Prenatal tobacco smoke exposure and neurological impairment at 10 years of age among children born extremely preterm: a prospective cohort.

OBJECTIVE: To determine the association between prenatal tobacco smoke exposure and neurological impairment at 10 years of age among children born extremely preterm (<28 weeks of gestation). DESIGN: The Extremely Low Gestational Age Newborn (ELGAN) Study, a prospective cohort. SETTING: Ten-year follow-up of extremely preterm infants born at 14 US hospitals between 2002 and 2004. METHODS: Prenatal tobacco smoke exposure was defined as a mother's report at enrolment of active (i.e. maternal) and passive smoking during pregnancy. Poisson regression with generalized estimating equations was used. Models adjusted for mother's age, race/ethnicity, education, insurance, pre-pregnancy body mass index, US region, multiple gestation and infant's sex; and in sensitivity analysis, gestational age at delivery and clinical subtype of preterm birth, given their classification as intermediate and non-confounding variables. MAIN OUTCOMES: Neurological impairment at 10 years, epilepsy, cerebral palsy and cognitive impairment. RESULTS: Of 1200 ELGAN study survivors, 856 were assessed at 10 years of age with neurological outcomes, of whom 14% (118/856) had active tobacco exposure during pregnancy and 24% (207/852) had passive tobacco exposure. Compared with children who were not exposed prenatally to tobacco, children exposed to active tobacco use during pregnancy had a higher risk of epilepsy (14% versus 5%; adjusted relative risk: 1.68, 95% CI 1.45-1.92). This risk remained after adjustment for gestational age at delivery and clinical subtype of preterm birth. Prenatal tobacco smoke exposure was not associated with other assessed neurological outcomes, including cerebral palsy and multiple measures of cognitive impairment. CONCLUSIONS: Among children born extremely preterm, prenatal active tobacco smoke exposure was associated with an increased risk of epilepsy at 10 years of life. TWEETABLE ABSTRACT: Among infants born before 28 weeks of gestation, prenatal active tobacco smoke exposure was associated with an increased risk of epilepsy at 10 years of life.

Understanding the Early Biological Effects of Isoprene-Derived Particulate Matter Enhanced by Anthropogenic Pollutants.

