scholarly journals 272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES

2004 ◽  
Vol 16 (2) ◽  
pp. 256
Author(s):  
I. Hayasaka ◽  
N. Yoshimoto ◽  
Y. Mori ◽  
K. Suzuki ◽  
R. Honda ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Morula Stage ◽  
The One ◽  
Estradiol Levels

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

103 BIRTH OF CLONED PIGLETS DERIVED FROM AN OPTIMIZED IN VITRO BLASTOCYST PRODUCTION SYSTEM BY TRICHOSTATIN A TREATMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
K. Zhang ◽  
J. Li ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Culture Media ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Deacetylase Inhibitor ◽  
The One ◽  
Effective Group

The present study was designed to examine the effect of trichostatin A (TSA, a histone deacetylase inhibitor) treatment on in vitro developmental ability of pig cloned embryos and to evaluate the feasibility of producing piglets from these embryos. Cell lines were established from 40-day-old fetuses, and adult ear skin was used as nuclear donor. In vitro-matured oocytes from abattoir-derived sow ovaries were used as cytoplast recipients for micromanipulator-assisted somatic cell nuclear transfer (SCNT). Data were analyzed by using SPSS (11.0) with one-way ANOVA, and each experiment was replicated at least 3 times. In Experiment 1, immediately after simultaneous fusion and activation, the reconstructed couplets were randomly cultured in porcine zygote medium 3 (PZM3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) with 10 �g mL-1 cytochalasin B (CB), 10 �g mL-1 cycloheximide (CHX), and 0 nM, 5 nM, or 50 nM TSA for the first 4 h. Cloned embryos (fused reconstructed couplets) were moved to the same culture media but without CB and CHX and further cultured at 38.5�C, under 5% CO2, 5% O2, 90% N2 and 100% humidity. After incubation for a total of 8–14 h in 50 nM, 19–24 h in 50 nM or 5 nM, and 31–36 h in 50 nM TSA in PZM3 (0 nM TSA serves as control for each group), the embryos were further cultured in vitro without TSA in PZM3 for up to 168 h. Cleavage and blastocyst development rates, based on embryos cultured, were recorded at 48 and 168 h of IVC, respectively. Results showed that 50 nM TSA treatment for 19-24 h supported a higher blastocyst development rate than the control group [No. blastocysts/No. embryos cultured (mean � SEM): 107/258, 47.4 � 5.9% vs. 65/324, 20.0 � 2.3%, respectively; P < 0.05], whereas similar pre-implantation development was obtained between the other 3 test groups and the control. In Experiment 2, TSA-treated cloned embryos at the one-cell stage or blastocyst stage were transferred to recipients to examine the possibility of producing piglets. Ten cloned piglets (2 are healthy and 8 died shortly after birth) and one ongoing pregnancy were obtained from 3 recipients who received an average of 110 one-cell stage embryos, whereas 4 piglets originating from traditional cloning were produced from one recipient which received 28 traditional cloned blastocysts (produced from the effective group in Experiment 1) and 30 handmade but non-TSA-treated ones. Our data demonstrate that TSA treatment after SCNT in porcine can significantly improve the in vitro blastocyst production, and embryos treated with TSA could support full-term development and result in healthy offspring.

scholarly journals Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin

Reproduction ◽  
2003 ◽  
pp. 111-118 ◽  
Author(s):  
BD Higgins ◽  
MT Kane
Keyword(s):  
Gene Activation ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Sodium Dependent ◽  
Morula Stage ◽  
Zygotic Gene ◽  
Zygotic Gene Activation ◽  
The One ◽  
Inositol Uptake

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
C. Sauvegarde ◽  
D. Paul ◽  
R. Rezsohazy ◽  
I. Donnay
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mature Oocyte ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Immature Oocyte ◽  
Cell Stage ◽  
Morula Stage ◽  
Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

195 SUPPRESSION OF SETDB1 DURING BOVINE EMBRYONIC DEVELOPMENT RESULTS IN PRE-IMPLANTATION MORTALITY AND DECREASED TRANSCRIPTION OF TRANSPOSABLE ELEMENTS

