• 한국수정란이식학회지
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• 제26권4호
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• pp.287-296
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• 2011

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.

• 한국가축번식학회지
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• 제16권3호
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• pp.199-208
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• 1992

These studies were carried out to investigate optimal physological conditions for in vitro fertilization (IVF) of mouse ova. The unfertilized ova were obtained by superovulation from ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and pH and osmolality of basal media were adjusted with the supplementation of sodium bicarbonate and sodium chloride, respectively. The optimal pH, and osmolality of culture media and the optimum period of sperm preincubation were examined in fertilization in vitro of mouse ova and the subsequent culture in vitro of embryos. The pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryosafter 26hrs. of incubation with preincubated sperm were evaluated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The percentage of in vitro fertilized ova was highest (64.7%) in media of pH 7.1 and lowest (38.0%) in pH 6.7. No significant difference in % fertilized ova was found from the media of pH 7.1 to 7.5. Compared with the result from pH 7.1 medium, the pollyspermy was increased signifciantly (p<0.05) in the media of pH over 7.5 and below 6.9;, and the % degenerated ova was significantly (p<0.05) increased in the media of pH below 6.9. 2. The percentage of in vitro fertilized ova was highest (69.4%) in media of osmolality 330 mOsm and lowest (47.9%) in osmolality 250 mOsm. No significant difference in % fertilized ova was found from the media of osmolality 310 to 350 mOsm. Compared with the result from osmolality 330 mOsm in medium, the polyspermy aws increased significantly(p<0.05) in the media of osmolality over 350 mosmol and blow 290 mOsm, and the % degenerated ova was significantly (P<0.05) increased in the media of osmolality below 290 mOsm. 3. The percentate of in vitro fertlilized ova was highest (62.7%) in media of period sperm preincubation 180 min. and lowest (40.4%) in sperm preincubation 30 minutes. No significant difference in % fertilized ova was found from the media of sperm preincubation 120 to 180 minutes. Compared with the result from sperm preincubation 180 minutes in medium, the polyspermy was low differ no significantly(P<0.05) in the media of period sperm preincubation, and the % degenerated ova was signifciantly(P<0.05) increased in the media of sperm presincubation below 60 minutes.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.50-50
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• 2002

During fertilization, morphological and molecular events in male and female chromatin are precisely controlled in time. However, little information is available on onset of pronuclear formation and first S-phase entry in the pig following intracytoplasmic sperm injection. To assess species specific paternal effect on the pronuclear formation and initiation of first S-phase in the pig, we examined time of onset of male and female pronuclear formation and onset of DNA synthesis in the oocytes following pig or mouse sperm injection. (omitted)

• 한국수정란이식학회지
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• 제4권1호
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• pp.21-27
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• 1989

Hydrogel chambers made from polymerized 2-hydroxyethyl methacrylate were used for in chamber fertilization. To determine whether sperm motility was preserved in the Hydrogel chamber, chambers which have rabbit sperm or frozen-thawed bovine sperm were transplanted inside of mouse peritoneal cavity and sperm were observed after recovering the chambers in the due time. As a result, it was determined that preservation of sperm motility was good. To determine whether in chamber fertilization was possible, chambers which have rabbit oocytes and sperm were transplanted inside of mouse peritoneal cavity and eggs were observed after recovering the chambers at 84 hr of preservation. As a result, the fact that fertilization and culture was occurred inside of the chamber was determined.

• 한국가축번식학회지
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• 제16권3호
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• pp.209-215
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• 1992

These studies were carried out to overcome 2-cell block and in vitro development to blastocysts in vitro fertilization of mouse embryos. The unfertilized ova were obtained by superovulation in ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and the pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryos after 26hrs of incubation with preincubated sperm were evaluatated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The optimal ranges of pH and osmolality of culture media and of sperm preincubation time for in vitro development of in vitro fertilized ova to blastocyst were pH 7.1 to 7.3, 250 to 350 mosmol and 60 to 180 min, respectively. 2. With the media of pH 7.1, 310 mOsm and sperm preincubation period of 120min in another experiment of large sample size, the in vitro fertilized ova was found 66.5% and the in vitro development of in vitro fertilized ova to blastocyst was found 35.8%. From the above results it was concluded that the optimal conditions of pH and osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOam and 120min, respectively.

