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Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

ABSTRACT: The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross-bred gilts (n?=?38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute-1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n?=?8); (b) heterogeneous spherical and ovoid (n?=?8); (c) uniform ovoid (n?=?8); (d) heterogeneous ovoid and tubular (n?=?8); and (e) uniform tubular (n?=?6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography (UPLC)-MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC-MS and UPLC-MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC-MS and UPLC-MS data, 38 and 59 metabolites, respectively, differed (p?

SUBMITTER: Walsh SC

PROVIDER: S-EPMC7003770 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

Walsh Sophie C SC Miles Jeremy R JR Yao Linxing L Broeckling Corey D CD Rempel Lea A LA Wright-Johnson Elane C EC Pannier Angela K AK

Molecular reproduction and development 20191216 1

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross-bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute-1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morpholog ...[more]

PMID: 31840336

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Project description:BACKGROUND: We hypothesized that genes that are up-regulated in the uterine endometrium at the initiation of conceptus elongation in cattle, and that encode for secreted proteins, contribute to the composition of the uterine luminal fluid (ULF) and ultimately, drive conceptus elongation. The aims of this study were to: 1) screen endometrial transcriptomic data for genes that encode secreted proteins on Day 13; 2) determine temporal changes in the expression of these genes during the estrous cycle/early pregnancy; 3) determine if expression of these genes is affected by altered concentrations of progesterone (P4) in vivo and 4) determine if the protein products of these genes are detectable in ULF. RESULTS: Of the fourteen candidate genes examined, quantitative real-time PCR analysis revealed the expression of APOA1, ARSA, DCN, LCAT, MUC13, NCDN, NMN, NPNT, NXPH3, PENK, PLIN2 and TINAGL1 was modulated in the endometrium (P<0.05) as the estrous cycle/early pregnancy progressed. APOA1, DCN and NPNT expression was higher in cyclic compared to pregnant heifers, and pregnancy increased (P<0.05) the expression of LCAT, NCDN, NMN, PLIN2 and TINAGL1. The magnitude of the increase in expression of APOA1, PENK and TINAGL1 on Day 13 was reduced (P<0.05) in heifers with low P4. Furthermore, low P4 decreased (P<0.05) the expression of LCAT and NPNT on Day 7, while an early increase (P<0.05) in the expression of NXPH3 and PLIN2 was observed in heifers with high P4. The protein products of 5 of the candidate genes (APOA1, ARSA, LCAT, NCDN and PLIN) were detected in the ULF on either Days 13, 16 or 19 of pregnancy. CONCLUSION: Using a candidate gene approach, we determined that both P4 concentration and the presence of the conceptus alter endometrial expression of PLIN2, TINAGL1, NPNT, LCAT, NMN and APOA1. Comparison of the expression profiles of these genes to proteins detected in ULF during conceptus elongation (i.e., Days 13 through 19) revealed the presence of APOA1, ARSA, LCAT, NCDN as well as members of the PLIN family of proteins that may play roles in driving conceptus elongation in cattle.

Project description:Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern pre-implantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil (CO) vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many of the differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of pre-implantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and pre-implantation blastocyst growth and development in sheep. Ewes were mated at estrus to intact Suffolk rams and then assigned randomly to receive daily intramuscular (i.m.) injections from days 1.5 to 9 of either corn oil vehicle (CO) or 25 mg progesterone (P4) (Sigma Chemical Co., St. Louis, MO) in CO vehicle, and hysterectomized on either day 9 or day 12 of pregnancy (n=5 per day and treatment). Microarray hybridization was conducted with a bovine whole genome spotted oligonucleotide array. This 24,000-element array was developed by the Bovine Oligo Microarray Consortium at the University of Missouri in collaboration with Texas A&M University and Iowa State University. The array includes 16,846 probes designed from expressed sequence tags (ESTs) that were aligned to homologous vertebrate proteins and to the 6X bovine genome assembly. The probe set was supplemented with oligos designed from 703 predicted RefSeq genes, and 5943 reproductive tissue ESTs. A link to probe sequences and annotations is available at http://www.bovineoligo.org. The annotation included assigning a human gene symbol based on homology using BLAST. The oligos were synthesized by Illumina and spotted onto aminosilane-coated glass slides by Dr. David Galbraith's laboratory at the University of Arizona (http://www.ag.arizona.edu/microarray/BOM.html). Hybridizations were performed with the Genisphere Array 350 kit (Genisphere Inc., Hatfield, PA) using dual channel design with dye-swapping. Preparation of cDNA was performed according to the manufacturer's instructions. Briefly, endometrial total RNA (9 Âµg) from each ewe (n=5 per day and treatment) was reverse-transcribed with Superscript II (Invitrogen, Carlsbad, CA) with the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labeling reagents. Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was co-hybridized with a cDNA sample from the opposite treatment for direct comparison of differences between treatments.

Dataset Information

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

Publications

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

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