Medicine and Health Sciences Commons^™

Expression And Localization Of Nrf2/Keap1 Signalling Pathway Genes In Mouse Preimplantation Embryos Exposed To Free Fatty Acids., Grace Dionne, Michele D. Calder, Dean H Betts, Basim Abu Rafea, Andrew J Watson

Obstetrics & Gynaecology Publications

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence …

Atrx Deletion In Neurons Leads To Sexually Dimorphic Dysregulation Of Mir-137 And Spatial Learning And Memory Deficits., Renee J. Tamming, Vanessa Dumeaux, Yan Jiang, Sarfraz Shafiq, Luana Langlois, Jacob Ellegood, Lily R. Qiu, Jason P. Lerch, Nathalie G. Bérubé

Paediatrics Publications

ATRX gene mutations have been identified in syndromic and non-syndromic intellectual disabilities in humans. ATRX is known to maintain genomic stability in neuroprogenitor cells, but its function in differentiated neurons and memory processes remains largely unresolved. Here, we show that the deletion of neuronal Atrx in mice leads to distinct hippocampal structural defects, fewer presynaptic vesicles, and an enlarged postsynaptic area at CA1 apical dendrite-axon junctions. We identify male-specific impairments in long-term contextual memory and in synaptic gene expression, linked to altered miR-137 levels. We show that ATRX directly binds to the miR-137 locus and that the enrichment of the …

P38 Mapk Regulates Cavitation And Tight Junction Function In The Mouse Blastocyst., Christine E Bell, Andrew J Watson

Obstetrics & Gynaecology Publications

UNLABELLED: Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8-16 cell stage, but its role has not …

Gonadotropin-Releasing Hormone Plasticity: A Comparative Perspective., T J Stevenson, T P Hahn, S A Macdougall-Shackleton, G F Ball

Brain and Mind Institute Researchers' Publications

Gonadotropin-releasing hormone 1 (GnRH1) is a key regulator of the reproductive neuroendocrine system in vertebrates. Recent developments have suggested that GnRH1 neurons exhibit far greater plasticity at the cellular and molecular levels than previously thought. Furthermore, there is growing evidence that sub-populations of GnRH1 neurons in the preoptic area are highly responsive to specific environmental and hormonal conditions. In this paper we discuss findings that reveal large variation in GnRH1 mRNA and protein expression that are regulated by social cues, photoperiod, and hormonal feedback. We draw upon studies using histochemistry and immediate early genes (e.g., c-FOS/ZENK) to illustrate that specific …

Embryo Collection Induces Transient Activation Of Xbp1 Arm Of The Er Stress Response While Embryo Vitrification Does Not., Tamara Abraham, Christopher L Pin, Andrew J Watson

Obstetrics & Gynaecology Publications

Embryo cryopreservation has become a standard procedure in the practice of assisted reproduction. While routinely performed in IVF labs, the effects of embryo vitrification on the molecular mechanisms governing preimplantation development remain largely unknown. The endoplasmic reticulum stress (ER stress) response is an evolutionary conserved mechanism that cells employ to manage ER stress. ER stress can be defined as an imbalance between protein synthesis and secretion within the ER. The primary focus of this study was to investigate whether standard embryo manipulations, including embryo collection, culture and vitrification, result in activation of the ER stress pathway in vitro and to …

Intrauterine Growth Restriction Increases Fetal Hepatic Gluconeogenic Capacity And Reduces Messenger Ribonucleic Acid Translation Initiation And Nutrient Sensing In Fetal Liver And Skeletal Muscle., Stephanie R Thorn, Timothy Regnault, Laura D Brown, Paul J Rozance, Jane Keng, Michael Roper, Randall B Wilkening, William W Hay, Jacob E Friedman

Paediatrics Publications

Expression of key metabolic genes and proteins involved in mRNA translation, energy sensing, and glucose metabolism in liver and skeletal muscle were investigated in a late-gestation fetal sheep model of placental insufficiency intrauterine growth restriction (PI-IUGR). PI-IUGR fetuses weighed 55% less; had reduced oxygen, glucose, isoleucine, insulin, and IGF-I levels; and had 40% reduction in net branched chain amino acid uptake. In PI-IUGR skeletal muscle, levels of insulin receptor were increased 80%, whereas phosphoinositide-3 kinase (p85) and protein kinase B (AKT2) were reduced by 40%. Expression of eukaryotic initiation factor-4e was reduced 45% in liver, suggesting a unique mechanism limiting …

