• Clinical and Experimental Reproductive Medicine
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• 제46권2호
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• pp.60-66
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• 2019

Objective: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment. Methods: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean${\pm}$standard deviation) was compared. Results: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p= 0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p= 0.974). A strong correlation (r = 0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r = 0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads. Conclusion: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples.

• Anatomy and Cell Biology
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• 제57권1호
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• pp.119-128
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• 2024

Glucocorticoids play a physiologic role in the adult male reproductive functions, modulating gonadal steroid synthesis and spermatogenesis, through the glucocorticoid receptor (GR). The expression of GR has been described in several key testicular cell types, including somatic cells and early germ cell populations. Nothing is known on GR in human spermatozoa. Herein, we explored the GR expression and its possible role in normal and testicular varicocele semen samples from volunteer donors. After semen parameter evaluation by macro- and microscopic analysis, samples were centrifuged; then spermatozoa and culture media were recovered for further investigations. By western blotting and immunofluorescence analyses we evidenced for the first time in spermatozoa the presence of GR-D3 isoform which was reduced in sperm from varicocele patients. By treating sperm with the synthetic glucocorticoid dexamethasone (DEXA), we found that survival, motility, capacitation, and acrosome reaction were increased in both healthy and varicocele samples. GR involvement in mediating DEXA effects, was confirmed by using the GR inhibitor mifepristone (M2F). Worthy, we also discovered that sperm secretes different cortisol amounts depending on its physio-pathological status, suggesting a defence mechanism to escape the immune system attach in the female genital tract thus maintaining the immune-privilege as in the testis. Collectively, our data suggests a role for glucocorticoids in determining semen quality and function, as well as in participating on sperm immune defensive mechanisms. The novelty of this study may be beneficial and needs to take into account in artificial insemination/drug discovery aimed to enhancing sperm quality.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.1-7
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• 1993

The present study was designed to test the validity of the semen analysis(S/A) and the sperm penetration assay(SPA) as a prognostic indicator of male fertility in 123 patients undergoing in vitro fertilization(IVF). We attempted to correlate the traditional semen parameters or the extent of sperm penetration in SPA with the results of human IVF rate or cleavage rate. Poor correlation was found between the results of S/A and human IVF rate(sensitivity, 80.6% ;specificity, 46.7%; positive predictive value, 91.6%;negative predictive value, 25%). Conversely, good correlation was found between the results of SPA and human IVF rate(sensitivity, 100% ; specificity, 80% ;positive predictive value, 97.3% ;negative predictive value, 100%). Our results corroborate the conclusion that SPA can be a valuable tool as a prognostic indicator of male fertilizing ability.

• Clinical and Experimental Reproductive Medicine
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• 제22권3호
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• pp.273-278
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• 1995

Mammalian, including human, spermatozoa undergo morphological and physiological changes during sperm maturation. There were, these changes may affect the fertilization and embryonic development. In this study, we examined the pronucleus formation, pronucleus disappearance and embryonic development in the human oocytes fertilized by intracytoplasmic sperm injection (ICSI). The injected spermatozoa were grouped into ejaculated, epididymal and testicular by the collecting region. Among 363 metaphase II injected oocytes, 287(79.1%) oocytes were normally fertilized and displayed two pronuclei. There were no difference in the fertilization rates and in the pronucleus formation and pronucleus disappearance at 16, 20 and 24 hr after ICSI, among the each spermatozoa group. Also, at 64 hr, the appearance of embryonic development was similar. From these results it can be concluded that there was no difference of maturity among the sperm collected from ejaculated, epididymis and testis in the pronucleus formation and embryonic development. Therefore, testicular spermatozoa are successfully used with ICSI in IVF-ET program.

• Clinical and Experimental Reproductive Medicine
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• 제17권1호
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• pp.71-80
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• 1990

항정자항체를 검사할 수 있는 면역분석법개발을 위하여 immunoaffinity chromatography로 분리 정제한 정자표현항원을 microtiter plate에 고정화 시킨것과 효소와 화학발광체로 표지된 2차항체를 사용하여 ELISA법과 CIA법을 개발하고 이 방법의 이용 가능성을 검토하기 위하여 기존 방법인 Kibrick test법으로 임상소견이 다른 남성혈청을 분석 비교하여 다음과 같은 결과를 얻었다. 1. 인간정자 표면항원을 분리하기 위한 immunoaffinity column을 제작키 위하여 인간정자를 토끼에 주사하여 정자에 대한 항혈청을 생산하였으며 이를 Protein A-Sepharose column으로 분리 정제하여 CNBr activated Sepharose-4B에 coupling시켜 immunoaffinity column을 제작하였다. 이 column에 균질화된 정자를 반응시키고 SDS를 넣은 Tris-HCI buffer로 용출시켰을때 60KD정도의 분자량을 갖는 분획을 얻었다. 2. 분리 정제된 human IgG를 microtier plate에 농도를 달리하여 고정화 시키고 ELISA용 Goat anti-human IgG-HRP conjugate와 CIA용 Rabbit anti-human IgG-ABEI-H conjugate와 반응시켜 그 활성도를 측정하였던 바 농도에 따라 반응의 정도가 감소하였다. 3. ELISA법으로 양성혈청의 희석곡선을 작성하였을때 경사가 완만한 것과 급한 것의 두 종류의 경향을 띈 곡선으로 대별되었으며 완만한 경사를 나타내는 것에서 1:160 희석치에서 O.D가가 0.1이하를 음성, 0.1이상 0.2이하를 약양성(weak positive) 그리고 0.2이상을 양성으로 판별하였다. 4. ELISA, CIA 그리고 Kibrick test로 동일시료를 분석 비교하였던바 ELISA와 CIA는 거의 동일한 상관관계를 보였으나 Kibrick test와는 50% 수준만 일치함을 보였다.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.57-63
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• 1993

From September 1988 to August 1992, two different methods of preparing human sperm before intrauterine insemination(IUI) were compared using the semen samples of seventy-three infertile couples. The sperms were prepared by a swim-up after sperm washing or by a continuous percoll gradient technique. Fourteen of 35 women conceived during IUI cycles using a sperm washing and swim-up method (40%), and 12 of 38 women conceived during IUI cycles using a percoll gradient technique(31.6%). Among the group with male infertile etiologic factor only, one of 5 women conceived during sperm washing and swim-up cycles(20%); one of 4 women conceived during percoll gradient cycles(25%). On the contrary, among the group with cervical factor only, six of 10 women conceived during sperm washing and swim-up cycles (60%) ; Five of 17 women conceived during percoll gradient cycles(29.4%). It is suggested that sperm separation by sperm washing ar -up is a useful technique for intrauterine insemination in cervical infertility, and sperm separation in percoll gradient appears to be more valuable for intrauterine insemination of male subfertility.

• Clinical and Experimental Reproductive Medicine
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• 제46권1호
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• pp.8-13
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• 2019

Objective: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. Methods: Thirty men with a total motile spermatozoa count > 80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. Results: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p< 0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p< 0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p> 0.05). HA binding ability did not change after DGC using any of the four media. Conclusion: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

• 한국수정란이식학회지
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• 제10권3호
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.

• 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.