Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

Mouse ovaries exhibit a peri-ovulatory rise of ornithine decarboxylase and its product putrescine concurrent with oocyte maturation. Older mice exhibit a deficiency of both the enzyme and putrescine. Peri-ovulatory putrescine supplementation in drinking water increases ovarian putrescine levels, reduces embryo resorption and increases live pups in older mice. However, it is unknown if putrescine acts in the ovaries to improve oocyte maturation. This study examined the impact of putrescine supplementation during oocyte in vitro maturation (IVM) on the developmental potential of aged oocytes. Cumulus–oocyte complexes from 9–12-month-old C57BL/6 mice were subjected to IVM with or without 0.5 mM putrescine, followed by in vitro fertilisation and culture to the blastocyst stage. Putrescine supplementation during IVM did not influence the proportion of oocyte maturation, fertilisation or blastocyst formation, but significantly increased blastocyst cell numbers (44.5 ± 1.9, compared with 36.5 ± 1.9 for control; P = 0.003). The putrescine group also had a significantly higher proportion of blastocysts with top-grade morphology (42.9%, compared with 26.1% for control; P = 0.041) and a greater proportion with octamer-binding transcription factor 4 (OCT4)-positive inner cell mass (38.3%, compared with 19.8% for control; P = 0.005). Therefore, putrescine supplementation during IVM improves egg quality of aged mice, providing proof of principle for possible application in human IVM procedures for older infertile women.

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Maternal influence on oocyte collection and embryo development after transvaginal puncture and in vitro fertilisation in dairy cows

BSAP Occasional Publication ◽

10.1017/s0263967x00034054 ◽

2001 ◽

Vol 26 (2) ◽

pp. 433-435

Author(s):

S. Chastant-Maillard ◽

H. Quinton ◽

C. Douar ◽

J. Marchal ◽

C. Richard ◽

...

Keyword(s):

Embryo Development ◽

Inner Cell Mass ◽

Formation Rate ◽

Cell Mass ◽

Blastocyst Stage ◽

Cell Counting ◽

In Vitro Fertilisation ◽

Oocyte Collection

AbstractThe aim of the study was to evaluate differences between cows in the “quality” of their oocytes defined as their ability to support embryonic development. Ten cows from the same herd, all primiparous and non-pregnant were submitted for oocyte collection by Ovum-Pick Up (OPU). The oocytes were matured in vitro and fertilised with semen from the same bull. In vitro embryo development, both quantitatively (percentages showing cleavage and forming blastocysts) and qualitatively (differential cell counting in blastocysts) was determined at the blastocyst stage (Day 7). The number of oocytes collected, the number of blastocysts obtained and the blastocyst formation rate varied between cows (P<0.001). The mean percentage of inner cell mass cells tended to higher for embryos derived from one cow. These results provide evidence that the quality of the oocytes was influenced by their maternal origin. Follicular growth also varied between cows.

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The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7971-7976.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7971-7976

Author(s):

L M Whyatt ◽

A Düwel ◽

A G Smith ◽

P D Rathjen

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Es Cells ◽

Expression System ◽

Cell Mass ◽

Embryonic Stem ◽

Expression Vectors ◽

Developmental Control ◽

Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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

Cells ◽

10.3390/cells8101272 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1272 ◽

Cited By ~ 3

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.

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77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab77 ◽

2019 ◽

Vol 31 (1) ◽

pp. 164

Author(s):

A. E. Ynsaurralde Rivolta ◽

M. Suvá ◽

V. Alberio ◽

C. Vazquez Echegaray ◽

A. Guberman ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Monozygotic Twin ◽

Cell Number ◽

Control Group ◽

Cell Stage ◽

Exact Test ◽

Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

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Single cell profiling of transcriptomic changes during in vitro maturation of human oocytes

10.21203/rs.3.rs-292144/v1 ◽

2021 ◽

Author(s):

Hiroki Takeuchi ◽

Mari Yamamoto ◽

Megumi Fukui ◽

Tadashi Maezawa ◽

Mikiko Nishioka ◽

...