Mitogen-Activated Protein Kinase (Mapk) Pathways Mediate Embryonic Responses To Culture Medium Osmolarity By Regulating Aquaporin 3 And 9 Expression And Localization, As Well As Embryonic Apoptosis., Christine E Bell, Nathalie M K Larivière, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

BACKGROUND: In order to advance the development of culture conditions and increase the potential for supporting normal preimplantation embryo development in vitro, it is critical to define the mechanisms that early embryos utilize to survive in culture. We investigated the mechanisms that embryos employ in response to culture medium osmolarity. We hypothesized that mitogen-activated protein kinase (MAPK) pathways mediate responses to hyperosmotic stress by regulating Aquaporin (AQP) 3 and 9 expression as well as embryonic apoptosis.

METHODS: Real-time reverse transcription and polymerase chain reaction and whole-mount immunofluorescence were used to determine the relative mRNA levels and protein localization patterns of …

Effects Of Early Gestation Gh Administration On Placental And Fetal Development In Sheep., Casey D Wright, Ryan J Orbus, Timothy Regnault, Russell V Anthony

Paediatrics Publications

Ovine GH (oGH) is synthesized in placental tissue during maximal placental growth and development. Our objectives were to localize oGH mRNA in the placenta, and study the impact of exogenous GH on twin pregnancies during the normal window (35-55 days of gestational age; dGA) of placental expression. In situ hybridization localized oGH mRNA in uterine luminal epithelium but not in tissues of fetal origin. While maternal GH and IGF-I concentrations were increased (P

Bovine Oocytes And Early Embryos Express Staufen And Elavl Rna-Binding Proteins., M D Calder, P Madan, A J Watson

Obstetrics & Gynaecology Publications

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to …

Chronic Late-Gestation Hypoglycemia Upregulates Hepatic Pepck Associated With Increased Pgc1alpha Mrna And Phosphorylated Creb In Fetal Sheep., Paul J Rozance, Sean W Limesand, James S Barry, Laura D Brown, Stephanie R Thorn, Dan Loturco, Timothy Regnault, Jacob E Friedman, William W Hay

Paediatrics Publications

Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA …

The Expression Of Ovine Placental Lactogen, Star And Progesterone-Associated Steroidogenic Enzymes In Placentae Of Overnourished Growing Adolescent Ewes., Richard G Lea, Peter Wooding, Ian Stewart, Lisa T Hannah, Stephen Morton, Karen Wallace, Raymond P Aitken, John S Milne, Timothy Regnault, Russell V Anthony, Jacqueline M Wallace

Paediatrics Publications

Overnourishing pregnant adolescent sheep promotes maternal growth but reduces placental mass, lamb birth weight and circulating progesterone. This study aimed to determine whether altered progesterone reflected transcript abundance for StAR (cholesterol transporter) and the steroidogenic enzymes (Cyp11A1, Hsd3b and Cyp17). Circulating and placental expression of ovine placental lactogen (oPL) was also investigated. Adolescent ewes with singleton pregnancies were fed high (H) or moderate (M) nutrient intake diets to restrict or support placental growth. Experiment 1: peripheral progesterone and oPL concentrations were measured in H (n=7) and M (n=6) animals across gestation (days 7-140). Experiment 2: progesterone was measured to mid- …

Effect Of Serum And Cumulus Cell Expansion On Marker Gene Transcripts In Bovine Cumulus-Oocyte Complexes During Maturation In Vitro., Michele D Calder, Anita N Caveney, Marc-Andre Sirard, Andrew J Watson

Obstetrics & Gynaecology Publications

OBJECTIVE: To determine the distribution of transcripts encoding the FSH receptor (FSHr), LH receptor (LHr), connexin 43 (Cx43), cyclooxygenase-2 (COX-2), and prostaglandin E(2) receptors 2 and 3 (EP2 and EP3) within bovine cumulus-oocyte complexes (COCs) and denuded oocytes and investigate the influence of gonadotropins, serum, and cumulus cell expansion on the abundance of transcripts encoding these genes.

