Early rescue oocyte activation for activation-impaired oocytes with no second polar body extrusion after intracytoplasmic sperm injection

2021 ◽  
Author(s):  
Takashi Shibahara ◽  
Yuu Fukasaku ◽  
Naoko Hayashi ◽  
Nozomi Miyazaki ◽  
Hiroaki Kawato ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Activation ◽  
Second Polar Body ◽  
Polar Body Extrusion

scholarly journals Mitochondrial activity and cytoskeleton organization in three pronuclei oocytes after intracytoplasmic sperm injection

Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 319-325
Author(s):  
Tuğba Kotil ◽  
M. Ertan Kervancıoğlu ◽  
Gülçin Ekter Kanten ◽  
Gülden Tunalı ◽  
Seyhun Solakoğlu
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Intracytoplasmic Sperm Injection ◽  
Mitochondrial Membrane ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Mitochondrial Activity ◽  
Electron Microscopic ◽  
Second Polar Body ◽  
Polar Body Extrusion

SummaryDigyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

Intracytoplasmic sperm injection in the rat

Zygote ◽  
1998 ◽  
Vol 6 (2) ◽  
pp. 143-147 ◽  
Author(s):  
D. Dozortsev ◽  
T. Wakaiama ◽  
A. Ermilov ◽  
R. Yanagimachi
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Survival Rates ◽  
Experimental Conditions ◽  
Injection Techniques ◽  
Partial Zona Dissection ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Activation Rates ◽  
Pronuclear Formation

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5–1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 197 ◽  
Author(s):  
SP Flaherty ◽  
D Payne ◽  
NJ Swann ◽  
CD Matthews
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Sperm Head ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Metaphase Chromosomes ◽  
Polar Bodies ◽  
Abnormal Fertilization ◽  
Second Polar Body ◽  
Sperm Chromosomes

The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 245-256 ◽  
Author(s):  
Tetsuo Ono ◽  
Eiji Mizutani ◽  
Chong Li ◽  
Teruhiko Wakayama
Keyword(s):  
Room Temperature ◽  
Polar Body ◽  
Methylation Status ◽  
Cost Effective ◽  
Oocyte Activation ◽  
Male Gametes ◽  
Effective Manner ◽  
Cell Embryo ◽  
Cauda Epididymidis ◽  
Second Polar Body

SummaryThe development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale
Keyword(s):  
Kinase Activity ◽  
Phase 1 ◽  
Polar Body ◽  
Calcium Transients ◽  
Phase 2 ◽  
Phase 3 ◽  
First Polar Body ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Maturation Promoting Factor

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

scholarly journals Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes

1992 ◽  
Vol 102 (3) ◽  
pp. 457-467 ◽  
Author(s):  
J.Z. Kubiak ◽  
M. Weber ◽  
G. Geraud ◽  
B. Maro
Keyword(s):  
Kinase Activity ◽  
Polar Body ◽  
Metaphase Arrest ◽  
Phosphorylated Proteins ◽  
Mouse Oocytes ◽  
Sequence Of Events ◽  
M Phase ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Centrosomal Proteins

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 69-76 ◽  
Author(s):  
S.A. Ock ◽  
J.S. Bhak ◽  
S. Balasubramanian ◽  
H.J. Lee ◽  
S.Y. Choe ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chromosomal Abnormality ◽  
Polar Body ◽  
Ultrastructural Changes ◽  
Time Interval ◽  
Oocyte Activation ◽  
Group 4 ◽  
Group 5 ◽  
Bovine Oocytes

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes

1993 ◽  
Vol 104 (3) ◽  
pp. 861-872 ◽  
Author(s):  
M.S. Szollosi ◽  
J.Z. Kubiak ◽  
P. Debey ◽  
H. de Pennart ◽  
D. Szollosi ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Histone H1 ◽  
Sperm Chromatin ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Spindle Rotation ◽  
Polar Body Extrusion ◽  
Pronuclear Formation

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Xin Tan ◽  
An Peng ◽  
Yong-Chao Wang ◽  
Yue Wang ◽  
Qing-Yuan Sun
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Second Polar Body

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte–second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

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