• Development and Reproduction
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• v.18 no.4
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• pp.213-224
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• 2014

Previously we have shown that human abdominal adipose derived-stem cells (ADSCs) could aggregate during the high-density culture in the presence of human serum (HS). In the present study, we observed that human cord blood serum (CBS) and follicular fluid (HFF) also induced aggregation. Similarly, porcine serum could induce aggregation whereas bovine and sheep sera induced little aggregation. qRT-PCR analyses demonstrated that, compared to FBS-cultured ADSCs, HS-cultured cells exhibited higher level of mRNA expression of CLDN3, -6, -7, -15, and -16 genes among the tight junction proteins. ADSCs examined at the time of aggregation by culture with HS, BSA, HFF, CBS, or porcine serum showed significantly higher level of mRNA expression of JAM2 among JAM family members. In contrast, cells cultured in FBS, bovine serum or sheep serum, showed lower level of JAM2 expression. Immunocytochemical analyses demonstrated that the aggregates of HS-cultured cells (HS-Agg) showed intense staining against the anti-JAM2 antibody whereas neither non-aggregated cells (HS-Ex) nor FBS-cultured cells exhibited weak staining. Western blot results showed that HS-Agg expressed JAM2 protein more prominently than HS-Ex and FBS-cultured cells, both of latter reveled weaker intensity. These results suggest that the aggregation property of ADSCs during high-density culture would be dependent on the specific components of serum, and that JAM2 molecule could play a role in the animal sera-induced aggregation in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.532-537
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• 2008

In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.59-67
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• 1999

To make up the medium for quantitative selection of capacitated-sperm through sucrose layer, the effects of BSA, caffeine, heparin and progesterone on sperm swim-up migration and movement were examined. And the results obtained were as follows; 1. BSA of 4mg/ml in bMSS stimulated sperm migration and movement, and attracted capacitated-sperm. 2. Caffeine of 5mM in bMSS containing 4mg/ml BSA stimulated sperm movement and attracted capacitated-sperm. 3. Heparin of 20$\mu\textrm{g}$/ml in bMAA containing both 4mg/ml BSA and 5mM caffeine stimulated movement and capacitation of sperm. 4. Progesterone of 50$\mu\textrm{g}$/ml in bMSS containing all 4mg/ml BSA, 5mM caffeine and 20$\mu\textrm{g}$/ml heparin (BCHP-MSS) attracted capacitated-sperm. 5. Effect of BCHP-MSS on sperm on sperm attraction was not different from effect of 10% follicular fluid solution (FF-MSS) on sperm swim-up separation. In conclusion, bMSS with 4mg/ml BSA, 5mM caffeine, 20$\mu\textrm{g}$/ml heparin and 50$\mu\textrm{g}$/ml progesterone(BCHP-MSS) was a optimal condition for selection of capacitated-sperm through sucrose layer.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.2
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• pp.123-131
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• 1991

In order to study the growth and maturation of ovarian follicle, the localization and activity of alkaline phosphatase(ALPase) and adenosine triphosphatase(ATPase) of the granulosa cells and theca layer were examined according to the follicle size, the follicle state and the ovarian cyclic phase in pig. Theca interna of the small follicles was more heavyly localized with reaction product by the activites of ALPase and ATPase than that of the large follicles. It is assumed that, as the follicles proceed to growth and maturation, antrum formation is the result of the follicular fluid accumulation by means of active transport by the activities of ALPase and ATPase in theca interna. The activities of ALPase and ATPase in atretic follicles were higher than those of normal follicles. These results imply that the mechanisms of follicle maturation and atresia are different according to the phase of ovarian cycle.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.11
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• pp.1559-1563
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• 2002

The ovarian follicular fluid was found to contain steroidogenesis stimulatory protein similar to albumin from human and buffalo. Therefore, the albumins from various species, commercial and purified, were studied for their steroidogenic effect on progesterone secretion by granulosa cells from buffalo ovaries, during culture. A dose of $20{\mu}g$ of bovine serum albumin was optimum to exhibit maximum progesterone secretion on day 6 of culture, in medium ($350{\mu}l$) containing $10^5$ cells. Among commercial albumins, chicken albumin showed highest effect on progesterone secretion, which was followed by albumins from goat, bovine, human, sheep and rat, respectively at day 6 of culture. The albumins were also purified from blood serum of buffalo, goat and rat using salt fractionation, ion-exchange chromatography, gel filtration and SDS-PAGE. The highest stimulatory effect on progesterone secretion was shown by albumin purified from buffalo blood serum and lowest by that from rat blood. Comparatively the buffalo and goat albumins were more biologically active than commercial albumins. The presence of some active molecules conjugated with freshly purified albumins may be responsible for better stimulatory effect.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.135-142
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• 2003

