scholarly journals Human Cumulus Cells in Long-Term In Vitro Culture Reflect Differential Expression Profile of Genes Responsible for Planned Cell Death and Aging—A Study of New Molecular Markers

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1265
Author(s):  
Błażej Chermuła ◽  
Wiesława Kranc ◽  
Karol Jopek ◽  
Joanna Budna-Tukan ◽  
Greg Hutchings ◽  
...  
Keyword(s):  
Cell Death ◽  
In Vitro Culture ◽  
Expression Analysis ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Genetic Profile

In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.

scholarly journals Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

2017 ◽  
Vol 114 (29) ◽  
pp. E5796-E5804 ◽  
Author(s):  
Ye Yuan ◽  
Lee D. Spate ◽  
Bethany K. Redel ◽  
Yuchen Tian ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Fourfold Increase

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called “FLI medium,” improves nuclear maturation of oocytes in cumulus–oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus–oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

scholarly journals How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Salvatore Giovanni Vitale ◽  
Paola Rossetti ◽  
Francesco Corrado ◽  
Agnese Maria Chiara Rapisarda ◽  
Sandro La Vignera ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Fertilization Rate ◽  
In Vitro Models ◽  
The One ◽  
High Level

Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment.

Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis

2011 ◽  
Vol 23 (8) ◽  
pp. 990 ◽  
Author(s):  
Shan Liu ◽  
Huai L. Feng ◽  
Dennis Marchesi ◽  
Zi-Jiang Chen ◽  
Avner Hershlag
Keyword(s):  
Embryo Development ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Beneficial Effects ◽  
Nuclear Maturation ◽  
High Concentrations ◽  
Vitro Development

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus–oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B + M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75–7500 mIU mL–1) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIU mL–1) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P > 0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 7500 mIU mL–1 Gn; however, when the concentration was increased to 75 000 mIU mL–1, the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).

scholarly journals “Naturalization” of Routine Assisted Reproductive Technologies by In Vitro Culture of Embryos with Microvibration: Sex Ratio, Body Length, and Weight of 2,456 Live-Birth Deliveries after Transfer of 9,624 Embryos In Vitro Cultured in Static System and with Microvibration

2017 ◽  
Vol 2017 ◽  
pp. 1-7
Author(s):  
Vladimir Isachenko ◽  
Karl Sterzik ◽  
Evgenia Isachenko ◽  
Robert Maettner ◽  
Plamen Todorov ◽  
...  
Keyword(s):  
Sex Ratio ◽  
In Vitro Culture ◽  
Body Length ◽  
Live Birth ◽  
Assisted Reproductive Technologies ◽  
Age Groups ◽  
Reproductive Technologies ◽  
Static System ◽  
Mechanical Agitation

Aim was to determine whether there is any difference in the sex ratio, body length, and body weight of 2,456 deliveries after transfer of 9,624 embryos derived using in vitro culture under static and mechanical microvibration conditions. Pronuclear embryos from 4435 patients were cultured in vitro under two different conditions: without (n=4821) and with mechanical agitation (n=4803). Sex ratio, body length, and weight of 2,456 live-birth deliveries after transfer of 9,624 embryos were noted. The proportion of males at birth was significantly associated with mode of in vitro culture of embryos only among women aged 40 years and older. The rate “body length” was significantly associated with mode of in vitro culture of embryos only among women aged 29 and younger. In the same time, among twins, this ratio positively associated with in vitro culture of embryos under microvibration only among women aged 30–34 years as well as ≥40 years and negatively among women aged 35–39 years. It was concluded that birth weight of infants was positively associated with mode of in vitro culture of embryos under microvibration among women of all age groups. This trial registration number is ISRCTN13773904, registered 6 April 2016.

scholarly journals Vitrification of immature oocytes of goats in Bangladesh

2019 ◽  
Vol 35 (1-2) ◽  
pp. 7-12
Author(s):  
MN Sharif ◽  
SM Choudhury ◽  
MM Rahman ◽  
MM Rahman ◽  
NS Juyena ◽  
...  
Keyword(s):  
Culture Medium ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Step Procedure ◽  
Inverted Microscope ◽  
Maturation Rate ◽  
Humidified Air

