131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 213
Author(s):  
V. Ghaffarilaleh ◽  
F. Ghafari ◽  
M. Teresa-Paramio ◽  
A. Fouladi-Nashta
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Apoptotic Cell ◽  
Cell Mass ◽  
Differential Staining ◽  
Embryo Viability ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Large Size

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.

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 Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle

2016 ◽  
Vol 37 (5) ◽  
pp. 3189
Author(s):  
Paula Alvares Lunardelli ◽  
Luciana Simões Rafagnin Marinho ◽  
Camila Oliveira Rosa ◽  
Amauri Alcindo Alfieri ◽  
Marcelo Marcondes Seneda
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Embryo Viability ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Trophectoderm Cells ◽  
Preimplantation Stage ◽  
First Time

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P < 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P < 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 h after fertilization and from those produced from follicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattle embryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at preimplantation stage.

scholarly journals Disruption of Ini1 Leads to Peri-Implantation Lethality and Tumorigenesis in Mice

2001 ◽  
Vol 21 (10) ◽  
pp. 3598-3603 ◽  
Author(s):  
Cynthia J. Guidi ◽  
Arthur T. Sands ◽  
Brian P. Zambrowicz ◽  
Tod K. Turner ◽  
Delia A. Demers ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Germ Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Tumor Formation ◽  
Embryo Viability ◽  
Early Embryonic Lethality ◽  
Poorly Differentiated ◽  
Rhabdoid Tumors

ABSTRACT SNF5/INI1 is a component of the ATP-dependent chromatin remodeling enzyme family SWI/SNF. Germ line mutations ofINI1 have been identified in children with brain and renal rhabdoid tumors, indicating that INI1 is a tumor suppressor. Here we report that disruption of Ini1 expression in mice results in early embryonic lethality. Ini1-null embryos die between 3.5 and 5.5 days postcoitum, and Ini1-null blastocysts fail to hatch, form the trophectoderm, or expand the inner cell mass when cultured in vitro. Furthermore, we report that approximately 15% ofIni1-heterozygous mice present with tumors, mostly undifferentiated or poorly differentiated sarcomas. Tumor formation is associated with a loss of heterozygocity at the Ini1 locus, characterizing Ini1 as a tumor suppressor in mice. Thus, Ini1 is essential for embryo viability and for repression of oncogenesis in the adult organism.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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.

scholarly journals In vitro fertilization and culture alters chromatin accessibility in the mouse inner cell mass

2018 ◽  
Vol 110 (4) ◽  
pp. e378
Author(s):  
E. Ruggeri ◽  
E. Grow ◽  
X. Liu ◽  
A. Donjacour ◽  
P. Rinaudo
Keyword(s):  
In Vitro Fertilization ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Chromatin Accessibility ◽  
Vitro Fertilization

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

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.

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&amp;M University.

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