• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.361-368
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• 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
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• v.26 no.2
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• pp.105-110
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.353-360
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• 2002

Although successful pronuclear formation and apposition were seen in porcine oocytes following mouse sperm injection, little is known on the morphology of male and female pronuclei following sperm injection. The objective of this study is to describe the ultrastructure of porcine zygote following murine sperm injection in relation to the chronology of pronuclear S phase. At 40h ~ 44h following in vitro maturation, Cumulus cells were removed in TCM-HEPES with 0.1% hyaluronidase. Then, spermatozoa was injected into the cytoplasm of oocytes. After. injection, all oocytes were transferred to NCSU23 medium and cultured at 39$^{\circ}C$ under 5% $CO_2$ in air. Oocytes were fixed in 2% glutaraldehyde in Dulbeccos phosphate-buffered saline and observed by Transmission Electron Microscopy. Nuclear precursor bodies were observed in each pronucleus. A cluster of large and small granules was attached in the nucleolus precursor body. After the apposition of male and female chromatin, chromatin condensation was observed throughout the nucleoplasm and nucleolus precursor bodies and condensed chromatin in contact with clusters of small and large granules and the nuclear envelope were found in apposed pronuclear regions. These results suggest that non-species specific nuclear cytoplasmic interactions take place during pronuclear formation and apposition following sperm injection.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.7-11
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• 2013

Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

• Development and Reproduction
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• v.3 no.1
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• pp.39-44
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• 1999

In the present study, we investigated the effect of maturity and motility of spermatozoa on the formation of pronuc-leus and subsequent developmental capacity of the human embryo in vitro. The fertilization was performed by means of intracytoplasmic sperm injection (ICSI) in HEPES-buffered m-TCM-199 medium. In the first part of the experiment, motile or im-motile human spermatozoa ejaculated were injected into cumulus-enclosed human oocytes matured in vivo. Significantly (p<0.002) higher proportion of oocytes that was injected with motile spermatozoa formed 2 pronuclei than the oocytes injected with immotile spermatozoa (79.8% vs 51.7%). In the second part of the experiment, cumulus-enclosed human oocytes matured in vivo were injected with motile or immotile spermatozoa collected from testes. There was no difference between motile and immotile spermatozoa. In the third part of the experiment, using modified Tyrode's medium containing 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin and 10% human follicular fluid, we found that the development of oocytes that formed 2 pronuclei were able to develop to 9-16 cells regardless of maturity and motility of spermatozoa.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.339-347
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• 2001

Sperm-mediated DNA transfer has a potential to markedly simplify techniques for the generation of transgenic animals. The exogenous DNA transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently introduced in the production of transgenic animals. In this study, the developmental competence and tile expression rates of transgene were investigated after injection of spermatozoon or sperm head with enhanced green fluorescent protein (EGFP) gene into the mature porcine oocytes. The porcine oocytes were injected with intact sperm, membrane-disrupted sperm or sperm head. After injection. embryos were cultured in NCSU23 medium up to the blastocyst stage, and the developmental competence and expression rates were studied. The developmental rate (67.0%) of sperm injection group was higher than that (59.7%) of sperm head injection group, and the rates of EGFP expression were also significantly different between sperm injection and sperm head injection groups (42.1 vs 20.0%) (F＜0.05). In the porcine oocytes injected with sperm treated with different methods of membrane disruption, the removal of sperm membrane did not alter the developmental competence of embryos. The rate of blastocysts at 7 days after injection with intact and membrane disrupted sperm were 15.0 and 14.2%, respectively. The EGFP expression rates, 38.4% in embryos injected with frozen-thawed sperm was higher than that, 22.4% of embryos injected with the Triton X-100 treated sperm. Prior to injection, sperm were cultured in different EGFP gene concentrations from 0.Ol to 1ng/u${mu}ell$. However, no significant difference in developmental rates of embryos among different concentrations of EGFP gene were observed. The highest expression rate of EGFP gene, 37.4% was obtained from the embryos injected with spermatozoa treated with 0.1 ng/${mu}ell$ EGFP gene. These results suggested that exogenous DNA could be attached to the membrane disrupted sperm, and that these sperm could be used as a vector carrying foreign DNA into embryos.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.56-56
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• 2001

This study was carried out to investigate on the improvement of fertilizing and developing ability of in vitro matured oocytes from individuals of bulls, sperm type, pretreatment of sperm or oocytes obtained by intracytoplasmic sperm injection(ICSI). 1. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated individual of bulls were 73.9%-87.0% and 33.3%-60.9%, respectively. 2. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated fresh and frozen sperm, tail-cutting and tail-scoring sperm were 82.0%, 78.0%, 42.2%, 51.1% and 56.0%, 42.0%, 17.8%, 22.2% respectively. and these values of fresh sperm injection were higher than that of frozen sperm, tail-cutting and tail-scoring. 3. The male pronuclear formation and developmental rates of oocytes obtained by sperm pretreated heparin, BFF(bovine follicula fluid), His, Ca Ionophore(Ⅰ) and Ⅰ ＋ caffeine methods were 66.7%-82.2% and 33.3%-60.6%, respectively. and these values of treatment of Ⅰ＋ caffeine were higher than that of other methods. 4. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated with or without zona pellucida were 80.0%, 72.0% and 46.0%, 36.0%, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.4
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• pp.156-162
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• 2015

Objective: To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). Methods: The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). Results: The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). Conclusion: The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.