Lactogenic Activity Research Articles

Effects of steroid and lactogenic hormones on islets of Langerhans: a new hypothesis for the role of pregnancy steroids in the adaptation of islets to pregnancy

Adaptive changes that occur in islets of Langerhans during pregnancy include enhanced insulin secretion, insulin synthesis, beta-cell proliferation, gap-junctional coupling among beta-cells, and glucose oxidation. We have determined that elevated lactogenic activity is directly responsible for these changes in beta-cell function. Recently, we showed that two of the principal adaptive characteristics (insulin secretion and beta-cell proliferation) of rat pregnancy peaked on day 15 and returned to control levels by day 20. As placental lactogen remains elevated during late gestation, it was of interest to determine whether pregnancy steroids could reverse the effects of lactogen on islets. In this study, rat islets were cultured with progesterone, estradiol, rat PRL (rPRL), or combinations of these hormones (progesterone and rPRL, estradiol and rPRL, or progesterone and estradiol and rPRL). Insulin secretion was examined for 8 days, and beta-cell proliferation by 2-bromo-5'-deoxyuridine (BrdU) incorporation on days 4 and 8. rPRL treatment resulted in a time-dependent increase in insulin secretion that was 3-fold greater than that from control islets by day 8. Progesterone and estradiol had minimal effects on insulin secretion. Estradiol had no effect on the increased insulin secretion observed with rPRL during the first 6 days and a small inhibitory effect on days 7 and 8. Although progesterone treatment had no effect on the increased insulin secretion induced by rPRL during the first 3 days, it subsequently resulted in a decline in insulin secretion to that from control islets. The combination of progesterone and estradiol was more effective than either steroid by itself in reversing the effects of rPRL on insulin secretion. Similar results were obtained in the BrdU labeling experiments: 1) a 7-fold increase in the number of BrdU-labeled nuclei per islet was observed after culture in the presence of rPRL; and 2) estradiol had a small inhibitory effect on the increased BrdU labeling observed with rPRL; however, 3) progesterone completely reversed the effect of rPRL on islet beta-cell division. These results demonstrate that progesterone counterregulates the effects of PRL on insulin secretion and islet beta-cell division. The temporal changes observed in islets in vitro under the influence of PRL and progesterone mimic those seen in islets during pregnancy. We conclude that progesterone, which increases in the later stages of gestation, is the primary hormone responsible for counteracting the stimulatory effects of elevated lactogenic activity on islets during late pregnancy.

Evidence for a Role of the Liver in the Luteotropic Action of Prolactin in Rats1

Recent studies indicate that the liver may participate in growth-promoting and lactogenic activity of prolactin (PRL). Accordingly, our study was designed to determine whether the liver participates in the luteotropic activity of the hormone in rats. Normally cycling Long-Evans rats received infusions of solvent or 50 micrograms ovine (o) PRL per day into either the external jugular vein (EJV) or the hepatic portal vein (HPV) via osmotic minipumps. The modes of delivery were continuous, 2 pulses of 8 h each per day (2-8), or 4 pulses of 2 h each per day (4-2). Observation of daily vaginal smears was used to classify rats as either cyclic or predominantly diestrus (PD). In a second study, rats fitted with osmotic minipumps received oPRL or vehicle as above, and serial blood samples were obtained for measurement of serum oPRL and rat (r) PRL concentrations. Estrous cycles of normal length were observed in 9 of 11 rats (82%) receiving solvent infusions. All 8 animals receiving continuous oPRL infusions were classified as PD, regardless of site of infusion. In addition, in 4 of 5 (80%) HPV-infused and 5 of 6 (83%) EJV-infused rats on the 2-8 pulse schedule, the smear patterns were classified as PD. However, when rats received 4-2 EJV, 9 of 13 animals (69%) were classified as cyclic, whereas 9 of 11 rats (82%) were classified as PD when oPRL was similarly pulsed into the HPV. Differences in serum concentrations of oPRL between oPRL-treated and vehicle-infused rats were significant only for continuously infused animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy.

