Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.     

Effects of glucose, glycerol, 3-hydroxybutyrate, insulin, and leptin on placental growth hormone secretion in placental explants

Placental growth hormone (PGH) is secreted from the syncytiotrophoblast in increasing amounts during pregnancy. The physiology and regulation of PGH is not well known; however, low glucose levels appear to stimulate PGH liberation IN VITRO and IN VIVO. PGH appears to have lipolytic effects, and inverse correlations between maternal body mass index and serum PGH levels have been reported. Therefore, substances related to maternal adipose tissue metabolism could influence PGH secretion. The effect of insulin, glycerol, 3-hydroxybutyrate (3-OHB), and leptin on PGH and human placental lactogen (hPL) secretion from cultured placental explants was studied. In glucose-free media, PGH content increased upto 237.5+/-28.4% of control media (p<0.001, ANOVA). Insulin levels were without effect on PGH secretion, as were 3-OHB, leptin, and glycerol at 0.02 mmol/l. Glycerol at 0.2 mmol/l increased PGH in all of the placental explants studied (n=8; mean increase 27.3+/-7.1%), and this difference was significantly different from the control explants (p=0.004). The liberation of hPL to culture media was different from PGH and was influenced by glucose and insulin. In conclusion, the absence of glucose profoundly increased PGH secretion in cultured placental explants. Addition of glycerol in physiologically relatively high concentrations showed a less pronounced stimulatory effect.     

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy.

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PGH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20 % with glucose loading. Correlation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not suppressed by oral glucose loading in non-diabetic mothers.     

Differential effects of placental lactogen,growth hormone and prolactin on rat liver ornithine decarboxylase activity in the perinatal period

Ovine placental lactogen, (oPL), ovine growth hormone, (oGH), and ovine prolactin, (oPRL) are present in high concentrations in the fetal circulation late in gestation. To determine if these hormones stimulate the activity of ornithine decarboxylase (ODC), an enzyme widely implicated in the control of cellular growth, rat fetuses were injected $\text{in utero}$ with 100 μg of oPL, oGH, oPRL, rat growth hormone (rGH) or rat prolactin (rPRL) and ODC activity in the livers, hearts, and brains of the fetuses was measured 2, 4, and 6 hours after injection. OPL stimulated fetal liver ODC activity by 282 ± 45% (mean ± SEM) as compared to litter mates injected with buffer alone but oGH, oPRL, rGH and rPRL had no effect on fetal liver ODC activity. However, in neonatal rats 24–48 hours old all five hormones significantly increased liver ODC activity. ODC activities in the hearts and brains of the fetuses and neonates were unaffected by any of the five hormones. In other experiments 50 μg of oPL significantly stimulated fetal liver ODC activity while 250 μg of oGH were without effect. However 25 μg of oGH significantly stimulated liver ODC activity in rat pups 1–2 days after birth. These results suggest that oPL, by its stimulation of ODC activity, has somatotropic effects in the fetus and that rat liver ODC activity becomes responsive to growth hormone and prolactin in the perinatal period.     

Placental lactogen not growth hormone and prolactin regulates secretion of progesterone in vitro by the 40-45 day ovine corpus luteum of pregnancy

The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL.     

Regulation of baboon fetal adrenal androgen production by adrenocorticotropic hormone, prolactin, and growth hormone

Factors other than adrenocorticotropic hormone (ACTH) are thought to influence fetal adrenal steroidogenesis during primate pregnancy. Therefore, we determined the effects of prolactin (Prl), growth hormone (GH), and human chorionic gonadotropin (hCG) as well as ACTH on steroid secretion by collagenase-dispersed baboon fetal adrenal cells. Adrenal glands were obtained from seven baboon (Papio anubis) fetuses following cesarean section at Day 100-107 of gestation (term = Day 184). Tissue was minced with a fine scissors and cells were dispersed with 0.2% collagenase, then washed with Medium 199 containing penicillin/streptomycin. Cells (0.5 X 10(4)) were placed in 4 ml Medium 199 with or without 10 nmol ovine Prl, ovine GH, or ACTH, or 50 nmol hCG. After 18 h incubation (37 degrees C), cells were separated by centrifugation and the quantities of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) secreted into the medium were determined. In controls, DHA secretion [224 +/- 96 ng/(24 h X 10(5) cells] was greater (P less than 0.05) than that of DHAS (20 +/- 12) and F (14 +/- 12). Adrenocorticotropic hormone, Prl, and GH stimulated (P less than 0.05) DHA secretion by 370% +/- 71%, 215% +/- 61%, and 292% +/- 73%, respectively; hCG was not effective. Due primarily to the relatively low secretion rates, DHAS and F secretion were not altered by hormonal treatment. Moreover, addition of 20 nmol progesterone to the medium in the presence or absence of ACTH did not influence F production. These findings indicate that the baboon fetal adrenal at midgestation does not utilize placental progesterone for F synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.     

