Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells.

Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

Detection of early placental hormone production in embryo transfer cycles lacking a corpus luteum.

PURPOSE: This study sought to identify the initiation of placental hormonal production as defined by the production of endogenous estradiol (E2) and progesterone (P4) in a cohort of patients undergoing programmed endometrial preparation cycles with single embryo transfers resulting in live-born singletons. METHODS: In this retrospective cohort study, patients undergoing either programmed frozen-thawed embryo transfer (FET) with autologous oocytes or donor egg recipient (DER) cycles with fresh embryos were screened for inclusion. Only patients who underwent a single embryo transfer, had a single gestational sac, and a resultant live-born singleton were included. All patients were treated with E2 patches and intramuscular progesterone injections. Main outcome measures were serial E2 and P4, with median values calculated for cycle days 28 (baseline), or 4w0d gestational age (GA), through 60, or 8w4d GA. The baseline cycle day (CD) 28 median value was compared to each daily median cycle day value using the Wilcoxon signed rank test. RESULTS: A total of 696 patients, 569 using autologous oocytes in programmed FET cycles and 127 using fresh donor oocytes, from 4/2013 to 4/2019 met inclusion criteria. Serum E2 and P4 levels stayed consistent initially and then began to increase daily. Compared to baseline CD 28 E2 (415 pg/mL), the serum E2 was significantly elevated at 542 pg/mL (P < 0.001) beginning on CD 36 (5w1d GA). With respect to baseline CD 28 P4 (28.1 ng/mL), beginning on CD 48 (6w6d GA), the serum P4 was significantly elevated at 31.6 ng/mL (P < 0.001). CONCLUSION: These results demonstrate that endogenous placental estradiol and progesterone production may occur by CD 36 and CD 48, respectively, earlier than traditionally thought.

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3ß-hydroxysteroid dehydrogenase (3ßHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows.

Steroid hormone synthesis in mitochondria.

Mitochondria are essential sites for steroid hormone biosynthesis. Mitochondria in the steroidogenic cells of the adrenal, gonad, placenta and brain contain the cholesterol side-chain cleavage enzyme, P450scc, and its two electron-transfer partners, ferredoxin reductase and ferredoxin. This enzyme system converts cholesterol to pregnenolone and determines net steroidogenic capacity, so that it serves as the chronic regulator of steroidogenesis. Several other steroidogenic enzymes, including 3ß-hydroxysteroid dehydrogenase, 11ß-hydroxylase and aldosterone synthase also reside in mitochondria. Similarly, the mitochondria of renal tubular cells contain two key enzymes participating in the activation and degradation of vitamin D. The access of cholesterol to the mitochondria is regulated by the steroidogenic acute regulatory protein, StAR, serving as the acute regulator of steroidogenesis. StAR action requires a complex multi-component molecular machine on the outer mitochondrial membrane (OMM). Components of this machine include the 18 kDa translocator protein (TSPO), the voltage-dependent anion chanel (VDAC-1), TSPO-associated protein 7 (PAP7, ACBD3), and protein kinase A regulatory subunit 1α (PKAR1A). The precise fashion in which these proteins interact and move cholesterol from the OMM to P450scc, and the means by which cholesterol is loaded into the OMM, remain unclear. Human deficiency diseases have been described for StAR and for all the mitochondrial steroidogenic enzymes, but not for the electron transfer proteins or for the components of the cholesterol import machine.

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3ß in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.

[Morphofunctional characteristics of the fetoplacental complex in pregnant women with an exacerbation of herpes virus infection and pathomorphological changes in fetal organs].

Impaired placental cholesterol synthesis in a pregnant woman with an exacerbation of herpesvirus infection is characterized by disturbances in placental hormone biosynthesis, causing severe morphological changes in fetal organs (liver, adrenals, lungs, and thymus).

[Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma].

BACKGROUND AND OBJECTIVE: Human ribonuclease inhibitor (hRI) extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s-hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells. METHODS: The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay (ELISA). The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density (MVD) were assessed. Normal saline (NS), V-pLNCX, and V-pLNCX-hri were used as controls. RESULTS: The infection efficiency of V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 microg/mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group (0.249 microg/mL) than in the NS group (0.035 microg/mL), V-pLNCX group (0.028 microg/mL) and V-pLNCX-hri group (0.169 microg/mL) (P<0.01). The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups (P>0.01). CONCLUSIONS: V-pLNCX-s-hri can effectively infect B16 cells and induce high expression of hRI. V-pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.