2015 ◽  
Vol 27 (1) ◽  
pp. 188
Author(s):  
M. D. Snyder ◽  
J. H. Pryor ◽  
K. J. Veazey ◽  
M. D. Peoples ◽  
G. L. Williamson ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Transposable Elements ◽  
Fluorescent Microscopy ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Small Interfering Rnas ◽  
Treatment Groups ◽  
Commercial Supplier ◽  
Morula Stage ◽  
Student’S T

Organization of chromatin structure by the combinatorial patterns of DNA methylation and post-translational histone modification is essential for the establishment and maintenance of proper transcriptional programs that result in the coordination of embryonic development. We previously observed that suppression of transcripts encoding SET domain, bifurcated 1 (SETDB1) using small interfering RNAs (siRNA) is embryonic lethal, with SETDB1-suppressed embryos (n = 361) arresting immediately before the blastocyst stage (blastocyst rate: Control 44.9 ± 4.9% and NULL injected 25.7 ± 6.0%). Studies in rodents indicate SETDB1 is a crucial regulator of transposable elements and that the precise epigenetic regulation of these elements is a key aspect of transcriptional programs controlling pluripotency and placentation. To better characterise the molecular basis of the observed mortality, we analysed expression of the bovine Long Interspersed Nuclear Element 1 family (LINE1) of transposable elements via quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). Mature bovine oocytes were obtained from a commercial supplier (De Soto Biosciences, Seymour, TN, USA) and IVF performed by standard laboratory protocol. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes divided into 3 different treatment groups: non-injected control (CNTL), non-targeting siRNA injected control (siNULL), and zygotes injected with siRNAs targeting SETDB1 (siSETDB1). Each embryo was injected with ~100 pL of siRNAs (10 µM) in fluorescent dextran solution. All zygotes were verified as injected by fluorescent microscopy and then cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) medium supplemented with 4 mg mL of BSA (Probumin, EMD Millipore, Darmstadt, Germany). Groups of embryos (15–20) from each treatment were lysed at the 4-cell, 8-cell, and morula stages, RNA extracted, and analysed by RT-qPCR using GAPDH and YWHAZ as reference genes. A two-way ANOVA and a Student's t-test were used to analyse the results from the RT-qPCR. As expected, siSETDB1-injected morulae displayed dramatic reduction in the level of Setdb1 transcripts as compared to siNULL control (96%; P < 0.05). Preliminary analysis of LINE1 transcripts at the morula stage indicated siSETDB1-injected embryos displayed a 75% reduction compared to the siNULL. Whether alteration in LINE1 regulation contributes to the developmental arrest and embryonic mortality of siSETDB1-injected embryos is under investigation.

190 EXPRESSION OF GENES ASSOCIATED WITH WINGLESS-RELATED MOUSE MAMMARY TUMOUR VIRUS SIGNALING IN THE PRE-IMPLANTATION BOVINE EMBRYO

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
P. Tribulo ◽  
J. I. Moss ◽  
P. J. Hansen
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
Wnt Signalling ◽  
Mammary Tumour ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Mouse Mammary Tumour Virus ◽  
Ct Value ◽  
Morula Stage ◽  
Canonical Wnt

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔCT values, which were calculated by subtracting the CT value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the CT value of the sample. The relative transcript abundance was calculated as the 2ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development. Research was supported by USDA-NIFA 2011-67015-30688.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

IVM of mouse fully grown germinal vesicle oocytes upon a feeder layer of selected cumulus cells enhances their developmental competence

2019 ◽  
Vol 31 (6) ◽  
pp. 1068
Author(s):  
Federica Cavalera ◽  
Milena Simovic ◽  
Mario Zanoni ◽  
Valeria Merico ◽  
Silvia Garagna ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Development Rate ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Developmental Competence ◽  
Mouse Oocytes ◽  
Feeder Layers ◽  
Morula Stage ◽  
Bidirectional Exchange

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus–oocyte–complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.

scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

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