201 SUPPRESSION OF Suz12 IN BOVINE PREIMPLANTATION EMBRYOS VIA CYTOPLASMIC SMALL INTERFERING RNA INJECTION

2011 ◽  
Vol 23 (1) ◽  
pp. 200
Author(s):  
G. L. Williamson ◽  
J. H. Pryor ◽  
K. Tessanne ◽  
M. C. Golding ◽  
C. R. Long
Keyword(s):  
Embryonic Development ◽  
Small Interfering Rna ◽  
Geometric Mean ◽  
Developmental Time ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Embryo Morphology ◽  
Interfering Rna

The proper removal of gametic epigenetic marks and coordinated reestablishment of the epigenome are critical to mammalian embryonic development. Suz12 is a member of Polycomb repressive complex 2, known to catalyse trimethylation of lysine 27 on histone 3 (H3K27me3; Pasini et al. 2004); Suz12 has also been shown to interact with Suv39h1 for proper trimethylation of H3K9 (de la Cruz et al. 2007). Our objective in this study was to suppress expression of Suz12 via cytoplasmic injection of small interfering RNA (siRNA) targeted at this gene, and to evaluate the effect on embryo development rates. Bovine zygotes were produced in vitro via standard laboratory procedures. Nineteen hours post-fertilization, presumptive zygotes (n = 3979) were divided into 3 treatment groups: noninjected control (CTL), or injected with a fluorescent dextran marker combined with either a nontargeting siRNA (NULL) or an Suz12-targeting siRNA (SUZ). Embryos were cultured in Bovine Evolve (Zenith Biotech, Canada) with 4 mg mL–1 of BSA (Probumin, Millipore, Billerica, MA) and collected at the 4-cell, 8-cell, morula, and blastocyst stages. Ribonucleic acid was isolated with an RNeasy® Mini Kit (Qiagen, Valencia, CA) from 3 replicates of pooled embryos at each stage (4-cell, n = 15; 8-cell, n = 20; morula, n = 10), except for the blastocyst stage [2 samples (n = 10) collected from the CTL and NULL groups, and 1 sample (n = 3) from the SUZ group]. Each RNA sample was reverse-transcribed into cDNA and diluted for use by quantitative real-time PCR. Relative gene expression levels from each sample were calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA), adjusted according to individual PCR efficiencies for each primer pair (R2 > 0.95) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA), to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). Our data indicate that Suz12 expression was suppressed to undetectable levels in SUZ-treated zygotes at all embryo stages analysed. The blastocyst rate of the SUZ group was extremely low (0.88 ± 0.16% SEM) compared with the CTL (19.87 ± 0.36% SEM) and NULL (5.09 ± 0.36% SEM) groups. Morphologically, SUZ morulae appeared fragmented with fewer larger cells than expected, whereas the NULL and CTL morulae seemed to develop normally. We presume the loss of Suz12 expression during this important developmental time is detrimental to embryo morphology and results in a decreased rate of blastocyst formation. Because of this decrease, we were able to collect only 3 SUZ blastocysts from a total of 227 injected. The microinjection procedure also contributed to significant (P < 0.05) decreases in blastocyst rates of the injected groups as compared with the CTL. Future experiments will explore potential alterations in histone methylation, as well as other epigenetic modifiers in bovine preimplantation embryos, to further elucidate the role of the epigenome in early embryonic development.

120 EMBRYO SURVIVAL FOLLOWING LIPID-BASED TRANSFECTION OF 1-CELL STAGE BOVINE EMBRYOS WITH SMALL INTERFERING RNA (siRNA) FRAGMENTS AND/OR DNA

2006 ◽  
Vol 18 (2) ◽  
pp. 168 ◽  
Author(s):  
M. Bertolini ◽  
L. R. Bertolini ◽  
S. G. Petkov ◽  
K. R. Madden ◽  
J. D. Murray ◽  
...  
Keyword(s):  
Embryo Development ◽  
Small Interfering Rna ◽  
Blastocyst Stage ◽  
Qualitative Assessment ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Survival ◽  
Interfering Rna ◽  
Higher Doses

The RNA interference (RNAi) technology is a powerful tool for studies in functional genomics. The aim of this study was to evaluate the effects of a cationic lipid-based small interfering RNA (siRNA) and/or DNA delivery to 1-cell-stage bovine embryos on survival to the blastocyst stage. In vitro-produced (IVP) embryos were generated according to Bertolini et al. 2002 (Theriogenology 58, 973), and cloned embryos were produced by the handmade cloning technique (Vajta et al. 2003 Biol. Reprod. 68, 571) using green fluorescent protein (GFP)-expressing fibroblast cells as nuclear donors. Lipofections were performed on zona-free 1-cell-stage IVP embryos at 24–28 h post-fertilization by exposure to 1% (v/v) Lipofectamine 2000 (Invitrogen Co., CA, USA), 0.002% (w/v) GFP plasmid (pEFGP-N1, Clontech Laboratories, CA, USA) and/or various doses of siRNA GFP-specific siRNA oligonucleotide (Invitrogen) or DNA methyltransferase 1 (Dnmt1)-specific siRNA fragments for 60 min at 39°C, according to 5 treatment groups: (1) zona-intact IVP embryos (controls), (2) zona-free control embryos (controls for embryo development after zona removal), (3) embryos treated with GFP + GFP-siRNA at 0, 50, 100, 200, 400, or 800 nm, (4) embryos treated with Dnmt1-siRNA at 0, 50, 100, 250, or 500 nm, and (5) cloned embryos (positive controls for GFP expression). After treatment, embryos were in vitro-cultured in a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256) for 7 days. Cleavage and developmental rates to at least 8-cell and to blastocyst stages were assessed at 48, 96, and 168 h post-fertilization (hpf), respectively. Data were analyzed by the chi-square test. Cleavage rates in embryos treated with higher doses of siRNA were lower than in all other groups (Table 1). Embryo survival to at least 8-cell stage at 48 h, based on cleavage, was similar among all treatments (data not shown), but survival to blastocyst stage was affected by higher doses of GFP- or Dnmt1-siRNA (Table 1). After a qualitative assessment by fluorescence microscopy at 168 hpf, 40 to 63% of GFP-transfected blastocysts showed various levels of fluorescence, irrespective of the siRNA treatments. Fragments of siRNA are known to be short-lived in cultured cells, although we are still uncertain of their behavior and effects in early bovine embryos. We are currently analyzing the effectiveness of the siRNA transfection in the early IVP and clone embryo. In conclusion, liposome transfection of 1-cell-stage embryos did not affect survival and development to the blastocyst stage. However, survival followed an siRNA dose-response effect, with doses higher than 400 nm appearing to be detrimental to embryo development, with a developmental arrest at or close to the embryonic genome activation period. Table 1. Developmental rate of bovine embryos following lipid-based transfection at the 1-cell-stage

109 THE ROLE OF FOLLISTATIN IN BOVINE EARLY EMBRYONIC DEVELOPMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 135
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Embryonic Development ◽  
Small Interfering Rna ◽  
Early Embryogenesis ◽  
Mrna Abundance ◽  
Early Embryonic Development ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Interfering Rna

We have previously demonstrated a positive association of follistatin mRNA abundance with bovine oocyte competence. Furthermore, exogenous follistatin supplementation during the early stages of in vitro bovine embryo development (before embryonic genome activation) can reduce time to first cleavage, increase proportion of embryos developing to the blastocyst stage, and increase trophectoderm cell numbers, suggesting a potential role for follistatin in bovine early embryonic development. However, the requirement of endogenous follistatin for early embryogenesis in cattle has not been directly tested. Thus, the aim of the present study was to determine the requirement of follistatin for early embryonic development using small interfering RNA (siRNA)- based knockdown procedures. Small interfering RNA corresponding to exons 2 (siRNA 2) and 3 (siRNA 3) of the bovine follistatin gene were synthesized, and the optimal dose of each siRNA resulting in maximal reduction in follistatin mRNA (at the 4-cell stage) following microinjection into presumptive zygotes was determined. Injection of follistatin siRNA 2 or siRNA 3 resulted in a >80% decrease in follistatin mRNA abundance in 4-cell embryos, but mRNA abundance for 5 housekeeping genes and the oocyte-specific gene JY-1 was not affected. Effects of follistatin siRNA injection on follistatin protein abundance were evaluated by immunofluorescence staining of 16-cell embryos. Follistatin immunoreactivity was dramatically reduced in siRNA-treated v. uninjected embryos. Upon validation, the effects of follistatin siRNA on early embryonic development were investigated. Cumulus–oocyte complexes were harvested from ovaries obtained from a local abattoir, matured and fertilized in vitro. Sixteen to 18 h following fertilization, denuded presumptive zygotes (25–30 per treatment, n = 4 replicates) were microinjected with (1) follistatin siRNA 2, (2) negative control (nonspecific) siRNA, (3) sham (water), or (4) served as uninjected controls. After injections, embryos were cultured in KSOM medium supplemented with 0.3% BSA. Proportions of embryos reaching the 2-cell stage within 30 h (early cleaving), 30–36 h (late cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Number of embryos reaching the 8–16-cell stage was recorded 72 h after fertilization, and embryos were cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% fetal bovine serum until day 7. Injection of follistatin siRNA 2 did not affect proportion of early and late cleaving embryos (21 v. 19% and 41 v. 37%) and total cleavage rate (80 v. 81%). However, injection of follistatin siRNA 2 decreased the proportion of embryos reaching the 8–16-cell stage (41 v. 59%) and percentage blastocyst development (12 v. 27%, P < 0.05). Experiments were repeated, and effects of follistatin siRNA 3 determined (25–30 embryos per treatment, n = 4 replicates). Similar results were obtained as for follistatin siRNA 2 injection. Results support a requirement of endogenous follistatin for bovine early embryogenesis.

129 KNOCKDOWN OF WBP1 DECREASES TROPHECTODERM FORMATION IN THE PRE-IMPLANTATION BOVINE EMBRYO

2017 ◽  
Vol 29 (1) ◽  
pp. 173
Author(s):  
M. S. Ortega ◽  
P. J. Hansen
Keyword(s):  
Embryonic Development ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Fold Change ◽  
Bovine Embryo

A single nucleotide polymorphism (SNP) in WBP1 has been previously associated with embryonic development to the blastocyst stage. WBP1 interacts with WW domain containing proteins including YAP1 from the hippo signalling pathway that is involved in trophectoderm (TE) formation. Here we tested whether reduction in mRNA abundance for WBP1 would reduce development to the blastocyst stage and formation of cells in the inner cell mass (ICM) and TE. Knockdown was performed using a GapmeR LNATM antisense oligonucleotide designed to target WBP1. A scrambled version of the same sequence was used as a control. Embryos were produced in vitro from slaughterhouse oocytes and bulls from Bos taurus and Bos indicus breeds. At 20 to 22 h after insemination (hpi), embryos were treated with 5 µM anti-WBP1 GapmeR (KD), 5 µM scrambled GapmeR (SC), or vehicle (CTL). At 72 to 75 hpi (the time of maximal WBP1 expression), groups of 18 to 20 embryos were collected from each treatment to evaluate WBP1 expression. Other cultured embryos (minimum of 50/treatment for each replicate) were cultured until Day 8 after insemination. Cleavage was assessed at Day 3 and blastocyst formation at Day 7 and 8. Embryos were collected at Day 8 to determine ICM and TE cell number by determining nuclear immunoreactive CDX2. All experiments were replicated 5 times. Fold change was calculated relative to the CTL group. Data were analysed by analysis of variance for gene expression and cell number, and through logistic regression for embryonic development. WBP1 expression was reduced (P = 0.04) in KD embryos compared to CTL (least squares means ± SEM: 1 ± 0.19 v. 0.64 ± 0.19 fold change) or SC (1.05 ± 0.19). There was no difference in expression between CTL and SC. Percent of embryos that cleaved was not affected by treatment (P > 0.05); however, percent of inseminated oocytes that became blastocysts tended to be lower in KD compared to CTL and SC at Day 7 (P = 0.09) [10.8 ± 2.8, 20 ± 3.0, and 16.3 ± 3.1% for KD, CTL, and SC, respectively] and 8 after insemination (P = 0.06) [13.7 ± 3.3, 24.2 ± 3.3, and 22.9 ± 3.6%]. Knockdown of WBP1 caused a reduction in number of total (P = 0.0004) and TE (P < 0.0001) cells with no effect on ICM cell number (P = 0.83). Total cell numbers for KD, SC, and CTL were 124.2 ± 6.4, 157.75 ± 7.4, and 124.28 ± 6.4 and numbers of TE cells were 59.7 ± 3.8, 90.0 ± 4.47, and 90.0 ± 4.4. Results show that reduction in mRNA for WBP1 decreases TE formation and tends to reduce competence of embryos to become blastocysts. This study was supported by USDA AFRI 2013–68004–20365.

scholarly journals 299 IN VITRO DEVELOPMENT OF IMMATURE PORCINE OOCYTES FERTILIZED IN VITRO TO THE BLASTOCYST STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 300
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
S.Y. Medvedev ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Significant Difference

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in this study. After in vitro maturation (IVM) for 48 h of cumulus-oocyte complexes, 75.4% (n = 442) of them extruded a visible polar body (PB). Most oocytes with a polar body (PB+ group) were found to be at metaphase II (M-II) stage (91.4%). Most oocytes without a visible polar body (PB− group, n = 144) appeared to be arrested at the germinal vesicle (GV) (41.6%) and first meiotic metaphase (M-I) (34.0%) stages. After IVF of oocytes (the day of IVF = Day 0), there was no significant difference between PB+ and PB− groups in rates of sperm penetration, monospermy, and oocyte activation after the penetration. Embryonic development was assessed by staining with 1% orcein. On Day 2, although there was no difference between the embryo cleavage in PB+ (n = 447) and PB− (n = 217) groups (47.0% and 35.9%, respectively), PB+ embryos had more cells than the PB− embryos (3.37 and 2.81 cells, respectively) (P < 0.05; ANOVA). On Day 4, the cleavage rate of PB+ embryos was higher than that of PB− embryos (45.4% and 24.3%, respectively), and PB+ embryos had more cells than the PB− embryos (8.26 and 6.0 cells, respectively) (P < 0.05; ANOVA). On Day 6, a significantly higher number of PB+ embryos developed to the blastocyst stage than that of the PB− embryos (34.6% and 20.7%, respectively) (P < 0.05). However, by subtracting the GV oocytes from the PB− group, there was no difference in blastocyst rates between the M-I arrested and M-II oocytes (35.3% and 34.6%, respectively). The number of blastomer nuclei in embryos obtained from the PB+ group (52.0) was significantly higher than that of the PB− group (29.1); however, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ significantly (1:1.9 and 1:2.2, respectively) (P < 0.05). Chromosome analysis revealed that PB+ blastocysts had significantly more diploid blastomeres (69.7%) than PB− blastocysts (44.0%), whereas PB− blastocysts had significantly more triploid cells (34.0%) compared with PB+ oocytes (8.4%)(P < 0.05; χ2 test). These results indicate that porcine oocytes arrested at the M-I stage undergo cytoplasmic maturation during culture and have the same ability to develop to blastocysts after IVF as M-II oocytes but with a lower cell number; the latter might be caused by the slower embryonic development.

scholarly journals Muscle-specific microRNA miR-206 promotes muscle differentiation

2006 ◽  
Vol 174 (5) ◽  
pp. 677-687 ◽  
Author(s):  
Hak Kyun Kim ◽  
Yong Sun Lee ◽  
Umasundari Sivaprasad ◽  
Ankit Malhotra ◽  
Anindya Dutta
Keyword(s):  
Antisense Oligonucleotide ◽  
Small Interfering Rna ◽  
Degradation Process ◽  
Microrna Target ◽  
C2c12 Myoblasts ◽  
Target Sites ◽  
The Many ◽  
Interfering Rna ◽  
In Vitro Transfection

Three muscle-specific microRNAs, miR-206, -1, and -133, are induced during differentiation of C2C12 myoblasts in vitro. Transfection of miR-206 promotes differentiation despite the presence of serum, whereas inhibition of the microRNA by antisense oligonucleotide inhibits cell cycle withdrawal and differentiation, which are normally induced by serum deprivation. Among the many mRNAs that are down-regulated by miR-206, the p180 subunit of DNA polymerase α and three other genes are shown to be direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program. The direct targets are decreased by mRNA cleavage that is dependent on predicted microRNA target sites. Unlike small interfering RNA–directed cleavage, however, the 5′ ends of the cleavage fragments are distributed and not confined to the target sites, suggesting involvement of exonucleases in the degradation process. In addition, inhibitors of myogenic transcription factors, Id1-3 and MyoR, are decreased upon miR-206 introduction, suggesting the presence of additional mechanisms by which microRNAs enforce the differentiation program.

scholarly journals In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

2009 ◽  
Vol 4 (1) ◽  
Author(s):  
Korakot Nganvongpanit ◽  
Patama Chaochird ◽  
Puntita Siengdee ◽  
Peraphan Pothacharoen ◽  
Kasisin Klunklin ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Human Chondrosarcoma ◽  
Interfering Rna

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

scholarly journals Molecular Determinants of Oocyte Competence: Potential Functional Role for Maternal (Oocyte-Derived) Follistatin in Promoting Bovine Early Embryogenesis

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2463-2471 ◽  
Author(s):  
Kyung-Bon Lee ◽  
Anilkumar Bettegowda ◽  
Gabbine Wee ◽  
James J. Ireland ◽  
George W. Smith
Keyword(s):  
Small Interfering Rna ◽  
Functional Role ◽  
Early Embryogenesis ◽  
Type I ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Cell Numbers ◽  
Positive Effects ◽  
Interfering Rna

Previous studies established a positive relationship between oocyte competence and follistatin mRNA abundance. Herein, we used the bovine model to test the hypothesis that follistatin plays a functional role in regulation of early embryogenesis. Treatment of early embryos with follistatin during in vitro culture (before embryonic genome activation) resulted in a dose-dependent decrease in time to first cleavage, increased numbers of blastocysts, and increased blastocyst total and trophectoderm cell numbers. To determine the requirement of endogenous follistatin for early embryogenesis, follistatin ablation/replacement studies were performed. Microinjection of follistatin small interfering RNA into zygotes reduced follistatin mRNA and protein and was accompanied by a reduction in number of embryos developing to eight- to 16-cell and blastocyst stages and reduced blastocyst total and trophectoderm cell numbers. Effects of follistatin ablation were rescued by culture of follistatin small interfering RNA-injected embryos in the presence of exogenous follistatin. To investigate whether follistatin regulation of early embryogenesis is potentially mediated via inhibition of endogenous activin activity, the effects of treatment of embryos with exogenous activin, SB-431542 (inhibitor of activin, TGF-β, and nodal type I receptor signaling) and follistatin plus SB-431542 were investigated. Activin treatment mimicked positive effects of follistatin on time to first cleavage and blastocyst development, whereas negative effects of SB-431542 treatment were observed. Stimulatory effects of follistatin on embryogenesis were not blocked by SB-431542 treatment. Results support a functional role for oocyte-derived follistatin in bovine early embryogenesis and suggest that observed effects of follistatin are likely not mediated by classical inhibition of activin activity.

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