Gene expression of placental hormones regulating energy balance in small for gestational age neonates.

OBJECTIVE: Fetal growth restriction is associated with an increased risk for metabolic and cardiovascular disease in later life. To further elucidate mechanisms that might be involved in the process of prenatal programming, we measured the adipokines leptin, resistin, and adiponectin and the GH-releasing hormone ghrelin in the placenta of small for gestational age (SGA) neonates. STUDY DESIGN: The control group included 24 placentas of appropriate for gestational age (AGA) newborns, in the study group were 16 placentas of SGA neonates. Gene expression of leptin, resistin, adiponectin, and ghrelin was examined. For hormones showing alterations in gene regulation placental protein expression was measured by Western blot. RESULTS: Placental mRNA expression of leptin was significantly increased in SGA placentas (p=0.0035, related to beta-actin). Protein concentration was increased, as well. There were no differences in placental resistin, adiponectin, or ghrelin gene expressions between SGA neonates and controls. Leptin was the only hormone to demonstrate a significant inverse correlation with birth weight (r=-0.44, p=0.01). Adiponectin correlated significantly with leptin (r=0.53, p=0.0023) and ghrelin (r=0.50, p=0.0045). CONCLUSIONS: Placental leptin gene expression and protein concentration showed the expected increase in the SGA group. Leptin was inversely correlated with birth weight. Positive correlation of adiponectin with leptin and ghrelin expression suggests an interaction between these hormones in the placenta. However, the unchanged expression of resistin, adiponectin, and ghrelin in SGA placentas and the absence of correlation with birth weight cast doubt whether these hormones produced in the placenta play a key role in fetal programming.

Nutritional influences on implantation and placental development.

The placenta is critical for nourishing the fetus throughout pregnancy, and also produces hormones that alter the metabolic functions of the mother. While the effects of nutrition on fetal development and long-term outcome have been very well documented, there are only a few reports based on studies in rat, sheep, and guinea pigs on how specific nutrients or general nutritional status affect the development of the blastocyst, its implantation, and the subsequent placenta. The data suggest that placental development is highly adaptable and that many types of compensation are possible for suboptimal nutrition.

Analysis of placental regulation of hematopoiesis.

Placental hormones contribute to changes in maternal physiology, especially to changes in the blood system. Methods are described to express a placental hormone from a cloned cDNA by transfection into a mammalian cell line, to purify the hormone, and to assess the activities of the hormone in primary mouse bone marrow cell cultures. The example used in this chapter is prolactin-like protein F (PLP-F), a recently discovered mouse placental hormone that acts on the myeloid lineage. This hormone has been expressed at high levels in stably transfected Chinese hamster ovary cells. The protein is secreted from these cells after cleavage of the signal sequence and the addition of N-linked carbohydrate. A series of chromatographic steps are used to purify the protein to homogeneity, which is verified by gel electrophoresis and silver staining; the identity of the purified protein is confirmed by immunoblot analysis. Purified protein is then assayed by addition to primary bone marrow cells and scoring the growth and the differentiation of the megakaryocyte progenitor, colony forming unit-megakaryocyte.

In vitro induction of trophoblast from human embryonic stem cells.

Human embryonic stem (ES) cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we describe a method by using bone morphogenetic protein (BMP)4, a member of the transforming growth factor (TGF)-beta superfamily, to induce the differentiation of human ES cells to trophoblast. Immunoassays (as well as DNA microarray and reverse-transcription polymerase chain reaction analyses--data not shown) demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). This technique underscores fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.

Production of recombinant human placental variant growth hormone in Pichia pastoris.

cDNA encoding mature human placental variant growth hormone (HGH-V) was synthesized by retro-transcription polymerase chain reaction (RT-PCR) from total RNA recovered from human term-placenta and cloned in pBluescript plasmid (pBS) in Escherichia coli. cDNA was subcloned into pPIC9, fusing it to the flanking regulatory sequences of the Pichia pastoris alcohol oxidase 1 gene (AOX1) and finally introduced into the genome of this yeast by homologous recombination. The resulting new recombinant strain produced and secreted, towards the culture medium, mature HGH-V, whose activity was demonstrated in cell culture by the Nb2 proliferation assay.

