• 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.

• 대한약침학회지
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• 제18권4호
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• pp.20-25
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• 2015

Objectives: Recently, thread-embedding therapy (TET) has been widely applied in Korean medicine for cosmetic purposes such as reducing skin wrinkles. An inserted thread was reported to have induced continuous stimulation, followed by support for connective tissue regeneration. However, the potential role of TET in hair-growth has not yet been reported. Methods: We designed this study to evaluate whether TET has a hair-growth-promoting effect. C57 black 6 (C57BL/6) mice were divided into three groups: normal saline-treated, minoxidil-treated, and thread-embedded groups. Normal saline or 5% minoxidil was topically sprayed on the dorsal skin of the mice once a day for 16 days. Medical threads were embedded into the dorsal skin of the mice in a single application. Hair growth activity was evaluated by using dermoscopic and microscopic observations. Sections of the dorsal skin were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7), and fibroblast growth factor-5 (FGF-5) were detected by using immunohistochemical staining. A reverse transcription-polymerase chain reaction (RT-PCR) analysis was adopted to measure the messenger RNA (mRNA) expressions of FGF-7 and FGF-5. Results: TET enhanced anagen development in the hair follicles of C57BL/6 mice. The expressions of BrdU and PCNA, both of which imply active cellular proliferation, were increased by using TET. Moreover, TET increased the expression of FGF-7, an anagen-inducing growth factor, while decreasing the expression of FGF-5, an anagen-cessation growth factor, both at the protein and the mRNA levels. Conclusion: TET enhanced hair re-growth in C57BL/6 mice. TET regulated the expressions of anagen-associated growth factors and activated the proliferation of hair follicular cells in depilated skin lesions. Considering its long-lasting effect, TET may be a good alternative therapeutic for the treatment of alopecia.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.

• 한국발생생물학회지:발생과생식
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• 제18권3호
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• pp.145-152
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• 2014

This study was performed to investigate the effect of of transdermal testosterone gel (TTG) on controlled ovarian stimulation (COS) and IVF outcomes and ovarian morphology according to pretreatment duration in poor responders. A total of 120 women were recruited for this pilot study. They were randomized into control, 2 weeks, 3 weeks or 4 weeks TTG treatment groups. For three TTG treatment groups, 12.5 mg TTG was applied daily for 2 weeks, 3 weeks or 4 weeks in preceding period of study stimulation cycle. After 3 weeks of TTG pretreatment, significant increase of antral follicle count (AFC) and significant decreases of mean follicular diameter (MFD) and resistance index (RI) value of ovarian stromal artery were observed (p=0.026, p<0.001, p<0.01, respectively). The total dose of rhFSH administered for COS significantly decreased after 3 and 4 weeks TTG treatment both compared with control group (p<0.001, p<0.001). The numbers of oocytes retrieved and mature oocytes were significanty higher in 3 and 4 weeks TTG treatment groups than control group (p<0.001, p<0.001 in the number of oocytes retrieved; p<0.001, p<0.001 in the number of mature oocytes). The clinical pregnancy rate and live birth rate were increased only in 4 weeks TTG treatment group compared with control group (p=0.030 and p=0.042, respectively). These data demonstrated that TTG pretreatment for 3 to 4 weeks increases AFC and ovarian stromal blood flow, thereby potentially improving the ovarian response to COS and IVF outcome in poor responders undergoing IVF/ICSI.

• 대한두경부종양학회지
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• 제20권2호
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• pp.128-134
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• 2004

Background and Objectives: Angiogenesis is the process of new blood vessel development from preexisting vessel. Angiogenenesis has been considered to be essential for the growth and expansion of a solid tumor. Vascular endothelial growth factor (VEGF), known as one of the most important vascular permeability factors, induces proliferation of endothelial cells, stiumulates angiogenesis, and increases vascular permeability. Several recents reports have documented that VEGF overexpression is associated with poor clinical outcomes in many maligmancies. The aims of this study were to determine whether microvessel density and VEGF expression are related to clinicopathologic factors such as age, sex, tumor size, tumor stage, and prognostic factors and to evaluate the relationship between VEGF expression and angiogenesis in benign and malignant thyroid tumors. Materials and Methods: The subjects were 65 patients (27 with papillary carcinoma, 27 with adenomatous hyperplasia, 11 with follicular adenoma) who underwent thyroidectomy from 1995 to 2001. Imuunohistochemistry was used to detect VEGF expression and microvessel density (MVD) in paraffin-embedded thryoid tumor specimens. Results: The intensity of the VEGF expression did not show stastically difference between benign and malignant thyroid tumors. There was no apparent correlation between VEGF expression and age, tumor size, T stage or scores of the AGES, AMES and MACIS systems. The neo-microvessel density was higher in the maligant tumor than the benign tumors. Also, higher neo-microvessel density was associated with metastases of the lymph nodes and scores of the AMES and AGES systems. Conclusion: Our results suggest that neo-microvessel vessel density may be a significant prognostic factor in the thyroid papillary carcinoma. But the VEGF expression does not appear to be an significant independent prognostic factor for thyroid papillary carcinoma.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• 한국수정란이식학회지
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• 제28권3호
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• pp.237-241
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• 2013

