65 THE EFFECTS OF CUMULUS CELLS ON DEVELOPMENTAL RATES OF VITRIFIED MOUSE OOCYTES IN C57BL/6J STRAIN

2013 ◽  
Vol 25 (1) ◽  
pp. 180
Author(s):  
N. Kashiwazaki ◽  
N. Kohaya ◽  
K. Fujiwara ◽  
K. Furui ◽  
J. Ito
Keyword(s):  
Ethylene Glycol ◽  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Analysis Data ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Mouse Oocytes ◽  
Free Media

Unfertilized oocytes are one of the most desired germ-cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including IVF and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. We have recently reported that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence (Kohaya et al. 2011 J. Reprod. Dev. 57, 675–680). Since the previous study was carried out using ICR mice (closed colony), we examined whether our protocol can be applied for C57BL/6J mice (inbred strain), which are commonly used for production of transgenic and knockout mice. The effect of cumulus cells on the ability of C57BL/6J mouse oocytes to be fertilized and develop in vitro was examined. Cumulus oocyte complexes (COC) derived from female mice with super ovulation were collected by flushing. Cumulus cells were removed for a portion of the oocytes (DO) using hyarulonidase. Oocytes from both treatment groups (COC and DO) were then vitrified according to the protocol we previously reported (Kohaya et al. 2011). After warming, vitrified COC and DO were used for IVF. All percentage data were subjected to arcsine transformation before statistical analysis. Data were analyzed by one-way ANOVA and Tukey’s test. Significance was considered at P < 0.05. The pronuclear formation rate of vitrified DO after IVF (20/58, 33.3%) was reduced compared with vitrified COC (55/90, 62.1%). Vitrified COC showed significantly (P < 0.05) higher developmental ability to develop into the 2-cell (50/90, 57.0%) and blastocyst stages (42/90, 45.9%) compared with vitrified DO [24.8% (16/58) and 18.4% (11/58), respectively]. The vitrified COC developed to term at a high success rate (51/90, 56.7%) being equivalent to the rate obtained with IVF using fresh COC (52/90, 57.8%). Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence. These findings will contribute to improve oocytes vitrification in not only experimental animals but also in clinical application in human infertility.

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 Reproductive Seasonality Affects In Vitro Embryo Production Outcomes in Adult Goats

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 873
Author(s):  
Joanna M.G. Souza-Fabjan ◽  
Lucas F.L. Correia ◽  
Ribrio I.T.P. Batista ◽  
Yann Locatelli ◽  
Vicente J.F. Freitas ◽  
...  
Keyword(s):  
Breeding Season ◽  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Developmental Competence ◽  
Reproductive Seasonality ◽  
Cleavage Rate

Reproductive seasonality may have a considerable influence on the efficiency of assisted reproductive technologies in seasonal species. This study evaluated the effect of season on cleavage, blastocyst rates and quality of in vitro produced (IVP) goat embryos. In total, 2348 cumulus–oocyte complexes (COCs) were recovered from slaughterhouse ovaries and subjected to the same IVP system throughout 1.5 years (49 replicates). The odds ratio (OR) among seasons was calculated from values of cleavage and blastocyst rates in each season. Cleavage rate was lower (p < 0.05) in spring (anestrus), in comparison with either autumn (peak of breeding season) or summer, while the winter had intermediate values. Furthermore, lower OR of cleavage was observed in spring. Blastocyst formation rate (from initial number of COCs) was higher (p < 0.05) in autumn (52 ± 2.5%) when compared with the other seasons (combined rates: 40 ± 1.9%). Moreover, its OR was higher (p < 0.05) in autumn compared to all other seasons and impaired in the spring compared to winter (OR: 0.54) and summer (OR: 0.48). Embryo hatchability and blastocyst cell number were similar (p > 0.05) among seasons. In conclusion, the breeding season leads to improved oocyte developmental competence, resulting in higher cleavage and blastocyst yield, whereas embryo quality remained similar throughout the years.

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.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

P–458 A computational biology approach to improve in-vitro folliculogenesis

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C D Berardino ◽  
N Bernabò ◽  
G Capacchietti ◽  
A Peserico ◽  
G Buoncuore ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Intracellular Signaling ◽  
Assisted Reproductive Technologies ◽  
Computational Modelling ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Paracrine Factors ◽  
Oocyte Growth ◽  
Topological Parameters