Transcription factors underlying the development and endocrine functions of the placenta.

The placenta has been the subject of extensive basic research efforts in two distinct fields. The developmental biology of placenta has been studied because it is the first organ to develop during embryogenesis and because a number of different gene mutations in mice result in embryonic lethality due to placental defects. The trophoblast cell lineage is relatively simple such that only two major, terminally differentiated cell types appear: an "invasive trophoblast" cell subtype such as extravillous cytotrophoblast cells in humans and trophoblast giant cells in mice, and a "transport trophoblast" cell subtype that is a syncytium (syncytiotrophoblast) in humans and mice. These two cell types also have been the focus of endocrinologists because they are the source of major placental hormones. Understanding the transcriptional regulation of placental hormone genes has given insights into the control of specificity of gene expression. Because most placental hormones are produced by very specific trophoblast cell subtypes, the transcriptional details promise to give insights into cell-subtype specification. The fields of developmental biology and molecular endocrinology appear to be meeting on this common ground with the recent discovery of key transcription factors. Specifically, the basic helix-loop-helix (bHLH) transcription factor Hand1 is essential for differentiation of trophoblast giant cells in mice and also regulates the promoter for the giant cell-specific hormone, placental lactogen I gene (Pl1). In contrast, formation of syncytiotrophoblast cells in mice is controlled by a distinct genetic pathway that is governed by the Gcm1 transcription factor, a homologue of the Drosophila glial cells missing gene. Human GCM I has been shown to regulate the activity of the placental-specific enhancer of the aromatase gene (CYP19), which is specifically expressed in syncytiotrophoblast. Together, these findings imply that some key transcription factors have the dual functions of controlling both critical cell fate decisions in the trophoblast cell lineage and later the transcription of cell subtype-specific genes unrelated to development.

Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition.

Summary In an earlier, companion, review, we concluded that cytokines produced by the placenta and associated membranes are likely to be involved in control of the processes of implantation and placental development (Bowen et al., 2002). In this review, we discuss evidence that cytokines continue to be part of a paracrine/autocrine regulatory network in the placenta and membranes throughout the mid and late stages of gestation. Cytokines are involved in regulation of placental growth during these later stages of pregnancy and also function to protect the fetus from pathological organisms. The evidence, while not entirely consistent, suggests that production of certain cytokines within the extraplacental membranes is altered during normal term parturition, whereas in the villous placenta evidence of labour-associated changes is much more equivocal. Roles for cytokines have been postulated in many facets of parturition, including expulsion of the fetus by uterine contractions, membrane rupture, and dilation of the cervix. Imbalances and disruptions to the cytokine milieu have been implicated in a number of diseases of pregnancy involving abnormalities of both placental growth/establishment and initiation of parturition. Cytokine secretion induced by intrauterine infection is associated with increased occurrence or severity of some neonatal diseases. This wealth of data supports the view that cytokines are an integral part of a functional regulatory/communication network operating within the placental-maternal unit during normal gestation.

Placental growth hormone and lactogen production by perifused ovine placental explants: regulation by growth hormone-releasing hormone and glucose.

The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 +/- 0.2 and 353.6 +/- 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.

A member of the nuclear factor-1 family is involved in the pituitary repression of the human placental growth hormone genes.

The human growth hormone (GH) gene family consists of five tandemly arranged and highly related genes, including the chorionic somatomammotropins (CSs), at a single locus on chromosome 17. Despite striking homologies in promoter and flanking DNA sequences, the genes within this locus have different tissue-specific patterns of expression: GH-N is expressed almost exclusively in the somatotrophs of the anterior pituitary; the remaining genes, including CS-A, are expressed in placental syncytiotrophoblast. Previously we proposed that active repression of the placental gene promoters in pituitary GC cells is mediated by upstream 'P' sequences and, specifically, a 263 bp region containing two 'P' sequence elements (PSE-A and PSE-B) and corresponding factors (PSF-A and PSF-B). We have now examined the possibility that PSF-A and PSF-B are members of the nuclear factor (NF)-1 family. Transcripts of NF-1A, NF-1C and NF-1X, but not of NF-1B, were readily detected in GC cells. High-affinity binding of NF-1 to PSE-B, but not to PSE-A, was confirmed by competition of DNA-protein interactions by using NF-1 DNA elements and antibodies. Functionally, a NF-1 element was able to substitute for PSE-B as a promoter-specific repressor in GC cells after gene transfer. However, there was a difference in the magnitude of repression exerted by the NF-1 and PSF-B elements on the CS-A promoter and, with the use of mutations, this difference was shown to be consistent with variations in NF-1-binding sequences. These results indicate that PSF-B, but not PSF-A, is a member of the NF-1 family, which participates in the PSF complex and in the repression of the CS-A promoter in pituitary GC cells.

