Ethanol Treatment Efficiently Induces Oocyte Activation and Further Embryonic Development in Rat.

Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Cζ (PLCζ) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca2+]i oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCζ complementary (c) RNA (1, 0.5 or 0.25 μg μL–1) into mare oocytes. The frequency of [Ca2+]i oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 μg μL–1 PLCζ cRNA (41.1 ± 5.3, 47 ± 4.0 and 55.4 ± 9.0, respectively). However, [Ca2+]i oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 μg μL–1 PLCζ cRNA (570.7 ± 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCζ (P > 0.05; range 97–100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 μg μL–1 PLCζ cRNA (3%) than for those injected with 1 μg μL–1 PLCζ cRNA (15%). Based on these results, microinjection of PLCζ may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca2+]i signalling in this species.

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A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

Zygote ◽

10.1017/s0967199408004875 ◽

2009 ◽

Vol 17 (1) ◽

pp. 29-36 ◽

Cited By ~ 5

Author(s):

Daisuke Sano ◽

Yuki Yamamoto ◽

Tomo Samejima ◽

Yasunari Seita ◽

Tomo Inomata ◽

...

Keyword(s):

Wistar Rats ◽

Strontium Chloride ◽

Oocyte Activation ◽

Ethanol Treatment ◽

Blastocyst Formation ◽

Sprague Dawley ◽

High Osmolarity ◽

Optimal Protocol ◽

Artificial Activation

SummaryIn nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

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Toxicity evaluation of ethanol treatment during in vitro maturation of porcine oocytes and subsequent embryonic development following parthenogenetic activation and in vitro fertilization

International Journal of Molecular Medicine ◽

10.3892/ijmm.2014.1924 ◽

2014 ◽

Vol 34 (5) ◽

pp. 1372-1380 ◽

Cited By ~ 5

Author(s):

SANGHOON LEE ◽

EUNHYE KIM ◽

SANG-HWAN HYUN

Keyword(s):

In Vitro Fertilization ◽

Embryonic Development ◽

In Vitro Maturation ◽

Ethanol Treatment ◽

Toxicity Evaluation ◽

Vitro Fertilization ◽

Parthenogenetic Activation

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Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Zygote ◽

10.1017/s0967199402004100 ◽

2002 ◽

Vol 10 (4) ◽

pp. 355-366 ◽

Cited By ~ 53

Author(s):

Kazuhiro Kikuchi ◽

Hans Ekwall ◽

Paisan Tienthai ◽

Yasuhiro Kawai ◽

Junko Noguchi ◽

...

Keyword(s):

Embryonic Development ◽

Oocyte Maturation ◽

Lipid Droplets ◽

Early Embryonic Development ◽

Oocyte Activation ◽

Energy Status ◽

Thin Sections ◽

Porcine Oocyte

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.

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Artificial oocyte activation improves later embryonic development in patients with a history of impaired fertilization

Fertility and Sterility ◽

10.1016/j.fertnstert.2017.07.447 ◽

2017 ◽

Vol 108 (3) ◽

pp. e147-e148

Author(s):

T. Takeuchi ◽

K. Sasamine ◽

S. Mizuta ◽

K. Yamaguchi ◽

Y. Takaya ◽

...

Keyword(s):

Embryonic Development ◽

Oocyte Activation ◽

History Of ◽

Artificial Oocyte Activation

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313 EFFECTS OF ETHANOL TREATMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) ON SPERM AFTER FORMATION AND THE MICROTUBULE ORGANIZATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab313 ◽

2005 ◽

Vol 17 (2) ◽

pp. 307

Author(s):

N. Fujinami ◽

Y. Hosoi ◽

H. Kato ◽

T. Mitani ◽

K. Matsumoto ◽

...

Keyword(s):

Embryonic Development ◽

Intracytoplasmic Sperm Injection ◽

Formation Rate ◽

Ethanol Treatment ◽

Microtubule Organizing Center ◽

Cleavage Rate ◽

Microtubule Organization ◽

Male And Female ◽

Bovine Oocytes ◽

Sperm Aster

The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171–178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that the sperm aster has an important role for the pronuclear movement to make the male and female pronuclei come into close apposition. From these results, it was concluded that oocyte activation after bovine ICSI promoted sperm aster formation and microtubule organization, and was effective to improve embryonic development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

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Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/s0102-09352007000200002 ◽

2007 ◽

Vol 59 (2) ◽

pp. 280-287 ◽

Cited By ~ 2

Author(s):

F. Perecin ◽

S.C. Méo ◽

C.L.V. Leal ◽

J.M. Garcia

Keyword(s):

Embryonic Development ◽

Developmental Competence ◽

Oocyte Activation ◽

Blastocyst Development ◽

Parthenogenetic Activation ◽

Cyclin Dependent Kinases ◽

Activation Rate ◽

Activation Rates ◽

Bovine Oocytes

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

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Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

Reproduction ◽

10.1530/rep.0.1260489 ◽

2003 ◽

pp. 489-499 ◽

Cited By ~ 20

Author(s):

SJ Bedford ◽

M Kurokawa ◽

K Hinrichs ◽

RA Fissore

Keyword(s):

Embryonic Development ◽

Intracellular Calcium ◽

Intracytoplasmic Sperm Injection ◽

Animal Species ◽

Mammalian Species ◽

Calcium Oscillations ◽

Large Animal ◽

Oocyte Activation ◽

Mouse Oocytes ◽

Species Specific

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

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