INTRODUCTION: Airborne fine particulate matter (PM2.5; particulate matter ≤ 2.5 µm in aerodynamic diameter) plays a key role in air quality, climate, and public health. Globally, the largest mass fraction of PM2.5 is organic, dominated by secondary organic aerosol (SOA) formed from atmospheric oxidation of volatile organic compounds (VOCs). Isoprene from vegetation is the most abundant nonmethane VOC emitted into Earth's atmosphere. Isoprene has been recently recognized as one of the major sources of global SOA production that is enhanced by the presence of anthropogenic pollutants, such as acidic sulfate derived from sulfur dioxide (SO2), through multiphase chemistry of its oxidation products. Considering the abundance of isoprene-derived SOA in the atmosphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. Although previous studies have examined the toxicological effects of certain isoprene-derived gas-phase oxidation products, to date, no systematic studies have examined the potential toxicological effects of isoprene-derived SOA, its constituents, or its SOA precursors on human lung cells. SPECIFIC AIMS: The overall objective of this study was to investigate the early biological effects of isoprene-derived SOA and its subtypes on BEAS-2B cells (a human bronchial epithelial cell line), with a particular focus on the alteration of oxidative stress- and inflammation-related genes. To achieve this objective, there were two specific aims.1. Examine toxicity and early biological effects of SOA derived from the photochemical oxidation of isoprene, considering both urban and downwind-urban types of chemistry.2. Examine toxicity and early biological effects of SOA derived directly from downstream oxidation products of isoprene (i.e., epoxides and hydroperoxides). METHODS: Isoprene-derived SOA was first generated by photooxidation of isoprene under natural sunlight in the presence of nitric oxide (NO) and acidified sulfate aerosols. Experiments were conducted in a 120-m3 outdoor Teflon-film chamber located on the roof of the Gillings School of Global Public Health, University of North Carolina at Chapel Hill (UNC-Chapel Hill). BEAS-2B cells were exposed to chamber- generated isoprene-derived SOA using the Electrostatic Aerosol in Vitro Exposure System (EAVES). This approach allowed us to generate atmospherically relevant compositions of isoprene-derived SOA and to examine its toxicity through in vitro exposures at an air-liquid interface, providing a more biologically relevant exposure model. Isoprene-derived SOA samples were also collected, concurrently with EAVES sampling, onto Teflon membrane filters for in vitro resuspension exposures and for analysis of aerosol chemical composition by gas chromatography/electron ionization-quadrupole mass spectrometry (GC/EI-MS) with prior trimethylsilylation and ultra-performance liquid-chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry equipped with electrospray ionization (UPLC/ESI-HR-QTOFMS). Isoprene-derived SOA samples were also analyzed by the dithiothreitol (DTT) assay in order to characterize their reactive oxygen species (ROS)-generation potential.Organic synthesis of known isoprene-derived SOA precursors, which included isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene-derived hydroxyhydroperoxides (ISOPOOH), was conducted in order to isolate major isoprene-derived SOA formation pathways from each other and to determine which of these pathways (or SOA types) is potentially more toxic. Since IEPOX and MAE produce SOA through multiphase chemistry onto acidic sulfate aerosol, dark reactive uptake experiments of IEPOX and MAE in the presence of acidic sulfate aerosol were performed in a 10-m3 flexible Teflon indoor chamber at UNC-Chapel Hill. Since the generation of SOA from ISOPOOH (through a non-IEPOX route) requires a hydroxyl radical (•OH)-initiated oxidation, ozonolysis of tetramethylethylene (TME) was used to form the needed •OH radicals in the indoor chamber. The resultant low-volatility multifunctional hydroperoxides condensed onto nonacidified sulfate aerosol, yielding the ISOPOOH-derived SOA needed for exposures. Similar to the outdoor chamber SOAs, IEPOX, MAE- and ISOPOOH-derived SOAs were collected onto Teflon membrane filters and were subsequently chemically characterized by GC/EI-MS and UPLC/ESI-HR-QTOFMS as well as for ROS-generation potential using the DTT assay. These filters were also used for resuspension in vitro exposures.By conducting gene expression profiling, we provided mechanistic insights into the potential health effects of isoprene-derived SOA. First, gene expression profiling of 84 oxidative stress- and 249 inflammation-associated human genes was performed for cells exposed to isoprene-derived SOA generated in our outdoor chamber experiments in EAVES or by resuspension. Two pathway-focused panels were utilized for this purpose: (1) nCounter GX Human Inflammation Kit comprised of 249 human genes (NanoString), and (2) Human Oxidative Stress Plus RT2 Profiler PCR Array (Qiagen) comprised of 84 oxidative stress-associated genes. We compared the gene expression levels in cells exposed to SOA generated in an outdoor chamber from photochemical oxidation of isoprene in the presence of NO and acidified sulfate seed aerosol to cells exposed to a dark control mixture of isoprene, NO, and acidified sulfate seed aerosol to isolate the effects of the isoprene-derived SOA on the cells using the EAVES and resuspension exposure methods. Pathway-based analysis was performed for significantly altered genes using the ConsensusPathDB database, which is a database system for the integration of human gene functional interactions to provide biological pathway information for a gene set of interest. Pathway annotation was performed to provide biological pathway information for each gene set. The gene-gene interaction networks were constructed and visualized using the GeneMANIA Cytoscape app (version 3.4.1) to predict the putative function of altered genes. Lastly, isoprene-derived SOA collected onto filters was used in resuspension exposures to measure select inflammatory biomarkers, including interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) genes, in BEAS-2B cells to ensure that effects observed from EAVES exposures were attributable to particle-phase organic products. Since EAVES and resuspension exposures compared well, gene expression profiling for IEPOX-, MAE- and ISOPOOH-derived SOA were conducted using only resuspension exposures. RESULTS AND CONCLUSIONS: Chemical characterization coupled with biological analyses show that atmospherically relevant compositions of isoprene-derived SOA alter the levels of 41 oxidative stress-related genes. Of the different composition types of isoprene-derived SOA, MAE- and ISOPOOH-derived SOA altered the greatest number of genes, suggesting that carbonyl and hydroperoxide functional groups are oxidative stress promoters. Taken together, the different composition types accounted for 34 of the genes altered by the total isoprene-derived SOA mixture, while 7 remained unique to the total mixture exposures, indicating that there is either a synergistic effect of the different isoprene-derived SOA components or an unaccounted component in the mixture.The high-oxides of nitrogen (NOx) regime, which yielded MAE- and methacrolein (MACR)-derived SOA, had a higher ROS-generation potential (as measured by the DTT assay) than the low- NOx regime, which included IEPOX- and isoprene-derived SOA. However, ISOPOOH-derived SOA, which also formed in the low- NOx regime, had the highest ROS-generation potential, similar to 1,4-naphthoquinone (1,4-NQ). This suggests that aerosol-phase organic peroxides contribute significantly to particulate matter (PM) oxidative potential. MAE- and MACR- derived SOA showed equal or greater ROS-generation potential than was reported in prior UNC-Chapel Hill studies on diesel exhaust PM, highlighting the importance of a comprehensive investigation of the toxicity of isoprene-derived SOA. Notably, ISOPOOH-derived SOA was one order of magnitude higher in ROS-generation potential than diesel exhaust particles previously examined at UNC-Chapel Hill. As an acellular assay, the DTT assay may not be predictive of oxidative stress; therefore, we also focused on the gene expression results from the cellular exposures.We have demonstrated that the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the redox-sensitive activation protein-1 (AP-1) transcription factor networks have been significantly altered upon exposure to isoprene-derived SOA. The identification of Nrf2 pathway in cells exposed to isoprene-derived SOA is in accordance with our findings using the DTT assay, which measures the thiol reactivity of PM samples as a surrogate for their ROS-generation potential. Specifically, our results point to the cysteine-thiol modifications within cells that lead to activation of Nrf2-related gene expression.However, based on our gene expression results showing no clear relationship between DTT activity and the number of altered oxidative stress-related genes, the DTT activity of isoprene-derived SOA may not be directly indicative of toxicity relative to other SOA types. While activation of Nrf2-associated genes has been identified with responses to oxidative stress and linked to traffic related air pollution exposure in both toxicological and epidemiological studies, their implicit involvement in this study suggests that activation of Nrf2-related gene expression may occur with exposures to all sorts of PM types.By controlling the exposure time, method, and dose we demonstrated that among the SOA derived from previously identified individual precursors of isoprene-derived SOA, ISOPOOH-derived SOA alters moreoxidative stress related genes than does IEPOX-derived SOA, but fewer than MAE-derived SOA. This suggests that the composition of MAE-derived SOA may be the greatest contributor to alterations of oxidative stress-related gene expression observed due to isoprene-derived SOA exposure. Further study on induced levels of protein expression and specific toxicological endpoints is necessary to determine if the observed gene expression changes lead to adverse health effects. In addition, such studies have implications for pollution-control strategies because NOx and SO2 are controllable pollutants that can alter the composition of SOA, and in turn alter its effects on gene expression. The mass fraction of different components of atmospheric isoprene derived SOA should be considered, but altering the fraction of high- NOx isoprene-derived SOA (e.g., MAE derived SOA) may yield greater changes in gene expression than altering the fraction of low- NOx isoprene derived SOA types (ISOPOOH- or IEPOX-derived SOA). Finally, this study confirms that total isoprene-derived SOA alters the expression of a greater number of genes than does SOA derived from the tested precursors. This warrants further work to determine the underlying explanation for this observation, which may be uncharacterized components of isoprene-derived SOA or the potential for synergism between the studied components.

