• Korean Journal of Animal Reproduction
• /
• v.14 no.2
• /
• pp.101-106
• /
• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Korean Journal of Animal Reproduction
• /
• v.22 no.2
• /
• pp.195-202
• /
• 1998

We demonstrated, for the first time, pronuclear formation and apposition in porcine ooc-ytes following intracytoplasmic injection of porcine, human, bovine and mouse spermatozoon. Microtubule organization and chromatin configuration were investigated in these oocytes during pronuclear apposition. Following intracytoplasmic injection of porcine spermatozoon, the microtubular aster was organized from the neck of spermatozoon, and filled the whole cytoplasm. This male derived microtubules appear to move both pronuclei to the center of oocytes. In contrast, following injection of spermatozoa from different species such as human, bovine and mouse, microtubules were organized from the cortex of the oocytes and concentrated to the pronuclei, which seems to move both male and female pronuclei to the center of oocyte. This organization is similar to what has been shown in the parthenogenetically activated por-cine oocytes. These results suggested that the porcine, human, bovine and mouse sperm chromatin can be formed pronucleus and apposited in the center of oocytes in the absence of male derived microtubule when they were injected into porcine oocytes.

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.361-368
• /
• 2002

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilization. After 44h in vitro maturation, spermatozoa was injected into the cytoplasm of oocytes. After injection, all oocytes were transferred to NCSU23 medium and cultured at 39'E under 5% CO2 in air. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8 to 9 h following the injection of porcine sperm, and 6 to 8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte center. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. These results suggested that DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, and the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.1-7
• /
• 2002

The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Tris­buffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The sperm­ich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM­199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU­23 medium and inseminated in mTBM medium showed superior in­vitro fertilization compared to those matured in mTCM­199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.

• Korean Journal of Animal Reproduction
• /
• v.23 no.3
• /
• pp.263-270
• /
• 1999

This study was conducted to investigate effects of L-ascorbic acid and selenium on maturation, fertilization, and development ablity of porcine follicular oocytes in vitro. When the follicular oocytes were cultured in the media containing 0, 62.5, 100 and 300 $\mu$M of L-ascorbic acid for 40~44h, the percentages of germinal vesicle breakdown were 86.8, 92.9, 91.7 and 92.6% respectively, and the nuclear maturation rates (M II) were 44.7, 57.1, 52.8 and 53.7%. The nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). When the follicular oocytes were cultured at 0, 0.4, 0.8, and $1.5\mu$M of selenium for 40~44h, the nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium, the incidence of male pronuclear formation was significantly increased (p＜0.05) and polyspermy rate was significantly decreased (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium increased the clevage rate, morula and blastocyst rate (p＜0.05). These results suggested that the addition of L-ascorbic acid and selenium to maturation medium increase the nuclear maturation rates, male pronuclear formation and normal embryonic development: in porcine oocytes matured and fertilized in vitro.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.2
• /
• pp.164-167
• /
• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Journal of Embryo Transfer
• /
• v.25 no.2
• /
• pp.97-101
• /
• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.3
• /
• pp.301-309
• /
• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.

• Development and Reproduction
• /
• v.4 no.1
• /
• pp.7-12
• /
• 2000

At fertilization, sperm penetrates into oocyte, male and female pronuclei are fused together, and mitotic division follows. However, little information is available on the interactive roles and dynamic processes between cytoplasmic and nuclear components during the pronuclear formation, migration and cell division. The assisted reproductive technologies such as, intracytoplasmic sperm injection (ICSI) and round spermatid injection(ROSI) could provides new treatments for the male infertility as well as tools for the study of basic mechanism during fertilization. Nuclear transfer can also provide a mechanism on the interactive roles between nucleus and cytoplasm since the process includes nuclear reprogrammming of differentiated cells in the enucleated oocytes. Recently, I have investigated developmental processes in porcine oocytes following fertilization parthenogenesis, ICSI, ROSI and nuclear transfer using indirect immunocytochemical and electron microscopic studies. The results could provide an insight into biological questions related with epigenesis as well as strategies for the enhancement of embryology in general such as ICSI and nuclear transfer.

• Journal of Animal Science and Technology
• /
• v.47 no.3
• /
• pp.363-370
• /
• 2005

Experiments were conducted to determine the effects of beta-mercaptoethanol ($\beta$-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and $\beta$-ME (0, 25, 50 and 100 ${\mu}$M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 ${\mu}$M $\beta$-ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing $\beta$-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.