• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2000년도 춘계심포지움
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• pp.64-75
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• 2000

During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period (1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor (hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1519-1523
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• 2015

Infection of the uterine cervix by human papilloma viruses (HPV) may be associated with cervical pre-cancer and invasive cervical carcinoma if left untreated. With advance in molecular techniques, it has become easier to detect the resence of HPV DNA long before the appearance of any lesion. This study concerned cervical scrape samples of 310 married non-pregnant women attending a gynecology outpatient department for both Pap and PCR testing to detect HPV DNA. Nested PCR using primers for L1 consensus gene with My9/My11 and GP6+/GP5+followed by multiplex PCR were carried out to detect HPV 16 and HPV18. Result: HPV prevalence was 11.9% out of which 3.67% cases of negative for intra-epithelial lesion or malignancy (NILM) and in 71.1% (27/38) of atypical cervical smears were HPV positive. There was increasing trend of high-risk-HPV positivity (HR HPV 16 and 18), from 20% in benign cytology (NILM) to 42.9 % in LSIL, 71.41% in HSIL and 100% in SCC. There was highly significant association of HPV infection with cervical lesion ($x^2=144.0$, p<0.01) and also with type specific HPV prevalence ($x^2=7.761^*$, p<0.05).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2000년도 수정란의 위생적 처리·검사 및 특수가축의 수정란이식 기술 심포지움
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• pp.64-75
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• 2000

During the last three decades considerable advances has been made in goat embryo production and transfer technology. The Korean native black goat is the most useful domestic ruminant in this country for biological investigation and application because it has a lot of merits such as relatively short generation period(1 vs 2 year for a cow), easy of handling, well adaptation, high fertility, convenient and inexpensive. This article covers the methods of superovulation, estrus synchronization, embryo collection and transfer techniques, pregnancy diagnosis and subsequent pregnancy and kidding rates for the production of transgenic Korean native black goats. More than one hundred goat kids have been produced as a result of our transgenic goat project via microinjection of foreign gene into pronuclei, in vitro culture, transfer of various stages of fresh and frozen-thawed microinjected embryos into oviducts or uteri of recipient does. We have got two transgenic goats carrying a transgene targeting the expression of recombinant human granulocyte colony stimulating factor(hG-CSF) to the mammary gland so far. Since collection and transfer of embryos in this species is usually accomplished by laparotomy, exteriorization of the reproductive tract for surgical embryo collection leads to the formation of post-operative adhesions. Nonsurgical or laparoscopic technique to reduce adhesions from repeated surgeries has great advantages in improving embryo production and transfer especially from valuable donors. We will discuss this later.

• 대한수의학회지
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• 제61권3호
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• pp.25.1-25.7
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• 2021

The axolotl has extraordinary regeneration capacity compared to other vertebrates. This remarkable potential has been attributed to its life-long neoteny, characterized by the exhibition of embryonic characteristics at the adult stage. A recent study provided a detailed morphological analysis of the sperm morphology of the Ambystoma mexicanum using routine and detailed histological techniques. The primary purpose of the present study is to describe a simple and inexpensive method for evaluating the morphology of axolotl sperm. In this study, spermatophore structures were collected and spread on slides and air-dried. The slides were stained with periodic acid Schiff, toluidine blue, Masson's trichrome, Giemsa, Spermac, and Diff-Quik dye for a morphological examination. The slides were coated with gold/palladium for a scanning electron microscopy examination. The sperm of the axolotl consisted of an elongated head, a neck, and a flagellum covered with an undulating membrane. The lengths of the midpiece, tail, and head were 8.575 ㎛, 356.544 ㎛, and 103.661 ㎛, respectively. In the flagellum part, the wavy membrane structure, whose function has not been explained, surrounds the tail. The data obtained from this study will constitute an important step in designing future research on the reproductive and regeneration capacity of the axolotl.

• 한국수정란이식학회지
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• 제17권2호
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• pp.153-162
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• 2002