Cadmium body burden and increased blood pressure in middle-aged American Indians: the Strong Heart Study.

Cadmium (Cd) is an environmental pollutant that has been associated with cardiovascular disease in populations, but the relationship of Cd with hypertension has been inconsistent. We studied the association between urinary Cd concentrations, a measure of total body burden, and blood pressure in American Indians, a US population with above national average Cd burden. Urinary Cd was measured using inductively coupled plasma mass spectrometry, and adjusted for urinary creatinine concentration. Among 3714 middle-aged American Indian participants of the Strong Heart Study (mean age 56 years, 41% male, 67% ever-smokers, 23% taking antihypertensive medications), urinary Cd ranged from 0.01 to 78.48 µg g-1 creatinine (geometric mean=0.94 µg g-1) and it was correlated with smoking pack-year among ever-smokers (r2=0.16, P<0.0001). Participants who were smokers were on average light-smokers (mean 10.8 pack-years), and urinary Cd was similarly elevated in light- and never-smokers (geometric means of 0.88 µg g-1 creatinine for both categories). Log-transformed urinary Cd was significantly associated with higher systolic blood pressure in models adjusted for age, sex, geographic area, body mass index, smoking (ever vs never, and cumulative pack-years) and kidney function (mean blood pressure difference by lnCd concentration (ß)=1.64, P=0.002). These associations were present among light- and never-smokers (ß=2.03, P=0.002, n=2627), although not significant among never-smokers (ß=1.22, P=0.18, n=1260). Cd was also associated with diastolic blood pressure among light- and never-smokers (ß=0.94, P=0.004). These findings suggest that there is a relationship between Cd body burden and increased blood pressure in American Indians, a population with increased cardiovascular disease risk.

