• Clinical and Experimental Reproductive Medicine
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• 제48권1호
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• pp.11-26
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• 2021

Advances in anticancer treatments have resulted in increasing survival rates among cancer patients. Accordingly, the quality of life after treatment, particularly the preservation of fertility, has gradually emerged as an essential consideration. Cryopreservation of embryos or unfertilized oocytes has been considered as the standard method of fertility preservation among young women facing gonadotoxic chemotherapy. Other methods, including ovarian suppression and ovarian tissue cryopreservation, have been considered experimental. Recent large-scale randomized controlled trials have demonstrated that temporary ovarian suppression using gonadotropin-releasing hormone agonists (GnRHa) during chemotherapy is beneficial for preventing chemotherapy-induced premature ovarian insufficiency in breast cancer patients. It should also be emphasized that GnRHa use during chemotherapy does not replace established fertility preservation methods. All young women facing gonadotoxic chemotherapy should be counseled about and offered various options for fertility preservation, including both GnRHa use and cryopreservation of embryos, oocytes, and/or ovarian tissue.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.213-222
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• 2023

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

• 한국수정란이식학회지
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• 제27권2호
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1247-1255
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• 2016

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, $40{\mu}g/mLs$ semen were standardized to find out the optimum dose and $20{\mu}g/mLs$ was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with $20{\mu}g/mL$ SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/$32.7{\mu}M$/$10^9$ spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

• 한국환경농학회지
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• 제36권4호
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• pp.256-262
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• 2017

식물 종자를 비롯한 각종 유전자원을 액체질소에 저장하면 유전형질 특성의 손실 없이 장기간 보존할 수 있다. 본 연구에서 보존조건이 까다로워 단명 종자로 분류되는 들깨 종자를 초저온 동결저장 방법으로 보존할 수 있는지를 조사하였다. 수집한 들깨 품종별 종자의 초기 발아율은 40-95% 수준으로 다양하였는데, 수분함량을 3-8%로 조절한 종자를 액체질소에 처리하여도 발아율은 감소하지 않았다. 종자의 수분함량이 4-5%인 종자의 초저온 처리 후 발아율이 가장 높았으며, 초기 발아율이 낮은 품종에서는 초저온 처리에 의해 발아율이 증가하기도 했다. 인위 노화처리에 따라 종자의 발아율과 ascorbate peroxidase 활성은 감소하였으며, 품종별로 종자의 활력 저하 정도는 크게 달리 나타났다. 대조 처리 종자와 비교하였을 때, 초저온 처리과정에 발생할 수 있는 산화스트레스가 들깨 종자의 활력을 저해하지는 않을 것으로 추정되었다. 따라서 들깨 종자를 4-5% 수분함량으로 건조시켜 초저온 동결 저장하면 활력 손실 없이 장기간 보존할 수 있을 것으로 판단되며, 노화가 급속히 진행되는 품종의 경우에는 고활력 자원을 확보하거나 휴면타파 처리 후 초저온 동결 보존하는 것이 유리할 것으로 생각된다.

• 생명과학회지
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• 제31권10호
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• pp.960-968
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• 2021

미세조류 연구는 18세기 후반부터 시작된 이후 생물산업에서 가장 중요한 생물자원으로 인식되어 왔다. 특히 미세조류의 산업 활용에 초점을 맞춘 식품/사료 및 생리 활성 화합물에 대한 초기 주요 연구 분야는 현재 대체 에너지 자원, 탄소 배출 저감 및 폐수 처리를 포함한 환경 연구 분야로 더욱 확대되고 있다. 하지만 그 산업적 활용의 중요성에도 불구하고 미세조류 배양의 장기 보존과 관련된 기초 연구 분야는 많은 주목을 받지 못하고 있다. 그러나 생물학적으로 활성을 띄는 안정적인 미세조류 배양체 보존은 이러한 미세조류의 산업적 활용을 더욱 부각시킬 수 있는 핵심적인 성공요소이다. 따라서 본 총설은 조류(algae)의 분류체계에서 가장 큰 분류군을 차지하는 녹조류(Chlorophyceae)를 포함하여 현재까지 개발된 다양한 최첨단 미세조류 냉동보존기술을 조사하였다. 또한, 국내 생물자원은행 및 국제 미세조류 자원은행에 기탁된 생물학적으로 활성을 띄는 미세조류 배양체를 보존·유지하기 수행하고 있는 보존 기술과 함께 동결보존 시 온도조절 효과, 보존제 효과 등 미세조류의 성공적인 동결보존 기술과 관련된 주요 요인들을 조사하였다. 본 연구를 통해 확인된 결과를 살펴보면, 미세조류의 형태 및 생리학적 다양성으로 인해 현재로서는 범용적으로 사용할 수 있는 표준 미세조류 장기 보존 방법이 없다는 것을 확인하였다. 따라서, 이러한 문제를 극복하기 위해서는 미세조류의 분류학적 체계를 명확하기 위한 종 특이적 바이오마커의 개발과 종 특이적 동결보존 방법에 기반한 체계적인 접근을 위한 기초 연구 분야에 대해 훨씬 더 많은 노력이 필요함을 확인하였다.

• 한국동물생명공학회지
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• 제36권2호
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• pp.106-115
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• 2021

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.

• Clinical and Experimental Reproductive Medicine
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• 제48권2호
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• pp.111-123
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• 2021

Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2% ±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.

• 한국해양생명과학회지
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• 제8권2호
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• pp.115-120
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• 2023

본 연구는 동자개 정자의 동결보존을 위해 동결보존제의 최적 농도와 적정 희석액을 구명하여 정자를 최상의 상태로 보존하여 인공종자를 생산하는데 목적이 있다. 실험은 3종의 희석액(I: 300 mM glycose, II: Kurokura extender, III: Li extender), 4종의 동결보존제(dimethyl sulfoxide, ethylene glycol, methanol and glycerol)와 4개의 동결보존제 농도(5, 10, 15, 20%)를 조합하여 대하여 조사하였다. 동결보존한 정자를 해동한 후 정자의 생존율과 정자활성지수로 동결보존제의 효과를 평가하였다. 희석액 III(Li extender)과 10과 15%의 methanol을 조합했을 때 동자개 정자의 생존율과 정자활성지수는 각각 66.9 ± 8.7, 67.3 ± 13.1%과 2.6 ± 0.4, 2.6 ± 0.5로 다른 희석액과 동결보존제보다 높았다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.72-72
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• 2003