Keyword(s):

Single Cell ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Maturation Stage ◽

Developmental Potential ◽

Human Oocytes ◽

Transcriptomic Changes

Abstract In vitro maturation of human oocytes is widely used for infertility treatment. However, the success rate of maturation varies depending on patients and molecular mechanisms underlying successful maturation remain unclear. Especially, gene expression profiles of oocytes at each maturation stage need to be revealed to understand the differential developmental abilities of oocytes. Here, we show transcriptomes of human oocytes during in vitro maturation by single cell RNA-seq analyses. Hundreds of transcripts dynamically altered their expression, and we identify molecular pathways and upstream regulators that may govern oocyte maturation. Furthermore, oocytes that are delayed in their maturation show distinct transcriptomes. Finally, we reveal genes whose transcripts are enriched in each maturation stage and that can be used for selecting an oocyte with a high developmental potential. Taken together, our work uncovers transcriptomic changes during human oocyte maturation and provides a molecular insight into the differential developmental potential of each oocyte.

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127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab127 ◽

2004 ◽

Vol 16 (2) ◽

pp. 186

Author(s):

J.O. Gjørret ◽

P. Maddox-Hyttel

Keyword(s):

Production Systems ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Embryo Cell ◽

Developmental Potential ◽

Cell Numbers ◽

Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to<0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P<0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

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145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

Reproduction Fertility and Development ◽

10.1071/srb09abs145 ◽

2009 ◽

Vol 21 (9) ◽

pp. 63

Author(s):

L. Ganeshan ◽

C. O'Neill

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Early Embryo ◽

Pluripotent Cells ◽

Drug Induced ◽

Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

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Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling

Cells ◽

10.3390/cells9061550 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1550 ◽

Cited By ~ 1

Author(s):

Marwa El Sheikh ◽

Ahmed Atef Mesalam ◽

Muhammad Idrees ◽

Tabinda Sidrat ◽

Ayman Mesalam ◽

...

Keyword(s):

Oxidative Stress ◽

In Vitro Maturation ◽

Inner Cell Mass ◽

Cell Mass ◽

Akt Signaling ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Vitamin B3 ◽

Cell Stage

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8–16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.

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Effect of anisomycin, a protein synthesis inhibitor, on thein vitrodevelopmental potential, ploidy and embryo quality of bovine ICSI embryos

Zygote ◽

10.1017/s0967199416000034 ◽

2016 ◽

Vol 24 (5) ◽

pp. 724-732 ◽

Cited By ~ 4

Author(s):

María Elena Arias ◽

Raúl Sánchez ◽

Ricardo Felmer

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Developmental Rate ◽

Fertilization Rate ◽

Terminal Deoxynucleotidyl Transferase ◽

Protein Synthesis Inhibitor ◽

Oocyte Activation ◽

Synthesis Inhibitor

SummaryIncreasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that ofin vitrofertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%,P< 0.05) and DMAP (28.6%,P> 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.

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Influence of vasoactive intestinal peptide (VIP), atrial natriuretic peptide (ANP) and insulin-like growth factor-I (IGF-I) on in vitro maturation of prepubertal and adult sheep oocytes

Zygote ◽

10.1017/s0967199400003361 ◽

1996 ◽

Vol 4 (04) ◽

pp. 343-348 ◽

Cited By ~ 14

Author(s):

S. Ledda ◽

L. Bogliolo ◽

G. Leoni ◽

P. Calvia ◽

S. Naitana

Keyword(s):

Growth Factor ◽

Atrial Natriuretic Peptide ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Optimal Conditions ◽

Factor I ◽

Igf I

Much effort has been focused on establishing optimal conditions for obtainingin vitromaturation of oocytes from different species with results comparable to those achieved afterin vivodevelopment (reviewed by Brackett, 1992). However, even though extraordinary progress has been made, thein vitrotechnology for oocyte maturation lags far behind thatin vivoand improvements are needed to increase the quantity and quality of the embryos produced from these matured oocytes.

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