Later Stages Of Gestation Research Articles

Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice.

Sonic hedgehog ( Shh) is crucial for organogenesis in the foregut. This study investigated the function of Shh at the late-gestational stage; during which, the esophagus continues to differentiate. We established cytokeratin 14 ( CK14)-Cre;Shhfl/fl mice in which the down-regulation of Shh in the epithelium occurred at approximately the same time as esophageal muscle conversion. Hematoxylin and eosin and immunohistochemical staining, with antibodies against keratin 14, Shh, patched 1 (Ptch1), Gli1, proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (αSMA), high-molecular-weight caldesmon (hCD), myogenin, paired box 7 (Pax7), β3-tubulin, and protein gene product 9.5 (PGP9.5), was performed to detect specific tissue dysplasia. Organ culture was conducted in vitro, and total mRNA was extracted to determine the transcriptional dysregulation. The esophagus of CK14-Cre;Shhfl/fl mice developed into an independent tube with an obvious dilatation at postnatal d 0.5. The number of cell layers and the expression of PCNA were decreased in mutant mice, compared with those in wild-type mice. The expression of hCD declined progressively in the middle, distal, and lower esophageal sphincter levels of the mutant esophagus from embryonic d 17.5, compared with the expression in wild-type littermates. Pax7 accumulation and myogenin reduction in mutant mice indicated that esophageal skeletal-myoblast progression was blocked. RNA sequencing analysis revealed a significant down-regulation of genes involved in proliferation and muscular motivation in CK14-Cre;Shhfl/fl mice. Thus, loss of Shh at the late-gestational stage leads to megaesophagus with reduced proliferation and a muscle development disorder in mice.-Jia, X., Min, L., Zhu, S., Zhang, S., Huang, X. Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice.

Copper Requirements of Pregnant Ewes under Conditions of the Arid Zone in Russia

The copper requirements of pregnant ewes in the environment of the arid zone in the Republic of Kalmykia have been studied. The organ and tissue analyses have shown that the copper quantitative concentrations in the fetal tissues and organs tend to vary depending on its age. The maximum copper contents are recorded in the bone tissue and the skin (1839.7 and 1714.08 μg%, respectively) of the fetuses of 100 days of age and in the bone tissue and the liver (1843.08 and 1261.04 μg%, respectively) of the 145-day-old fetuses. In the bone tissue, the percentage of the element was relatively steady during the period of the prenatal development and varied in the range of 1839.7–1843.08 μg%. The blood copper level has slightly increased for the survey period; the blood mass increased 5.84-fold. The total amount of copper in the blood of the fetus at the age of 100 days was 111.21 μg%, while it increased up to 717.33 μg% in the blood of the 145-day-old fetus. For the period of the fetal development, the copper contents in the muscle tissue and the sheepskin with wool decreased from 957.06 to 52.53 μg% and 1714.08 to 170.7 μg%, respectively, while the total amount of this element in these tissues decreased from 3531.07 to 1020.5 μg and from 1458.65 to 1309.05 μg, respectively. The liver copper content elevated with the gestational age by 37.14 μg%, it comprised 1261.04 μg% up to the age of 145 days, and the total content increased 3.1 times. The relative percentage and the total content of copper in the lungs increased by 77.29 μg% and 4.3 times, respectively, between 100 and 145 days of age generally due to the increase in the organ masses. The total copper content in the 100-day-old and 145-day-old fetuses comprised 6.95 and 9.74 mg, respectively. The copper doses at the stages of early (55 days), middle (100 days), and late (145 days) gestation of the Kalmyk fat-rumped meat sheep were determined to be 12.93, 14.02, and 15.34 mg, respectively. Taking into consideration the fact that the dietary copper absorbtion rate is in the range of 71.05–73%, the fat-rumped ewes entering early, middle, and late pregnancy should intake 18.2, 19.3, and 21 mg dietary copper, respectively. The dietary copper intake norms per 1 kg dry matter intake are 11.0, 7.9, and 8.0 mg; the copper intake norms per 1 kg live weight should be 0.30, 0.32, and 0.35 mg.