It is generally accepted that chronic stress impairs female reproduction. It negatively affects ovarian function and the number of ovulated oocytes. Chronic stress lowers the number of retrieved oocytes. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. The main corticosteroids are cortisol, cortisone, 11-deoxycortisol and corticosterone, cortisol being one of the most commonly used welfare and stress physiological indicator. In this study, we investigated the effect of cortisol level on progesterone patterns and ovulation in the dog. Cortisol and progesterone level of serum were analyzed by radioimmunoassay. The day of ovulation was considered as the day when serum progesterone concentration was 6.0~8.0 ng/ml. In vivo dog oocytes were collected by flushing oviducts of mixed-breed bitches at three days after ovulation. We classified dogs as having group 1 (cortisol level, 0 ${\leq}$ or < $2{\mu}g/dl$), group 2 (corisol level, 2 ${\leq}$ or < $4{\mu}g/dl$), group 3 (cortisol level, 4 ${\leq}$ or < $6{\mu}g/dl$) and group 4 (cortisol level, $6{\mu}g/dl$ ${\leq}$). The patterns of progesterone were not different in four cortisol groups. The average numbers of retrieved oocytes was not different in four cortisol groups. These results suggest that different cortisol levels on estrus dogs do not affect ovulation, number of ovulated oocytes and progesterone changes.

• Asian-Australasian Journal of Animal Sciences
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• 제11권6호
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• pp.655-660
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• 1998

To produce blastocysts more efficiently, it is required to identity accurately the factors involving embryonic cleavage in the chemically defined medium. Effects of pyruvate, lactate, calcium and protein sources on early cleavage of bovine follicular oocytes were investigated. The percentage of IVF embryos cleaved to ${\geq}$ 2-cell or ${\geq}$ 8-cell was higher in pyruvate (+) and lactate (+) (48 or 14%) than in pyruvate (-) and lactate (-) (22% or 4%), than in pyruvate (+) and lactate (-) (28% or 5%) and than in pyruvate (-) and lactate (+) (40% or 10%). Lactate was more effective than pyruvate during early cleavage of bovine embryos in the chemically defined medium. The percentage of IVF embryo cleaved to ${\geq}$ 2-cell and ${\geq}$ 8-cell in calcium (-) (19 and 6%) was significantly (p < 0.05) lower than in calcium (+) (78 and 45%). The percentage of embryos developed to ${\geq}$ 2-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (57, 57 and 57%). Also the percentage of A grade embryos developed to ${\geq}$ 2-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (40, 35 and 28%). The percentage of embryos developed to ${\geq}$ 8-cell showed no significant (p < 0.05) difference among BSA, 1 and 20% FBS (33, 23, and 22%). However, the percentage of A grade embryos developed to ${\geq}$ 8-cell in BSA (24%) was significantly (p < 0.05) higher than in 1 and 20% FBS (13 and 8%). The percentage of embryos developed to ${\geq}$ morula showed no significant (p < 0.05) difference among BSA, 1, 10 and 20% FBS (76, 76, 80 and 68%). The percentage of A grade embryos developed to ${\geq}$ morula in 10% FBS (59%) was significantly (p < 0.05) higher than 20% FBS (43%). The percentage of embryos developed to blastocyst showed no significant (p < 0.05) difference among BSA, 1, 10 and 20% FBS (34, 41, 43 and 32%). However, the percentage of A grade embryos developed to ${\geq}$ blastocysts in 10% FBS (25%) was significantly (p < 0.05) higher than in 20% FBS (8%).

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.334-339
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• 2007

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of $CaCl_2$ and 0.1 mM of $MgCl_2$ for $30{\mu}s$ using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of $H_2O_2$ in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and $100{\mu}M$ L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and $100{\mu}M$ L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and $200{\mu}M$ L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the $H_2O_2$ level in developing embryos in a dose-dependant manner. The $H_2O_2$ level (pixels/ embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and $200{\mu}M$ L-ascorbic acid, respectively. So, we recommend to supplement 50 or $100{\mu}M$ L-ascorbic acid in porcine in vitro culture medium.

• Applied Microscopy
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• 제40권4호
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• pp.211-218
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• 2010

포유동물의 난소는 호르몬과 감각신경 및 교감신경의 지배를 받아 난소 내 혈류의 조절, 스테로이드 호르몬 생성 및 난포의 발달과 관계된 난소의 고유기능이 조절되고 있다. 본 실험에서는 통상적인 추적자인 CT-HRP와 Pseudorabies바이러스(PRV)의 Bartha strain을 신경추적자로 이용하여 뇌하수체 후엽 및 난소와 연결된 중추신경부위를 밝히고자 하였다. 또 oxytocin을 난소 지배 신경축속에서 동정함으로써 신경축내 oxytocinergic neuron들의 존재를 확인하고, 이들이 배란을 중심으로 한 난소생식주기에 따라 보이는 중추 내 oxytocin신경세포의 변화를 조사하고자 하였다. Sprague-Dawley 흰쥐를 대상으로 난소 내에 PRV를 주사하고 48시간 후 실험동물들은 4% paraformaldehyde-lysine periodae로 고정하였으며, 뇌를 적출하여 $30{\mu}m$ 두께의 관상연속 절편을 만들어 CTHRP, PRV 및 oxytocin에 대한 삼중염색을 시행하였다. 본 실험 결과 후뇌에서부터 전뇌에 이르기까지 PRV에 양성반응을 보인 신경핵들이 관찰되어 난소를 지배하는 신경축을 구성할 수 있었다. 또 시상하부의 뇌실옆핵에서 oxytocin, PRV와 CT-HRP에 삼중으로 염색된 세포가 관찰됨으로써 신경내분비축과 자율신경축이 공동으로 기원하고 있다는 것을 형태학적으로 보여주었다. 따라서 oxytocin은 이 두 계통 내에서 호르몬의 역할과 신경전달물질의 역할을 겸할 것이라는 것을 추측할 수 있었다.