Expression of growth hormone and its receptor in the placental and feto-maternal environment during early pregnancy in sheep.

In a previous study we showed the existence of GH in the ovine placenta. We now supplement the information available on placental GH and describe the presence and distribution of GH receptor (GH-R) messenger RNA (mRNA) in uterine, fetal, and placental tissues during early pregnancy. GH mRNA was not detected in the placenta before day 27 (d27). Its expression peaked between d40 and d45 and fell after d55. GH mRNA was localized in the trophectoderm and syncytium. During the d35-d50 period, concentrations of GH in the maternal circulation were not increased. In umbilical blood, however, GH was detected from d35 and was presumed to be of placental origin, because GH mRNA was not detected in the fetal pituitary gland on d40. We report on GH-R mRNA expression in the placenta between d20-d120. The relative abundance of GH-R transcripts increased significantly between d25-d43. In the endometrium, GH-R mRNA was detected from d8-d120 of pregnancy and from d4-d16 of the cycle. GH-R mRNA was localized in the trophectoderm, fetal mesoderm, and maternal uterine stroma. In the fetal liver, GH-R mRNA was first detectable on d35. The results of this study indicate that between d35-d50 of pregnancy, the endometrium, placenta, and fetus are all potential targets for the placental GH.

Expression of the growth hormone gene in ovine placenta: detection and cellular localization of the protein.

In several species, placenta has been found to express GH-related proteins. In the ovine placenta, such a protein, ovine chorionic somatommamotropin, has been described, but its involvement in the fetal/placental growth process is not clearly established. The aim of this study was to investigate the occurrence of another GH-related peptide in the ovine placenta. Placental extracts (days 30-140 of pregnancy) showed GH immunoreactivity between days 35-70. SDS-PAGE analysis of these extracts indicated that this immunoreactivity corresponded to 22- and 28-kDa proteins. GH-like immunoreactivity was localized on cotyledonary frozen sections in the syncytium and the trophectoderm. Northern blot analysis of placental RNA showed the expression of GH-hybridizing transcripts migrating to the same position as that of GH pituitary messenger RNA (mRNA). Those transcripts were highly expressed between days 40 and 50. Their sequence analysis showed the existence of three GH mRNA (GHP1, GHP2, and GHP3). GHP1 is identical to pituitary GH mRNA and probably codes for the 22-kDa protein. GHP2 and GHP3 encode the same protein, which differs from GHP1 by four amino acids. This study establishes the expression of GH gene and GH-immunoreactive proteins in the ovine placenta.

[Effect of indomethacin on the lactogenic action of prolactin]. / Effet de l'indométhacine sur l'action lactogène de la prolactine.

The action of indomethacin on the lactogenic activity of prolactin has been evaluated usind the technique of rabbit mammary gland organ culture. Indomethacin is totally unable to inhibit prolactin action as estimated by lactose synthetase activity and casein synthesis. These data suggest, as opposed to previous works, that prostaglandins are not involved in the mechanism of prolactin action on lactogenesis.

Ontogeny and characterization of epidermal growth factor receptors on the fetal area of the sheep placenta.

Studies of the binding of 125I-labelled epidermal growth factor (EGF) to the ovine placenta were carried out on days 50, 90-100 and 140 of pregnancy. Membrane fractions were purified from the fetal area of the cotyledon. Two classes of binding sites were found. Their dissociation constants (Kd) were not significantly different for the three stages of pregnancy considered (high-affinity Kd 54-70.2 pmol/l; low-affinity Kd 12.2 to 19 nmol/l). However, the number of high-affinity binding sites on days 90-100 was significantly (P < 0.01) greater (146 +/- 19 fmol/mg protein) than on either day 50 or day 140 (respectively 74.2 +/- 1.26 and 56.3 +/- 5.6 fmol/mg protein). Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that the major part of the EGF was specifically cross-linked to a protein of molecular weight of 150 kDa and to lesser extent to 180 kDa and 130 kDa proteins. Membranes prepared from unfrozen tissues, in the presence of sodium iodoacetate to reduce endogenous enzymatic conversion of the 180 kDa form to the 150 and 130 kDa forms, still exhibited a major EGF-binding protein of 150 kDa. The occurrence of an increased number of EGF receptors at the period of rapid cotyledonary growth which coincides with the increase in placental hormonal secretions suggests that EGF has a role in the development of the ovine placenta.

Is oxytocin of conceptus origin involved in inhibition of luteal regression in early pregnancy in ewes?