Interleukin 22 prevents lipopolysaccharide- induced preterm labor in mice.

Preterm birth is widespread and causes 35% of all neonatal deaths. Infants who survive face potential long-term complications. A major contributing factor of preterm birth is infection. We investigated the role of interleukin 22 (IL22) as a potential clinically relevant cytokine during gestational infection. IL22 is an effector molecule secreted by immune cells. While the expression of IL22 was reported in normal nonpregnant endometrium and early pregnancy decidua, little is known about uterine IL22 expression during mid or late gestational stages of pregnancy. Since IL22 has been shown to be an essential mediator in epithelial regeneration and wound repair, we investigated the potential role of IL22 during defense against an inflammatory response at the maternal-fetal interface. We used a well-established model to study infection and infection-associated inflammation during preterm birth in the mouse. We have shown that IL22 is upregulated to respond to an intrauterine lipopolysaccharide administration and plays an important role in controlling the risk of inflammation-induced preterm birth. This paper proposes IL22 as a treatment method to combat infection and prevent preterm birth in susceptible patients.

Lysine Requirements of Healthy Pregnant Women are Higher During Late Stages of Gestation Compared to Early Gestation

Lysine is the first limiting amino acid in cereal proteins and is found mainly in animal-derived products. Current Dietary Reference Intake (DRI) recommendations extrapolate lysine requirements during pregnancy from nonpregnant adult data, and may underestimate true requirements. Our objective is to define a quantitative lysine requirement in healthy pregnant women and to determine whether requirements vary between 2 phases of gestation. Fourteen pregnant women in early (12-19 wk) and 19 women in late (33-39 wk) gestation were studied using the indicator amino acid oxidation technique. Individual lysine intakes (6-84 mg · kg-1 · d-1, deficient to excess) were tested on each study day as a crystalline amino acid mixture based on egg protein composition. Isonitrogenous diets maintained protein intake at 1.5 g · kg-1 · d-1 and calorie intake at 1.7 times resting energy expenditure during each study day. Phenylalanine and tyrosine intakes were held constant across all lysine intakes. Breath and urine samples were collected at baseline and isotopic steady state. Lysine requirements were determined by measuring the oxidation of L-[1-13C]-phenylalanine to 13CO2 (F13CO2). Biphase linear regression crossover analysis was used to determine a breakpoint (which represents the estimated average requirement, EAR) in F13CO2. The EAR for lysine during early gestation was determined to be 36.6 mg · kg-1 · d-1 (R2 = 0.484, upper 95% CI = 46.2 mg · kg-1 · d-1), similar to an earlier adult requirement of 36 mg · kg-1 · d-1. The EAR for lysine during late gestation was determined to be 50.3 mg · kg-1 · d-1 (R2 = 0.664, upper 95% CI = 60.4 mg · kg-1 · d-1), 23% higher than the current pregnancy DRI EAR recommendation of 41 mg · kg-1 · d-1. Our results suggest that lysine requirements are higher during late gestation compared to early gestation, and that current dietary lysine recommendations during late stages of pregnancy may be underestimated. The results have implications for populations consuming cereal-based diets as their primary source of protein. This trial was registered at clinicaltrials.gov as NCT01776931.

Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis.

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.

Zika Virus Infects Intermediate Progenitor Cells and Post-mitotic Committed Neurons in Human Fetal Brain Tissues

Zika virus (ZIKV) infection is associated with microcephaly in fetuses, but the pathogenesis of ZIKV-related microcephaly is not well understood. Here we show that ZIKV infects the subventricular zone in human fetal brain tissues and that the tissue tropism broadens with the progression of gestation. Our research demonstrates also that intermediate progenitor cells (IPCs) are the main target cells for ZIKV. Post-mitotic committed neurons become susceptible to ZIKV infection as well at later stages of gestation. Furthermore, activation of microglial cells, DNA fragmentation, and apoptosis of infected or uninfected cells could be found in ZIKV-infected brain tissues. Our studies identify IPCs as the main target cells for ZIKV. They also suggest that immune activation after ZIKV infection may play an important role in the pathogenesis of ZIKV-related microcephaly.