Inflammation in induced sputum after aluminium oxide exposure: an experimental chamber study.

INTRODUCTION: Workers in aluminium production are exposed to a complex mixture of particles and gases potentially harmful to the airways, among them aluminium oxide (Al2O3). With the use of an exposure chamber, we aimed to examine the effects of short-term controlled exposure to Al2O3 on lung function and inflammatory markers in healthy volunteers. METHODS: 15 men (age 19-31) were exposed in random order to clean air or Al2O3 particles (3.8-4.0 mg/m(3)) for 2 h including 30 min exercise (stationary bike, 75 W). The permissible exposure level (PEL) for Al2O3 by Occupational Safety and Health Administration, USA, is 5 mg/m(3) time weighted average (TWA). Sham and particle exposures were separated by at least 2 weeks. Spirometry was carried out, and induced sputum and blood samples were collected 48 h before and 4 and 24 h after exposure. RESULTS: Levels of sputum neutrophils (mean (±SEM)) was increased 24 h post-Al2O3 vs pre-Al2O3 exposure (43% (4) vs 31% (4), p=0.01) and the protein level of interleukin (IL)-8 had a 4.8 (0.9)-fold change increase 24 h after exposure (p<0.01). Following Al2O3 exposure, gene signatures in sputum were significantly increased related to several pathways. CONCLUSIONS: The present study suggests that controlled exposure to Al2O3 particles at levels below PEL (TWA) induces airway inflammation in healthy humans marked by elevated neutrophils and elevated IL-8. In addition, increased expression of genes associated with several biological processes was observed in sputum. Interestingly, inhaled Al2O3-induced effects were localised to the airways and not systemic.

Developmental Windows of Susceptibility to Inorganic Arsenic: A Survey of Current Toxicologic and Epidemiologic Data.

Globally, millions of people are exposed to elevated levels of inorganic arsenic (iAs) via drinking water. Exposure to iAs is associated with a wide range of negative health outcomes, including cancers, skin lesions, neurological impairment, cardiovascular diseases, and an increased susceptibility to infection. Among those exposed to iAs, the developing fetus and young children represent particularly sensitive subpopulations. Specifically, it has been noted in animal models and human populations that prenatal and early life iAs exposures are associated with diseases occurring during childhood and later in life. Recent epidemiologic and toxicologic studies have also demonstrated that epigenetic alterations may play a key mechanistic role underlying many of the iAs-associated health outcomes, including the carcinogenic and immunologic effects of exposure. This review summarizes some of the key studies related to prenatal and early life iAs exposure and highlights the complexities in isolating the precise developmental windows of exposure associated with these health outcomes.

Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

In vitro development and nutrient uptake by embryos derived from oocytes of pre-pubertal and adult cows.