This study describes the presence in and production by the ovine conceptus of an oxytocin-like peptide during the early stages of development. Oxytocin was measured by radioimmunoassay in ovine conceptuses from days 14 to 30 of pregnancy. Tissue concentrations of oxytocin increased from day 14 (24.8 +/- 5 pg/100 mg) until day 19 (122.9 +/- 52 pg/100 mg) and then decreased (3 +/- 1 pg/100 mg). Oxytocin was released into culture medium by day-15 ovine conceptuses at a rate of 262 +/- 55 pg/24 h. Reverse-phase high-performance liquid chromatography (HPLC) analysis of peptides extracted from a pool of ovine conceptuses was conducted using chromatographic conditions developed to separate oxytocin from other nonapeptides. Radioimmunoassay of HPLC fractions for oxytocin revealed an immunoactive conceptus peptide in a single fraction at the same retention time as chromatographed authentic oxytocin. Radioimmunoassay and chromatographic data therefore suggest that this oxytocin-like peptide is similar, if not identical, to authentic oxytocin. Concentrations of oxytocin in conceptus tissue were maximal during the period of inhibition of luteal regression (days 14-19). It is proposed that conceptus oxytocin is involved in the maintenance of luteal function in early pregnancy.

IGF-I and IGF-II receptors in the sheep placenta: evolution during the course of pregnancy.

The role of IGFs in placental growth is poorly understood. IGF-II receptors have been characterised in the ovine placenta and used extensively for radioreceptor assay, but their evolution during placental development has not been considered. In this study, binding sites for IGF-I were characterised in the ovine cotyledon by binding and cross-linking studies and the evolution of the number of IGF-I and IGF-II receptors on placentae collected on days 50, 75, 100 and 140 of pregnancy were compared. IGF-I bound onto placental membranes with a mean association constant of 1.7 nM-1 except on day 50 when a lower association constant was observed (0.8 nM-1). Scatchard analysis of the displacement curves led to a single binding site model. IGF-II was as potent as IGF-I at displacing the binding of 125I-labelled IGF-I on those membranes, whereas insulin cross-reaction was only 1%. IGF-II bound on our placental membrane preparations with the characteristics described previously and neither IGF-I nor insulin was able to displace this binding. Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that IGF-I was linked to a protein with a molecular weight of about 135,000 Da and IGF-II to a protein of 250,000 Da. The mean +/- S.E.M. number of IGF-I receptors was significantly higher on days 50 and 75 than on days 100 and 140 (154 +/- 12, 105 +/- 11 vs 65 +/- 4, 48 +/- 3 fmol/mg, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogenic activity of high molecular weight forms of insulin-like growth factor-II in amniotic fluid.

Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

Ovine chorionic somatomammotrophin (oCS) production by isolated cotyledon cells from sheep in early and mid gestation: auto-regulation by recombinant oCS.

We report the ability of sheep placental cotyledonary cells, isolated at different periods of pregnancy (40 to 90 days) to produce ovine chorionic somatomammotrophin (oCS) in in vitro culture conditions. This oCS production increased gradually with stage of pregnancy. Endogenous oCS net production by isolated placental cells was increased, in a dose-dependent manner, by addition of recombinant oCS (roCS). This effect was not observed after addition of recombinant ovine growth hormone. The roCS effect was more potent on cells collected during early pregnancy. Specific immunoprecipitation of oCS revealed that roCS treatment was associated with an increased dose-dependent incorporation of [35S]methionine-[35S]cysteine. These findings provide evidence that oCS may act in a paracrine/autocrine manner to up-regulate its own production during early gestation. We suggest that this autoregulation may be associated with morphological and functional differentiation of the trophoblast during the growth of the placenta.

Human placental growth hormone--a review.

Placental growth hormone (PGH) is the product of the GH-V gene, predominantly expressed in the syncytiotrophoblast layer of the human placenta. PGH differs from pituitary growth hormone by 13 amino acids and possesses one glycosylation site. It has high somatogenic and low lactogenic activities. In the maternal circulation from 12-20 weeks up to term, PGH gradually replaces pituitary growth hormone, which becomes undetectable. PGH is secreted by the placenta in a non-pulsatile manner. This continuous secretion appears to have important implications for physiological adjustment to gestation and especially in the control of maternal IGF1 levels. PGH secretion is regulated in vitro and in vivo by glucose. Lower maternal levels of PGH are observed in pregnancies with fetal growth retardation. PGH is one example of a trophoblast hormone, which allows maternal metabolic adaptation to pregnancy. In addition, our recent data on its expression in invasive extravillous trophoblasts suggest that the physiological role of PGH might also include a direct influence of this hormone on placental development via an autocrine or paracrine mechanism.

