153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca2+ Fluorophore in Cumulus Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 216
Author(s):  
H. J. McLennan ◽  
M. L. Sutton-McDowall ◽  
S. Heng ◽  
J. G. Thompson
Keyword(s):  
Xenopus Oocytes ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Molecular Probes ◽  
Vitelline Membrane ◽  
Oocyte Activation ◽  
Sperm Binding ◽  
Significant Difference ◽  
The Media

During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P < 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca2+ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca2+ at the time of oocyte activation.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Oocyte Growth ◽  
North West ◽  
Significant Difference ◽  
Cytoplasmic Maturation

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

scholarly journals Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 725-735
Author(s):  
Julieta Gabriela Hamze ◽  
María Jiménez-Movilla ◽  
Raquel Romar
Keyword(s):  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
In Vitro Fertilisation ◽  
Sperm Binding ◽  
Fertilisation Rate ◽  
Gamete Recognition ◽  
Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

229 EXPRESSION OF mRNA ENCODING GLYCOLYTIC ENZYMES IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FSH

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
E. S. Caixeta ◽  
P. Ripamonte ◽  
M. F. Machado ◽  
R. B. da Silva ◽  
C. Price ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
In Vitro Maturation ◽  
Housekeeping Gene ◽  
Cumulus Cells ◽  
Glycolytic Enzymes ◽  
Mrna Abundance ◽  
Relative Expression ◽  
Significant Difference

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.

160 Exposure to Cadmium Affects Oocyte and Embryo Competence in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 219
Author(s):  
C. De Canditiis ◽  
N. Pagano ◽  
V. Franco ◽  
I. Paradiso ◽  
É. C. Dos Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Dapi Staining ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Vitro Model ◽  
Hoechst Staining

There is a growing worldwide concern regarding the increased release of the heavy metal cadmium (Cd) in the environment, due to several industrial processes, as it is known to affect health. Among other heavy metals, Cd is widely recognised to influence the reproductive system at different levels, interfering with both gametes and embryo functions in several species (Thompson and Bannigan, 2008 Reprod. Toxicol. 25, 304-315). The in vitro model can be used to mimic environmental conditions allowing us to evaluate their effect on oocyte maturation and early embryo development. Therefore, the aim of this study was to evaluate the influence of different Cd concentrations on nuclear maturation, apoptosis in cumulus cells, and cleavage and blastocyst yields in cattle. For this purpose, abattoir-derived bovine oocytes were in vitro matured, fertilized, and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). In particular, oocytes were matured with 0 (control; n = 126), 0.1 μM (n = 139), 1 μM (n = 134), and 10 μM of Cd (n = 135), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. For each replicate, after 22 h of maturation, a representative sample of oocytes (n = 10 per each group) was used to evaluate nuclear maturation by 4′,6-diamidino-2-phenylindole (DAPI) staining and another sample (n = 10 per each group) to assess cumulus-cells complex apoptosis by TUNEL/Hoechst staining (Pocar et al. 2005 Reproduction 130, 857-868). The remaining oocytes were in vitro fertilized and cultured with 0 (n = 106), 0.1 μM (n = 119), 1 μM (n = 114), and 10 μM (n = 115) Cd. The experiment was repeated 3 times. On Day 8 post-IVF, the blastocyst yields were recorded. Differences among groups were analysed by ANOVA, with the least significant difference method used as a post hoc test. Data are presented as means ± SE. Unexpectedly, the exposure of oocytes to Cd during IVM did not affect the percentage of oocytes undergoing nuclear maturation (on average 96.3 ± 2.3). In contrast, concentrations of 1 and 10 μM Cd increased the percentage of apoptotic cumulus-cells in cumulus–oocyte complexes (COC) compared with the control (3.4 ± 0.4, 10.6 ± 1.8, 15.0 ± 0.9, 16.7 ± 4.0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.05). It is worth pointing out that with the highest concentration, cumulus expansion did not occur and cumulus cells appeared detached from the oocyte. Likewise, 1 and 10 μM Cd decreased cleavage rates compared with the control (68.7 ± 1.8, 54.3 ± 5.0, 58.5 ± 4.2 and 2.8 ± 2.6, respectively, with 0, 0.1, 1, and 10 μM Cd; P < 0.01). Finally, blastocyst yields decreased when oocytes were treated with 0.1 μM Cd and no development to blastocyst was observed at the 2 higher concentrations (35.1 ± 1.7, 26.2 ± 3.1, 0, 0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.01). In conclusion, exposure to Cd during maturation negatively affects bovine COC, as indicated by the increased apoptotic index in cumulus cells, without influencing the nuclear maturation process. Furthermore, the presence of Cd during in vitro fertilization and culture severely impairs both the fertilization and post-fertilization embryo development.

