• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.41-41
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• 2001

Chromatin configuration and microtubule assembly were determined in porcine maturing and activated oocytes following intracytoplasmic sperm injection. Microtubule localization was confirmed using a mouse monoclonal antibody to $\alpha$-tubulin and detected using a fluorescent labeled goat anti-mouse secondary antibody. DNA was stained with propidium iodide. The image of microtubules and chromatin was captured using laser scanning confocal microscope. In germinal vesicle stage oocyte, sperm chromatin remained condensation and sperm derived microtubules were not observed at 8 to 12 h after sperm injection. At 24 h after injection, the sperm nucleus developed to the metaphase chromatin along the metaphase structure of female nucleus. In some metaphase I stage oocytes, sperm chromatin decondensed at 8 h to 12 h after injection, sperm aster was seen soon after sperm injection. At 24 h after sperm injection into metaphase I stage oocyte, male chromatin developed to the metaphase chromatin while female chromatin extruded first polar body and formed the metaphase chromatin. At 12 to 15 h after sperm injection into preactivated oocytes, condensed sperm nucleus was located in close proximity of female pronucleus. However, the condensed nucleus did not fuse with female pronucleus. In preactivated ocytes, injected sperm remained condensation, a few sperm organized small microtubular aster. Instead, maternal derived microtubules were organized near the female chromatin, which seem to move condensed male chromatin near to the female pronucleus. These results suggest that sperm nuclear decondensing activity and nucleation activity of centrosome during fertilization are cell cycle dependent. In absence of male functional centrosome, female origin centrosome takes over the role of microtubule nucleation for nuclear movement.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.447-456
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• 1999

Objective: The purpose of this study was to evaluate outcome of intracytoplasmic sperm injection (ICSI) using epididymal and testicular sperm in patients with azoospermia. Methods: From March, 1993 to May, 1999, a retrospective clinical analysis was done of a total of 140 cycles in 112 patients who underwent ICSI. Subjects were divided into three groups: ejaculated-ICSI group included 42 cycles in 34 patients with ejaculated sperm who underwent ICSI due to severe oligospermia and past history of failed or poor fertilization in the previous in vitro fertilization and embryo tranfer (IVF-ET) cycles, microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection (MESA-ICSI) group included 50 cycles in 42 patients with congenital absence of the vas deferens (CAVD) or unreconstructable obstructive azoospermia and testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) group included 48 cycles in 36 patients with no spermatozoa which can be retrieved from epididymis or non-obstructive azoospermia. Results: Normal two-pronuclear fertilization rates were similar in three groups: 64.4% for ejaculated-ICSI group, 59.4% for MESA-ICSI group and 60.4% for TESE-ICSI group. The pregnancy rates were 26.2%, 26.0% and 25.0% respectively. There were no significant differences in the fertilization, cleavage, and clinical pregnancy rates among ICSI cycles using ejaculated, epididymal and testicular sperm. Conclusion: Epididymal and testicular sperm obtained in azoospermic patients can fertilize oocyte successfully and may lead to be similar fertilization rates and clinical pregnancy rates to ejaculated sperm.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.359-364
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• 1999

Recent advances in intracytoplasmic sperm (ICSI) and round spermatid injection (ROSI) would provide exciting opportunities not only for the male infertility but also for studying gamete physiology during fertilization and early development. Furthermore, intracytoplasmic sperm injection could be used to produce transgenic animal (Perry et al., 1999). However, it is not clear in the fertilization processes in mammalian oocytes following intracytoplasmic injection of spermatozoon, isolated sperm head or round spermatid. (omitted)

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.12-12
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• 2001

To get insight into the nature of foreign mitochondria and syngamy during mammalian fertilization we compared fertilization processes in porcine oocytes following microinjection of porcine or mouse spermatozoa. Pronuclear movement, sperm mitochondria, and DNA synthesis were imaged with propidium iodide, mitotracker, and BrdU under confocal laser scanning microscope. Intracytoplasmic injection of either porcine or mouse spermatzoon activated porcine oocytes without additional parthengenetic stimulation. Foreign mitochondria in either mouse or porcine sperm midpiece were introduced into porcine oocytes following sperm injection, but rapidly disappeared from the actively developing porcine oocytes. BrdU experiment showed new DNA synthesis in porcine oocytes following injection of mouse spermatozoon or sperm head. At 24 h after injection of mouse isolated sperm head or a spermatozoon, mitoic metaphase was seen in oocyte, but they did not go to normal cell division (Table). These results suggest that pronuclear formation, foreign mitochondria disruption, DNA synthesis and syngamy formation during fertilization are not species specific processes.(Table Omitted).

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Asian-Australasian Journal of Animal Sciences
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• v.4 no.3
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• pp.251-254
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• 1991

Immobilized (killed) mouse spermatozoa or sperm head were microinjected into mouse oocytes matured in vivo and cultured for 72h in vitro. When non-capacitated spermatozoon was injected, oocytes that developed to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 27.8 (15/54) and 3.7% (2/54), respectively. When non-capacitated sperm head was injected. development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.3 (16/75) and 8.0% (6/75), respectively. When capacitated spermatozoon was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 21.4 (15/70) and 4.3% (3/70), respectively. When capacitated sperm head was injected, development to $${\geq_-}$$ 2-cell and $${\geq_-}$$ 4-cell was 29.9 (35/117) and 10.3% (12/117), respectively. In contrast, none developed beyond 4-cell in the sham-operated group. The results of this study demonstrated that mouse oocytes matured in vivo can undergo normal appearing cleavage to 4-cell stage by dead-sperm injection. Sperm treatment prior to injection did not affect the ability of mouse oocytes to cleave in vitro.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.1-6
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• 1993

This experiment was conducted to determine the effects of gossypol injection spermatogenesis of mice. Gossypol was injected into the stroma of testes(TS) and the doses of gossypol injected were 5, 10 and 15mg per kg of body weights, respectively. The number of sperm and the weight of testes were gradually reduced(P<0.01) from 2 to 4 weeks after gossypol treatment in all groups of mice treated with gossypol, compared with the control group. The rates of malformation(loss of proacrosome, damage of midpiece and breaking of tail) of sperm were significantly(P<0.01) increased at 2 and 3 weeks after the injection of 10 or 15mg of gossypol. However, the weight of testes and the number of normal sperm were gradually increased and the malformation rate of sperm was decreased between 4 and 6 weeks after injection of 5mg of gossypol. The results of this experiment indicated that probably ireeversible suppression of spermatogenesis could be brought about easily and immediately by the single injection of gossypol into TS.