229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI

2009 ◽  
Vol 21 (1) ◽  
pp. 212
Author(s):  
F. A. Garcia-Vazquez ◽  
K. Aviles-Lopez ◽  
C. Matas
Keyword(s):  
Sperm Head ◽  
Oocyte Activation ◽  
Treatment Groups ◽  
Sperm Penetration ◽  
P 16 ◽  
Low Incidence ◽  
High Level ◽  
Pronuclear Formation ◽  
Sperm Decondensation

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment. Supported by MEC (AGL2006-03495).

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
N. Maedomari ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Oocyte Meiosis ◽  
Freeze Dried ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pre Treatment ◽  
Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

scholarly journals Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 301-305 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Naoya Bandoh ◽  
Shinobu Nakahira ◽  
She-Hoon Oh ◽  
Seiji Tsuboi
Keyword(s):  
High Performance ◽  
Sperm Nucleus ◽  
Treatment Groups ◽  
Intracellular Content ◽  
Sperm Penetration ◽  
Rat Eggs ◽  
Pronuclear Formation ◽  
Glutamyl Transpeptidase ◽  
Increased Activity ◽  
Sperm Decondensation

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 ± 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 ± 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 ± 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 ± 74.1 nmol/egg in unfertilised eggs, 146.0 ± 50.0 nmol/egg in DSH eggs and 39.7 ± 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

Pronuclear formation and intracellular glutathione content of in vitro-matured porcine oocytes following in vitro fertilisation and/or electrical activation

Zygote ◽  
1995 ◽  
Vol 3 (3) ◽  
pp. 273-281 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Todd T. Stumpf ◽  
Thomas C. Cantley ◽  
Nam-Hyung Kim ◽  
Billy N. Day
Keyword(s):  
Electrical Stimulation ◽  
Electrical Activation ◽  
In Vitro Fertilisation ◽  
Oocyte Activation ◽  
Intracellular Content ◽  
Intracellular Glutathione ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pronuclear Formation

SummaryPronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 249-257 ◽  
Author(s):  
Seiji Kito ◽  
Yuki Ohta
Keyword(s):  
Calcium Concentration ◽  
Detrimental Effect ◽  
Choline Chloride ◽  
Positive Control ◽  
Basic Medium ◽  
Vitro Fertilization ◽  
Medium Osmolarity ◽  
Sperm Penetration ◽  
Pronuclear Formation

SummaryTo optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm–ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230–305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25–75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

2002 ◽  
Vol 57 (8) ◽  
pp. 2093-2104 ◽  
Author(s):  
Byung-Ki Kim ◽  
Sang-Chan Lee ◽  
Kwang-Sun Lee ◽  
Bok-Kyu Lee ◽  
Chang-Hee Han ◽  
...  
Keyword(s):  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

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.

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 172 ◽  
Author(s):  
M. J. Sanchez-Calabuig ◽  
P. Beltran-Brena ◽  
E. Martinez-Nevado ◽  
D. Rizos ◽  
J. F. Perez-Gutierrez ◽  
...  
Keyword(s):  
Bottlenose Dolphin ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Sperm Chromatin ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 149 ◽  
Author(s):  
A. Quiñones Martorello ◽  
G. Rios ◽  
A. Cano ◽  
R. H. Alberio
Keyword(s):  
Zona Pellucida ◽  
Calcium Influx ◽  
Oocyte Activation ◽  
High Concentration ◽  
Hoechst 33342 ◽  
Polar Bodies ◽  
Dmso Concentration ◽  
Sperm Penetration ◽  
Bovine Oocytes

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

scholarly journals Defective sperm head decondensation undermines the success of ICSI in the bovine

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 307-318 ◽  
Author(s):  
Luis Águila ◽  
Ricardo Felmer ◽  
María Elena Arias ◽  
Felipe Navarrete ◽  
David Martin-Hidalgo ◽  
...  
Keyword(s):  
Calcium Oscillations ◽  
Sperm Head ◽  
Oocyte Activation ◽  
Bovine Sperm ◽  
Bovine Oocytes ◽  
Mouse Eggs ◽  
Do So ◽  
Nuclear Decondensation

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
N. Maedomari ◽  
K. Kikuchi ◽  
M. Fahrudin ◽  
N. Nakai ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
First Polar Body ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
And Control

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

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