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Temporal regulation of mRNAs for select bone morphogenetic proteins (BMP), BMP receptors and their associated SMAD proteins during bovine early embryonic development: effects of exogenous BMP2 on embryo developmental progression
© Lee et al.; licensee BioMed Central Ltd. 2014
Received: 25 February 2014
Accepted: 7 July 2014
Published: 15 July 2014
We previously demonstrated embryotrophic actions of maternal (oocyte-derived) follistatin during bovine early embryogenesis. Classical actions of follistatin are attributed to inhibition of activity of growth factors including activins and bone morphogenetic proteins (BMP). However, temporal changes in BMP mRNA in early bovine embryos and the effects of exogenous BMP on embryo developmental progression are not understood. The objectives of present studies were to characterize mRNA abundance for select BMP, BMP receptors and BMP receptor associated SMADs during bovine oocyte maturation and early embryogenesis and determine effects of addition of exogenous BMP protein on early development.
Relative abundance of mRNA for BMP2, BMP3, BMP7, BMP10, SMAD1, SMAD5, ALK3, ALK6, ALK2, BMPR2, ACVR2A and ACVR2B was determined by RT-qPCR analysis of germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes and in vitro produced embryos collected at pronuclear, 2-cell (C), 4C, 8C, 16C, morula and blastocyst stages. Effects of addition of recombinant human BMP2 (0, 1, 10 and 100 ng/ml) during initial 72 h of embryo culture on early cleavage (within 30 h post insemination), total cleavage, development to 8C-16C and blastocyst stages and blastocyst mRNA abundance for markers of inner cell mass (NANOG) and trophectoderm (CDX2) were also determined.
Abundance of mRNA for BMP2, BMP10, SMAD1, SMAD5, ALK3, ALK2, BMPR2 and ACVR2B was elevated in MII oocytes and/or pronuclear stage embryos (relative to GV) and remained elevated through the 8C -16C stages, whereas BMP3, BMP7 and ALK2 mRNAs were transiently elevated. Culture of embryos to the 8C stage in the presence of α-amanitin resulted in increased abundance for all of above transcripts examined relative to untreated 8C embryos. Effects of addition of exogenous BMP2 on early cleavage rates and rates of development to 8C-16C and blastocyst stages were not observed, but BMP2 treatment increased blastocyst mRNA for CDX2 and NANOG.
Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis.
Oocyte developmental competence was defined by Sirard et al.  as the capacity of the oocyte to resume meiosis, cleave after fertilization, help promote embryonic development and implantation, and bring a pregnancy to term in good health . Our previous studies support a positive functional role for maternal (oocyte-derived) follistatin in bovine oocyte competence. Follistatin mRNA is positively associated with developmental competence in two distinct bovine models of egg quality [2, 3]. Furthermore, follistatin supplementation during the first 72 h of bovine embryo culture (until embryonic genome activation) enhanced proportion of embryos that cleaved early and proportion of embryos developing to the blastocyst stage in a dose dependent fashion . Follistatin treatment also increased total blastocyst cell numbers specifically through an increase in trophectoderm (TE) cells, with no effect on numbers of inner cell mass (ICM) cells and increased blastocyst mRNA for the TE specific transcription factor CDX2 . We also observed similar effects of follistatin treatment on early cleavage and rates of development to blastocyst stage for rhesus monkey embryos , demonstrating potential translational relevance of results from the bovine model system. However, the mechanisms responsible for stimulatory effects of follistatin on multiple indices of bovine early embryonic development to date still remain elusive.
Follistatin was initially classified as a high affinity binding protein inhibiting activin action . However, stimulatory effects of exogenous activin treatment on early cleavage and blastocyst rates for bovine embryos were also observed suggesting a potential alternative mechanism of follistatin action . Follistatin can also bind and regulate activity of multiple additional TGFβ superfamily members including BMPs [6–8]. A prominent role for BMPs in regulation of patterning of early embryos has been described . Furthermore, BMPs have been implicated in regulation of trophoblast differentiation in human embryonic stem cells . However, less is known about their role during preimplantation embryonic development, particularly in farm species.
In this study, we investigated temporal changes in mRNA abundance for multiple BMP, BMP receptors and their associated SMADs in bovine oocytes and embryos and the source of such transcripts (maternal versus embryonic) in bovine embryos coincident with embryonic genome activation. We also determined the effect of exogenous BMP2 on multiple endpoints relevant to bovine early embryonic developmental progression. Results demonstrate dynamic regulation of maternal mRNAs for BMP system components during bovine early embryonic development and effects of exogenous BMP2 treatment on indices of cell lineage determination in bovine blastocysts. Results also suggest embryotropic actions of follistatin on bovine early embryogenesis are likely not mediated by antagonism of BMP2 signaling.