Autologous embryo–cumulus cells co-culture and blastocyst transfer in repeated implantation failures: a collaborative prospective randomized study

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 173-180 ◽  
Author(s):  
M. Benkhalifa ◽  
A. Demirol ◽  
T. Sari ◽  
E. Balashova ◽  
M. Tsouroupaki ◽  
...  
Keyword(s):  
Embryo Development ◽  
Randomized Study ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Prospective Randomized Study ◽  
Human Embryos ◽  
Feeder Cells ◽  
Positive Role ◽  
Significant Difference

SummaryIn repeated implantation failure, the co-culture of human embryos with somatic cells has been reported to promote the improvement of embryos quality, implantation and pregnancy rate. It was reported that feeder cells can be more beneficial to the oocyte and embryo by detoxifying the culture medium and supporting embryo development via different pathways. In this study, 432 patients, each with a minimum of three repeated implantation failures, were accepted for a prospective randomized study with or without autologous cumulus cell embryo co-culture and transfer at day 3 or day 5–6. We also investigated the expression of leukaemia inhibitor factor (LIF) and platelet activating factor receptor (PAF-R) on day 3 confluent cumulus cells. The statistic analysis of the data showed significant difference of implantation and clinical pregnancy rates between classical culture and day 3 compared with co-culture and day 5–6 transfer. The molecular analysis showed that cumulus cells express the LIF and the PAF-R genes and confirmed the possible positive role of growth factors and cytokines in early embryo development. Embryo co-culture systems with autologous cells can be beneficial in routine in vitro fertilization for embryo selection and implantation improvement. More molecular investigations need to be done to improve elucidation of the complex dialogue between the embryo and feeder cells prior to implantation and to understand the involved biological function and molecular process during embryo development.

Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis

2017 ◽  
Vol 8 (2) ◽  
pp. 61-67
Author(s):  
Harsha K Bhadarka ◽  
Nayana H Patel ◽  
Kruti B Patel ◽  
Nilofar R Sodagar ◽  
Yuvraj D Jadeja ◽  
...  
Keyword(s):  
Time Lapse ◽  
Mean Value ◽  
Primary Objective ◽  
Sperm Cryopreservation ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Day 3 Embryo

ABSTRACT Aim In recent past, many studies had come up with the combination of time-lapse (TL) imaging of embryo morphokinetics as a noninvasive means for improving embryo selection and in vitro fertilization (IVF) success. The primary objective of the study was to find out if there is significant variation in morphokinetics of embryos with different implantation potential and also to study the effect of sperm freezing on time points of embryogenesis events in embryos with implantation potential. Materials and methods Kinetic data and cycle outcomes were analyzed retrospectively in 142 patients who had undergone IVF/intracytoplasmic sperm injection (ICSI) cycles using semen with normal parameters and embryo transfer (ET) on day 3. For the surety of specificity of morphokinetics, only cases with single ET cycles were included in the study. Timing of specific events, from the point of ICSI, was determined using TL imaging. Kinetic markers like time to syngamy (t-pnf), t2, time to two cells (c), 3c (t3), 4c (t4), 5c (t5), 8c (t8), tMor, CC2, CC3, t5–t2, t5–t4, s1, s2, and s3 were calculated. The cleavage synchronicity from the 2–8 cell stage (CS2–8), from 4 to 8 cell stage (CS4–8), and from 2 to 4 cell stage (CS2–4) were calculated as defined elsewhere. Deoxyribonucleic acid replication time ratio (DR) was also included in the comparison. Analysis of variance test was used for comparison of the mean timing of cell division and cell cycle intervals. Results Morphokinetics t-pnf, t2, t8, CC2, S2, S3, CS2–8, CS4–8, and CS2–4 differed significantly between embryos with and without implantation potential, when embryos were developed using fresh semen, while t3, t4, t5, CC2, S2, t5–t2, CS2–4, and DR differed significantly between the embryos with and without implantation potential when frozen semen was used. No significant difference was found in mean value of any of the above-stated parameters when comparison was done between implanted embryos fertilized by either fresh or cryopreserved sperm. Conclusion Many morphokinetics parameters of embryogene­sis vary significantly between embryos with different ability to implant; therefore, the criteria developed in our IVF lab can be useful for selection of suitable embryo even at day 3 of development with more chances of implantation. Clinical significance Study indicates necessity of development of individualized selection model based on morphokinetics for every IVF lab and also confirms freezing as an important tool for fertility preservation of males as it does not affect events of embryogenesis. How to cite this article Bhadarka HK, Patel NH, Patel KB, Sodagar NR, Jadeja YD, Patel NH, Patel MN, Patel AV, Patel DH, Patel JS. Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis. Int J Infertil Fetal Med 2017;8(2):61-67.

