Dataset Information

Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

ABSTRACT: Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and their interaction. The expression was higher in nonpregnant than in pregnant heifers at Day 18. The conceptus transcripts abundance of FGFR2 and FGFR3 were significantly lower at Day 15 than 18. In the endometrium, FGF1 protein abundance significantly decreased from Day 12 onwards and FGF2 protein abundance showed a minor, but a significant increase at Day 15 in comparison to Days 12 and 18. We concluded that the decrease in FGF1 mRNA expression in pregnant heifers at Day 18 points towards a potential contribution of FGF1 in the preimplantation process. Additionally, successful embryo elongation might require a spatiotemporal FGF2 protein increase in the endometrium.

SUBMITTER: Chiumia D

PROVIDER: S-EPMC7084457 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

Chiumia Daniel D Schulke Katy K Groebner Anna E AE Waldschmitt Nadine N Reichenbach Horst-Dieter HD Zakhartchenko Valeri V Bauersachs Stefan S Ulbrich Susanne E SE

International journal of molecular sciences 20200226 5

Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and thei ...[more]

PMID: 32111034

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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.

Project description:The aim was to examine the effect of sire fertility status on conceptus-induced changes in the bovine endometrial transcriptome. To generate elongated conceptuses, Day 7 blastocysts produced in vitro using frozen-thawed sperm from Holstein Friesian bulls (3 High fertility, HF and 3 Low fertility, LF) were transferred in groups of 5-10 into synchronized heifers (n = 7 heifers per bull) and recovered following slaughter on Day 15. Day 15 endometrial explants recovered from the uterine horn ipsilateral to the corpus luteum were recovered from synchronized cyclic heifers (n = 4). Explants from each heifer were co-cultured for 6 h in RPMI medium alone (Control) or with 100 ng/ml ovine recombinant interferon tau (IFNT) or with a single conceptus from each HF or LF bull. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq and the resulting data were analyzed with R software. The numbers of differentially expressed genes (DEG; FDR<0.05) were: HF vs. Control: 956; LF vs. Control: 1021; IFNT vs. Control: 1301; HF vs. LF: 2. Unsurprisingly, the majority of DEG (658) were common to all comparisons and were related to IFNT-induced changes in the endometrium. Prior to applying the adjusted p-value, there were 700 DEG between HF and LF, with 191 and 509 genes more expressed in HF or LF, respectively (p < 0.05). Overrepresentation analysis of KEGG pathways (FDR<0.05), revealed that DEG with higher expression in LF were involved in cell cycle and proteolysis, while those upregulated DEG by HF conceptuses were strongly associated with immune process pathways, such as TNF, NF-kappa B, cytokine-cytokine receptor interaction, and TLR signaling. These pathways were also enriched by DEG upregulated by IFNT compared to the Control. Furthermore, only the HF, and not the LF group, affected the expression of most genes in these pathways (p < 0.05) according to a negative binomial regression model. Finally, a weighted gene co-expression network analysis revealed two clusters of co-expressed genes associated with the HF conceptuses (p < 0.05), which were also enriched for the aforementioned pathways. In conclusion, HF conceptuses, similar to IFNT treatment, stimulated multiple pathways involved in immune response, which were apparently not affected by LF conceptuses.

Dataset Information

Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

Publications

Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

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