Oocyte collection, in vitro maturation, in vitro fertilization and embryo culture
Bovine oocytes used for all described experiments were obtained from ovaries harvested at a local abattoir. Aspiration and in vitro maturation of oocytes, in vitro fertilization and culture of embryos was performed as previously described . Cumulus-oocyte complexes (aspirated from 2–7 mm visible follicles) with > 4 compact cumulus cell layers and homogeneous cytoplasm were matured in TCM 199 [supplemented with 0.2 mM sodium pyruvate, 5 mg/ml gentamicin sulfate, 6.5 mM L-glutamine, 156 nM bovine LH (Sioux Biochemical, Sioux Center, Iowa), 15.6 nM bovine FSH (Sioux Biochemical), 3.67 nM 17β-estradiol and 10% v/v defined FBS (Hyclone, Logan, UT)] for 24 h at 38.5°C, 5% CO2 in air with maximum humidity. For in vitro fertilization, matured oocytes were washed and co-incubated with sperm for 20 h in fertilization medium (114 mM NaCl, 25 mM NaHCO3, 3.2 mM KCl, 0.34 mM NaH2PO4, 0.183 mM penicillin-G, 16.6 mM sodium lactate, 0.5 mM MgCl2·6H2O, 2.7 mM CaCl2·2H2O, 0.2 mM sodium pyruvate, 6 mg/ml BSA and 1.5 U of heparin) at 38.5°C, 5% CO2 in air with maximum humidity. After cumulus cell removal, presumptive zygotes were washed and cultured in potassium simplex optimization medium (KSOM; EMD Millipore, Billerica MA) supplemented with 0.3% BSA for 72 h. Embryos were then washed and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until day 7.
Temporal changes in BMP system mRNA abundance in bovine oocytes and early embryos
Sequence of primers for real time RT-PCR for TGFβ superfamily members and receptors, CDX2 and NANOG
Genbank accession number
F: 5′- CCACAAACCCGCCATATCTC -3′
R: 5′- TAGCACCCTCTAACACCTCTG -3′
Effect of transcriptional inhibition on BMP system mRNA abundance
To determine the effects of inhibition of transcription on mRNA abundance in in vitro derived embryos, presumptive zygotes were cultured in serum free KSOM with 0.3% BSA with or without the addition of 50 μg/ml α-amanitin (Sigma, St. Louis, MO). The 8C embryos were then collected at 52 h post insemination (n = 4 pools of 10 embryos per group) and placed in lysis buffer and snap frozen and stored as above until RNA isolation. Half the RNA was subjected to reverse transcription using oligo(dT) primers as described above for use in quantification of polyadenylated transcripts for genes of interest. The remaining RNA was transcribed using random hexamers for quantification of adenylated and deadenylated (total) transcripts for genes of interest. The cDNA produced was subjected to qPCR for the BMP system components as described above. Data were normalized relative to abundance of endogenous control (RPS18).
Effect of BMP2 supplementation on bovine embryo developmental progression
To examine the effect of BMP2 supplementation on early cleavage rate (assessed 30 hpi), rate of development to 8C-16C stage (assessed 72 hpi), blastocyst rate (assessed 7 d post insemination) and the abundance of mRNA for the TE marker (CDX2) and ICM marker (NANOG) in resulting blastocysts, presumptive zygotes were cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10 or 100 ng/ml BMP 2 (30 presumptive zygotes per group, 4 replicates). The 8C-16C stage embryos were then separated 72 h post fertilization and cultured in fresh KSOM medium (minus exogenous BMP2) supplemented with 0.3% BSA and 10% FBS until d 7. Blastocysts were harvested at d 7 post fertilization (n = 4 pools of 5 blastocysts each per treatment) and lysed and frozen as above until RNA isolation and RT-qPCR analysis as described above.
All data were analyzed using one way ANOVA in SAS followed by Fishers Protected Least Significant Difference Test to determine differences between means. For embryo culture experiments, % data were arc-sin transformed prior to analysis. Data are presented as mean ± SEM.
