The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or -196 degrees C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for I h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 degrees C (11%) and +25 degrees C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (>= 10 spikes/h) was higher in the non-dried control group (79%; P < 0.05) than in the FD groups (58, 55, and 54% for storage at -196, +4, and +25 degrees C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12 h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P < 0.05) resumed meiosis than those injected with +25, +4 and -196 degrees C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P < 0.05) than those in all the FD groups (34, 27, and 28% for storage at -196, +4, and +25 degrees C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or -196 degrees C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.ArticleTHERIOGENOLOGY. 71(3):543-552 (2009)journal articl

Multiple aster formation is frequently observed in bovine oocytes retrieved from 1 day-stored ovaries

We have recently reported that multiple aster formation after IVF was one of the factors negatively affecting to developmental competence of vitrified-warmed bovine matured oocytes, and that a short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF can be involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to Day 8 blastocysts when compared with those from fresh ovaries (37 vs 63%). Immunostaining against -tubulin 10 h post-IVF revealed that the higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than fresh ovaries (31 vs 15%). Treatment of post-IVM oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 vs 32% in control group). However, the suppressing effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 vs 37% in control group). These results suggest that higher incidence of multiple aster formation by bovine ovary storage was not responsible for decreased developmental competence of IVF oocytes.ArticleZYGOTE.24(1):115-120(2015)journal articl

A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 degrees C for 24 h. After ICSI, oocytes were treated with 5 mu M ionomycin for 5 min, 7% ethanol for 5 or 10 min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 mu g/mL cycloheximide for 5 h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3 h. Across the activation regimens, the cleavage rates of ICSI oocytes (45% to 77%) were higher than those of parthenogenetically activated oocytes (11% to 21%; P 0.05). Higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen using two triggers for single calcium increase effectively improved blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.ArticleTHERIOGENOLOGY. 72(4):453-460 (2009)journal articl

Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year

ArticleMolecular Reproduction and Development. 75(5): 890-894 (2008)journal articl

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.ArticleTHERIOGENOLOGY. 77(5):908-915 (2012)journal articl

Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization

Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified–warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus–oocyte complexes with β-mercaptoethanol (βME) and l-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified–warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without βME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with βME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified–warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.ArticleZYGOTE. 22(4):476-482 (2014)journal articl

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.ArticleTHERIOGENOLOGY. 74(6):1028-1035 (2010)journal articl

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium unproved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol. 15% DMSO and 0 5 M sucrose When post-warm blastocysts were cultured in inSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0 05) by the addition of 10 mu M Y-27632 (94 9 +/- 2 4%, mean +/- SEM) compared to a control (78 0 +/- 6 0%) Conversely. after 48 h of culture, there were no significant differences in hatching late (62.8 +/- 11 1 vs 59 6 +/- 9.4%) and mean total cell number (135 2 +/- 13 1 vs. 146 7 +/- 13 3) In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91 7 +/- 3 8 vs 54 7 +/- 8 9%. P < 0 05). with no difference in mean total cell number of blastocysts (230 0 +/- 23 0 vs 191 2 +/- 22 2, P = 0 23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2. or Day 4 (and remained present until Day 8). resulting in no improvement in blastocyst yield compared to a control group (7.5 +/- 2 1, 31 4 +/- 2 3, 36 2 +/- 3.2. and 28.6 +/- 6.9%. respectively) In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warmingArticleTHERIOGENOLOGY. 73(8):1139-1145 (2010)journal articl