본 연구는 사람의 시험관아기 프로그램에서 수정 후 1일째의 전핵 등급이 체외 수정란의 발달에 미치는 영향을 조사하였다. 정상적인 시험관 아기 시술을 시행한 실례 환자를 대상으로 과배란을 유기하여 배란 직전의 난자를 채취하여 정자를 주입한 다음 체외 수정을 유도하였다. 수정 유도 18시간 후 전핵과 핵인의 형태에 따라 전핵의 등급을 1 및 2등급으로 나누어 각각 3일 동안 체외 배양을 실시하여, 체외 수정란의 형태에 따라 1, 2 및 3등급으로 분류하였든 바, 전핵의 등급에 따라 각각 1등급 체외수정란의 발달율은 1등급 전핵란에서는 83.5%로서 2등급 전핵란의 5.5%보다 유의적으로 (P<0.05) 높았다. 1등급 및 2등급 전핵란에서 수정 후 3일째에 5-세포기 이상의 단계로 발달하는 경우는 각각 85.1% 및 24.5%를 나타내었고, 평균 할구수는 7.4$\pm$2.1 및 4.1$\pm$3.5개를 나타내어 발달 능력에서 1등급 전핵란이 2등급 전핵란보다 유의적으로 (P<0.05) 높게 나타났다.

• The Korean Journal of Zoology
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• v.18 no.2
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• pp.61-70
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• 1975

여포액이 여포안에서는 난자의 성숙을 억제하지만 여포액을 일단 여포 밖으로 내어 배양액을 섞거나 혹은 여포액만으로 난자를 배양할 때 난자는 성숙분열을 일으키는 것을 보아 왔다. 저자들은 소의 여포액 내의 성분중 어떤 것 들이 난자 성숙에 유효한 가를 밝히기 위하여 본 실험을 행하였다. 그 결과는 다음과 같다. 첫째, 여포액을 투석하여 얻은 dialysable fraction을 이용하여 난자를 배양하였을 때 높은 성숙율을 얻었다. 반대로 non-dialysable fraction를 포함하는 기본배양액 내에서는 난자의 성숙율은 극히 낮았으며 퇴화하는 경향이 있었다. 둘째, paper chromatography 의 방법으로 dialysable fraction이 포함하는 free amino acid를 조사한 결과 8가지의 amino acid가 동정되었다. 이 amino acid를 기본배양액에 섞어 배양액으로 만들어 난자를 배양했을 때 난자성숙율이 90%이상이었고 제 2차 중기까지 도달한 난자는 30시간 이내에 10% 정도였다. 셋째, 상기의 amino acid가 들어있는 배양액에 다시 biotin를 첨가하여 배양하면 48시간내에 제 2차 분열 중기까지 도달하는 난자가 60%에 이르렀다. 위 결과로 보아 여포액은 in vitro인 경우 난자의 성숙을 유발하며 성숙을 유발하는 주요 성분은 여포액내에 포함되어 있는 몇가지 amino acid와 biotin과 같은 vitamine B가 될 것이라는 결론을 얻었다.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.267-271
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• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.323-331
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• 1999

The effects of insulin-like growth factor-I (IGF-I) and cumulus cells during in vitro maturation in porcine oocytes were examined. When follicular oocytes were cultured in medium with different concentrations of IGF-I, maturation rates were 60, 61 and 62 and 72% for 0, 15 and l0ng/$m\ell$ IGF-I. In medium with 10ng/$m\ell$ IGF-I, maturation rates were not significantly difference between oocytes with (68%) and without (52%) cumulus cells during the culture. In medium with-out IGF-I, however, the maturation rates in oocytes with cumulus cells (63%) was significantly (P＜0.05) higher than oocytes without cumulus cells (32%). On the other hand, when IGF-I was added for first 24 h period or later 24 h period of culture, maturation rates were higher in oocytes with (61 and 49%) that than without (49 and 45%) cumulus cells. In experiment used medium without fetal calf serum (FCS) and porcine follicular fluid (PFF), the maturation rates in oocytes with cumulus cells for 48 h (48 and 67%) or first 24 h (46 and 63%) period after culture were significantly (P＜0.01) higher than in oocytes without cumulus cells (16 and 18%) in the presence or absence of IGF-I. These results indicated that cumulus cells is essential on maturation in vitro in porcine oocytes, but IGF-I can promote oocytes maturation of oocytes without cumulus cells in medium with FCS and PFF.