Thyroid hormone regulates protease expression and activation of Notch signaling in implantation and embryo development.

A clinical association between thyroid dysfunction and pregnancy complications has been extensively reported; however, the molecular mechanisms through which TH might regulate key events of pregnancy have not been elucidated yet. In this respect, we performed in vivo studies in MMI-induced hypothyroid pregnant mice, evaluating the effect of hypothyroidism on the number of implantation sites, developing embryos/resorptions and pups per litter, at 4.5, 10.5, 18.5 days post-coitum (dpc) and at birth. We also studied the expression of major molecules involved in implantation and placentation, such as the proteases ISPs, MMPs, TIMPs and Notch pathway-related genes. Our results demonstrate that hypothyroidism may have a dual effect on pregnancy, by initially influencing implantation and by regulating placental development at later stages of gestation. To further elucidate the role of TH in implantation, we performed in vitro studies by culturing 3.5 dpc blastocysts in the presence of TH, with or without endometrial cells used as the feeder layer, and studied their ability to undergo hatching and outgrowth. We observed that, in the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching by upregulating the expression of blastocyst-produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. These results clearly indicate that TH is involved in the bidirectional crosstalk between the competent blastocyst and the receptive endometrium at the time of implantation.

Congenital Malformations Attributed to Prenatal Exposure to Cyclophosphamide.

Cyclophosphamide (CPA) remains one of the most widely prescribed anticancer drugs. It is also used in the treatment of rheumatoid arthritis, childhood nephrotic syndrome and systemic lupus erythematosus. It is a potent immunosuppressive agent. It is commonly used in blood and bone marrow transplantation. With the growing trend among women postponing childbearing, the number of women who are diagnosed with breast cancer is also increasing thus escalating the chances of exposure of the unborn child to antineoplastic drugs. A review of the literature provides strong evidence for the teratogenic effects on infants prenatally exposed to CPA. Both sporadic case reports and larger case series have demonstrated that babies with cyclophosphamide embryopathy are afflicted with intrauterine growth restriction, small for gestational age, and craniofacial malformations including eye anomalies, cleft/arched palate, hydrocephaly, micrognathia, low set microtia, hearing defects, craniosynostosis, and facial asymmetry. Also observed in these cases are limb defects such as radial, ulnar and tibial hypoplasia, club foot, digital defects of the hand and feet as well as vertebral fusion, brevicolis, and occasional Sprengel's deformity. These anomalies vary in consistency of occurrence and severity of the phenotype across cases and lack the specificity of thalidomide embryopathy or rubella embryopathy. However, they do occur is no longer in doubt. First trimester of pregnancy seems to be particularly susceptible to fetal malformations, although CPA effects on fetuses of later stages of gestation (hearing defects, growth restriction for example) are also reported occasionally. One of the major concerns from a mechanistic point of view is our inability to dissect the teratogenic effects of CPA from those of other drugs administered together with CPA as combination therapy. Animal experiments have been of particular value in that they are able to circumvent the numerous extraneous variables inherent to human case reports. They have also revealed the detrimental effects of CPA on gametes, preimplantation embryos, organogenesis as well as their potential teratogenic mechanisms. Of particular importance are the role of genetic polymorphisms, male mediated teratogenesis, ovarian failure, preimplantation embryo loss, epigenetic modifications, proxidant-antioxidant imbalance, autophagy, apoptosis, microRNAs and postclosure neural tube defects induced by CPA -all of which are areas for further research in CPA teratogenesis.

Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood

Maternal inflammation during pregnancy can have detrimental effects on embryonic development that persist during adulthood. However, the underlying mechanisms and insights in the responsible cell types are still largely unknown. Here we report the effect of maternal inflammation on fetal microglia, the innate immune cells of the central nervous system (CNS). In mice, a challenge with LPS during late gestation stages (days 15–16–17) induced a pro-inflammatory response in fetal microglia. Adult whole brain microglia of mice that were exposed to LPS during embryonic development displayed a persistent reduction in pro-inflammatory activation in response to a re-challenge with LPS. In contrast, hippocampal microglia of these mice displayed an increased inflammatory response to an LPS re-challenge. In addition, a reduced expression of brain-derived neurotrophic factor (BDNF) was observed in hippocampal microglia of LPS-offspring. Microglia-derived BDNF has been shown to be important for learning and memory processes. In line with these observations, behavioral- and learning tasks with mice that were exposed to maternal inflammation revealed reduced home cage activity, reduced anxiety and reduced learning performance in a T-maze. These data show that exposure to maternal inflammation during late gestation results in long term changes in microglia responsiveness during adulthood, which is different in nature in hippocampus compared to total brain microglia.

