Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs.

Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process.     

Virulence of H5N1 virus in mice attenuates after in vitro serial passages

Background

Human cytomegalovirus (HCMV) is the most common pathogen in uterus during pregnancy, which may lead to some serious results such as miscarriage, stillbirth, cerebellar malformation, fetus developmental retardation, but its pathogenesis has not been fully explained. The hypofunction of extravillous cytotrophoblast (EVT) invasion is the essential pathologic base of some complications of pregnancy. c-erbB-2 is a kind of oncogene protein and closely linked with embryogenesis, tissue repair and regeneration. Matrix metalloproteinase (MMP) is one of the key enzymes which affect EVT migration and invasion function. The expression level changes of c-erbB-2, MMP-2 and MMP-9 can reflect the changes of EVT invasion function.

Results

To explore the influence of HCMV on the invasion function of EVT, we tested the protein expression level changes of c-erbB-2, MMP-2 and MMP-9 in villous explant cultured in vitro infected by HCMV, with the use of immunohistochemistry SP method and western blot. We confirmed that HCMV can reproduce and spread in early pregnancy villus; c-erbB-2 protein mainly expressed in normal early pregnancy villous syncytiotrophoblast (ST) remote plasma membrane and EVT, especially remote EVT cell membrane in villous stem cell column, little expressed in ST proximal end cell membrane and interstitial cells; MMP-2 protein primarily expressed in early pregnancy villous EVT endochylema and rarely in villous trophoblast (VT), ST and interstitial cells; MMP-9 protein largely expressed in early pregnancy villous mesenchyme, EVT and VT endochylema. Compared with control group, the three kinds of protein expression level in early pregnancy villus of virus group significantly decreased (P < 0.05).

Conclusion

HCMV can infect villus in vitro and cause the decrease of early pregnancy villous EVT's invasion function.     

Proteins-markers of placental insufficiency

The proteomic analysis of amniotic fluids of women with physiological pregnancy and pregnancy, complicated by placental insufficiency has been carried out on the second and the third trimesters. The differences in protein patterns between physiological pregnancy and pregnancy complicated by placental insufficiency seen during these gestation periods include: i) the absence of peroxiredoxin 2, epidermal fatty acid binding protein, and haptoglobin in placental insufficiency; ii) appearance of several proteins absent in physiological pregnancy: hippocalcin-like protein 1, CDC37-like protein, NKG2D ligand 2 (II trimester), CDC37-like protein, NKG2D ligand 2 (III trimester). These differences in the amniotic fluid proteome, obviously, have pathogenetic importance for the development of the placental insufficiency. The revealed proteins of distinction may serve as markers of this obstetrical pathology.     

Use of Wheat Germ Lectin-Sepharose for isolating pregnancy-specific protein

Specific pregnancy protein was obtained from retroplacental blood serum by saline fractionation, affinity chromatography on Wheat germ Lectin-Sepharose, gel filtration on Sephadex G-200, hydrophobic chromatography on Phenyl-Sepharose, and ion exchange chromatography on DEAE-Sephacel. The purified preparation of specific pregnancy protein is highly suitable for preparing antisera and the development of immuno- and radioassays of the protein in question.     

A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. L-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (L-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-alpha, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of alpha-tocopherol, a PKC-alpha inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-alpha. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.     

Some Physiological Adjustments Relating to Nutrition in Pregnancy

Information from human and animal studies clearly indicates that a number of adjustments in protein, fat, iron and calcium metabolism occur during pregnancy. Recognition of these adjustments has led to a reappraisal of nutrient requirements during pregnancy. The existence of significant protein storage during pregnancy is called into question; in contrast, the evidence suggests that the major storage component is fat, perhaps in anticipation of the energy costs of lactation. In women who will not lactate, this storage is of no benefit and is an etiologic factor in obesity. From a consideration of iron metabolism in pregnancy, it is concluded that it is better to prevent iron depletion in non-pregnant women, especially that associated with excessive menstrual loss, than to administer iron therapy to pregnant women.     

早孕因子研究进展

早孕因子既是一种微量蛋白又是一种妊娠相关蛋白,其纯品的获得尤为重要。简要论述了早孕因子的本质和功能,并就早孕因子在超早期妊娠诊断中的实际功能价值进行了深入探讨;结合早孕因子的国内外研究进展和生理生化特点提出了2种蛋白质提纯方案;针对当前早孕因子提纯及检测方法中存在的问题进行了讨论。     

A genetic polymorphism in a functional domain of human pregnancy zone protein: the bait region. Genomic structure of the bait domains of human pregnancy zone protein and alpha 2 macroglobulin.

Genomic clones containing the exons coding for the bait domain of human pregnancy zone protein and alpha 2 macroglobulin were isolated and fragments containing the bait exons were sequenced. It is shown that the bait domains of both alpha 2 macroglobulin and pregnancy zone protein are encoded by two exons, with conserved exon/intron boundaries. A genetic polymorphism showing either a Met or a Val residue as the sixth amino acid of the pregnancy zone protein bait domain was detected with the rare Met allele showing a gene frequency of 0.065.     

Protein metabolism in the mouse during pregnancy and lactation.

