• 한국가축번식학회지
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• 제26권4호
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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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• 제25권3호
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• pp.331-340
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• 1998

본 연구에서는 돼지 난자내에 돼지정자, 여러 가지 처리된 정자두부 (1% Triton, 0.1% Trypsin, 100mM NaOH)및 이종정자 (소, 쥐, 사람)를 미세 주입한 후 난 활성과 웅성 전핵형성, 전핵의 이동 및 배발달을 관찰하였다. 전자현미경으로 관찰한 결과 Triton X-100을 처리하였을 때 첨체가 제거되었으나 핵 주변 물질은 제거되지 않았고 Trypsin 또는 NaOH를 처리 할 경우 핵주변 물질 (perinuclear material)이 제거됨을 볼 수 있었다. 돼지 난자는 정자, 정자두부 및 Triton X-100을 처리한 정자두부의 주입을 통해 난 활성이 유도되었으며 쥐, 소, 사람의 정자를 주입하였을 때 난 활성이 유도되고 정상적인 전핵형성이 이루어졌다. 그러나 정자꼬리나 Trypsin 또는 NaOH에 의해 정자 핵주변 물질(perinuclear material)이 제거된 정자두부를 주입하였을때는 난 활성은 야기되지 않았다. 유사분열 및 2-세포기까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었으나 이 종정자를 주입한 난자에서는 관찰되지 않았다. 또한 상실배 및 배 반포까지 정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었다. 이러한 결과는 돼지에서 정자 및 정자두부의 미세주입에 의해 수정이 이루어지는 것을 시사하며 수정시 정자유래의 난할성인자는 정자 핵주변 물질(porinuclear material)에 존재하며 종특이적이지 않다는 것을 보여주는 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제26권2호
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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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).

• 한국수정란이식학회지
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• 제22권1호
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• pp.1-7
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• 2007

본 연구는 체외 성숙된 난자와 동결 융해 정자를 이용한 돼지의 체외 수정 과정에서 난구 세포의 존재가 정자 침투율, 웅성전핵 형성률 그리고 후기배로의 체외 발육에 미치는 영향을 알아보기 위하여 수행되었다. 돼지 난소로부터 난자-난구세포 복합체를 채취하여 eCG/hCG, 10% 돼지 난포액, epidermal growth factor 등이 첨가된 TCM 199 배양액에서 44시간 배양하여 체외 성숙을 유도하였다. 성숙 배양 후 난구 세포를 제거한 난자와 난구 세포가 부착되어 있는 난자를 돼지 동결 융해정액을 이용하여 5mM caffeine과 10mM calcium chloride를 함유한 mTBM배양액에서 8시간 체외 수정하였다. 체외 수정 후 난자를 고정, 염색하여 정자 침투율과 웅성전핵 형성률을 조사하였고(실험 $1{\sim}3$) 일부 수정란을 North Carolina State University-23 배양액에서 체외 수정 후 156시간 배양하여 후기배로의 발육능을 검토하였다(실험 3). 실험 1에서는 정자 농도를 $7.5{\times}10^5/ml$로 조정하여 나화 난자와 난구 세포 부착난자에서 정자 침투율 및 웅성전핵 형성률을 조사하였다. 실험 2에서는 난구 세포 부착 난자의 체외 수정에 적합한 정자 농도를 구하기 위해 2, 3, 4, 및 $5{\times}10^6/ml$의 농도로 난자를 수정한 후 정자 침투율 및 웅성전핵 형성률을 조사하였다. 실험 3에서는 나화 난자 및 난구 세포 부착 난자를 각각 $7.5{\times}10^5/ml$의 정자 농도로 체외 수정한 후 후기배로의 발육률을 조사하였다. 실험 1의 결과 정자 침투율은 나화 난자에 비해 난구 세포 부착 난자에서 유의적으로 감소되었다(35.2% vs. 77.4%; p<0.01). 실험 2에서 다양한 정자 농도에 의한 정자 침투율과 정상 수정률을 바탕으로 판단했을 때 $4.6{\times}10^6/ml$의 정자 농도가 다른 정자 농도에 비해 난구 세포부착 난자의 체외 수정에 적합한 것으로 나타났다. 체외 수정과정에서 난구 세포 부착된 상태로 수정된 난자는 나화 난자에 비해 유의적으로(p<0.05) 높은 분할률(48.8% vs. 58.9%), 배반포 형성률(11.0% vs. 22.8%)과 배반포 세포수$(22{\pm}2\;vs.\;29{\pm}2)$를 나타내었다. 본 연구의 결과로부터 돼지의 체외 수정과정에서 난구 세포의 존재는 정자 침투를 저해하지만 분할률, 배반포 형성률 및 배반포의 세포수를 증가시키는 것으로 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Applied Microscopy
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• 제19권1호
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• pp.70-88
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• 1989

The storage sac of U. unicinctus can be divided into two parts morphologically as well as functionally ; one is proximal and the other distal storage sac. It is because of the seasonal morphological change of the storage sac caused by sperm accumulation in the storage sac. The proximal storage sac contains the mature sperm with the dumbbell-shaped acrosome and well developed one or two mitochondria in the middle piece, whilst the sperm of hemispherical acrosome associate closely with an accessory cell in the distal storage sac. This means that the sperm do not perform the synchronous development in a storage sac, which is not the case of sperm development in the coelomic cytophorus. In addition, the basal membrane of the distal storage sac is different from that of the proximal storage sac in term of tissue formation. Connective tissues, acces-sory cells and small lumen develop on the basal membrane of the distal sto-rage sac, which is well contrasted with the thin basal membrane of the proximal storage sac. The function of the storage sac is discussed in rela-tion of the sperm development and the seasonal change.

• 한국발생생물학회지:발생과생식
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• 제19권1호
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• pp.1-10
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• 2015

The purpose of the present study was to examine the seminiferous epithelium cycle of Bombina orientalis using a light microscope. The cycle was divided into a total of 10 stages, according to the morphological characteristics of the cells. The spermatogenetic cells included primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatid and sperm. At stage I, the primary spermatogonia was located closer to basal lamina of the seminiferous tubule without spermatocyst formations. Especially at the stage II, the secondary spermatogonia were located in the spermatocyst. The primary and secondary spermatocytes were found from stages III to VI. The secondary spermatocytes were smaller in size than the primary spermatocytes, but they had thicker nucleoplasm and smaller nuclei. The round-shaped, early sperm cells were formed in stage VII, and further divided at stage VIII to have more concentrated nucleoplasm before division to matured sperm cells. At stage X, the matured sperm cells emerged from the spermatocyst. Considering the above results, this study presented the special characteristics in the generation and type of sperm formation. The germ cell formation occurred in various stages, like the perspectives of Franca et al (1999), ultimately, providing taxonomically useful information.

• 한국수정란이식학회지
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• 제25권2호
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• pp.97-101
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• 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.

• 한국수정란이식학회지
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• 제33권4호
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• pp.229-235
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• 2018

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.