Abstract 346: Glucose Inhibits Cardiomyocyte Maturation Through Nucleotide Biosynthesis

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is known to be associated with congenital heart disease. However, little is known about the mechanism how blood glucose impacts heart formation. Using a chemically-defined human pluripotent stem cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic and electrophysiological levels via nucleotide biosynthesis through the pentose phosphate pathway. Even though blood glucose level stays stable in utero during normal pregnancy, glucose uptake by fetal cardiac tissue was found drastically reduced at late gestational stages in the mouse. Interestingly, perturbation of glucose dynamics during gestation in a murine model of diabetic pregnancy promoted mitosis and inhibited maturation of fetal cardiomyocytes. Therefore, this observation suggests that the metabolic switch is not only to meet the energy demand but also to induce a genetic program to facilitate cardiac maturation, providing a possible mechanistic basis for the congenital heart disease in diabetic pregnancy.

P 45 Does apolipoprotein B (apo B) level in early pregnancy predict excessive gestational weight gain and adverse pregnancy outcome?

Introduction Excessive maternal gestational weight gain (GWG) is a risk factor for several maternal and fetal complications. Lipid metabolism like apo B is linked to obesity and maintenance of pregnancy via placental cholesterol uptake and steroid hormone production. Additionally, evidence is increasing that apo B containing lipoproteins directly influence angiogenesis via modulation of the VEGF-receptor 1. Maternal ApoB levels are associated with adverse outcome of pregnancy e. g. in preeclampsia and IUGR at onset of the disease. Objectives The hypothesis of the study was that apo B level early in pregnancy are associated with excessive GWG and predictive for adverse outcomes. Material and methods A retrospective study, including 547 pregnant women, was conducted. According to IOM recommendations on GWG, there were 95 women with inadequate GWG, 171 with adequate GWG and 281 with excessive GWG. Apo B levels from early pregnancy serum samples were measured by ELISA. The Kruskal–Wallis-test was used in order to compare the apo B levels between the three GWG groups. Apo B levels were also assessed for the prediction of adverse pregnancy outcomes like SGA/ IUGR and hypertension. Results There was no significant difference in apo B levels between the different GWG groups. Compared to the women with adequate GWG neither women with inadequate GWG had differing apo B levels (p = 0,6135) nor had women with excessive GWG (p = 0,0515). We found significant higher levels of apoB in overweight and obese patients as compared to normal BMI (p Conclusions Apo B cannot be used as a marker for identifying the risk of excessive GWG or adverse pregnancy outcomes early in pregnancy and therefore remains differential only in later stages of gestation.

Placental gene activity of significant angiogenetic factors in the background of intrauterine growth restriction

Placental vascular endothelial growth factor A (VEGF-A) gene and endoglin gene are both overexpressed in placental samples obtained from pregnancies with intrauterine growth restriction compared to normal pregnancies. In the background of these changes a mechanism can be supposed, in which the increased endoglin activity in intrauterine growth restriction (IUGR) leads to impaired placental circulation through an antioangiogenetic effect. This results in the development of placental vascular dysfunction and chronic fetal hypoxia. It is chronic hypoxia that turns on VEGF-A as a compensatory mechanism to improve fetal vascular blood supply by promoting placental blood vessel formation. Although the maternal serum placental growth factor (PlGF) level is a potential predictor for both IUGR and praeeclampsia, placental PlGF gene activity may be less of an active in the regulation of placental circulation in IUGR pregnancies during the later stages of gestation. Orv. Hetil., 2017, 158(16), 612-617.