The present study compared the developmental potential and uptake of nutrients by embryos from pre-pubertal and adult cows. Oocytes retrieved from ovaries of 5 to 7 month old calves and adult cows were matured and fertilized in vitro. Embryos were cultured in SOFaa to the blastocyst stage (7 days post-insemination). At successive stages of development, rates of glucose and pyruvate uptake were measured non-invasively by microfluorescence for individual embryos. Fertilization was equivalent in embryos from pre-pubertal and adult cows (P > 0.05), however development to blastocyst was significantly lower in embryos from pre-pubertal cows (9.8% versus 33.7%, respectively; P < 0.05). Total blastocyst cell number was not different between pre-pubertal and adult material (P > 0.05). Glucose uptake was exponential (pre-pubertal, r = 0.82; adult, r = 0. 82; P < 0.05), with an increase in uptake beyond the 8- to 16-cell stage. Glucose uptake was significantly lower in embryos from pre-pubertal cows at the 2- to 4-cell stages (1.5 versus 3.0 pmoles/embryo/hr; P < 0.05), but was equivalent to the adult cow at all other stages of development (P > 0.05). Pyruvate uptake was low until the blastocyst stage. Pyruvate uptake by embryos from pre-pubertal cows was significantly different to adult cows at the 1-cell stage (2.7 versus 4.6 pmoles/embryo/hr, respectively; P < 0. 05) and 2- to 4-cell stages (4.9 versus 3.6 pmoles/embryo/hr, respectively; P < 0.05). Pyruvate uptake was equivalent in the two groups in the later stages of development (P > 0.05). Perturbations in the uptake of nutrients by embryos from pre-pubertal cows were most likely due to the presence of a high proportion of developmentally incompetent embryos. Further, embryos from pre-pubertal cows that did develop to the blastocyst were as viable as blastocysts from adult cows with respect to nutrient uptakes and total cell number.

The effects of post-weaning progestagen treatment (Regumate) of early-weaned primiparous sows on subsequent reproductive performance.

This study investigated the effects of feeding the orally active progestagen, altrenogest (Regumate) post-weaning on the subsequent reproductive performance of early weaned sows. Ninety (90) Large White/Landrace first parity sows were randomly assigned to three treatments. Treatment 1 (EW) and treatment 3 (CW) sows were weaned on day 12 and day 24 post-partum, respectively while treatment 2 sows (EW-R) were weaned on day 12 post-partum and received an individual daily dose of 20 mg of Regumate on days 13 to 24 post-partum inclusive. Each sow was mated naturally at least twice at the first post-weaning or post-treatment oestrus and slaughtered on days 25-28 of pregnancy to determine the number of corpora lutea and embryos. Regumate-to-oestrus and weaning-to-oestrus intervals were similar for EW-R and CW sows (6.2 vs. 5.6 days). However, both intervals were significantly shorter (P < 0.01) than the weaning-to-oestrus interval of EW sows (7.3 days). An excellent synchronization of oestrus was achieved with Regumate treatment with 97% of treated sows in oestrus within 7 days of Regumate withdrawal compared with 64% for EW sows (P < 0.01) and 87% for CW sows (P > 0.05). Treatment with Regumate resulted in a significant increase in ovulation rate (16.9 vs. 15.4 and 14.9 for treatments EW-R, EW and CW, respectively; P < 0.05) and a non-significant increase in early embryonic survival (77% vs. 68% vs. 68% for treatments EW-R, EW and CW, respectively; P > 0.05). These results indicate that Regumate feeding is a potential management tool to alleviate the diminished reproductive performance associated with early weaning regimes since it leads to successful control of oestrus, higher ovulation and embryo survival rates and thus a greater potential litter size.

Ultrasonically guided transvaginal oocyte recovery from calves treated with or without GnRH.

We investigated the effect of GnRH given after gonadotropin stimulation on follicle growth and oocyte quality in young calves in a transvaginal oocyte recovery program. A 60 mg MPA pessary was inserted into each of nineteen 5-mo-old Friesian calves for 7 d; on Day 5 they received 140 mg, s.c. FSH (Folltropin) and 200 IU, i.m. PMSG and on Day 8 ten of the calves received 40 micrograms, i.m. GnRH (Fertagyl). Follicles were measured and aspirated on Day 9 using an ultrasound unit with a 6 MHz transvaginal probe (Toshiba). Oocytes from individual calves were recovered, graded and cultured in maturation media for 2 h (+GnRH group) or 22 h (-GnRH group), then fertilized and cultured for 6 d in SOF containing 0.8% BSA and amino acids. Oocyte viability (Class A,B or C) and embryo morphology were recorded. This procedure was repeated on the 19 calves plus 5 others 1 m.o. later, after random allocation to their respective groups. Approximately 70% of the calves responded to gonadotropin stimulation (> 2 follicles over 5 mm in diameter). Calves receiving GnRH tended to have both a higher number of follicles > 2 mm in diameter (27.1 vs 18.7) and of aspirated follicles (22.0 vs 14.1); however, there was a large variability between individuals (0 to 83 follicles and 0 to 73 aspirated). The total number of oocytes collected (10.8 vs 10.9) was not affected by GnRH treatment, probably due to the poor recovery rates in the highly stimulated calves from the +GnRH group, but GnRH did improve the proportion of viable oocytes (6.5 vs 4.1) due to a lower number of Class E oocytes (1.4 vs 4.5; P < 0.05). In the GnRH group, 40% of the viable oocytes had matured at the time of collection versus 0% in the group not treated with GnRH. The necessity of different culture runs between times and treatments prevented any meaningful comparison between groups for embryo development. Following the transfer of 19 morula/blastocyst-stage embryos to recipients, 3 pregnancies were detected by ultrasound examination on Day 60, with 1 oocyte originating from the +GnRH group and 2 from the -GnRH group.