본 연구에서는 한우의 신속한 증식을 위한 번식기술 개발을 위해 발정이 재귀되지않은 무발정 한우의 번식장애 치료효과와 공태기를 감축하기 위한 난소실질내 PGF／sub 2$\alpha$처치효과를 알아보았다. 1. 번식장애의 감별진단 결과는 검사두수 43두중 둔성발정 또는 발정발견의 잘못이 18두 (41.9%)로 가장 많았고, 난소기능정지 14두 (32.6%), 난포낭종 4두(9.3%), 영구황체 3두 (7.0%), 자궁내막염 2두 (4.7%), 자궁축농증과 황체낭종은 각각 1두(2.3%)로 나타났다. 2. 둔성발정 또는 발정발견의 잘못 18두를 PGF／sub 2$\alpha$／ 25 mg으로치료하여 18두가 발정이 발현되었고 이중 16두 임신되었다. 3. 난소기능정지의 경우는 GnRH 200$\mu\textrm{g}$으로 치료하여 10두중 8두가 발정이 발현되어 이중 7두가 임신이 되었으며, 난포낭종도 역시 CnRH 200$\mu\textrm{g}$으로 치료하여 4두중 3두가 발정이 발현되어서 인공수정후 임신이 되었다. 4. 영구황체 3두와 황체낭종 1두는 각각 PGF／sub 2$\alpha$／ 25 mg으로 치료하여 3두 모두 발정이 발현되어 수정후 임신이 되었다. 5. 자궁내막염 2두는 PGF／sub 2$\alpha$／25mg과 항생제 요법을 병행한 결과 2두 모두 발정이 발현되어서 인공수정후 임신이 되었다. 6.공태기를 줄이기 위한 방법으로 분만 후 20일이 경과한 소에 대해 14일 간격으로 PGF／sub 2$\alpha$/의 난소실질내 투여시 공여동물 23두중 17두에서 임신이 확인되어 74%의 수태율을 보였다. 또한 11일 간격으로 PGF／sub 2$\alpha$／ 를 투여한 군에서는 17두중 16두가 임신되어 94%의 높은 수태율을 보여 공태기를 더 단축할 수 있는 우수한 처리법임을 알 수 있었다.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.55-66
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• 2002

배아줄기세포(embryonic stem cell, ESC)는 미분화상태로 지속적인 계대가 가능하며, 정상 핵형과 전 분화능(pluripotency)을 가져 생체내-외에서 분화 유도시 삼배엽성의 모든 세포로 분화 가능하다. ESC를 feeder 세포 없이 부유배양하면 배아체(embryoid body, EB)를 형성하고, 초기 배아 발생과 유사한 분화 양상을 갖는다. ESC의 분화 유도가 초기배아 발생처럼 생식호르몬(GTH: FSH, LH; steroids)의 영향을 받는지는 불명하다. 본 연구는 ESC가 분화과정중 생식호르몬처리에 의해 그들 수용체가 발현되는가를 알아보고자 하였다. 순계혈통 생쥐인 C57BL/6J에서 과배란 유도후 포배를 수획하고, 유사분열적으로 불활성화된 feeder 세포와 공배양하여, 계대배양 하는 중 배아줄기세포주(JHYl)를 확립하였다. JHY1의 alkaline phosphatase 활성과 SSEA-1, 3, 4 발현을 통해 ESC임을 확인하였다. Feeder 세포 없이 ESC를 계대배양 후 호르몬처리(FSH LH E$_2$, P$_4$, T)하에서 5일 동안 부유배양하여 배아체를 형성시키고, 이후 7일 동안 부착배양하여 분화를 유도하였다. GTH와 스테로이드의 수용체 발현 실험에서 ESC에 E$_2$ 처리에 의한 LHR의 발현 증가를 제외한 나머지 호르몬 처리군에서 ESC보다 낮은 생식호르몬의 수용체 발현이 관찰되었다. 생식호르몬을 농도별 수용체 발현 정도는 증감되지 않았다. 미분화 ESC 표지유전자인 Oct-4는 호르몬 처리군에서도 발현되었다. 각 배엽의 표지유전자들(영양세포, handl; 외배엽성, keratin와fgf-5; 중배엽성, enolase와 $\alpha$ -globin; 내배엽성, gata-4와 $\alpha$ -fetoprotein) 등의 발현 양상을 조사한 결과 호르몬 처리후 내배엽성 표지유전자외에는 발현 증가가 관찰되지 않았다. 즉 생식호르몬에 의해 gata-4, $\alpha$-fetoprotein의 발현이 증가되는 것으로 보아 내배엽성 계열로의 분화 유도가 이루어진 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제30권3호
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• pp.241-248
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• 2003

Objective: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. Methods: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. Results: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. Conclusion: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.121-130
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• 1994

Expression of gonadotropin releasing hormone(GnRH) has been described in the rat ovary. It remains, however, unkown whether GnRH is synthesized as a prohormone. Therefore, this study was performed to verify the expression of pro-GnRH by in situ hybridization and further to investigate the effect of gonadotropin on GnRH or GnRH mRNA in rat ovary by immunohistochemical and in situ hybridization techniques. Adult female Sprague-Dawely rats were used and the estrous cycle was synchronized by intraperitoneal injection of pregnant mare's serum gonadotropin(PMSG). Ovaries were fixed with 4% paraformaldehyde and embedded with G.C.T. compound and cut by cryostat. For immunohistochemistry, avidin-biotin peroxidase complex(ABS) method was employed and for in situ hybridization, $^{35}S$-end labeled oligonucleotide was used and followed by autoradiography. By in situ hybridization using GnRH oligomer and GAP(GnRH associated protein) oligomer, GnRH mRNA and GAP mRNA were co-localized in the fullicular cells, luteal cells, interstitial cells and theca cells. GnRH or GnRH mRNA signals in the ovary increased by human chorionic gonadotropin(hCG) injection. At the 3 and 6 hrs after hCG injection, the number of GnRH and GnRH mRNA containing cells increased rapidly and the density of GnRH and GnRH mRHA culminated at 9 hrs after heG injection. With the follicular development, the high expression of GnRH and GnRH mRNA was also observed within the follicles. After ovulation, the density of GnRH or GnRH mRNA decreased in the follicles but increased in the corpus lutea.