Early neonatal loss of inhibitory synaptic input to the spinal motor neurons confers spina bifida-like leg dysfunction in a chicken model.

ABSTRACTSpina bifida aperta (SBA), one of the most common congenital malformations, causes lifelong neurological complications, particularly in terms of motor dysfunction. Fetuses with SBA exhibit voluntary leg movements in utero and during early neonatal life, but these disappear within the first few weeks after birth. However, the pathophysiological sequence underlying such motor dysfunction remains unclear. Additionally, because important insights have yet to be obtained from human cases, an appropriate animal model is essential. Here, we investigated the neuropathological mechanisms of progression of SBA-like motor dysfunctions in a neural tube surgery-induced chicken model of SBA at different pathogenesis points ranging from embryonic to posthatch ages. We found that chicks with SBA-like features lose voluntary leg movements and subsequently exhibit lower-limb paralysis within the first 2 weeks after hatching, coinciding with the synaptic change-induced disruption of spinal motor networks at the site of the SBA lesion in the lumbosacral region. Such synaptic changes reduced the ratio of inhibitory-to-excitatory inputs to motor neurons and were associated with a drastic loss of γ-aminobutyric acid (GABA)ergic inputs and upregulation of the cholinergic activities of motor neurons. Furthermore, most of the neurons in ventral horns, which appeared to be suffering from excitotoxicity during the early postnatal days, underwent apoptosis. However, the triggers of cellular abnormalization and neurodegenerative signaling were evident in the middle- to late-gestational stages, probably attributable to the amniotic fluid-induced in ovo milieu. In conclusion, we found that early neonatal loss of neurons in the ventral horn of exposed spinal cord affords novel insights into the pathophysiology of SBA-like leg dysfunction.

Transrectal Doppler sonography of uterine blood flow in ewes during pregnancy

In sheep, there is a lack of information on the behaviour of hemodynamic indices and parameters of blood flow velocity of the uterine artery during pregnancy, making it impossible to determine the real meaning of the values found and their probable relevance in normal physiological or pathological states. The objective of the present study was to evaluate the blood flow velocity parameters and hemodynamic indices of the uterine artery in ewes (18) during pregnancy (33). Based on non-invasive colour Doppler sonography, we evaluated the peak systolic velocity (PS), end diastolic velocity (ED), time-averaged maximum and minimum velocity in a cardiac cycle (TAMAX and TAMEAN), pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (S/D), heart rate (HR), arterial diameter (AD) and the blood flow volume (BFV). Examinations started on day 28 and continued at two-week intervals until parturition. The Doppler parameters and the diameter of the uterine artery underwent significant changes during pregnancy. In the evaluated animals, indices related to resistance of the uterine artery, namely the impedance of blood flow, decreased throughout the initial, middle and late stages of gestation (PI: 1.15, 1.04, 0.97; RI: 0.61, 0.59, 0.57; S/D: 2.68, 2.52, 2.39, respectively) (p < 0.05). In contrast, the contents related to higher uterine irrigation increased during gestation (PS, ED, TAMAX, TAMEAN, HR, AD and BFV) (p < 0.05). No differences were found between the means of the variables in relation to the right and left uterine arteries and between single and multiple pregnancies in the hemodynamic indices. The present study is the first to demonstrate changes in uterine hemodynamics throughout pregnancy in sheep.

Detection of Placental Proteomes at Different Uterine Positions in Large White and Meishan Gilts on Gestational Day 90.