DESIGN: Prospective controlled animal study.

SETTING: University research laboratory.

PATIENT(S): Animal models for human studies.

INTERVENTION(S): Cumulus-oocyte complexes were treated in culture with serum and gonadotropin-supplemented media to examine the effects to mRNA transcript levels.

MAIN OUTCOME MEASURE(S): Variation in mRNA transcript levels. …

Responsiveness Of Bovine Cumulus-Oocyte-Complexes (Coc) To Porcine And Recombinant Human Fsh, And The Effect Of Coc Quality On Gonadotropin Receptor And Cx43 Marker Gene Mrnas During Maturation In Vitro., Michele D Calder, Anita N Caveney, Lawrence C Smith, Andrew J Watson

Obstetrics & Gynaecology Publications

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine …

The Gamma-Subunit Of The Na-K-Atpase As A Potential Regulator Of Apical And Basolateral Na+-Pump Isozymes During Development Of Bovine Pre-Attachment Embryos., L C Barcroft, S E Gill, A J Watson

Obstetrics & Gynaecology Publications

Expression and activity of the Na-K-ATPase within the basolateral membrane domains of the trophectoderm epithelium provide the driving force for accumulation of Na(+) and Cl(-) across the nascent epithelium, mediating fluid movement into the forming blastocoel. Within the trophectoderm of the bovine blastocyst, multiple isozymes of the Na-K-ATPase are expressed. Immunolocalization has demonstrated that the alpha1-isozyme localizes within the basolateral membrane, whereas the alpha 3-isozyme localizes to the apical cell margins. Gene-specific RT-PCR and wholemount indirect immunofluorescence confocal laser scanning microscopy were used to examine expression of the Na-K-ATPase gamma-subunit (a regulatory subunit of the Na-K-ATPase) throughout development of bovine …

Sensitivity Of Bovine Blastocyst Gene Expression Patterns To Culture Environments Assessed By Differential Display Rt-Pcr., D R Natale, P A De Sousa, M E Westhusin, A J Watson

Obstetrics & Gynaecology Publications

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine …

Cyclooxygenase-2 And Prostaglandin E(2)(Pge(2)) Receptor Messenger Rnas Are Affected By Bovine Oocyte Maturation Time And Cumulus-Oocyte Complex Quality, And Pge(2) Induces Moderate Expansion Of The Bovine Cumulus In Vitro., M D Calder, A N Caveney, M E Westhusin, A J Watson

Obstetrics & Gynaecology Publications

Expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) receptor 2 (EP2) are necessary for rodent cumulus expansion in vivo. Prostaglandin E(2) receptor 3 (EP3) has been detected in bovine preovulatory follicles and corpora lutea. The current experiments examined the effect of PGE(2) on bovine cumulus expansion in vitro and expression of COX-2, EP1, EP2, EP3, and EP4 mRNAs in bovine cumulus-oocyte complexes (COCs) at 0, 6, 12, 18, and 24 h time points during maturation in vitro. Concentrations of PGE(2) above 50 ng/ml resulted in moderate cumulus expansion of bovine COCs, but expansion did not occur in the absence of …

Expression Of Pthrp And Pthr (Pth/Pthrp-R) Mrnas And Polypeptides In Bovine Ovary And Stimulation Of Bovine Blastocyst Development In Vitro Following Pthrp Treatment During Oocyte Maturation., P H Watson, M E Westhusin, A J Watson

Obstetrics & Gynaecology Publications

Parathyroid hormone related protein (PTHrP) and its receptor have well-established roles in the development and regulation of many tissues, including bone and mammary gland. The objectives of this study were: (1) to characterize the distribution of mRNAs encoding parathyroid hormone (PTH)-related protein (PTHrP) and receptor (PTHR) in bovine ovary; (2) to characterize the distribution of PTHrP and PTHR polypeptides in bovine ovary; (3) to examine the influences of PTHrP (1-141) treatment during bovine oocyte maturation in vitro on blastocyst development. mRNAs encoding PTHrP and PTHR were detected by in situ hybridization methods in oocytes, and granulosa cells in all follicles …