Up-regulation of maternal islet function is essential to accommodate the increased demand for insulin during pregnancy. Previously, we suggested that lactogenic activity regulates islet function during pregnancy. However, this hypothesis was based on the effect of homologous PRLs on islets, since the homologous placental lactogens (or islets) were unavailable. In this study we examine the direct effects of homologous placental lactogens (PL), PRL, and GH on insulin secretion and B-cell division in rat, mouse, and human islets in vitro. Neonatal rat islets were cultured for 8 days in the presence of 0-1000 ng/ml rat PL-I (rPL-I), rPRL, or rGH. Media were changed daily, and the insulin concentration was determined. rPL-I and rPRL (500 ng/ml) treatment resulted in a 2-fold increase in insulin secretion. rGH (1000 ng/ml) elicited a 30% increase in insulin secretion. Similarly, cell replication, as indicated by BrdU incorporation into B-cells, was increased 4-fold in the presence of rPL-I and rPRL. The ED50 for insulin secretion and 5'-bromo-2'-deoxyuridine (BrdU) incorporation was 70 ng/ml for rPL-I and 150 ng/ml for rPRL. Similarly, in adult rat islets, insulin secretion was increased 1.6-fold, and B-cell replication increased 3-fold in the presence of the lactogenic hormones. Neonatal mouse islets were cultured for 8 days in the presence of 500 ng/ml mouse (m) PL-I, mPL-II, mPRL, or mGH. mPL-I, mPL-II, and mPRL treatment resulted in a 2-fold increase in insulin secretion. mGH elicited a 30% increase in insulin secretion. BrdU incorporation into B-cells was increased 3-fold in the presence of mPL-I and mPRL and 2-fold in the presence of mPL-II. Adult human islets were cultured for 8 days in the presence of 1 microgram/ml human (h) PL, hPRL, or hGH. For human islets isolated from six pancreata obtained from females, hPL (138 +/- 10%), hPRL (133 +/- 9%), and hGH (117 +/- 3%) significantly increased insulin secretion compared to that from control islets. This study compares the direct effects among homologous PLs, PRLs, and GHs on insulin secretion and B-cell division in rat, mouse, and human islets. The results indicate that placental lactogen directly regulates islet function in several species and is probably the principal hormone responsible for the increased islet function observed during normal pregnancy.

Growth hormone expression in human Burkitt lymphoma serum-free Ramos cell line.

A human nonpituitary cell line grown under serum-free (sf) conditions (sfRamos Burkitt lymphoma cell line) has been reported to secrete a 29K PRL-like peptide which acts as an autocrine growth factor. Conditioned medium from these cells was examined for lactogenic activity using the Nb2 bioassay and RIAs specific for human GH (hGH) and hPRL. SfRamos conditioned medium stimulated the growth of Nb2 cells. Anti-hGH monoclonal antibodies but not anti-hPRL inhibited the mitogenic effect of sfRamos conditioned medium on Nb2 cells. Immunoreactive hGH but not hPRL was detected by RIA. Immunoprecipitation with anti-hGH polyclonal antibody followed by Western blot analysis with anti-hGH monoclonal antibody revealed a specific 22K band with the same mobility as pituitary hGH. Northern blot analysis with an hGH complementary DNA (cDNA) probe revealed a 1.0-kilobase transcript migrating coincident with pituitary hGH messenger RNA. A less abundant, 1.6-kilobase transcript was also observed. Reverse transcriptase-polymerase chain reaction using specific primers for the hGH cDNA generated the predicted 248-base pair band. Polymerase chain reaction sequencing of this fragment revealed sequence identity to the hGH-N cDNA, demonstrating conclusively the expression of the hGH-N gene in the sfRamos cell line.

The effect of progestins on prolactin receptor gene transcription in human breast cancer cells.

The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nM of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 nM or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland development.

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes.

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated by the use of labelled ovine prolactin) was increased 2-3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.

Evidence that the N-terminus of human growth hormone is involved in expression of its growth promoting, diabetogenic, and insulin-like activities.

Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.

Placental prolactin-like proteins in rodents: Variations on a structural theme

The placenta of many species of mammals produces and secretes placental lactogen (PL), a polypeptide hormone with structural and functional properties similar to pituitary prolactin (PRL). In rodents, the placenta produces more than one PL and also other proteins with structural homology to PRL (reviewed by Duckworth et al., 1988; Ogren and Talamantes, 1988). Unlike the PLs, these other PRL-like proteins appear to bind only weakly or not at all to lactogenic receptors (PRL receptors) and have little or no lactogenie activity. The physiological functions of the placental PRL-like proteins are not well understood. While there is good evidence that the PLs play a role in the preparation of the mammary gland for lactation, the nature of their lactogenic activities is not yet defined. For example, it is not known whether the PLs have effects on the mammary gland that differ qualitatively from those of pituitary PRL. In addition, receptors that bind PLs have been identified in a number of tissues other than the mammary gland (e.g., liver and ovary), but the effects of PLs on these potential target tissues have not been examined. Even less is known about the functions of the nonlactogenic PRL-like proteins, and with one exception, recep-