Both pituitary and placental growth hormone transcripts are expressed in human peripheral blood mononuclear cells (PBMC).

The hGH-V gene codes for a variant of human pituitary growth hormone (hGH-N) named placental growth hormone (hPGH). hPGH shares 93% amino acid identity with hGH-N. Until now the hGH-V gene was considered to be exclusively expressed in human placenta, where it replaces maternal circulating hGH-N at the end of pregnancy. In this study we investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis hGH-N, and hGH-V, gene expression in PBMC in men, women and pregnant women. We have demonstrated that hGH-N and hGH-V transcripts are simultaneously produced by PBMC in both men and women as well as pregnant women. The PBMC of a PIT-1-negative woman expressed only the hGH-V transcript, but not the hGH-N one as expected. In conclusion, hGH-V mRNA is expressed by cells other than the syncytiotrophoblast, is not regulated by PIT-1, and may be involved in immune regulation, as is pituitary GH.

Effects of glucose on placental hormones in the human term placenta in vitro.

Glucose intake during pregnancy results in a decrease in endogenous insulin-like growth factor binding protein-1 (IGFBP-1). However, the exact role of glucose on placental secretion of IGFBP-1 is unclear. This study was designed to investigate the direct effects of glucose on the production of IGFBP-1 and other placental hormones, using an isolated placental preparation. Using the dual recirculating perfusion system for an isolated human placenta lobule, a total of 43 experiments were performed over a duration of 6 hours. Twenty placentae were perfused with a medium containing 141 +/- 10 mg/dL (7.83 +/- 0.56 mmol/L) glucose (group I) and 23 placentae with 242 +/- 12 mg/dL (13.43 +/- 0.67 mmol/L) glucose (group II). Levels of insulin, glucose, lactate, insulin-like growth factor (IGF-I), IGFBP-1, human placental lactogen (hPL) and beta-human chorionic gonadotropin (beta-hCG) were measured at 30 minute intervals during perfusion. Insulin and IGF-I were barely detectable in the perfusates and their levels were not modulated by glucose. IGFBP-1 was predominantly detected in the maternal rather than the fetal compartment of the placental circulation. Glucose increased the levels of IGFBP-1 in the maternal circulation in groups I and II during the first two hours of perfusion (188 +/- 58% and 193 +/- 31%, respectively). However, during the subsequent 4 hour period, the increase in IGFBP-1 concentration was significantly higher in group II (926 +/- 427%) than in group I (428 +/- 216%) (p < 0.05). There was no difference in the levels of hPL or beta-hCG between the two groups in the maternal circulation. Thus, glucose stimulates the production of IGFBP-1 in the maternal circulation of a placenta in vitro. This increase in IGFBP-1 by glucose in vitro, as opposed to the decrease of IGFBP-1 in vivo, may be due to a lack of circulatory maternal insulin in the isolated placental preparation. These results also suggest that there may be a functional barrier within the placenta that prevents an increase in the level of IGFBP-1 in the fetal circulation.

The role of the placenta in fetal nutrition and growth.

The placenta plays a key role in the nutrition of the fetus. It mediates the active transport of nutrients and metabolic wastes across the barrier separating maternal and fetal compartments, as well as modifying the composition of some nutrients through its own metabolic activity. The function of the placenta is essential to the growth of a healthy fetus; it is becoming apparent that the activities of the placenta are in turn modulated by signals originating from the fetus. Communication between placenta and fetus is especially critical in intrauterine growth retardation. The importance of the interaction of factors like insulin-like growth factor and epidermal growth factor with their receptors is becoming increasingly clear.