.INTERVENTIONS:Measurements of PRL concentrations before and after chromatographic separations.RESULTS:The majority of serum PRL had an estimated MW of at least 669 kd in the first patient and approximately 171 kd in the second. During a pregnancy, a new form of PRL (MW 291 kd) appeared in the first patient's serum and persisted for at least 3 years. Immunoprecipitation and polyacrylamide gel electrophoresis under reducing and denaturing conditions revealed that the largest form of PRL (MW 669 kd) was composed mostly of 25 kd glycosylated PRL; intermediate forms (171 kd and 291 kd) were composed of roughly equal portions of 25 kd glycosylated PRL and 23 kd nonglycosylated PRL, whereas "little" PRL in these patients was composed primarily of 23 kd nonglycosylated PRL. Injection of the first patient's serum into rats demonstrated that the human PRL (hPRL) immunoreactivity was cleared from the serum more slowly than the PRL from sera containing predominantly little hPRL; after stimulation with thyrotropin-releasing hormone in the second patient, serum PRL concentrations decayed more slowly than observed in normal subjects.CONCLUSIONS:Large forms of serum PRL are at least partially glycosylated. These large forms are heterogeneous, both within and among patients. Delayed clearance may account for increased serum PRL concentrations in patients with macroprolactinemia.

Human decidual tissue, the specialized endometrium of the luteal phase of the menstrual cycle and pregnancy, synthesizes and releases prolactin, relaxin, renin, and at least three insulinlike growth factor (IGF)-binding proteins. Although the physiologic roles of these decidual protein hormones during pregnancy are unclear, numerous studies suggest that the hormones may act locally to affect the function of the placenta, decidua, and fetal membranes. In addition, the synthesis and release of these hormones appear to be regulated locally by factors produced by the placenta, decidua, and fetal membranes.

Studies from this laboratory indicate that the synthesis and release of prolactin from human decidual cells are regulated by factors released by the placenta, decidua and fetal membranes. A 23.5 kD protein (decidual prolactin-releasing factor, PRL-RF) has been purified to homogeneity from human placental tissue and placental conditioned medium. The releasing factor stimulates an acute release of prolactin that occurs within the first few minutes of exposure and a prolonged release, secondary to new hormone synthesis, that begins about 6-8 hours later. In addition, the synthesis and release of decidual prolactin are stimulated by IGF-I, insulin and relaxin, each acting through distinct plasma membrane receptors. In contrast, the synthesis and release of decidual prolactin are inhibited by arachidonic acid, lipocortin I and a 35-45 kD decidual protein (prolactin-releasing inhibitory factor) that has been partially purified from decidual conditioned medium. Studies of the second messengers involved in the regulation of decidual prolactin release strongly suggest that decidual prolactin release may be mediated, at least in part, by activation of phosphoinositide metabolism and stimulation of adenylate cyclase. The demonstration that the synthesis and release of decidual prolactin are regulated by PRL-RF, IGF-I, insulin, relaxin, arachidonic acid, PRL-IF and lipocortin I strongly suggests that there is novel autocrine/paracrine feedback regulation between the placenta, fetal membranes, and decidua in the regulation of decidual prolactin.

Human decidual tissue synthesizes and secretes a protein that is identical to pituitary prolactin in its chemical, biological and immunological properties. Nevertheless, the factors that regulate the synthesis and release of prolactin from the decidual tissues appear to be different to those regulating the synthesis and release of pituitary prolactin. Studies from our laboratory over the past few years indicate that the synthesis and release of decidual prolactin are regulated, at least in part, by factors released by placenta, fetal membranes and decidua. The placenta releases a 23.5 KMr protein [decidual prolactin-releasing factor (PRL-RF)] that stimulates a rapid release of prolactin within the first few minutes of exposure and a sustained, prolonged, increase in the synthesis and release of prolactin beginning 6–8 h after exposure. The acute release ofprolactin in response to PRL-RF is inhibited by decidual prolactin release-inhibitory factor (PRL-IF), a 35–45 K Mr protein that is released by the decidua. The secondary increase in the synthesis and release of prolactin in response to PRF-RF is blocked by lipocortin I, which is synthesized by both the placenta and decidua. IGF-I, insulin and relaxin also stimulate the synthesis and release of prolactin. However, the stimulation in response to these factors does not occur until 24–48 h after exposure. The cellular mechanisms involved in the release of decidual prolactin are as yet unknown. However, recent studies implicate activation of adenylate cyclase, phospholipase C-mediated phosphoinositide hydrolysis and phospholipase A2-mediated arachidonic acid release in the regulation of prolactin release. The finding that the synthesis and release of decidual prolactin are regulated, at least in part, by PRL-RF IGF-I, insulin, relaxin and lipocortin I strongly suggests that there is novel feedback regulation between the placenta, fetal membranes, and decidua in the regulation of the synthesis and release of decidual prolactin.