Occurrence of a growth hormone-releasing hormone-like messenger ribonucleic acid and immunoreactive peptide in the sheep placenta.

Growth hormone releasing factor (GHRH) has been described in the rat, mouse and human placentae. This study reports the presence of an immunoreactive GHRH activity (IR-GHRH) in the ovine placenta. This activity was detected by radioimmunoassay from day 50 (D50) until the end of pregnancy. Higher IR-GHRH concentration in placental tissue was observed on days 100 (543 +/- 123 pg/g) and 140 (550 +/- 62 pg/g) and, when compared with the GHRH content of the ovine hypothalamus (1.2 ng/hypothalamus), represents a considerable amount of GHRH per placenta (a mean of 200 ng). Perifused placenta explants released IR-GHRH in vitro at a mean rate of 200 pg/g/h. Depolarization by 55 mM KCl increased the IR-GHRH concentration of the perifusion media 1.7 times over basal values. The elution position of GHRH immunoreactivity in the gel filtration chromatography profiles was the same for placenta and hypothalamus extracts and lay very near to the molecular weight of bovine GHRH. Northern blot hybridization analysis revealed the existence of a placental transcript whose size (0.75 kb) was comparable to the size of the ovine hypothalamus and rat placenta GHRH transcripts. Hybridization signal was observed at each stage studied from D50 until D120 of pregnancy. This study demonstrated the existence of a IR-GHRH peptide in the ovine placenta.

Composition of uterine flushings from Large White and prolific Chinese Meishan gilts.

This study examined differences in selected components of uterine secretions from Large White and prolific Chinese Meishan gilts during the oestrous cycle or early pregnancy. Total recoverable protein, uteroferrin (measured as acid phosphatase activity), acyl aminopeptidase, calcium, sodium, potassium, immunoglobulins A and G, glucose, fructose, oestradiol-17 beta, and prostaglandins F2 alpha (PGF2 alpha) and E2 (PGE2) in uterine flushings were measured. During the oestrous cycle, breed effects were detected only for total protein (P = 0.07), which tended to be higher for Large White gilts. However, for pregnant gilts, total recoverable glucose (P less than 0.05), fructose (P less than 0.05) sodium (P less than 0.05), immunoglobulin A (P less than 0.01), PGF (P less than 0.01), PGE (P less than 0.01), and acyl aminopeptidase (P less than 0.05) were greater in uterine flushings from Meishan gilts. Only uteroferrin was higher (P = 0.06) in uterine flushings from Large White gilts. Concentrations of prolactin were higher (P less than 0.05) in plasma from cyclic or pregnant Meishan gilts, but concentrations of total oestrogens and progesterone were not affected by pregnancy status or breed. These results suggest that Meishan conceptuses, individually or collectively, are more stimulatory to endometrial secretion and/or transport of the components of histotroph into the uterine lumen, or that the endometrium of Meishan gilts is more sensitive to conceptus signals responsible for the accumulation of histotroph in the uterine lumen.

Aspects of the antiluteolytic activity of the conceptus during early pregnancy in ewes.

Experiments were undertaken to determine whether the conceptus renders a corpus luteum resistant to the luteolytic action of prostaglandin F2 alpha (PGF2 alpha), and modulates release of this prostaglandin by the uterus of early pregnant ewes. Prostaglandin F2 alpha was luteolytic when administered to indomethacin-treated ewes on d 10 and 11 of the estrous cycle. The same PGF2 alpha treatment was not luteolytic when applied on d 19 and 20 of pregnancy in ewes treated with indomethacin. Pulsatile release of PGF2 alpha (measured by 15-keto-13,14-dihydro PGF2 alpha-PGF2 alpha plasma level, PGFM) was observed between d 14 and 16 of the cycle but not during the same period of pregnancy. Ablation of the conceptus on d 17 resulted in progressive restoration of PGFM surges and subsequent luteolysis. Estradiol-17 beta (E2-17 beta) administration on d 12 of the cycle induced earlier PGFM surges and luteal regression. The same E2-17 beta treatment administered on d 14, 19 and 33 of pregnancy failed to induced PGFM pulses and luteolysis. In the absence of the conceptus (surgical ablation), E2-17 beta treatment was luteolytic (PGFM surges) on d 17 but not on d 33. We conclude that the conceptus controls the amount and pattern of PGF2 alpha released by the uterus, as well as the sensitivity of the uterus to E2-17 beta as early as d 14 of pregnancy. Simultaneously, an embryonic protective effect takes place at the luteal level.