• Clinical and Experimental Reproductive Medicine
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• 제24권2호
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• pp.153-165
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• 1997

In male reproducible health, fertility and IVF (in-vitro fertilization), semen analysis has been most important. Semen analysis can be divided into concentration, motional and morphological analysis of sperm. The existing method which was developed earlier to analyze semen concentrated on the sperm motility analysis. To provide more useful and precise solutions for clinical problems such as infertility, semen analysis must include sperm morphological analysis. But the traditional tools for semen analysis are subjective, imprecise, inaccurate, difficult to standardize, and difficult to reproduce. Therefore, with the help of development of microcomputers and image processing techniques, we developed a new sperm morphology analyzer to overcome these problems. In this study the agreement on percent normal morphology was studied between different observers and a computerized sperm morphology analyzer on a slide-by-slide basis using strict criteria. Slides from 30 different patients from the SNUH andrology laboratory were selected randomly. Microscopic fields and sperm cells were chosen randomly and percent normal morphology was recorded. The ability of sperm morphology analyzer to repeat the same reading for normal and abnormal cells was studied. The results showed that there was no significant bias between two experienced observers. The limits of agreement were 4.1%${\sim}$-3.8%. The Pearson correlation coefficient between readers was 0.79. Between the manual and sperm morphology analyzer, the same findings were reported. In this experiments the slides were stained by two different methods, PAP and Diff-Quik staining methods. The limits of agreement were 7.2%${\sim}$-5.7% and 6.0%${\sim}$-6.3%, respectively. The Pearson correlation coefficients ware 0.76 and 0.91, respectively. The limits of agreement was tighter below 20% normal forms. In the experiments of repeatability, 52 cells stained by PAP and Diff-Quik staining methods were analyzed three times in succession. Estimating pairwise agreement, the kappa statistic for the pairs were 0.76, 0.81, 0.86, and 0.75, 0.88, 0.88 respectively. In this study it was shown that there was good agreement between manual and computerized assessment of normal and abnormal cells. The repeatability and agreement per slide of computerized sperm morphology analyzer was excellent. The computer's ability to classify normal morphology per slide is promising. Based on results obtained, this system can be of clinical value both in andrology laboratories and IVF units.

• 생명과학회지
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• 제29권5호
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• pp.621-630
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• 2019

암컷의 자궁에서 일어나는 수정은 새로운 생명의 시작점이다. 암컷의 번식기관은 난소, 난관, 자궁, 자궁경부 및 질로 구성되어 있으며, 이들기관은 발정주기에 따라 생리학적인 역할이 조절된다. 자궁은 수정란의 발달과 착상이 이루어지는 곳이기 때문에, 수정란과 자궁 환경의 상호작용은 안정적인 임신을 위한 필수적인 조건으로 알려져있다. 자궁내막은 자궁의 한 부분으로써 이들의 형태학적인 특징은 호르몬에 의해 반복적으로 변화되며, 자궁내막으로부터 분비되는 자궁액 역시 그 특징이 변화하게 된다. 최근, 자궁내막 및 자궁액 내 포함된 대량의 단백질을 단백체학과 생물정보학의 발전에 따라 검출할 수 있게 되었으며, 이러한 기술에 의해 번식학 발전을 가속화하고 있다. 대량의 단백질 정보는 성호르몬 신호기전 및 혈관신생과 같은 이론 등을 깊게 연구할 수 있는 도구로써 이용되고 있다. 본 총설에서는 자궁내막의 재구성, 자궁선 및 자궁액에 대한 기초적인 생물학적인 지식을 바탕으로, 단백체학과 생물정보학을 활용한 자궁내막 및 자궁액 연구에 대해서 소개하고자 한다. 또한, 생물정보학 도구를 활용하여 단백체학에서 탐색된 자궁내막 및 자궁액 관련 단백질들의 상호작용 알아보는 방법에 대해서도 소개하였다. 따라서, 본 총설의 내용은 발정주기동안 자궁내막 안에서 일어나는 새로운 세포 신호기전을 탐색하는데 큰 도움이 될것이라 생각된다.