Within-litter uniformity in pigs is a major factor affecting piglet survival and growth performance. We know that Meishan (MS) gilts have higher piglet survival rate than Large White (LW) gilts because their foetal weight is less varied. To understand the molecular basis for placental nutritional transport during the late stages of gestation in LW and MS, we employed the isobaric tags for relative and absolute quantification (iTRAQ) method to investigate alterations in the placental proteomes of LW and MS gilts on gestational day 90. Investigation of foetal weight at different uterine positions revealed that the foetal and placental weights as well as the foetal concentration of glucose were significantly higher in LW gilts positioned towards the utero-tubal junction than in those positioned toward the cervix; however, no such differences were observed in MS gilts, and MS gilts had a greater uniformity in foetal weight on day 90 of gestation. Comparisons of the proteomes between placentas positioned toward the cervix and those positioned toward the utero-tubal junction identified 38 differentially expressed proteins in the two breeds. These proteins play a central role in nutrient transport and metabolism, as well as in transcriptional and translational regulation. Of particular interest is the finding that the placentas of LW gilts showed 14 differential expression of proteins mainly related to lipid transport and energy metabolism (including solute carrier family 27, mitochondrial trifunctional protein, and NADH dehydrogenase [ubiquinone] flavoprotein 2), but only 2 proteins in MS gilts. In contrast, the differentially expressed proteins in MS gilts were primarily involved in transcriptional and translational regulation (such as ribosome-sec61 and 40S ribosomal protein S23), with a few related to glucose and coenzyme transport and metabolism (including glucose transport protein and ferrochelatase). Our results revealed that placental lipid and energy metabolism might play a crucial role in the regulation of foetal weight, based on uterine position in two distinct pig breeds. These findings provide a deeper understanding of placental efficiency that can be utilized to provide a new method to enhance the efficiency of livestock production.

Placental gene expression of the placental growth factor (PlGF) in intrauterine growth restriction

Objective: We analyzed changes in gene expression of placental growth factor (PIGF) in human placental samples obtained postpartum from pregnancies with IUGR.Methods: During a twelve-month study period representing the calendar year of 2012 placental samples from 101 pregnancies with IUGR and from 140 normal pregnancies were obtained for analysis of a potential difference in PIGF gene expression.Results: There was no significant difference in gene activity of the PIGF gene between the IUGR versus normal pregnancy groups (Ln2α: 0.92; p < 0.06). Within the IUGR group, no fetal gender-dependent differences were seen in placental PIGF gene expression (Ln2α: 0.72; p = 0.05). Placental PIGF gene activity was significantly lower in fetuses with more severe IUGR versus less severe cases (Ln2α: −1.49; p < 0.03).Conclusion: We found no difference in gene expression of PIGF in placental samples obtained from IUGR pregnancies versus normal pregnancy suggesting the absence of a direct role of PIGF gene activity in the development of defective angiogenesis in IUGR during the later stages of gestation. However, in more severe cases of intrauterine growth restriction PIGF expression does show a significant decrease indicating its potential role in the profound defect in angiogenesis in these cases.

Prenatal decision-making in the second and third trimester in trisomy 21-affected pregnancies.

Down syndrome (DS) is the most common chromosome abnormality among live-born infants and the most frequent genetic cause of intellectual disability. The majority of pregnancies affected by DS are terminated. The decision concerning whether or not to continue a pregnancy following the prenatal diagnosis of DS is complex and amongst others, motivated by attitudes towards termination, socioeconomic factors, and ultrasound findings. In Germany, termination of pregnancy (TOP) is a legal option, even during the later stages of gestation. The aim of the present study was to evaluate the pregnancy outcomes as well as possible factors that influence the decisions made by women with trisomy 21-affected pregnancies. In our study 112 pregnancies affected by trisomy 21 were included. Our data confirm that most patients are more likely to terminate a trisomy 21-affected pregnancy [76 (67.9%) vs. 36 (32.1%) continued pregnancies]. Beyond that we found that women who continued their pregnancy tended to be at an advanced stage in their pregnancy at the time of karyotyping. With regards to factors from their medical history as well as sonographic findings there was no identifiable single factor that could distinguish between women that opted to continue or terminate their pregnancy.

Identification of the pregnancy-associated glycoprotein family (PAGs) and some aspects of placenta development in the European moose (Alces alces L.)

This study describes the identification and a broad-based characterization of the pregnancy-associated glycoprotein (PAG) genes expressed in the synepitheliochorial placenta of the Alces alces (Aa; N = 51). We used: (1) both size measurements (cm) of various Aa embryos/fetuses (crown-rump length) and placentomes (PLCs); (2) PCR, Southern and sequencing; (3) Western-blot for total placental glycoproteins; (4) deglycosylation of total cotyledonary proteins; and (5) double heterologous IHC for cellular immune-localization of the PAGs as pregnancy advanced (50–200 days post coitum). The crown-rump length and PLC size measurements permitted a novel pattern estimation of various pregnancy stages in wild Aa. The PLC number varied (5–21) and was the greatest at the mid and late stages of gestation in females bearing singletons or twins. The genomic existence of the identified PAG-like family was named AaPAG-L. Amplicon profiles of the AaPAG-L varied in the number and length (118–2000 bp). Southern with porcine cDNA probes confirmed specificity and revealed dominant AaPAG-L amplicons in males and females. Nucleotide sequences of the AaPAG-L amplicons shared 86.27% homology with the bovine PAG1 (bPAG1) gene. Amino acid AaPAG sequences revealed in silico 88.23% to 100% homology with the bPAG1 precursor. Western-blots revealed a dominant mature 55 kDa AaPAG fraction, and the major ∼48 kDa glycosylated form that was deglycosylated to ∼44 kDa. The AaPAG-Ls was immuno-localized to mono- and bi-nucleated trophectodermal cells (TRD–chorionic epithelium), where signal intensity resembled intense TRD proliferation within developing PLCs as pregnancy advanced. This is the first study identifying the AaPAG-L family in the largest representative among the Cervidae.

Interaction of steroid receptor coactivators and estrogen receptors in the human placenta.

Female sex steroid hormones such as estrogen and progesterone have a pivotal role in maintaining pregnancy in human and animals. Especially, estrogen exerts specific effects on the cardiovascular system and angiogenesis, and thus affects significantly on placentation. Although the functions of estrogen have been emphasized during pregnancy, their signaling pathways in the placenta have not been fully understood. In this study, estrogen signaling was evaluated according to gestational age. Human placenta samples were collected and divided into early preterm (n=10), late preterm (n=18), and term (n=20) groups. First, serum estrogen concentration and corticotropin-releasing hormone (CRH) mRNA expression, which is known as gestation clock gene, were increased following gestation age in our experimental condition, as we expected. Next, the expression of estrogen receptors (ERs) and steroid receptor coactivators (SRCs) in the placenta was evaluated. ERα (ESR1) and ERβ (ESR2) were expressed highly at term period compared with early preterm. In addition, SRC family including SRC1, SRC2, and SRC3 was expressed in the human placenta, and the levels of SRC1, SRC2, and SRC3 were increased in the placenta at the late stage of gestation. The interaction of ERs with SRCs was also examined, which was significantly enhanced at term period. In the immunostaining results, it was indicated that ERs and SRCs were all dominantly expressed in syncytiotrophoblast cells. These results suggested that SRC1, SRC2, and SRC3 were expressed and interact with ERs highly at the late stage of gestation, and may amplify the signaling of estrogen in the placenta to maintain pregnancy.

Reproductive pattern and sex hormones of Calotes emma Gray 1845 andCalotes versicolor Daudin 1802 (Squamata; Agamidae)

We monitored testicular and ovarian morphologies, seminiferous tubules, the sexual segments of the kidneys (SSK), follicular histologies, and male testosterone and female estradiol to define the reproductive pattern of Calotes emma and Calotes versicolor. Samples were collected monthly at Sakaerat Environmental Research Station in Thailand for 1 year. Testicular hypertrophies occurred at a time characteristic for each species, with their time course corresponding well to both active spermatogenesis and the hypertrophied SSK. Gravid females were also found at a time characteristic for each species. In active reproductive females, oviductal eggs were concomitantly encountered with ovarian vitellogenic follicles. The previtellogenic and vitellogenic follicles corresponded well to granulosa layer alterations. The distinct large pyriform cells were present in the granulosa layer of previtellogenic follicles but disappeared from the vitellogenic follicles. Male testosterone levels rose during testicular and SSK hypertrophies, and female estradiol levels increased during active reproductive stages of late vitellogenic follicles and gestation. We suggest that the reproductive patterns of C. emma and C. versicolor fall into the same reproductive pattern of annual continual reproduction, but that the time courses of such events are different in the 2 Calotes, and even in individuals of the same Calotes population.