Cryopreservation of oocytes and embryos by vitrification can have advantages in assisted reproductive technologies (ARTs) in mammals. The aim of this study was to establish an effective vitrification procedure and cryodevice for goat’s oocytes in Bangladesh. Cumulus oocyte complexes (COCs) were collected from ovaries from slaughterhouse. COCs with more than 3 layers of cumulus cells were selected. COCs were vitrified by two-step procedure using 7.5% and 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA), loaded on Cryotop or French mini-straw, then directly plunged into liquid nitrogen (LN2). Then the COCs containing Cryotop or French mini-straws were warmed in 0.25 M sucrose and 20% FBS-supplemented tissue culture medium (TCM) 199 followed by in vitro culture in 50 μl droplets of bicarbonate-buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 h at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. The in vitro maturation rate of goat’s oocytes after vitrification and warming was 39.3 ± 6.8%, 31.3 ± 9.4%, 61.6 ± 14.2% when using Cryotop (cryodevice), French mini-straws and without vitrification (control), respectively. Maturation rate was significantly higher (P<0.05) without vitrification. It is suggested that both Cryotop and French mini-straw are efficient cryodevices for vitrification of goat’s oocytes and further investigation is required to optimize the protocol for vitrification and warming procedure for the satisfactory survival of goat’s oocytes. The Bangladesh Veterinarian (2018) 35(1&2): 7-12

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

71 The Effect of Two Different In Vitro Culture Media and Mice Embryo Groupings on Hatchability After 24 Hours of Culture

2018 ◽  
Vol 30 (1) ◽  
pp. 174
Author(s):  
N. C. Negota ◽  
M. L. Mphaphathi ◽  
L. P. Nethenzheni ◽  
T. L. Rammutla ◽  
N. R. Serota ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Significant Difference ◽  
Autocrine Signalling ◽  
Blastocyst Hatching

Mammalian blastocysts must hatch out from the zona pellucida before implantation. In vitro embryo culture and grouping of mice blastocysts are conducive options of assisted reproductive technologies (ART) to speed up the hatching rate of mice embryos. The number of embryos per unit volume has the greatest impact on hatching rates due to autocrine signalling. The study aimed to determine the effect of two in vitro culture (IVC) media (TCM-199 and Ham’s F10) and embryo groupings (1, 2, 3, and 4 embryos per 50-µL droplet) after 24 h of culture on hatching rate. Breeds of C57BL/6 (n = 10) and BALB/c (n = 10) were raised until they reached maturity and bred naturally to produce the first filial generation. The photoperiod was 14 h of light followed by 10 h of darkness in the breeding house, and feed and water were provided ad libitum. Female mice were superovulated using eCG and hCG. The first filial generations from 2 breeds were used for the collection of 160 blastocysts and randomly allocated into 2 IVC media (80 embryos for TCM-199 and 80 embryos for Ham’s F10) and again subjected to 4 embryo groupings (1, 2, 3, and 4 embryos per droplet) treatments. Four replicates were done per treatment group. The general linear model of Minitab version 17 (Minitab Inc., State College, PA, USA) was used to analyse the data. The hatching rate of blastocyst stage was significantly higher for TCM-199 (56.9 ± 27.2) compared with Ham’s F10 (50.0 ± 35.1%). The comparison of all embryo groupings, 1 (20.0 ± 40.5), 2 (28.8 ± 29.7), 3 (59.1 ± 38.8), and 4 (43.8 ± 32.4%) per 50-µL droplet showed significant differences, irrespective of IVC medium and breed. In TCM-199, groupings of 1 (20.0 ± 41.0), 2 (30.0 ± 29.9), 3 (63.3 ± 40.3), and 4 (42.5 ± 33.5%) had a significant difference on blastocyst hatching percent. In Ham’s F10, groupings of 1 (20.0 ± 41.0), 2 (27.5 ± 30.2), 3 (55.0 ± 37.9), and 4 (45.0 ± 32.0%) were significantly different on blastocyst hatching rate. However, an increase in hatching rate was observed for the interaction of media and embryo groupings and especially when embryos were increased per droplet in all breeds. In conclusion, the use of TCM-199 and grouping of 3 embryos per 50-µL droplet during culture had the highest hatching rate compared with the use of Ham’s F10.

The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model

2017 ◽  
Vol 8 (4) ◽  
pp. 411-417 ◽  
Author(s):  
M.-A. Sirard
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Genetic Model ◽  
Culture Media ◽  
Reproductive Technologies ◽  
The Body ◽  
Model Systems ◽  
Gene Perturbations

Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

Neither gonadotropin nor cumulus cell expansion is needed for the maturation of competent porcine oocytes in vitro

2021 ◽  
Author(s):  
Bethany K Redel ◽  
Lee D Spate ◽  
Ye Yuan ◽  
Clifton N Murphy ◽  
R Michael Roberts ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Cumulus Cell ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Key Indicator ◽  
Cytoplasmic Competence

Abstract In vitro maturation of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: 1) Basal (−gonadotropins (GN)-FLI); 2) -GN + FLI (supplement of FGF2, LIF, and IGF1); 3) + GN-FLI; 4) + GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared to the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.

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