Protein synthesis was measured in vivo in the whole body and in a number of individual tissues in mice at various stages of pregnancy and lactation. The absolute rate of protein synthesis in the whole body increased from 640 mg/day in virgin mice to 1590 mg/day by day 18 of pregnancy, and to 2100 mg/day by day 15 of lactation. Large proportions of these increments were contributed by the rapidly growing foetuses and placentae in the pregnant animals and by protein synthesis in the mammary glands during lactation. In addition, a substantial stimulation of growth and protein synthesis was also observed in the liver and the gastrointestinal tract. Gastrocnemius muscle showed no changes in protein metabolism, indicating that in the well-fed mouse this tissue is not required to play a role as a protein reserve during pregnancy and lactation.     

Changes in protein turnover during gestation in the foetuses,placentas, liver,muscle and whole body of rats given a low-protein diet

(1)Protein synthesis and content have been studied in skeletal muscle, liver, foetuses and placentas of pregnant rats given a protein-deficient diet. Changes which occurred during the anabolic and subsequent catabolic phases of pregnancy are compared with those in well-fed pregnant and in protein-deficient non-pregnant rats. (2) The normal increase in liver protein did not occur during pregnancy in the protein-deficient group. (3) Protein deficiency affected protein content of the placenta earlier and more severely than that of the foetus. (4) Rates of protein synthesis in liver, placentas and foetuses were enhanced above control values by protein deficiency. (5)_Muscle protein increased normally during the anabolic phase of pregnancy but fell during the catabolic phase, unlike values for weel-fed animals. (6) Muscle protein synthesis rates rose by similar amounts in well-fed and protein-deficient animals during the anabolic phase of pregnancy. The fall to starting values during the catabolic phase was sharper and earlier in protein-deficient animals, which could reduce demands on the body amino acid pool by an amount equivalent to over 50% of the needs for protein deposition in foetuses and placentas. Thus, changes in muscle protein synthesis in both anabolic and catabolic phases of pregnancy may afford some protection to foetal protein synthesis.     

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.     

Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy.

The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.     

The impact of maternal protein restriction during rat pregnancy upon renal expression of angiotensin receptors and vasopressin-related aquaporins

Background  

Maternal protein restriction during rat pregnancy is known to impact upon fetal development, growth and risk of disease in later life. It is of interest to understand how protein undernutrition influences the normal maternal adaptation to pregnancy. Here we investigated the mechanisms regulating renal haemodynamics and plasma volume during pregnancy, in the context of both normal and reduced plasma volume expansion. The study focused on expression of renal angiotensin receptors (ATR) and vasopressin-related aquaporins (AQP), hypothesising that an alteration in the balance of these proteins would be associated with pregnancy per se and with compromised plasma volume expansion in rats fed a low-protein diet.     

Increased urinary excretion of nephrin, podocalyxin, and βig-h3 in women with preeclampsia

Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, βig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r(2) = 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. βig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. βig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary βig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder.     

Desmosomes are reduced in the mouse uterine luminal epithelium during the preimplantation period of pregnancy: a mechanism for facilitation of implantation

Dynamic regulation of intercellular junctions is an essential aspect of many developmental, reproductive, and physiological processes. We have shown that expression of the desmosomal protein desmoplakin decreases in the luminal uterine epithelium during the preimplantation period of pregnancy in mice. By the time of implantation (between Days 4.5 and 5 of pregnancy), desmoplakin protein can barely be detected by SDS-PAGE and Western blotting, and by immunocytochemistry, it is restricted to well-spaced, punctate dots at the apicolateral junction. Using confocal XZ series and electron microscope quantitation, both the density and distribution of desmosomes along the lateral cell surfaces of luminal epithelial cells were observed to change during early pregnancy. On Day 1 of pregnancy, desmosomes were found at high density in the apicolateral junctional complex, being present here in 79% of ultrathin sections examined, whereas on Day 5, the density was much reduced (present in only 18% of ultrathin sections examined). Desmosomes were found along the lateral surfaces, at or below the level of the nucleus, in 15% of ultrathin sections examined on Day 1 of pregnancy but in only 1% on Day 5. Desmoplakin mRNA declined during the first 4-5 days of pregnancy, along with the protein, suggesting that these changes are controlled at the level of mRNA. This study shows that desmosomes are regulated during early pregnancy, and we propose that a reduction in desmosome adhesion facilitates penetration of the luminal epithelium by trophoblast cells at implantation.     

The use of the probit model to predict pregnancy status of buffalo based on physio-chemical properties of estrual mucus

Improper timing of artificial insemination with respect to ovulation is one of the major factors hampering the conception rate in buffalo. The present study was an attempt to relate physio-chemical changes in estrual mucus to subsequent pregnancy status in order to find their optimal values for determining the time for artificial insemination (AI). Serum estradiol, total protein and dry matter contents of estrual mucus were evaluated to predict the subsequent pregnancy in 36 buffalo during October 1988 to February 1989. Serum estradiol was determined by radioimmunoassay (RIA); spinnbarkeit, dry matter and total protein were determined by standard methods. Multivariate probit analyses were carried out to relate these variables to subsequent pregnancy status. Elasticity and protein concentration were significantly related to prediction probability of pregnancy status, and they predicted the pregnancy status 86% of the times correctly (P < 0.05). The probability of pregnant animals being correctly classified was 0.76, whereas the corresponding value for non-pregnant animals was 0.95. The present study demonstrated the possibility of using such a statistical model on mucus characteristics for determining proper AI time for better conception rates in Nili-Ravi water buffalo.     

Cellular localization of an embryonic interferon,ovine trophoblastin and its mRNA in sheep embryos during early pregnancy

The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.     

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.