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 …

Stress-Inducible Phosphoprotein 1 Has Unique Cochaperone Activity During Development And Regulates Cellular Response To Ischemia Via The Prion Protein., Flavio H Beraldo, Iaci N Soares, Daniela F Goncalves, Jue Fan, Anu A Thomas, Tiago G Santos, Amro H Mohammad, Martin Roffé, Michele D Calder, Simona Nikolova, Glaucia N Hajj, Andre L Guimaraes, Andre R Massensini, Ian Welch, Dean H Betts, Robert Gros, Maria Drangova, Andrew J Watson, Robert Bartha, Vania F Prado, Vilma R Martins, Marco A M Prado

Obstetrics & Gynaecology Publications

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the …

Endogenous Folate Accumulation In Oocytes And Preimplantation Embryos And Its Epigenetic Implications., Mellissa R W Mann, Andrew J Watson

Obstetrics & Gynaecology Publications

No abstract provided.

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 …

Ouabain Stimulates A Na+/K+-Atpase-Mediated Sfk-Activated Signalling Pathway That Regulates Tight Junction Function In The Mouse Blastocyst., Holly Giannatselis, Michele Calder, Andrew J Watson

Obstetrics & Gynaecology Publications

The Na(+)/K(+)-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na(+)/K(+)-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and …

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 …

Snai1 And Snai2 Are Asymmetrically Expressed At The 2-Cell Stage And Become Segregated To The Te In The Mouse Blastocyst., Christine E Bell, Andrew J Watson

Obstetrics & Gynaecology Publications

SNAI1 and SNAI2 are transcription factors that initiate Epithelial-to-Mesenchymal cell transitions throughout development and in cancer metastasis. Here we show novel expression of SNAI1 and SNAI2 throughout mouse preimplantation development revealing asymmetrical localization of both SNAI1 and SNAI2 in individual blastomeres beginning at the 2-cell stage through to the 8-cell stage where SNAI1 and SNAI2 are then only detected in outer cells and not inner cells of the blastocyst. This study implicates SNAI1 and SNAI2 in the lineage segregation of the trophectoderm and inner cell mass, and provides new insight into these oncogenes.

Genomic Rna Profiling And The Programme Controlling Preimplantation Mammalian Development., Christine E Bell, Michele D Calder, Andrew J Watson

Obstetrics & Gynaecology Publications

Preimplantation development shifts from a maternal to embryonic programme rapidly after fertilization. Although the majority of oogenetic products are lost during the maternal to embryonic transition (MET), several do survive this interval to contribute directly to supporting preimplantation development. Embryonic genome activation (EGA) is characterized by the transient expression of several genes that are necessary for MET, and while EGA represents the first major wave of gene expression, a second mid-preimplantation wave of transcription that supports development to the blastocyst stage has been discovered. The application of genomic approaches has greatly assisted in the discovery of stage specific gene expression …

Preimplantation Embryo Programming: Transcription, Epigenetics, And Culture Environment., Veronique Duranthon, Andrew J Watson, Patrick Lonergan

Obstetrics & Gynaecology Publications

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed …

Pp2cdelta (Ppm1d, Wip1), An Endogenous Inhibitor Of P38 Mapk, Is Regulated Along With Trp53 And Cdkn2a Following P38 Mapk Inhibition During Mouse Preimplantation Development., Jenny A Hickson, Barry Fong, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

Preimplantation embryos utilize mitogen-activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cdelta (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cdelta proteins are expressed throughout mouse preimplantation development. Treatment …

Na/K-Atpase Beta1 Subunit Expression Is Required For Blastocyst Formation And Normal Assembly Of Trophectoderm Tight Junction-Associated Proteins., Pavneesh Madan, Keeley Rose, Andrew J Watson

Obstetrics & Gynaecology Publications

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and …

Mouse Preimplantation Embryo Responses To Culture Medium Osmolarity Include Increased Expression Of Ccm2 And P38 Mapk Activation., Barry Fong, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

BACKGROUND: Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK) occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM) was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2). The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation …

Na+/K+ -Atpase Regulates Tight Junction Formation And Function During Mouse Preimplantation Development., Michelle I Violette, Pavneesh Madan, Andrew J Watson

Obstetrics & Gynaecology Publications

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment …

P38 Mitogen-Activated Protein Kinase (Mapk) First Regulates Filamentous Actin At The 8-16-Cell Stage During Preimplantation Development., Andrew J M Paliga, David R Natale, Andrew J Watson

Obstetrics & Gynaecology Publications

BACKGROUND INFORMATION: The MAPK (mitogen-activated protein kinase) superfamily of proteins consists of four separate signalling cascades: the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPK); the ERKs (extracellular-signal-regulated kinases); the ERK5 or big MAPK1; and the p38 MAPK group of protein kinases, all of which are highly conserved. To date, our studies have focused on defining the role of the p38 MAPK pathway during preimplantation development. p38 MAPK regulates actin filament formation through the downstream kinases MAPKAPK2/3 (MAPK-activated protein kinase 2/3) or MAPKAPK5 [PRAK (p38 regulated/activated kinase)] and subsequently through HSP25/27 (heat-shock protein 25/27). We recently reported that 2-cell-stage murine …