scholarly journals 236LOCALIZATION OF INTERFERON-TAU IN BOVINE EMBRYOS AND CUMULUS CELLS BY CONFOCAL MICROSCOPY

2004 ◽  
Vol 16 (2) ◽  
pp. 239 ◽  
Author(s):  
K.M. Johnson ◽  
X. Alvarez ◽  
H.M. Kubisch
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Regulation Of Gene Expression ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Interferon Tau ◽  
Mdbk Cells

Interferon-tau (IFN-t) is a protein secreted by the conceptus of ruminant species and thought to be the primary signal in maternal recognition of pregnancy. Experiments were conducted to detect IFN-t in bovine oocytes, cumulus cells and embryos by use of immunocytochemistry and laser-scanning confocal microscopy. Embryos were produced by in vitro fertilization of in vitro-matured oocytes. Oocytes and embryos were fixed in formaldehyde at various stages of development, and stored in PBS until staining and microscopy. Cumulus cells were stripped from immature oocytes and cultured in M-199 (10% fetal calf serum) on coverslips treated with poly-D-lysine. They were divided into four treatment groups: (1) without hormones (control), (2) with the addition of FSH, (3) estradiol, or (4) FSH and estradiol. Bovine MBDK cells (ATCC CCL 22) and primary fibroblasts were cultured as controls on coverslips but without addition of hormones. A polyclonal antibody raised against bovine IFN-t was used, followed by a secondary conjugated antibody (AlexaFluor 488, Molecular Probes, Eugene OR). Actin was stained with phalloidin (AlexaFluor 568, Molecular Probes, Eugene OR). Cumulus, MDBK cells and fibroblasts were further stained with propidium iodine to visualize nuclei. Imaging was performed on a Leica laser-scanning confocal microscope. IFN-t was detected in hatched and unhatched Day 7 and Day 9 blastocysts, where its expression was restricted to the trophectoderm. IFN-t was also found in Day 5 and 6 morulae, but not at earlier stages. Furthermore, IFN-t was detected in the cumulus cell masses of oocytes before and after IVM, but not in the oocyte itself. Controls, in which the primary antibody was omitted, were negative regardless of developmental stage. IFN-t was also found in cultured cumulus cells regardless of whether hormones had been added to the medium;; however, the protein was localized in the nuclei of cells only if they had been cultured with FSH, whereas in cells cultured with estrogen alone or without hormones IFN-t was restricted to the cytoplasm. In contrast, no IFN-t was detected in MDBK cells or fibroblasts. These results extend previous findings by showing that IFN-t is expressed as early as the morula stage. Moreover, these results demonstrate that IFN-t is also produced by cumulus cells where FSH appears to initiate a translocation of IFN-t into the nucleus, suggesting a role in regulation of gene expression.

scholarly journals Assessment of Intracellular Calcium and Plasmalemmal Membrane Potential in Cryopreserved Metaphase II Mouse Oocytes

2020 ◽  
Author(s):  
Omar Farhan Ammar ◽  
Therishnee Moodley
Keyword(s):  
Membrane Potential ◽  
Xenopus Oocytes ◽  
Channel Blocker ◽  
Oocyte Activation ◽  
Human Oocytes ◽  
Mouse Oocytes ◽  
Failed Fertilization ◽  
Intracellular Ca ◽  
In Vitro Ageing

Abstract Objectives: Ca2+ is critical for normal oocyte activation and fertilization, and any alteration to the Ca2+ homeostasis may lead to failed fertilization or even cell death. It has been shown that intracellular Ca2+ is increased in bovine and human oocytes when cultured in vitro. Additionally, ATP sensitive potassium channels have been characterised recently in human and Xenopus oocytes. Glibenclamide a KATP channel blocker was shown to protect human oocytes from Ca+2 overloading via inhibition of plasmalemmal KATP channels. This research note aims to demonstrate the effects of oxidative stress and in vitro ageing on the intracellular Ca+2 and plasmalemmal membrane potential dynamics in cryopreserved metaphase II (MII) mouse oocytes. Also, this study aims to show if glibenclamide (a KATP channel blocker ) has a role in regulating intracellular Ca+2 and plasmalemmal membrane potential through KATP channels in cryopreserved metaphase II mouse oocytes.Results: our data did not show an increase in intracellular Ca2+ in untreated cryopreserved mouse oocytes loaded with Fluo-3 AM dye. However, an increase in the plasmalemmal membrane potential was noticed (hyperpolarization). Glibenclamide has shown no significant effect on Ca2+ and plasmalemmal membrane potential.

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