Endothelin as a neuroprotective factor in the olfactory epithelium.

In mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ETB receptors, while ETA receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ETA and ETB receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ETB-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ETB and indirectly by limiting non neuronal cells death through ETA.

Insulin but not leptin protects olfactory mucosa from apoptosis.

The mammalian olfactory mucosa (OM) is continually renewed throughout life. Owing to their position in the nasal cavity, OM cells are exposed to multiple insults, including high levels of odourants that can induce their death. OM regeneration is therefore essential to maintain olfactory function, and requires the tight control of both cell death and proliferation. Apoptosis has been implicated in OM cell death. Olfaction is one of the senses involved in food intake and depends on individual nutritional status. We have previously reported the influence of hormones related to nutritional status on odour perception and have shown that the OM is a target of insulin and leptin, two hormones known for their anti-apoptotic properties. In the present study, we investigated the potential anti-apoptotic effect of these metabolic hormones on OM cells. Both Odora cells (an olfactive cell line) and OM cells treated with etoposide, a p53 activity inducer, exhibited mitochondrial-dependent apoptosis that was inhibited by the pan-caspase inhibitor zVAD-fmk. Insulin, but not leptin, impaired this apoptotic effect. Insulin addition to the culture medium reduced p53 phosphorylation, caspase-3 and caspase-9 cleavage, and caspase-3 enzymatic activity induced by etoposide. The apoptotic wave observed in the OM after interruption of the neuronal connections between the OM and the olfactory bulb by bulbectomy was impaired by intranasal insulin treatment. These findings suggest that insulin may be involved in OM cellular dynamics, through endocrine and/or paracrine-autocrine effects of circulating or local insulin, respectively.

Nutritional status modulates behavioural and olfactory bulb Fos responses to isoamyl acetate or food odour in rats: roles of orexins and leptin.

Food odours are major determinants for food choice, and their detection depends on nutritional status. The effects of different odour stimuli on both behavioural responses (locomotor activity and sniffing) and Fos induction in olfactory bulbs (OB) were studied in satiated or 48-h fasted rats. We focused on two odour stimuli: isoamyl acetate (ISO), as a neutral stimulus either unknown or familiar, and food pellet odour, that were presented to quiet rats during the light phase of the day. We found significant effects of nutritional status and odour stimulus on both behavioural and OB responses. The locomotor activity induced by odour stimuli was always more marked in fasted than in satiated rats, and food odour induced increased sniffing activity only in fasted rats. Fos expression was quantified in periglomerular, mitral and granular OB cell layers. As a new odour, ISO induced a significant increase in Fos expression in all OB layers, similar in fasted and satiated rats. Significant OB responses to familiar odours were only observed in fasted rats. Among the numerous peptides shown to vary after 48 h of fasting, we focused on orexins (for which immunoreactive fibres are present in the OB) and leptin, as a peripheral hormone linked to adiposity, and tested their effects of food odour. The administration of orexin A in satiated animals partially mimicked fasting, since food odour increased OB Fos responses, but did not induce sniffing. The treatment of fasted animals with either an orexin receptors antagonist (ACT-078573) or leptin significantly decreased both locomotor activity, time spent sniffing food odour and OB Fos induction in all cell layers, thus mimicking a satiated status. We conclude that orexins and leptin are some of the factors that can modify behavioural and OB Fos responses to a familiar food odour.

Expression of insulin system in the olfactory epithelium: first approaches to its role and regulation.

