373 OOCYTE-ACTIVATING CAPACITY OF BOVINE SPERMATOGENIC CELLS AND ACTIVATION PROTOCOLS FOR INTRACYTOPLASMIC INJECTION OF BOVINE ROUND SPERMATIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 302
Author(s):  
C. Kani ◽  
M. Takenaka ◽  
T. Muneto ◽  
M. Yamamoto ◽  
T. Horiuchi
Keyword(s):  
Germ Line ◽  
Oocyte Activation ◽  
Spermatogenic Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
Mouse Oocytes ◽  
Intracytoplasmic Injection ◽  
Bovine Oocytes ◽  
Activation Capacity

In vitro spermatogenesis can be applied to generate spermatids or spermatozoa and produce a genetically modified male germ line. Intracytoplasmic injection of the spermatids or spermatozoa is an important technique for effective production of offspring. The objective of this study is to evaluate oocyte-activation capacity of bovine spermatids or spermatozoa and to determine the effective activation treatment for in vitro development of bovine oocytes injected with round spermatids. Cryopreserved testicular spermatogenic cells and cauda epididymal spermatozoa obtained from a 1-year-old Japanese bull were used. In the first experiment, we injected bovine round (ROS) and elongated (ELS) spermatids, or testicular (TES) and cauda epididymal (CES) spermatozoa into mouse oocytes to examine their oocyte-activating capacity. The presence of pronuclei within whole-mounted oocytes was observed 4 h after injection. In the second experiment, we injected similar spermatids and spermatozoa into bovine oocytes without additional activation, and examined cleavage and blastocyst development. In the third experiment, bovine oocytes injected with ROS were activated with 7% ethanol or 5 �M ionomycin for 5 min (1 � Et or 1 � Iono) immediately after injection; some were further activated repeatedly at 3 h after injection (2 � Et or 2 � Iono), and some of these were subjected to 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h after the second activation (2 � Et + DMAP or 2 � Iono + DMAP). Data were analyzed by the chi-square test in all experiments. The vast majority of bovine ROS failed to activate mouse oocytes (activation rate 10%). Activation rates of mouse oocytes injected with bovine ELS, TES, and CES were 61%, 75%, and 91%, respectively. The results suggest that oocyte-activation capacity is acquired during transformation from ROS to ELS. Cleavage and blastocyst rates of bovine oocytes injected with CES (59% and 19%, respectively) were significantly higher (P < 0.05) than the rates obtained with injections of TES (37% and 9%) and ROS (5% and 0%) without additional activation. However, cleavage and blastocyst rates of bovine oocytes injected with ROS in the groups of 2 � Et + DMAP (80% and 19%) and 2 � Iono + DMAP (76% and 19%) were significantly higher (P < 0.05) than those in the groups of 1 � and 2 � Et (37% and 2%, 59% and 4%), 1 � and 2 � Iono (10% and 7%, 22% and 4%), or those receiving a sham injection and activated with 2 � Iono + DMAP (43% and 4%). These results demonstrate that intracytoplasmic injection of ROS with repeated Et or Iono activation followed by DMAP treatment is more efficient than single or double Et or Iono activation.

332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23

2007 ◽  
Vol 19 (1) ◽  
pp. 281 ◽  
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Cell Number ◽  
Additional Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Chi Square ◽  
Parthenogenetic Embryo ◽  
Pig Oocyte

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P < 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P < 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality. Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa This work was supported by grants ISS-CS11 and Fondazione Cariplo.

scholarly journals Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 295-302 ◽  
Author(s):  
Walt Yamazaki ◽  
Christina Ramires Ferreira ◽  
Simone Cristina Méo ◽  
Cláudia Lima Verde Leal ◽  
Flávio Vieira Meirelles ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Standard Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Reconstructed Embryos ◽  
Developmental Capacity ◽  
Bovine Oocytes

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

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

2007 ◽  
Vol 59 (2) ◽  
pp. 280-287 ◽  
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.

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Osamu Okitsu ◽  
Shuji Yamano ◽  
Toshihiro Aono
Keyword(s):  
Human Sperm ◽  
Human Spermatozoa ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Bovine Sperm ◽  
Female Pronucleus ◽  
Sperm Factor ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Daniel Szöllösi ◽  
Renata Czołowska ◽  
Ewa Borsuk ◽  
Maria S. Szöllösi ◽  
Pascale Debey
Keyword(s):  
Nuclear Envelope ◽  
Transcriptional Activity ◽  
Chromatin Condensation ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
Female Pronucleus ◽  
Mouse Fetuses

SummaryNuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in Go phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25–45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These ‘pronuclei’, similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

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.

scholarly journals Spontaneous Mutagenesis Is Enhanced in Apex Heterozygous Mice

2004 ◽  
Vol 24 (18) ◽  
pp. 8145-8153 ◽  
Author(s):  
Jessica Huamani ◽  
C. Alex McMahan ◽  
Damon C. Herbert ◽  
Robert Reddick ◽  
John R. McCarrey ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Germ Line ◽  
Excision Repair ◽  
Spontaneous Mutant ◽  
Spermatogenic Cells ◽  
Ap Endonuclease ◽  
Repair Activity ◽  
Base Excision ◽  
Mutant Frequency

ABSTRACT Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex +/−) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex +/− lacI + mice compared to frequencies from Apex +/+ lacI + littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex +/− lacI + mice were significantly elevated twofold compared to levels for 9-month-old Apex +/+ lacI + control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

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