Mrnas Encoding Aquaporins Are Present During Murine Preimplantation Development., H Offenberg, L C Barcroft, A Caveney, D Viuff, P D Thomsen, A J Watson

Obstetrics & Gynaecology Publications

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) …

Differential Involvement Of Na(+),K(+)-Atpase Isozymes In Preimplantation Development Of The Mouse., D J Macphee, D H Jones, K J Barr, D H Betts, A J Watson, G M Kidder

Obstetrics & Gynaecology Publications

Na(+),K(+)-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the alpha1 and beta1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional alpha subunit isoform (alpha3) and the remaining two beta subunit isoforms are also present in preimplantation embryos. Whereas alpha3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for beta2 and beta3 mRNAs. Immunoblot analyses confirmed that these subunits are …

Impact Of Bovine Oocyte Maturation Media On Oocyte Transcript Levels, Blastocyst Development, Cell Number, And Apoptosis., A J Watson, P De Sousa, A Caveney, L C Barcroft, D Natale, J Urquhart, M E Westhusin

Obstetrics & Gynaecology Publications

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte …

Effects Of Superovulated Heifer Diet Type And Quantity On Relative Mrna Abundances And Pyruvate Metabolism In Recovered Embryos., C Wrenzycki, P De Sousa, E W Overström, R T Duby, D Herrmann, A J Watson, H Niemann, D O'Callaghan, M P Boland

Obstetrics & Gynaecology Publications

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, …

Trophectoderm Differentiation In The Bovine Embryo: Characterization Of A Polarized Epithelium., L C Barcroft, A Hay-Schmidt, A Caveney, E Gilfoyle, E W Overstrom, P Hyttel, A J Watson

Obstetrics & Gynaecology Publications

Blastocytst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na-K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, beta-catenin, and the tight junction associated protein, zonula occludens protein 1, in pre-attachment bovine embryos, in vitro. Immunocytochemistry and gene specific reverse transcription--polymerase chain reaction methods were used. Transcripts for E-cadherin and …

Transient Expression Of A Translation Initiation Factor Is Conservatively Associated With Embryonic Gene Activation In Murine And Bovine Embryos., P A De Sousa, A J Watson, R M Schultz

Obstetrics & Gynaecology Publications

In the present study the abundance of mRNAs for eukaryotic translation initiation factors eIF-1A (formerly known as eIF-4C), -2alpha, -4A, -4E, and -5 was examined in in vivo-derived mouse embryos throughout preimplantation development using a semiquantitative reverse transcription-polymerase chain reaction assay. Although the mRNA profile for each gene is unique, only mRNA for eIF-1A transiently increases during embryonic gene activation (EGA) at the 2-cell stage, and this was confirmed by an independent hybridization-based assay. In in vitro-developed bovine embryos, mRNA for eIF-1A was transiently detected at the 8-cell stage, when the major activation of the genome occurs in this species. …

Bovine Oviductal And Embryonic Insulin-Like Growth Factor Binding Proteins: Possible Regulators Of "Embryotrophic" Insulin-Like Growth Factor Circuits., Q A Winger, P De Los Rios, V K Han, D T Armstrong, D J Hill, A J Watson

Obstetrics & Gynaecology Publications

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs …

A Growth Factor Phenotype Map For Ovine Preimplantation Development., A J Watson, P H Watson, M Arcellana-Panlilio, D Warnes, S K Walker, G A Schultz, D T Armstrong, R F Seamark

Obstetrics & Gynaecology Publications

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst …

Possible Roles Of Insulin And Insulin-Like Growth Factors In Rat Preimplantation Development: Investigation Of Gene Expression By Reverse Transcription-Polymerase Chain Reaction., X Zhang, G M Kidder, A J Watson, G A Schultz, D T Armstrong

Obstetrics & Gynaecology Publications

The sensitive mRNA phenotyping technique of reverse transcription-polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine …