144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO

2007 ◽  
Vol 19 (1) ◽  
pp. 189
Author(s):  
Y. H. Choi ◽  
H. D. Harding ◽  
A. D. Obermiller ◽  
K. Hinrichs
Keyword(s):  
Embryonic Development ◽  
Expression Pattern ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Cell Mass ◽  
Population Variation ◽  
Early Embryonic Development

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

scholarly journals The second lineage differentiation of bovine embryos fails in the absence of OCT4/POU5F1

2021 ◽  
Author(s):  
Kilian Simmet ◽  
Mayuko Kurome ◽  
Valerie Zakhartchenko ◽  
Horst-Dieter Reichenbach ◽  
Claudia Springer ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
Lineage Differentiation

The mammalian blastocyst undergoes two lineage segregations, i.e., formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) the remaining pluripotent lineage. To clarify expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9 and 12 blastocysts completely derived ex vivo by staining for OCT4, NANOG, SOX2 (EPI) and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost of NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show, that OCT4 is required cell-autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos

2019 ◽  
Vol 31 (12) ◽  
pp. 1758 ◽  
Author(s):  
Elaine M. Carnevale ◽  
Elizabeth S. Metcalf
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Inner Cell Mass ◽  
Early Stage ◽  
Cell Mass ◽  
Quality Parameters ◽  
Early Embryo ◽  
Developmental Competence ◽  
Limited Information

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for &gt;15 years.

63 QUALITY OF BOVINE EMBRYOS PRODUCED IN VITRO FROM IMMATURE OOCYTES TREATED WITH A SUBLETHAL HYDROSTATIC PRESSURE

2013 ◽  
Vol 25 (1) ◽  
pp. 179
Author(s):  
C. Díez ◽  
B. Trigal ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
E. Correia ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Pressure Treatment ◽  
Cell Counts ◽  
Development Rates ◽  
Bovine Oocytes

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P < 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P > 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine. RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

scholarly journals Colony-Stimulating Factor 2 (CSF-2) Improves Development and Posttransfer Survival of Bovine Embryos Produced in Vitro

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5046-5054 ◽  
Author(s):  
Bárbara Loureiro ◽  
Luciano Bonilla ◽  
Jeremy Block ◽  
Justin M. Fear ◽  
Aline Q. S. Bonilla ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Colony Stimulating Factor ◽  
Bovine Embryos ◽  
Epigenetic Effects ◽  
Trophectoderm Cells ◽  
Stimulating Factor

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30–35 of gestation. Moreover, treatment with CSF2 from either d 1–7 or 5–7 after insemination reduced pregnancy loss after d 30–35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

scholarly journals Effect of serum-containing and serum-free culture medium-mediated activation of matrix metalloproteinases on embryonic developmental competence

2019 ◽  
Vol 64 (No. 12) ◽  
pp. 473-482
Author(s):  
Sang Hwan Kim ◽  
Jong Taek Yoon
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Serum Free Medium ◽  
Vitro Fertilization ◽  
Serum Free ◽  
Foetal Bovine Serum

In this study, we examined whether serum-free and serum-containing media affect matrix metalloproteinase (MMP) activity with respect to embryonic development, and whether MMP expression is correlated with the development of in vitro fertilized eggs. When oocytes were cultured in serum-free medium (containing polyvinylpyrrolidone) and serum (foetal bovine serum)-containing medium, the generation of meiosis 2 (MII) oocytes was 76% and 87.5%, respectively (P &lt; 0.05). After in vitro fertilization using mature oocytes, we observed 39.72% and 64.05% of cleaved oocytes in serum-free and serum-containing groups, respectively (P &lt; 0.05). Our analysis revealed differential expression and activity of MMPs. The serum-containing group showed high MMP-9 activity during oocyte maturation and development of in vitro produced embryos, with particularly high activity in the inner cell mass zone of the embryos. Therefore, this study suggests that the presence or the absence of serum will affect the activity of MMPs, which can be used to measure the rate of embryonic development.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro

2015 ◽  
Vol 27 (3) ◽  
pp. 557 ◽  
Author(s):  
Yan Zhu ◽  
Ya-Hong Jiang ◽  
Ya-Ping He ◽  
Xuan Zhang ◽  
Zhao-Gui Sun ◽  
...  
Keyword(s):  
Embryonic Development ◽  
G Protein ◽  
Embryo Development ◽  
Expression Patterns ◽  
Critical Role ◽  
Α Subunit ◽  
G Protein Signalling ◽  
Early Embryos ◽  
Development In Vitro

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription–polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

236 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATOR REGULATORS IN ASSOCIATION WITH BMP15 ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 207
Author(s):  
M. F. Machado ◽  
M. F. G. Nogueira ◽  
R. B. Gilchrist ◽  
M. L. Sutton-McDowall ◽  
D. G. Mottershead ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Oocyte Competence ◽  
Significant Difference

BMP15 is a promising peptide to improve oocyte competence; also, addition of cyclic adenosine monophosphate modulator (cAMP) regulators prevents spontaneous maturation in vitro and promotes embryo development. We aimed to assess embryo development after prematuration [pre-in vitro maturation (IVM)] with IBMX and Forskolin (FSK) and maturation in the presence or absence of a purified pro mature region of BMP15. Immature cumulus-oocyte complexes (COC) were cultured in vitroMat (IVF Vet Solutions, Adelaide, Australia) plus 4 mg mL–1 fatty acid free-BSA and rhFSH (0.1 IU mL–1), then divided into the following treatment groups: 1) spontaneous IVM: 24 h of IVM; 2) spontaneous IVM + BMP15: 24 h of IVM in the presence of BMP15 (100 ng mL–1); 3) Pre 2 h: pretreatment with IBMX (500 µM; Sigma-Aldrich) and FSK (100 µM; Sigma-Aldrich) for 2 h following 24 h maturation; and 4) Pre 2 h + BMP15: pretreatment with IBMX and FSK for 2 h following 24 h maturation in the presence of BMP15 (100 ng mL–1). After maturation, oocytes were inseminated and zygotes were cultured for 5 days in VitroCleave (IVF Vet Solutions, Adelaide, Australia) and transferred into VitroBlast (IVF Vet Solutions, Adelaide, Australia) until blastocyst assessment (Days 7 and 8). Zona-intact embryos were retrieved to assess differential staining of trophectoderm and inner cell mass. Data were transformed into a logarithm and analysed by 1-way ANOVA and post hoc least significant difference using SigmaStat software (SPSS Inc., San Jose, CA, USA; P < 0.05). There was no difference among groups on cleavage rates or blastocyst rates at Day 7; however, both Pre 2 h treatments increase hatched blastocyst rates at Day 8 of embryo development (Table 1). Supplementation with BMP15 increased total blastocyst rates at Day 8, regardless of pretreatment with IBMX+FSK (Table 1). Our data demonstrate that embryos from oocytes matured in the presence of BMP15 or pretreated with IBMX+FSK increase trophectoderm and total cell numbers; however, no differences were observed for inner cell mass. We conclude that Pre 2 h treatment or BMP15 increase embryo development; however, no effect of cAMP regulators in association with BMP15 on embryo development was observed. Table 1.Embryo development Supported by FAPESP (project numbers: 2012/1073-8; 2013/12960-9; 2013/05083-1; 2012/50533-2).

scholarly journals Effects of recombinant human follicle-stimulating hormone on embryo development in mice

2005 ◽  
Vol 288 (5) ◽  
pp. E845-E851 ◽  
Author(s):  
L. J. Edwards ◽  
K. L. Kind ◽  
D. T. Armstrong ◽  
J. G. Thompson
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Ovarian Stimulation ◽  
Inner Cell Mass ◽  
Follicle Stimulating Hormone ◽  
Cell Mass ◽  
Nuclear Staining ◽  
Insulin Like Growth Factor ◽  
Stimulating Hormone

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 × CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 ± 5.1%) and 20 (77.3 ± 5.6%) IU rhFSH groups compared with control embryos (96.7 ± 1.0%). The number of inner cell mass cells was reduced ( P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

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