Developmental potential of bovine oocytes matured in vitro or in vivo

Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P<0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos. Table 1 The viability of vitrifield-thawed bovine embryos using various containers

Download Full-text

In vitro maturation alters gene expression in bovine oocytes

Zygote ◽

10.1017/s0967199415000672 ◽

2016 ◽

Vol 24 (4) ◽

pp. 624-633 ◽

Cited By ~ 12

Author(s):

Paulo R. Adona ◽

Cláudia L.V. Leal ◽

Fernando H. Biase ◽

Tiago H. De Bem ◽

Lígia G. Mesquita ◽

...

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Regulatory Networks ◽

Developmental Competence ◽

Protein Protein Interactions ◽

Developmental Potential ◽

Oocyte Competence ◽

Bovine Oocytes

SummaryGene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein–protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein–protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

Download Full-text

The neural crest population responding to endothelin-3 in vitro includes multipotent cells

Journal of Cell Science ◽

10.1242/jcs.110.14.1673 ◽

1997 ◽

Vol 110 (14) ◽

pp. 1673-1682 ◽

Cited By ~ 1

Author(s):

J.G. Stone ◽

L.I. Spirling ◽

M.K. Richardson

Keyword(s):

Neural Crest ◽

Schwann Cells ◽

Cell Types ◽

Developmental Potential ◽

Colony Assay ◽

Cellular Targets ◽

Multipotent Cells ◽

Endothelin 3

The peptide endothelin 3 (EDN3) is essential for normal neural crest development in vivo, and is a potent mitogen for quail truncal crest cells in vitro. It is not known which subpopulations of crest cells are targets for this response, although it has been suggested that EDN3 is selective for melanoblasts. In the absence of cell markers for different precursor types in the quail crest, we have characterised EDN3-responsive cell types using in vitro colony assay and clonal analysis. Colonies were analysed for the presence of Schwann cells, melanocytes, adrenergic cells or sensory-like cells. We provide for the first time a description of the temporal pattern of lineage segregation in neural crest cultures. In the absence of exogenous EDN3, crest cells proliferate and then differentiate. Colony assay indicates that in these differentiated cultures few undifferentiated precursors remain and there is a low replating efficiency. By contrast, in the presence of 100 ng/ml EDN3 differentiation is inhibited and most of the cells maintain the ability to give rise to mixed colonies and clones containing neural crest derivatives. A high replating efficiency is maintained. In secondary culture there was a progressive decline in the number of cell types per colony in control medium. This loss of developmental potential was not seen when exogenous EDN3 was present. Cell type analysis suggests two novel cellular targets for EDN3 under these conditions. Contrary to expectations, one is a multipotent precursor whose descendants include melanocytes, adrenergic cells and sensory-like cells; the other can give rise to melanocytes and Schwann cells. Our data do not support previous claims that the action of EDN3 in neural crest culture is selective for cells in the melanocyte lineage.

Download Full-text

137 L-carnitine improves developmental potential of bovine oocytes exposed to high lipid concentrations during in vitro maturation

Reproduction Fertility and Development ◽

10.1071/rdv34n2ab137 ◽

2022 ◽

Vol 34 (2) ◽

pp. 306

Author(s):

G. Catandi ◽

E. Carnevale

Keyword(s):

In Vitro Maturation ◽

Developmental Potential ◽

High Lipid ◽

Bovine Oocytes

Download Full-text

TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810422115 ◽

2018 ◽

Vol 115 (44) ◽

pp. E10370-E10378 ◽

Cited By ~ 21

Author(s):

Miranda L. Bernhardt ◽

Paula Stein ◽

Ingrid Carvacho ◽

Christopher Krapp ◽

Goli Ardestani ◽

...

Keyword(s):

Rational Design ◽

Culture Media ◽

Mammalian Development ◽

Developmental Potential ◽

Store Depletion ◽

Offspring Growth ◽

Mechanistic Basis ◽

Genetic Mouse Models

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm–egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

Download Full-text

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽

10.3390/cells10113111 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3111

Author(s):

Po-Yu Lin ◽

Denny Yang ◽

Chi-Hsuan Chuang ◽

Hsuan Lin ◽

Wei-Ju Chen ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Embryonic Cells ◽

Cell Stage ◽

Developmental Potential ◽

Functional Features ◽

Early Mouse ◽

Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Download Full-text

64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab64 ◽

2004 ◽

Vol 16 (2) ◽

pp. 154

Author(s):

H.S. Park ◽

M.Y. Lee ◽

S.P. Hong ◽

J.I. Jin ◽

J.K. Park ◽

...

Keyword(s):

Nuclear Transfer ◽

Electric Pulse ◽

Serum Starvation ◽

Cleavage Rate ◽

Developmental Potential ◽

In Vitro Development ◽

Significant Difference ◽

Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

Download Full-text

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

10.21203/rs.3.rs-73275/v2 ◽

2020 ◽

Author(s):

Vera A van der Weijden ◽

Meret Schmidhauser ◽

Mayuko Kurome ◽

Johannes Knubben ◽

Veronika L Flöter ◽

...

Keyword(s):

Signaling Pathways ◽

Early Stage ◽

Developmental Competence ◽

Molecular Signaling ◽

Developmental Potential ◽

Stage Of Development ◽

Molecular Signaling Pathways ◽

Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Download Full-text

Effect and possible mechanisms of melatonin treatment on the quality and developmental potential of aged bovine oocytes

Reproduction Fertility and Development ◽

10.1071/rd16223 ◽

2017 ◽

Vol 29 (9) ◽

pp. 1821 ◽

Cited By ~ 20

Author(s):

Shuang Liang ◽

Jing Guo ◽

Jeong-Woo Choi ◽

Nam-Hyung Kim ◽

Xiang-Shun Cui

Keyword(s):

In Vitro Fertilisation ◽

Cortical Granule ◽

Melatonin Treatment ◽

Developmental Potential ◽

Exogenous Melatonin ◽

Atp Production ◽

Quality Deterioration ◽

Oocyte Aging ◽

Bovine Oocytes

After reaching the metaphase II (MII) stage, unfertilised oocytes undergo a time-dependent process of quality deterioration referred to as oocyte aging. The associated morphological and cellular changes lead to decreased oocyte developmental potential. This study investigated the effect of exogenous melatonin supplementation on in vitro aged bovine oocytes and explored its underlying mechanisms. The levels of cytoplasmic reactive oxygen species and DNA damage response in bovine oocytes increased during in vitro aging. Meanwhile, maturation promoting factor activity significantly decreased and the proportion of morphologically abnormal oocytes significantly increased. Melatonin supplementation significantly decreased quality deterioration in aged bovine MII oocytes (P < 0.05). Additionally, it decreased the frequency of aberrant spindle organisation and cortical granule release during oocyte aging (P < 0.05). In the melatonin-supplemented group, mitochondrial membrane potential and ATP production were significantly increased compared with control. Furthermore, melatonin treatment significantly increased the speed of development of bovine oocytes to the blastocyst stage after in vitro fertilisation and significantly decreased the apoptotic rate in the blastocysts (P < 0.05). The expression of Bax and Casp3 in the blastocysts was significantly reduced after treatment with melatonin, whereas expression of Bcl2 significantly increased (P < 0.05). In conclusion, these findings suggest that supplementation of aged bovine oocytes with exogenous melatonin improves oocyte quality, thereby enhancing the developmental capacity of early embryos.

Download Full-text