Results and discussion
Temporal regulation of BMP mRNA abundance during oocyte maturation and early embryogenesis
Effect of embryo culture in the presence of the transcriptional inhibitor α-amanitin on BMP, BMP receptor and SMAD1 and SMAD5 mRNA abundance
Effects of α-Amanitin treatment on mRNA abundance in bovine 8C embryos
RT with oligo dT
RT with random hexamers
1.15 ± 0.27a
9.08 ± 1.19b
1.03 ± 0.16
1.77 ± 0.17
1.17 ± 0.35a
7.33 ± 1.27b
1.02 ± 0.12
1.45 ± 0.17
1.10 ± 0.27a
3.19 ± 0.76b
1.25 ± 0.49
1.30 ± 0.21
1.02 ± 0.11a
2.85 ± 0.60b
1.15 ± 0.36
3.26 ± 0.88
1.02 ± 0.12a
3.36 ± 0.66b
1.17 ± 0.40
2.96 ± 1.31
1.04 ± 0.19a
3.82 ± 0.25b
1.07 ± 0.23
1.59 ± 0.31
1.23 ± 0.37a
9.41 ± 0.72b
1.06 ± 0.22
1.42 ± 0.31
1.12 ± 0.25a
5.43 ± 0.71b
1.05 ± 0.20
1.38 ± 0.16
1.27 ± 0.50a
5.73 ± 1.34b
1.02 ± 0.13
0.86 ± 0.09
Effect of BMP 2 supplementation on embryo development and trophectoderm and inner cell mass marker mRNA abundance in resulting blastocysts
A growing body of evidence suggests that oocyte secreted factors can enhance oocyte developmental competence and embryo developmental progression [14, 15, 23]. Our previous studies support a role for endogenous, oocyte-derived follistatin in promoting bovine embryo developmental progression and cell allocation to the TE lineage . Results were obtained with follistatin treatment during initial stages of embryo culture (d 1–3) up to embryonic genome activation. To our knowledge, effects of exogenous BMP2 treatment during culture of bovine embryos have not been previously reported. Addition of BMP2 or BMP4 during in vitro maturation did not impact rates of meiotic maturation and cumulus expansion or rates of embryonic development to blastocyst stage following in vitro fertilization . However, evidence supports a role for oocyte-derived GDF9 and BMP15 in promoting oocyte developmental competence as addition of these growth factors exogenously during in vitro maturation can enhance rates of development to the blastocyst stage and blastocyst cell allocation to TE [14, 15]. Thus, levels of endogenous BMP2 available during in vitro maturation and embryo culture may not be limiting to embryo developmental capacity in vitro as reflected by rates of development to the blastocyst stage. However, the current studies do demonstrate that bovine embryos can respond to BMP2 stimulation during initial 3 d of culture in vitro with an increase in blastocyst mRNA for NANOG and CDX2 measured 4 d later and demonstrate effects of BMP2 treatment on indices of cell allocation mediated well after treatment administration. The mechanisms responsible for increased blastocyst NANOG and CDX2 mRNA in response to BMP2 stimulation are not known. However, BMP4 mediated induction of CDX2 mRNA expression  and promoter activity  have been described in other cell lines and in human ES cells and signaling for both BMP2 and BMP4 is mediated via SMAD1/5 . However, SMAD2/3 pathways are linked to NANOG promoter regulation in embryonic stem cells and antagonistic to BMP signaling and differentiation to TE fate . While the addition of BMP2 had no effect on the proportion of embryos that cleaved early or developed to the 16C or blastocyst stages, the highest dose of BMP2 did increase both NANOG and CDX2 mRNA compared to control embryos at the blastocysts stage. NANOG is a marker of the ICM of the blastocysts and is important for maintaining pluripotency of the cells in the ICM . In contrast CDX2 is a marker for TE cells and is in fact required for the establishment of the TE in the blastocysts of mice and cattle [28, 29]. NANOG and CDX2 also cross regulate each other to promote proper blastocyst formation . As BMP2 supplementation increases mRNA for both markers it is possible that BMP2 enhances the differentiation of the two cell types, rather than promoting one cell type over the other.
In summary, results of present studies demonstrate pronounced temporal regulation of mRNA for select BMP ligands, type I and II receptors and cognate intracellular signaling molecules during bovine early embryonic development. This study was limited in that it only examined changes in abundance rather than accompanying changes in protein abundance. However, to our knowledge, temporal changes in abundance of mRNA for specific BMP including BMP2, BMP3, BMP7 and BMP10 at specific stages of bovine early embryonic development have not been examined previously. While stimulatory effects of BMP2 on early cleavage and development to 8C to 16C and blastocyst stages were not noted in response to BMP2 supplementation of culture media (at doses tested) during first 72 h of development, increased mRNA for CDX2 and NANOG was detected in resulting blastocysts, demonstrating a functional BMP2 signaling system in early bovine embryos. Furthermore, distinct results observed in present studies suggest that embryotrophic actions of follistatin reported in previous studies  likely are not linked to inhibition of endogenous BMP2 activity.
This project was supported by the National Institute of Child Health and Human Development of the National Institutes of Health under award number R01HD072972.
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