Several molecular forms of immunoreactive human (h) PRL with differences in biological activities have been described. To investigate these differences in PRL secretion, we have characterized the molecular forms of circulating PRL in postpartum women during breastfeeding (n = 5) and sleep (n = 5) and in eumenorrheic women with galactorrhea (n = 5) after TRH stimulation (n = 4), metaclopramide infusion (n = 5), and morphine administration (n = 2). All provocative testing in eumenorrheic women were carried out during the early follicular phase of the menstrual cycle (days 1-5). PRL variants were identified and semiquantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography. Mean basal levels of PRL were significantly elevated in nursing women (P less than 0.01) compared to those in nonlactating postpartum women and women with galactorrhea. The relative proportions of PRL variants [glycosylated PRL with an apparent mol wt of 25K and a nonglycosylated species (hPRL) with an apparent mol wt of 23 K] were similar for all three groups. A third immunoreactive PRL variant with an apparent mol wt of 21K was identified in nonlactating postpartum women. With breastfeeding or sleep, PRL concentrations increased more than 4-fold and were accompanied by a shift toward the lower mol wt, nonglycosylated species. In response to TRH (200 micrograms, iv), PRL levels increased by 493 +/- 86% (mean +/- SE) over basal levels. This acute release of PRL was positively correlated (r = 0.06074; P less than 0.001) with a significant increase in secretion of the hPRL species (P less than 0.05). During metaclopramide infusion, a similar increment in hPRL was observed during acute PRL release. Administration of the opiate agonist morphine was associated with a small increment in PRL release and did not alter the proportions of PRL variants in the circulation. Because glycosylation is a posttranslational step in the processing of PRL, our studies suggest that the acute release of PRL in response to physiological and pharmacological secretagogues may reflect the output of more recently synthesized PRL from the pituitary. The physiological role of the 21K PRL species remains to be determined.

Prolactins in glycosylated and nonglycosylated form were isolated from 218 second- and third-trimester amniotic fluid samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose paper, and probed with anti-human prolactin serum and protein A labeled with iodine 125. The glycosylated and nonglycosylated prolactin bands were exposed by autoradiography. The bands were analyzed by densitometry, and the ratio of glycosylated to total (glycosylated + nonglycosylated) prolactin was calculated (glycosylated/total ratio) and compared with matched prolactin radioimmunoassay measurements. Both radioimmunoassay-measured and nonglycosylated prolactin decreased from the second to the third trimester whereas glycosylated prolactin concentrations remained unchanged. Concomitantly, glycosylated/total prolactin ratios increased significantly (p less than 0.001). The correlation of radioimmunoassay-measured prolactin was closer to nonglycosylated prolactin (r2 = 0.32, p = 0.00001) than to glycosylated prolactin (r2 = 0.02, p = 0.06). We suggest that the proportion of amniotic fluid glycosylated prolactin increases with length of gestation and that glycosylation of prolactin is inversely related to total prolactin secretion. Furthermore, prolactin's immunoreactivity correlated more closely to the nonglycosylated-prolactin variant, suggesting that radioimmunoassay of prolactin poorly recognizes the glycosylated prolactin form.

In order to study the possible differential effects of the nonglycosylated and glycosylated forms of prolactin on insulin content and secretion in pancreatic islets, neonatal rat pancreatic islets were exposed for 6 days in vitro to 2 μg/ml of nonglycosylated ovine prolactin (oPRL), or to 2 μl of glycosylated oPRL (G-oPRL). oPRL stimulated a significant increase (p < 0.01) in the total amount of insulin released into the medium over the 6 day culture period; however, G-oPRL had no effect. Islets cultured for 6 days in the presence of oPRL showed no increase in the amount of DNA per islet. However, there was a significant (p < 0.007) increase in the amount of total protein synthesized by the islets exposed to oPRL. These findings suggest that the effect of oPRL on neonatal rat pancreatic islet cells is a nonspecific effect. The nonglycosylated form of PRL may play a role in B-cell function by promoting protein synthesis, which results in augmented insulin synthesis.

The receptor-binding properties of monomeric nonglycosylated human PRL (hPRL), glycosylated hPRL that does not bind to Concanavalin-A-Sepharose (G1-hPRL) and glycosylated hPRL that binds to Concanavalin-A-Sepharose (G2-hPRL) were tested in the lactating rabbit mammary gland RRA for lactogenic hormones. Variations in the glycosylation pattern of G-hPRL altered its receptor-binding properties, suggesting that the site of glycosylation may be proximal to the receptor-binding region. Relative potencies for the displacement of [125I]hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 40%, and 26%, respectively. Relative potencies for displacement of [125I]G1-hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 44%, and 69%, respectively; however, the displacement curve for G2-hPRL was not parallel to the others. When G2-hPRL was radiolabeled, there was no specific binding to lactogenic receptors. The presence of PRL receptor subtypes and/or kinetic cooperativity was suggested by the complexity of the binding isotherms. The immunoreactivities of the PRLs were tested in a homologous RIA, using polyclonal antiserum. The modification of the glycosylation pattern of hPRL significantly altered the RIA values for PRL. When hPRL was used as the radiotracer, the percent cross-reactivities of hPRL, G1-hPRL, and G2-hPRL were approximately 100%, 23%, and 17%, respectively. When G1-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 135%, and 54% for hPRL, G1-hPRL, and G2-hPRL; however, the displacement curve for hPRL was not parallel to those of the glycosylated hPRLs. When G2-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 32%, and 37% for hPRL, G1-hPRL, and G2-hPRL. These data point out that the glycosylation heterogeneity of hPRL is a factor that affects the diagnostic accuracy of hPRL determinations. Specific RIAs for each PRL are needed so that we can have valid and reliable measurements of each PRL isoform and consequently gain a better understanding of PRL's complex biological role.