The regulation of ovine placental lactogen the role of the fetal hypothalamic-pituitary axis

The possible r?le of the fetal hypothalamic-pituitary axis in regulating the secretion of ovine placental lactogen (oPL) was investigated in chronically-catheterised ewes and fetuses in late pregnancy. Intravascular administration of agents to fetuses that significantly increased fetal prolactin concentrations (chlorpromazine 6.25 mg;thyrotrophin releasing hormone, 10 micrograms), significantly reduced fetal prolactin concentrations (bromocriptine, 0.033 mg/h), or significantly reduced fetal growth hormone (GH) concentrations (somatostatin, 2.5 micrograms/min), had no effect on maternal or fetal oPL concentrations. Mean fetal levels of prolactin or GH in late gestation could not be correlated with oPL concentrations, although fetal hypophysectomy prevented the normal prepartum fall in oPL concentrations.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

Active immunization of ewes against ovine placental lactogen increases birth weight of lambs and milk production with no adverse effect on conception rate

In two experiments, 16 Booroola-Assaf and 35 Assaf ewe-lambs were actively immunized at 5 months of age against recombinant ovine placental lactogen (oPL). At 9 months of age, the ewe-lambs were mated for the first time and then introduced into a frequent mating-system. Anti-oPL antibody titers, reproductive performance, maternal serum levels of oPL during pregnancy, lamb birth weight and milk production of the ewes were followed in the immunized ewes and in their non-immunized control counterparts. All the immunized ewes developed anti-oPL antibodies, which interfered with oPL bioactivity in an in vitro cell proliferation assay. Conception rates did not differ (P>0.05) between immunized and non-immunized ewes. Abundant antibody-bound non-active oPL detected in sera of immunized ewes by western blotting indicated enhanced oPL production by the placenta following immunization. An increase (P<0.02) in serum oPL bioactivity, but not immunoreactivity, was observed in the immunized ewes in late gestation relative to control ewes. The average litter size was 1.83 and 1.32 lambs born per ewe lambing in the first and second experiments, respectively. Average birth weights of lambs born to the immunized ewes were higher (P<0.01) than for lambs born to control ewes by 10, 17 and 39% for those born as singles, twins and triplets, respectively. Immunized ewes produced 19 and 33% more milk (P<0.02) than the control ewes in the first 3.5 months of the first and second lactations, respectively. These findings do not suggest a role for oPL in maternal recognition of pregnancy, but they strongly suggest important roles for oPL in fetal growth and mammogenesis. Immunization of ewes against oPL may thus represent a novel practical technique for enhancing birth weights of lambs born to prolific sheep, as well as milk production by both dairy and mutton ewes.     

Ovine placental lactogen stimulation of ornithine decarboxylase activity in brain and liver of neonatal rats

Ovine placental lactogen (oPL), growth hormone (oGH), prolactin (oPRL) and human placental lactogen (hPL) were administered intracisternally (ic) or intraperitoneally (ip) to 17 day old rats and brain and liver ODC activities determined four hours later. When given ic, oPL, oGH and oPRL caused significant increases in brain ODC activity, while hPL had no significant effect. After ip administration, oPL and oGH also caused a significant increase in brain as well as liver ODC activity but oPRL and hPL were without significant effect. The stimulation of polyamine metabolism by oPL together with earlier reports of its potent somatotropic effects and its high concentration in the fetus supports the hypothesis that oPL may be important in the regulation of fetal growth.     

Ruminant placental lactogens act as antagonists to homologous growth hormone receptors and as agonists to human or rabbit growth hormone receptors

Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined.     

Placental lactogen as a regulator of luteal cells function during pregnancy in sheep

The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods. Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates. After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis. Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE2 or PGF2alpha. Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation. There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy. Simultaneous oPL treatment with PGE2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy. In contrast, PGF2alpha alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF2alpha on Days 70 and 95 of gestation protected against luteolytic action of PGF2alpha. The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF2alpha) observed on Days 70 and 95 of gestation.     

Regulation of growth hormone secretion in human trophoblastic cells in culture

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Isolation and characterization of the porcine growth hormone gene

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.     

Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor

The structure of the ternary complex between ovine placental lactogen (oPL) and the extracellular domain (ECD) of the rat prolactin receptor (rPRLR) reveals that two rPRLR ECDs bind to opposite sides of oPL with pseudo two-fold symmetry. The two oPL receptor binding sites differ significantly in their topography and electrostatic character. These binding interfaces also involve different hydrogen bonding and hydrophobic packing patterns compared to the structurally related human growth hormone (hGH)-receptor complexes. Additionally, the receptor-receptor interactions are different from those of the hGH-receptor complex. The conformational adaptability of prolactin and growth hormone receptors is evidenced by the changes in local conformations of the receptor binding loops and more global changes induced by shifts in the angular relationships between the N- and C-terminal domains, which allow the receptor to bind to the two topographically distinct sites of oPL.     

Evolution of the sensitivity of isolated adipocytes of ewes to the lipolytic effects of different stimuli during pregnancy and lactation

Four successive biopsies of omental adipose tissue were performed at 43,100,140 days and during the 3rd week of lactation on 6 "Pré-Alpes" ewes. Using isolated adipocyte incubation, we studied the evolution of both basal lipolysis and stimulated lipolysis in response to different stimuli during these physiologic periods. The basal lipolysis increased from 53 +/- 10 micrograms glycerol/4 hr incubation/g total lipids (TL) at 43 days of pregnancy and 55 +/- 11 micrograms/4 hr/g TL at 100 days of pregnancy to a maximum value of 204 +/- 10 micrograms/4 h/g TL observed one week before parturition. Basal lipolysis remained at a high level during lactation: 153 +/- 27 micrograms/4 hr/g TL. The sensitivity of the fat cells to the lipolytic effects of isoproterenol, theophylline and adenosine-deaminase evolved with profiles comparable to that observed for basal lipolysis. The threshold concentration of stimuli necessary to observe an effect was decreased and the maximum response was increased. Bovine growth hormone (bGH) did not exhibit a direct lipolytic effect during pregnancy and lactation. Nevertheless, bGH provoked a significant potentiation of 20% of the lipolysis stimulated by isoproterenol and theophylline at 43 and 100 days of pregnancy. Ovine placental lactogene hormone (oPL) did not modulate, directely or undirectely, lipolysis.