Decidual-Type Prolactin Expression by the Human Myometrium

The human myometrium, in addition to the decidualized endometrium of the late luteal phase and of pregnancy, has been proposed as a second source of uterine PRL, since immunoreactive PRL was found in supernatants from myometrial explant cultures. We demonstrate here that: 1) the human (h) PRL gene is expressed in the myometrium in vivo; 2) myometrial PRL is identical to pituitary hPRL; 3) the encoding transcript differs from pituitary hPRL messenger (m) RNA but is homologous to decidual and IM-9-P3 lymphoid hPRL mRNA; and 4) the expression of myometrial hPRL mRNA is inhibited by progestin. hPRL mRNA was detected in freshly isolated myometrium by Northern blot hybridization and was larger than the pituitary message. Sequence and primer extension analyses revealed that the transcript is identical to pituitary hPRL mRNA downstream of the pituitary cap site and carries an extension of the 5'-untranslated region homologous to that of decidual/IM-9-P3 lymphoid hPRL mRNA. This mRNA species results from alternative transcription initiation and comprises exon 1a of the hPRL gene, which is not transcribed in the pituitary. hPRL mRNA steady state levels and hPRL secretion increased dramatically when myometrial explants were maintained in long term culture. In addition to the 23,000 mol wt form, myometrial explants synthesized a glycosylated hPRL variant (G-hPRL) which was approximately 500 Daltons larger than pituitary G-hPRL but of similar size as lymphoid G-hPRL (26,500). Lactogenic activity of myometrial conditioned medium paralleled that of pituitary hPRL in the Nb2 lymphoma bioassay and was neutralized by the addition of monoclonal antibody to hPRL. hPRL secretion and hPRL mRNA abundance were not affected by estrogen but were markedly reduced by medroxy-progesterone acetate, which was maximally effective at a dose as low as 10(-10) M.

Fertility of Transgenic Female Mice Expressing Bovine Growth Hormone or Human Growth Hormone Variant Genes1

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypophysectomized Rats Depend on Residual Prolactin for Survival*

Hypophysectomized (Hypox) female Fischer 344 rats had 10-20% lactogenic activity in their serum when compared to controls by the Nb2 lymphoma proliferation assay. If such animals were treated daily with a rabbit antirat PRL serum, their serum lactogenic activity diminished further; severe anemia and immunological anergy developed; and death occurred within 8 weeks. In contrast, untreated Hypox animals increased gradually their serum lactogenic activity, starting on the 7th week after pituitary removal, which rose up to 50% of control levels by week 9. Hypox animals showed normochromic normocytic anemia, a grossly reduced immunocompetence, decreased body, thymus, spleen, adrenal, and ovary weights, and decreased DNA and RNA synthesis in the thymus, spleen, and bone marrow. However, the condition of Hypox animals did not deteriorate further over the 9-week experimental period. All the hematological deficiencies and decrease in organ weights observed in Hypox rats were normalized after grafting with syngeneic pituitaries (SPG). These effects of SPG could be inhibited by additional treatment with antirat PRL serum. Treatment of Hypox animals with ovine PRL had a restoring effect similar to SPG, which was not inhibited by additional antirat PRL serum treatment. Rat and ovine PRL and GH and human placental lactogen all stimulated the incorporation of 3H-thymidine by rat bone marrow cells in vitro. These results indicate that PRL has a multiple trophic effect and is capable of maintaining vital bodily functions for long periods of time.

6 Endometrial prolactin and implantation

The role of prolactin in human implantation and human endometrial function is still unclear. Synthesis of prolactin from human endometrium and decidua was first demonstrated in 1977. Prolactin mRNA isolation from term decidua subsequently confirmed expression of the prolactin gene in human endometrium. More recently, a glycosylated form of prolactin has been isolated from both the human pituitary and human endometrium. It appears that predominately glycosylated prolactin is secreted in the late luteal phase of the menstrual cycle, with increasingly greater amounts of non-glycosylated prolactin secreted as pregnancy advances. The biological significance and regulation of prolactin glycosylation is uncertain, although glycosylated ovine prolactin has only 20-33% of the lactogenic activity of ovine prolactin. Pituitary lactotropes, of ectodermal origin, produce pituitary prolactin and are regulated by dopamine, oestradiol and thyrotropin releasing hormone. Decidual cells, of mesodermal origin, are not influenced by these pituitary secretogogues. Progesterone and calcium both appear to stimulate pituitary and decidual prolactin secretion, while arachidonic acid seems to inhibit decidual prolactin release. More recently, insulin like growth factor-1, a polypeptide growth factor implicated in the oestrogen promoted growth reproductive tissues, has been found in high concentration in the pig uterus and has been shown to stimulate the synthesis and secretion of prolactin from human decidua. Prolactin receptors have not been reported on endothelium of blood vessels and no clear evidence exists that endometrial prolactin may modulate the secretion of endothelin, endothelium derived relaxing factor or other potentially important substances controlling menstruation. Prolactin does appear necessary for the normal production of macrophage activating factors, including interferon, and may have a local immunomodulatory role upon human implantation for this reason. Human alpha 2-interferon has sequence homology to ovine trophoblast protein-1 and is found in high concentration in human pregnancy and fetal tissues. There is some evidence that interferon may reduce human endometrial prolactin secretion, but whether endometrial or decidual prolactin is critical to human implantation awaits further study.

Effects of Heterologous Growth Hormones on Hypothalamic and Pituitary Function in Transgenic Mice

The expression of the mouse metallothionein-I (mMT) promoter/human growth hormone (hGH) fusion gene in transgenic mice leads to female sterility and major alterations in the function of the hypothalamic-adenohypophyseal system. These alterations include increases in median-eminence norepinephrine turnover and plasma LH levels, and a decrease in plasma prolactin levels in intact males, and an increase in median-eminence dopamine turnover combined with the suppression of plasma prolactin levels in ovariectomized females. To further characterize these changes and to determine whether they are due to the lactogenic or somatotropic activity of hGH, we have studied hypothalamic and pituitary function in transgenic mice expressing mMT/hGH, mMT/hGH.B 'variant', or mMT/bGH fusion genes. In males, the expression of the hGH.B gene was associated with a reduction in pituitary prolactin release in vitro and an increase in LH response to LHRH stimulation, while the bGH transgene did not affect any of the examined parameters of LH and prolactin release. Median-eminence norepinephrine turnover was increased in each of the three lines of transgenic males, while median-eminence dopamine turnover was reduced only in animals expressing the hGH.B gene. In ovariectomized females, plasma LH was suppressed by hGH variant expression, while median-eminence norepinephrine turnover was suppressed in both hGH.B and bGH animals. The turnover of dopamine was increased in the median eminence of females expressing either of the human genes (hGH or gHG.B) and reduced in the median eminence of ovariectomized bGH females. We conclude that the hGH.B gene is weakly lactogenic in mice, and that the chronic stimulation of either GH receptors (by bGH) or both GH and prolactin receptors (by hGH or hGH.B) can lead to profound alterations in the metabolism of hypothalamic neurotransmitters and pituitary hormone release.

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.

Studies on the thyroid in transgenic mice expressing the genes for human and bovine growth hormone

The thyroid glands of transgenic mice (TM) expressing the genes for human (h) and bovine (b) growth hormone (GH) were studied. The percentages of larger follicles in hGH TM of either sex were significantly greater than in the corresponding normal littermates, and follicles ranging up to 350 microns in diameter were present in male hGH TM. In contrast, thyroid follicles were only slightly enlarged in male bGH TM, and were unchanged in female bGH TM. The serum concentrations of T4 were significantly decreased in male bGH TM and not altered in the other groups. Serum concentrations of T3 were slightly, but significantly increased in female hGH TM and female bGH TM, but were unaffected in male TM of either type. Since the principal difference between these foreign GHs in rodents is the additional lactogenic activity of human GH, these results may indicate that the effects of prolactin can influence the development of the thyroid.

Characterization of Glycosylated Bovine Placental Lactogen and the Effect of Enzymatic Deglycosylation on Receptor Binding and Biological Activity

Bovine placental lactogen (bPL) is a glycoprotein hormone that has both somatogenic and lactogenic properties. Purified preparations of the hormone contain many isoforms that are separated by isoelectric focusing. The sequence for bPL contains one consensus site for an N-linked oligosaccharide and many potential sites for O-linked sugars. To determine whether the isoforms are the result of differences in glycosylation, the oligosaccharide portion of bPL was partially characterized. In addition, a number of the isoforms were isolated and enzymatically deglycosylated to determine the effect of O- and N-linked glycosylation on biological activity. Biological activity was assessed in a somatotropin radioreceptor assay and also in the Nb2 lymphoma lactogenic bioassay. The structure of N-linked oligosaccharide was found to be sialylated and triantennary and appeared to be the same for all of the different charge isomers. Compositional analysis suggested that O-linked oligosaccharides were also present. Treatment of the intact hormone with neuraminidase resulted in the loss of some, but not all, of the isoforms, suggesting that a large degree of the charge heterogeneity is due to posttranslational modifications unrelated to glycosylation. Enzymatic removal of N-linked oligosaccharides from native bPL resulted in a 1.2-2.3-fold increase in binding to the somatotropin receptor, whereas receptor binding was unaffected by enzymatic removal of O-linked oligosaccharide. Lactogenic activity was affected very little by the removal of either type of oligosaccharide. The data suggests that glycosylation of bPL may have a small effect on receptor specificity, but that overall its presence does not dramatically affect receptor binding or biological activity.