Food odours are major determinants for food choice; their detection is influenced by nutritional status. Among different metabolic signals, insulin plays a major role in food intake regulation. The aim of the present study was to investigate a potential role of insulin in the olfactory mucosa (OM), using ex vivo tissues and in vitro primary cultures. We first established the expression of insulin receptor (IR) in rat olfactory mucosa. Transcripts of IR-A and IR-B isoforms, as well as IRS-1 and IRS-2, were detected in OM extracts. Using immunocytochemistry, IR protein was located in olfactory receptor neurones, sustentacular and basal cells and in endothelium of the lamina propria vessels. Moreover, the insulin binding capacity of OM was quite high compared to that of olfactory bulb or liver. Besides the main pancreatic insulin source, we demonstrated insulin synthesis at a low level in the OM. Interestingly 48 h of fasting, leading to a decreased plasmatic insulin, increased the number of IR in the OM. Local insulin concentration was also enhanced. These data suggest a control of OM insulin system by nutritional status. Finally, an application of insulin on OM, aiming to mimic postprandial insulin increase, reversibly decreased the amplitude of electro-olfactogramme responses to odorants by approximately 30%. These data provide the first evidence that insulin modulates the most peripheral step of odour detection at the olfactory mucosa level.

[Demonstration and evolution of chorionic gonadotropin in ewes]. / Mise en évidence et évolution d'une gonadotrophine chorionique chez la brebis.

Occurrence of ovine Chorionic Gonadotropin (oCG) is demonstrated in placenta and amniotic fluid with the use of a radioreceptor assay (corpus luteum membranes) in ewes. Identification of oCG is possible as early as 15th day of pregnancy. It should be secreted at a constant rate, and its maximum concentration is recorded on the 130th day. This hormonal factor might be one of the major components accountable for high progesteronemia observed during the 100 last days of pregnancy.

Comparative studies of prostaglandins F2 alpha and E2 in late cyclic and early pregnant sheep: in vitro synthesis by endometrium and conceptus effects of in vivo indomethacin treatment on establishment of pregnancy.

Endometrial concentrations of prostaglandins F2 alpha (PGF2 alpha) and E2 (PGE2) were measured by specific radioimmunoassay in sheep, on day 14 of estrous cycle or pregnancy, during luteolysis (Day 16 of the cycle), and after implantation (Day 23 of pregnancy): concentrations observed on day 14 of cycle and pregnancy were similar. During luteolysis, on day 16 of cycle, a consistent drop was noticed. If luteal regression did not occur, as a consequence of the presence of an embryo, endometrial concentrations of PGF2 alpha on day 23, were twice those of day 14, and PGE2 remained unchanged. In vitro 2 hour incubations of endometrial caruncular tissue from 14 days cyclic or pregnant ewes resulted in de novo synthesis of PG which could be increased by Arachidonic Acid and inhibited by Indomethacin; during the first 30 min of incubation, the PGF2 alpha synthesis was comparable for both endometrial tissues, whereas PGE2 synthesis was twice as great in pregnant endometrium. Fourteen and 23 day conceptuses had high PGF2 alpha and PGE2 concentrations which were not due to maternal PG sequestration: de novo PG synthesis which could be inhibited by Indomethacin was observed in incubated 14 day old embryos. Treatment of pregnant ewes from day 7 to day 22 after mating, either with Indomethacin (300 mg s.c. daily) or with Acetylsalicylic Acid (1 g I.V. daily) resulted in a sharp diminution of endometrial PG concentration and release, with no apparent effect on the establishment of pregnancy. These results tend to ascribe a less important role to PG during early pregnancy in sheep as compared with rodents, in terms of embryonic growth and implantation.

Effects of systemic administration of indomethacin to cyclic ewes on endometrial concentrations of prostaglandins effects on estrous cycle length and on progesterone, luteinizing hormone and prolactin patterns.

Experiments were designed to evaluate in cyclic sheep the effects of systemic administration of a prostaglandin synthetase inhibitor (Indomethacin). Indomethacin (100 mg, 3 times daily, S.C.) was administered from day 7 of the estrous cycle for 16 days to five ewes in which the cycle was synchronized as well as the cycles of five control ewes. All control ewes had cycles of approximately 17 days duration, but three of five Indomethacin treated ewes showed no estrous behavior before their slaughter at 23 days after induced ovulation. Autopsy revealed normal corpora lutea which had not undergone luteolysis, as confirmed by progesterone determination in blood. The two remaining Indomethacin treated ewes showed an estrous behavior on day 19 and 20 respectively together with a "preovulatory surge" of luteinizing hormone and prolactin which was not followed by follicular rupture. These results show that inhibition of PGF2 alpha synthesis by systemic administration of Indomethacin to the ewe is able to prevent luteolysis. When luteolysis did occur however, it was not followed by an ovulation despite a normal gonadotropin surge, indicating that inhibition of prostaglandin synthesis by systemic administration of Indomethacin interferes with the luteolysis and follicle rupture processes.