Mitogen-Activated Protein Kinase (Mapk) Blockade Of Bovine Preimplantation Embryogenesis Requires Inhibition Of Both P38 And Extracellular Signal-Regulated Kinase (Erk) Pathways., Pavneesh Madan, Michele D Calder, Andrew J Watson

Obstetrics & Gynaecology Publications

Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the …

Rgs14 Is A Mitotic Spindle Protein Essential From The First Division Of The Mammalian Zygote., Luke Martin-Mccaffrey, Francis S Willard, Antonio J Oliveira-Dos-Santos, David R C Natale, Bryan E Snow, Randall J Kimple, Agnieszka Pajak, Andrew J Watson, Lina Dagnino, Josef M Penninger, David P Siderovski, Sudhir J A D'Souza

Obstetrics & Gynaecology Publications

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle …

Deletion Of The Na/K-Atpase Alpha1-Subunit Gene (Atp1a1) Does Not Prevent Cavitation Of The Preimplantation Mouse Embryo., L C Barcroft, A E Moseley, J B Lingrel, A J Watson

Obstetrics & Gynaecology Publications

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the …

Aquaporin Proteins In Murine Trophectoderm Mediate Transepithelial Water Movements During Cavitation., Lisa C Barcroft, Hanne Offenberg, Preben Thomsen, Andrew J Watson

Obstetrics & Gynaecology Publications

Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms. We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development. AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages. In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells. In contrast, AQP 9 was predominantly observed within the apical membrane domains of the …

Rac-1 And Iqgap Are Potential Regulators Of E-Cadherin-Catenin Interactions During Murine Preimplantation Development., David R Natale, Andrew J Watson

Obstetrics & Gynaecology Publications

Adherens junction formation is fundamental for compaction and trophectoderm differentiation during mammalian preimplantation development. We recently isolated an IQGAP-2 cDNA from a differential display-polymerase chain reaction screen of bovine preimplantation developmental stages. IQGAP-1 and -2 proteins mediate E-cadherin-based cell-to-cell adhesion through interactions with beta-catenin and the Rho GTPases, rac1 and cdc42. Our study demonstrates IQGAP-1,-2, rac-1 and cdc42 mRNAs are present throughout murine preimplantation development. IQGAP-1 and rac-1 protein distribution changes from predominantly plasma membrane associated to predominantly cytoplasmic as the embryo progresses through cleavage divisions and compaction to the blastocyst stage.

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 …

Regulation Of Blastocyst Formation., A J Watson, L C Barcroft

Obstetrics & Gynaecology Publications

Preimplantation or pre-attachment development encompasses the "free"-living period of mammalian embryogenesis, which directs development of the zygote through to the blastocyst stage. Blastocyst formation is essential for implantation, establishment of pregnancy and is a principal determinant of embryo quality prior to embryo transfer. Cavitation (blastocyst formation) is driven by the expression of specific sets of gene products that direct the acquisition of cell polarity within the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Critical gene families controlling these events include: the E-cadherin-catenin cell adhesion family, …

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 …

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 …

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 …

Na/K-Atpase-Mediated 86rb+ Uptake And Asymmetrical Trophectoderm Localization Of Alpha1 And Alpha3 Na/K-Atpase Isoforms During Bovine Preattachment Development., D H Betts, L C Barcroft, A J Watson

Obstetrics & Gynaecology Publications

This study evaluated Na/K-ATPase alpha 1- and alpha 3-subunit isoform polypeptide expression and localization during bovine preattachment development. Na/K-ATPase cation transport activity from the one-cell to blastocyst stage was also determined by measuring ouabain-sensitive 86Rb+ uptake. Both alpha1- and alpha 3-subunit polypeptides were detected by immunofluorescence to encircle the entire cell margins of each blastomere of inseminated zygotes, cleavage stage embryos, and morulae. Immunofluorescent localization of alpha1-subunit polypeptide in bovine blastocysts revealed an alpha1 immunofluorescence signal confined to the basolateral membrane margins of the trophectoderm and encircling the cell periphery of each inner cell mass (ICM) cell. In contrast, alpha …

Expression Of Genes Encoding Antioxidant Enzymes In Preimplantation Mouse And Cow Embryos And Primary Bovine Oviduct Cultures Employed For Embryo Coculture., M B Harvey, M Y Arcellana-Panlilio, X Zhang, G A Schultz, A J Watson

Obstetrics & Gynaecology Publications

Preimplantation embryos from a variety of mammalian species contrast markedly in their response to culture in vitro. Murine preimplantation embryos display a wider tolerance than other mammalian species to culture environments, and this has contributed to the development of several effective defined culture media. Embryo coculture on somatic cells remains the most effective method of supporting reasonable rates of bovine preimplantation development in vitro. The patterns of gene expression for several antioxidant enzymes during preimplantation murine and bovine development were examined by use of the reverse transcription-polymerase chain reaction technique to determine whether the differential developmental capacity of mammalian preimplantation …

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 …

Preimplantation Development Of In Vitro-Matured And In Vitro-Fertilized Ovine Zygotes: Comparison Between Coculture On Oviduct Epithelial Cell Monolayers And Culture Under Low Oxygen Atmosphere., A J Watson, P H Watson, D Warnes, S K Walker, D T Armstrong, R F Seamark

Obstetrics & Gynaecology Publications

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.