Effect of immunisation against leukaemia inhibitory factor on the establishment of pregnancy in sheep.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is implicated in blastocyst implantation in mice and maintains the development of ovine embryos in culture. Previously, LIF mRNA and protein were demonstrated in the endometrium throughout the oestrous cycle and early pregnancy in the ewe. In this study pregnant ewes were passively immunised against human recombinant LIF with polyclonal antibodies raised in cows by active immunisation. Ewes were immunised during two stages of early pregnancy: blastocyst development to hatching, and blastocyst elongation to implantation. Only animals passively immunised against LIF showed detectable LIF antibodies in their sera and in uterine lumina flushings by radioimmunoassay and Western blot analysis. Pregnancy was confirmed by ultrasound on day 55 and a 33.5% non-significant decrease in pregnancy rate of anti-LIF treated animals was observed, when compared to animals in control groups (untreated or treated with bovine anti-keyhole limpet hemocyanin). Cows actively immunised with recombinant human LIF and exhibiting high levels of LIF antibodies in their sera at the time of blastocyst implantation also showed a reduced pregnancy rate in comparison to control animals. Although LIF may not be obligatory for implantation in ruminants it does appear to have a role during the establishment of pregnancy.

The collection of oocytes from bovine ovaries.

Various needle sizes (17- and 20-g) and aspiration pressures (25, 50, 75 and 100 mmHg) were used to aspirate a total of 5,827 ovarian follicles from bovine ovaries from a slaughterhouse source to assess the impact on the quantity and quality of recovered immature oocytes. The cumulus oocyte complexes (COC's) were graded according to the presence and consistency of cumulus cells surrounding the oocyte and the data analyzed using general linear models. Overall recovery rates and the recovery of oocytes considered viable for IVM/IVF procedures (Classes A, B and C) were both significantly higher using a 17-g needle than a 20-g needle (P < 0.01). As the vacuum pressure increased so did the recovery rate of the total number of oocytes, although the number of viable oocytes reached a maximum at a calculated vacuum pressure of 55 mmHg for the 17-g needle and 77 mmHg for the 20-g needle, with an increased incidence of denuded oocytes at higher vacuum pressures. In a second experiment conducted on 1, 473 follicles, no significant difference was found between 17-g double (flushing) and 17-g single lumen needles in the recovery rate of either the total number or number of viable oocytes when using a vacuum pressure of 50 mmHg.

Leukaemia inhibitory factor in endometrium during the oestrous cycle, early pregnancy and in ovariectomized steroid-treated ewes.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is essential for blastocyst implantation in mice and maintains the development of ovine embryos in culture. The expression of LIF was examined by northern blot analysis in endometrial tissue from cyclic (days 4-16) and pregnant (days 4-20) ewes, and the corresponding protein was immunolocalized. Expression of mRNA encoding LIF remained relatively constant throughout the oestrous cycle and was present during early pregnancy. A decrease in mRNA encoding LIF was observed during early pregnancy (on days 12-14) and expression was highest on days 16-20. Immunoreactive LIF was present in the cellular compartments of the endometrium throughout the oestrous cycle and early pregnancy, with maximal immunostaining in the caruncular and intercaruncular luminal epithelium, and moderate staining in the glandular epithelium and intercaruncular stroma. Immunoreactive LIF was also detected in the trophoblast cells of day 17 blastocysts. Separately cultured endometrial epithelial and stromal cells from pregnant animals both expressed mRNA encoding LIF. Ovariectomized steroid-treated ewes were studied to establish whether steroid hormones had a role in regulating endometrial LIF. Ewes treated with oestradiol alone showed lower concentrations of immunoreactive LIF in the endometrium in comparison to ovariectomized, control animals, while treatment of ovariectomized animals with both oestradiol and progesterone had a greater inhibitory effect on LIF immunolocalization. These studies demonstrate the presence of mRNA encoding LIF and protein throughout the oestrous cycle and early pregnancy and suggest that steroid hormones may be involved in their regulation.

Proadrenomedullin NH2-terminal peptide (PAMP)(12-20) has vasodepressor activity in the rat and cat.

Decreases in systemic arterial pressure in response to human proadrenomedullin NH2-terminal 20 peptide (hPAMP), a truncated analog, hPAMP(12-20), and human adrenomedullin (hADM) were compared in the rat and cat. The order of potency was hADM > hPAMP > hPAMP(12-20). hPAMP(12-20) was approximately 3-fold less potent than the full sequence peptide, hPAMP, and 10-fold less potent than the related peptide, hADM. The duration of the vasodepressor responses to hPAMP(12-20) and hPAMP were similar, and responses to both peptides were significantly shorter in duration than hADM. Vasodepressor responses to hPAMP(12-20), hPAMP, and hADM were greater in the rat when compared to responses to the peptides in the cat.

Catecholamine release mediates pressor effects of adrenomedullin-(15-22) in the rat.

Human adrenomedullin, a novel hypotensive peptide, contains a six-member ring structure similar to that found in calcitonin gene-related peptide and pancreatic amylin. Unlike the full-sequence peptide, human adrenomedullin-(15-22) [hADM-(15-22)], which contains the ring structure, increases systemic arterial pressure in the rat but not the cat. We undertook the present study to investigate the mechanism by which hADM-(15-22) increases systemic arterial pressure in the rat. Injection of hADM-(15-22) in doses of 10 to 300 nmol/kg i.v. increased systemic arterial pressure in a dose-dependent manner and was threefold less potent than norepinephrine when doses were compared on a nanomole basis. However, the ring structures of human calcitonin gene-related peptide and human amylin, human calcitonin gene-related peptide-(1-8) and human amylin-(1-8), respectively, had no significant effect on systemic arterial pressure in the rat. Pressor responses to hADM-(15-22) were reduced significantly after administration of phentolamine or reserpine. Responses to hADM-(15-22) were not altered by the angiotensin type 1 blocking agent DuP 753 or the endothelin-A/endothelin-B receptor blocking agent bosentan, and responses to hADM-(15-22) and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) were reduced after bilateral adrenalectomy. Pressor responses to DMPP were reduced by hexamethonium, whereas the nicotinic blocking agent had no effect on the pressor response to hADM-(15-22). These data suggest that increases in systemic arterial pressure in response to hADM-(15-22) in the rat are mediated by the activation of alpha-adrenergic receptors by catecholamines released from the adrenal medulla. The present data suggest that hADM-(15-22) releases catecholamines from the adrenal medulla by a noncholinergic mechanism.

Leukaemia inhibitory factor in human endometrium throughout the menstrual cycle.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine previously demonstrated to be essential for blastocyst implantation in mice. Samples of endometrium from normal cyclic women throughout the menstrual cycle were tested for LIF messenger RNA by Northern blot analysis and the corresponding protein was localised immunohistochemically with a polyclonal antibody to LIF. Western blot analysis detected a 45 kDa LIF protein in an extract from late secretory tissue. The expression of LIF messenger RNA transcript was detected only during the mid and late secretory phases of the cycle after day 20. Immunoreactive LIF was observed in all human endometrial samples. In the stroma there were moderate to high levels of immunohistochemical staining throughout the cycle with considerable variation between individuals but no cyclical variation. Epithelial staining, both luminal and glandular, was also present throughout the cycle but this was relatively low in the proliferative phase and strongest in the mid to late secretory phases. The marked cyclical changes of immunoreactive LIF in the human endometrial epithelium suggest a paracrine/autocrine role for LIF in endometrial function. Whether LIF is essential for implantation in the human remains to be established.

Relationships between follicle-stimulating hormone, follicle growth and ovulation rate in sheep.

The changes in follicle-stimulating hormone (FSH) concentration required to affect follicle growth and ovulation rate within individual ewes were examined. Relationships between peripheral FSH concentrations during the late-luteal and follicular phase and subsequent ovulation rates were investigated in 22 ewes from 4 breeds over 3 successive cycles (Experiment 1). Ewes were grouped as follows: Group 1 (n = 6), ewes exhibiting the same ovulation rate at each oestrous cycle: Group 2 (n = 5), ewes with three different ovulation rates at each oestrous cycle; and Group 3 (n = 11), ewes with the same ovulation rate at two oestrous cycles and a different ovulation rate on one occasion. Data from ewes in Group 1 and 3 provided estimates on the variation in FSH concentrations between cycles which were not large enough to alter ovulation rate (range, 0-67% variation in FSH concentration). In Group-2 ewes, there was no consistent association between increases in ovulation rate and the proportional increases in FSH concentrations. Differences in FSH concentrations were often less than those that did not alter ovulation rate in Group-I ewes. Furthermore, only 3 of 11 Group-3 ewes demonstrated high FSH concentrations associated with high ovulation rate (or low FSH concentrations and low ovulation rate) when compared with the concentrations found at the two cycles in which ovulation rate was similar. Hence, there was little evidence that FSH concentrations during the late-luteal and follicular phase are associated with changes in ovulation rate within individual ewes. In Experiment 2, follicles of similar size obtained from the same ewe (FecBFec+ and Romanov) showed markedly different responses in vitro to graded doses of FSH as measured by aromatase activity. It is concluded that, within a ewe, the large variability between gonadotrophin-dependent follicles in their requirement for FSH prevented the expression of any thresholds of ovarian response to FSH.

Human leukemia inhibitory factor improves the viability of cultured ovine embryos.

Embryos were collected from ewes on Day 6 after estrus (Day 0 = estrus), placed in M2 culture medium, and assigned to 1 of 4 treatment groups. Some embryos were transferred to recipient ewes on Day 6 of their estrous cycle either in pairs (group 1) or singularly (group 2) within 3 h of collection. The remaining embryos were individually cultured for 48 h in an atmosphere of 5% CO2 in humidified air in either synthetic oviduct fluid (SOF) medium (group 3) or SOF containing 1,000 U/ml of recombinant human leukemia inhibitory factor (hLIF) (SOF + hLIF: group 4). These embryos were then transferred to recipient ewes on Day 8 of their estrous cycle. The addition of hLIF to culture medium significantly improved the development of the embryos compared with control embryos prior to transfer (blastocysts hatching from the zona pellucida: group 3 = 16% vs. group 4 = 64%, p less than 0.05; those degenerative: group 3 = 27% vs. group 4 = 9%, p less than 0.05) and the subsequent pregnancy rates of the recipient ewes, receiving a single embryo, at Day 70 of pregnancy (group 3 = 16% vs. group 4 = 50%, p less than 0.05). The pregnancy rate of ewes given embryos cultured for 48 h in SOF + hLIF prior to transfer (50%; group 4) was similar to the group 2 ewes receiving a single embryo soon after collection (52%), but the pregnancy rate for both groups was significantly lower than that for the group 1 ewes receiving two embryos soon after collection (89%: 53% twins, 36% singles; p less than 0.05).

The effect of leukaemia inhibitory factor (LIF) on embryogenesis.

Leukaemia inhibitory factor (LIF) was originally identified as a haemopoetic factor that induced the differentiation of certain myeloid leukaemia cell lines. In contrast to this action, LIF was subsequently shown to inhibit the spontaneous differentiation of murine embryonic stem cells in culture, thus maintaining their pluripotency and ability to contribute to the germline of chimaeric mice. In the mouse, mRNA for LIF is expressed by the endometrial glands of the uterus coincident with the time of blastocyst implantation and receptors have been found on the preimplantation blastocyst. The signal for LIF expression appears to be of maternal origin, perhaps regulated by oestradiol. Recombinant LIF improves the development of murine and ovine blastocysts in culture although there is some species specificity with respect to the type of LIF that is bioactive. It is proposed here that LIF acts on the trophectoderm of the rapidly expanding blastocyst and improves the implantation rate of otherwise compromised embryos. Further studies in livestock should elicit therapeutic uses for LIF in embryo culture, embryo transfer and embryo survival in vivo.