Purification, partial characterization, and development of a specific radioimmunoassay for goat placental lactogen

Placental lactogen (PL) was isolated from goat cotyledonary tissue by a combination of mild alkaline extraction, anion and cation exchange chromatography, chromatofocussing and molecular filtration. The product, enriched 15,000-fold from the initial extract, was homogeneous when examined by SDS-gel electrophoresis (Mr 22,500) and isoelectricfocussing indicated a pI of 8.35 with a trace contaminant of pI 8.0. When assessed by relative binding activity in radioreceptor assays (RRA), goat PL exhibited somatotropic activity equivalent to 2.2 units/mg dry weight and lactogenic activity equivalent to 28.5 units/mg. A radioimmunoassay (RIA) for goat PL is described that is highly sensitive (190 pg/tube) and has acceptable repeatability within and between assays (6 and 13%, respectively). The assay is not affected by goat pituitary extracts or partly purified goat growth hormone and prolactin. Despite the marked increase in sensitivity of the RIA over that previously available when goat PL was measured by RRA, the hormone was not detected in jugular plasma of goats before Day 44 of pregnancy; concentrations increased thereafter and highest levels were measured during the last third of pregnancy in animals bearing triplets. Measurements by RIA are in general agreement with those obtained earlier in several studies in which RRAs were used. The hormone was detected in amniotic fluid. Maternal concentrations of goat PL declined before parturition and were undetectable by 18 h post partum.

Immunohistochemical co-localization of placental lactogen II and relaxin in the golden hamster (Mesocricetus auratus).

Two hormones with lactogenic activity are produced by the hamster placenta during the second half of pregnancy. One of these hormones, hamster placental lactogen II (haPL-II), has been well characterized; however, its cellular source is not known. In the present study, haPL-II was localized in placental tissues using a specific antibody and the avidin-biotin-peroxidase immunohistochemical technique. Because relaxin has been localized in the hamster placenta, it was of interest to determine if haPL-II and relaxin are localized in the same cells. haPL-II immunoactivity was observed in primary and secondary giant trophoblast cells of the placenta on Days 12, 14, and 15 of pregnancy. On Day 15 positive staining was also observed in large cells located within mesometrial arteries and in eosinophilic bodies associated with degenerating sheathed arteries of the decidua basalis. haPL-II-positive staining was not observed in placentae from Days 8 or 10 of pregnancy. On Day 14, haPL-II was colocalized with relaxin in 75% of the giant trophoblast cells observed. Therefore, it is probable that these hormones are synthesized and secreted by the same cell.

Site-Directed Mutations of Human Growth Hormone that Selectively Modify Its Lactogenic Activity and Binding Properties

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.

Biological activities of glycosylated and nonglycosylated porcine prolactin

Nonglycosylated and glycosylated porcine prolactin (PRL) were separated using concanavalin A-Sepharose CL-6B column chromatography and tested for mitogenic and lactogenic activities, as well as immunoaffinity and receptor binding characteristics compared to total (nonseparated) porcine PRL. Mitogenic activity, using Nb2 lymphoma cells, was 4- and 50-fold greater ( P < 0.01) for total PRL than nonglycosylated and glycosylated PRL, respectively. Glycosylated PRL had 64% higher ( P < 0.05) lactogenic activity than nonglycosylated or total PRL. In a homologous radioimmunoassay (RIA), displacement was greatest for total, followed by the nonglycosylated and glycosylated forms of PRL. Competitive inhibition of porcine [ 125I(total) PRL by radioinert total, nonglycosylated and glycosylated PRL in a homologous radioreceptorassay (RRA), indicated similar K a values for total and nonglycosylated PRL, but different receptor numbers, while radioinert glycosylated PRL had a higher K a, but bound fewer receptors. Therefore, glycosylated porcine PRL has greater lactogenic activity and higher binding affinity despite decreased mitogenicity, while nonglycosylated PRL had characteristics similar to total PRL. Results from the homologous RRA and the Nb2 assay suggest that both forms of PRL are necessary to achieve biological effects similar to those for total PRL. The two forms of PRL may have individual and collective effects, while changes in the ratio between these forms may influence physiologically diverse effects of PRL on target tissues.