• Journal of Radiation Protection and Research
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• 제31권1호
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• pp.31-35
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• 2006

Black mouse에 하나로 원자로의 BNCT시설을 이용하여 중성자(flux:1.036739E+09)를 머리를 정면으로 16 및 32 Gy 조사한 후 생물학적 효과를 관찰하는 일환으로 고환에 대한 물리학적 변화 및 조직 변화를 관찰하였다. 조사 후 90일이 경과한 후에 고환의 무게는 변화가 없었으나 고환의 부피는 약간 감소하였으며, 정자수도 감소하였다. 고환의 조직검사에서는 32 Gy 중성자 조사군에서 위축된 정세관의 수가 증가되었으며 stage VI에서의 정세관에서는 정조세포 및 비사기 정모세포가 고갈되어 있음을 알 수 있었다. 중성자 조사(32 Gy)후 고환의 손상이 장기간 경과 후에도 회복되지 않음을 알 수 있었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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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.

• Clinical and Experimental Reproductive Medicine
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• 제24권2호
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• pp.187-192
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• 1997

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

• 한국가축번식학회지
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• 제23권1호
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• pp.19-27
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• 1999

본 연구는 사람정자 추출물을 마우스 난모세포의 세포질내에 주입하여 단위발생에 미치는 영향을 구명하기 위하여 실시하였으며, 그 얻어진 결과는 다음과 같았다. 1. 마우스 난모세포가 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지로 각각 10 pl 씩 주입되었을 때, 전핵을 형성하고 제2극체를 방출하는 난모세포의 활성율은 각각 14.5, 9.8 그리고 14.9%였다. 칼슘농도별 난모세포의 활성율은 유의성이 인정되지 않았다 (P＞0.05). 2. 마우스난모세포에 가열하지 않은 사람정자 추출물 10 pl을 주입후 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 1.7 mM 칼슘 농도에서의 난모세포 활성율(51.8%)은 0 과 5mM 칼슘 농도에서의 난모세포 활성율보다 유의하게 높았다. 3. 마우스 난모세포에 가열처리한 사랍정자 추출물 10 pl을 주입 후 0, 1.7 그리고 5mM의 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 칼슘 농도별 난모세포 활성율은 유의성이 인정되지 않았다. 난모세포 활성율은 11.8∼17.0%의 범위에 있었다. 4. 마우스 난모세포에 가열하지 않은 사람 정자 추출물, 가열 처리한 사람 정자 추출물, 그리고 PBS 배지를 각각 l0 pl 씩 주입 후 1.7 mM 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 가열하지 않은 사람 정자 추출물을 주입한 난모세포의 활성율이 54.5% 로 제일 높았다. 처리별 난모세포의 활성율은 유의성이 인정되었다(P＜0.05), 5. 마우스 난모세포에 가열하지 않은 l일령과 6일령 정자 추출물을 각각 l0 pl 씩 주입 후 1.7mM 칼슘농도를 가지는 PBS 배지에서 각각 배양하였을 때, 1일령 정자 추출물을 주입한 난모세포의 활성율은 60.0%로 6일령 정자 추출물을 주입한 난모세포의 활성율 11.1% 보다 유의하게 높았다. 이상의 결과를 종합해 보면 가열하지 않은 사람정자 추출물에는 oscillin 과 같은 난모세포 활성인자가 존재한다는 것이 입증되었다.

• Applied Microscopy
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• 제28권2호
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• pp.193-205
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• 1998

The Urechis unicinctus sperm and spermatogenic cells prepared from the testis are investigated to identify $\alpha-tubulin$ of axoneme microtubules using mouse monoclonal $anti-\alpha-tubulin$ as the first Ab and Gold(10nm) conjugated goat anti-mouse IgG as the Ab marker. The Ag-Ab reaction analyzed excellently the localization of $\alpha-tubulin$ and the gold particles incorporated with the proximal and distal centrioles, manchette microtubules, and flagellum. The gold particles can be also observed in the spermatogenic cells while the cells are still in sperm ball which is composed of a somatic cell and spermatogenic cells. The sperm ball is the functional unit of sperm production in U unicinctus testis. The spermatids are developed from the spermatogenic cells in the sperm ball and released into the testis cavity through a cortical cytoplasmic opening. The spermatid architectures are similar with the mature sperm of the testis cavity in aspects of shape of discoid acrosome, degree of nuclear condensation and ring type of mitochondrion. However, the distal centriole connecting with the flagella can be observed from the mature sperm while the both proximal and distal centrioles reveal only in the spermatids. The proximal centriole is directly connected with nuclear outer membrane during the stage of nuclear condensation and oriented perpendicularly to the distal centriole whose axis coinciding with the longitudinal axis of the spermatozoon. There are indications that the distal centriole is intimately associated with the polymerization of the flagellum. The manchette microtubules appear during spermatid development but the mature sperm have round head and no conspicuous middle piece.