Abstract Study question Considering the complexity of mechanisms involved in mammalian ovarian folliculogenesis, how about improving the current in-vitro folliculogenesis (ivF) protocols to prolong individual reproductive chance? Summary answer Computational modelling approach based on network theory was used to manage complexity, improve ivF knowledge and discover new molecules to be targeted for innovating assisted-reproductive-technologies. What is known already: Over the past decades, based on the large ovarian-pool of immature-gametes availability, ivF systems were developed in several mammalian species to support oocyte growth in order to preserve human-fertility and contrast endangered species extinction. Only mouse live-births were obtained when primordial/primary follicles were cultured in-vitro, instead the oocyte differentiation is extremely slow in medium-sized mammals. Moreover, the degree of meiotic-competence is quite incomplete if compared to mice, because oocytes must proceed until late antral-follicle stage to acquire a complete developmental competence. These observations denote the importance to adopt further investigations for establishing a complete ivF protocol in translational mammal model. Study design, size, duration Two researchers expert on reproductive biology generated the Web of Science-Mammals-Made in-vitro folliculogenesis (WoS_MMivF) database including 1111 manuscripts published in peer-reviewed international papers indexed selected in Advanced Search of WoS “Core-collection” by carrying out an independent analysis. Two additional researchers verified the correctness of the records. Participants/materials, setting, methods WoS_MMivF network was built up using Cytoscape 2.6.3 software. The network was analyzed for topological parameters (closeness-centrality, betweenness-centrality and edge count) and to identify key controllers (Hub.BN). Bidimensional-kernel-density-estimation (2D KDE) identifies Hub.BN controllers; Search-Tool-for-the-Retrieval-of-Interacting-Genes/Proteins (STRING) were used to enrich the network with new proteins. Main results and the role of chance The analysis of topological parameters demonstrated that the network is scale-free according to Barabási-Albert-model with a high-degree of robustness-against-random-damage, great controllability and navigability. The network reproduces a coherent framework identifying cross-talking molecules playing a key role in the inter-follicular/intra (somatic and germinal compartment) dialogue. The network allows to organize signalling transduction events/molecules by stratifying them in three layers: input-layer recognizes molecules generating the information flux working as systemic endocrine (pituitary/chorion/enteric-related endocrine hormones) and local paracrine-factors (TGFbeta-superfamily-members and growth-factors) exerting either intrafollicular control or remote feedback on reproductive-cycle. Processing-layer presents molecules able to elaborate/amplify the endocrine/paracrine controllers of ovarian functions, including components of codified intracellular-signaling-pathways like PI3K, KIT and MAPK and second messengers cAMP and Ca2+. These cascades are necessary to promote in-vitro reproducible follicular functions and modulate steroidogenesis, representing molecular events stratified in the output-layer. STRING analysis allowed to extend the regulatory flow of information towards two major biological action contexts: metabolic-control (paracrine-factors and signal-transduction) and angiogenesis. Metabolic-control mediated by mTOR and its interactor cognates FOXO1, FOXO3/SIRT1 plays a key role for ivF, representing the energy sensors of the reproductive cells in hypothalamic-pituitary-ovarian-axis first regulating the status of follicle quiescence/activation and then fate of the structure (specialization or apoptosis). Limitations, reasons for caution - Wider implications of the findings: STRING identified mTOR as key pathway of folliculogenesis, which might act as a molecular-switch to be pharmacologically targeted for potential new in-vitro strategies modulating follicular fate. These results suggest that computational approach in biology might offer perspective in identifying unknown signals, implementing research questions and innovative protocols to face female-fertility. Trial registration number Not applicable

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
R. Krisher ◽  
A. Auer ◽  
K. Clark ◽  
K. Emsweller ◽  
S. Rogers ◽  
...  
Keyword(s):  
South Africa ◽  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Genetic Management ◽  
Blastocyst Development ◽  
Antidorcas Marsupialis

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P &lt; 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.

scholarly journals Metabolic markers of developmental competence for in vitro-matured mouse oocytes

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 475-483 ◽  
Author(s):  
Kimberly A Preis ◽  
George Seidel ◽  
David K Gardner
Keyword(s):  
Lactate Production ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Developmental Competence ◽  
Metabolic Markers ◽  
Oocyte Competence ◽  
Carbohydrate Consumption ◽  
Mouse Oocytes

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus–oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 μl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0–16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8–12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8–16 h of maturation, than by oocytes that did not fertilize (8–12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12–16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

Ex vivo early embryo development and effects on gene expression and imprinting

2005 ◽  
Vol 17 (3) ◽  
pp. 361 ◽  
Author(s):  
David K. Gardner ◽  
Michelle Lane
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Assisted Reproductive Technologies ◽  
Ex Vivo ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Laboratory Animals ◽  
Cell Physiology ◽  
Mammalian Embryo

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus. It has been demonstrated that conditions that alter gene expression, and in some instances the imprinting status of specific genes, have all previously been shown to adversely affect cell physiology. Thus, questions are raised regarding the aetiology of abnormal gene expression and altered imprinting patterns, and whether problems can be averted by using more physiological culture conditions. It is also of note that the sensitivity of the embryo to its surroundings decreases as development proceeds. Post compaction, environmental conditions have a lesser effect on gene function. This, therefore, has implications regarding the conditions used for IVF and the culture of the cleavage stage embryo. The developmental competence of the oocyte also impacts gene expression in the embryo, and therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. Furthermore, the genetics and dietary status of the mother have a profound impact on embryo development and gene expression. The significance of specific animal models for human assisted reproductive technologies (ART) is questioned, given that most cattle data have been obtained from in vitro-matured oocytes and that genes imprinted in domestic and laboratory animals are not necessarily imprinted in the human. Patients treated with ART have fertility problems, which in turn may predispose their gametes or embryos to greater sensitivities to the process of ART. Whether this is from the drugs involved in the ovulation induction or from the IVF, intracytoplasmic sperm injection or culture procedures themselves remains to be determined. Alternatively, it may be that epigenetic alterations are associated with infertility and symptoms are subsequently revealed through ART. Whatever the aetiology, continued long-term monitoring of the children conceived through ART is warranted.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Lalantha R. Abeydeera ◽  
Wei-Hua Wang ◽  
Thomas C. Cantley ◽  
Randall S. Prather ◽  
Billy N. Day
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Thiol Compound ◽  
Nuclear Maturation ◽  
Mean Differences ◽  
Pronuclear Formation

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, β-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 μM BME, 0.5 μg/ml LH, 0.5 μg/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20–22 h and then without hormonal supplements for an additional 20–22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5–6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78–89%). The mean differences in penetration rate (69–77%), polyspermy rate (31–40%), male pronuclear formation rate (93–96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32–39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13–15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.

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