• Journal of Embryo Transfer
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• v.24 no.3
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.1
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• pp.136-144
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• 2007

Biotae Orientalis has been widely used for treatment of relaxion of smooth muscle, gastrointestinal hemorrhage and alopecia in Oriental Medicine. This experiment examined the effect of an extracts, obtained from the acetone and MeOH extracts of dried or fresh Biotae Orientalis, on hair growing activity of the C57BL/6N mice after topical application to skin. First, We examined on hair growth activity of extracts of Biotae Orientalis compare to control and 1% minoxidil groups. Second, We investigated on the number of hair follicle and mast cells after topical application of extracts of the Biotae Orientalis to skin for 16 day. Third, We investigated immunoreactive density of vascular endothelial growth factor(VEGF), protein kinase C-${\alpha}$(PKC-${\alpha}$) and stem(mast) cell factor(SCF) in skin of C57BL/6N mice by immunohistochemical methods. I fourth investigated changes of subpopulation of splenocytes and thymocytes in C57BL/6N mice for 16day using laser flow cytometry. The results were as follows : Hair growing effect of acetone and MeOH extracts of dried and fresh Biotae Orientalis was observed in 70%, 90% and 60% in hair removed skin area in 16 day respectively. Immunoreactive density of VEGF and PKC-${\alpha}$ in skin of experimental groups was weakly stained compare to control group in 10 day. Immunoreactive density of stem cell factor in skin of experimental group was heavily stained compare to control group in 10 day. Splenic TH/TC Iymphocytes of lived MeOH extracts group significantly increased compare to control group. TH cells in thymic T lymphocytes were increased compare to control group. These experiment suggest that acetone and MeOH extracts of Biotae Orientalis may be used for topical treatment of alopecia areata.

• Development and Reproduction
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• v.7 no.1
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• pp.15-22
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• 2003

The Fas antigen (Fas) as a cell-surface receptor protein which mediates apoptosis-inducing signals plays an important role in the immune system. Expression of Fas mRNA is detected not only in lymphoid organs but also in the nonlymphoid organs. In the ovary, most of the follicles is known to undergo atreisa through apoptosis. However, the exact mechanism of atresia was not elucidated yet. Therefore, the purposes of the present study were to investigate the expression of Fas and Fas ligand in mouse ovary and to clarify the relationship between expression of Fas and Fas ligand and atresia of follicle. The result of RT-PCR demonstrated that Fas and Fas ligand mRNA was expressed in ovary, especially granulosa cells and oocytes. The immunohistochemistry showed that the granulosa cells and oocytes in growing follicles were stained for Fas and Fas ligand, but primordial follicles were not. Furthermore, Fas and Fas ligand were intensively stained in the atretic follicles As results of TUNEL staining to detect apoptotic cells in the ovaries, the number of TUNEL-positive (apoptotic) granulosa cells and oocytes increased in the atretic follicles compared to the healthy normal follicles. These results demonstrate that there is the positive relationship between expression of Fas and Fas ligand in granulosa cells and oocyies and apoptosis of them leading to atresia of follicles. It suggests that expression of Fas and Fas ligand could be associated with atresia of follicles in mouse ovary.

• Korean Journal of Environmental Biology
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• v.20 no.3
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• pp.224-231
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• 2002

Photoperiod (length of light per day) is a major factor in regulating reproductive function in golden hamsters. The information of photoperiod is transmitted to the reproductive endocrine system by melatonin. Thus the effects of melatonin aye investigated in male golden hamsters exposed to photoperiods. Paired testicular weights were markedly reduced in the animals housed in short photoperiod $(SP,\le{12\;hours\;day^{-1})$ and injected with melatonin in the evening, but not in long photoperiod $(LP,\le{12.5}\;hours\;day^{-1})$ and injected with melatonin in the morning. The histological examination of regressed testes showed reduction of tubular lumen diameter including the numbers of cells and Leydig cell number. The mean values of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) were also lowered in the sexually inactive animals than in the sexually active animals. Melatonin receptor was identified by reverse-transcription polymerase chain reaction (RT-PCR) and its expression was examined in various tissues to scrutinize the action site of melatonin. It turned out 309 nucleotides and was definitely expressed in hypothalamus and pituitary including spleen, retina, and epididymis. And gonadotropin releasing hormone (GnRH) gene, which is a key element in regulating reproduction, was identified by RT-PCR but the expression of GnRH was not modified by the treatment of melatonin. Taken together, photoperiod via melatonin indirectly affects reproductive endocrine system, possibly through the release of GnRH, not the synthesis of GnRH.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.113-120
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• 1989

In order to determine the sex of preimplantation embryos prior to transfer in cattle, a series of experiments were carried out using 45 Holstein donor cows to examine the ovarian response on the gonadotropin and PGF2${\alpha}$, and the morphology of fresh embryos or frozen/thawed embryos after deep freezing at -196$^{\circ}C$. The sexing of embryos treated with the medium containing H-Y antiserum(10%, v/v) and FITC anti-mouse IgG(10%, v/v) were analysed by chromosomal analysis, and the sex of the embryos which survived were ascertain after delivering the pups. The results obtained were summarized as follows ; 1. The average number of developed follicle and corpus luteum per cow were 13.5 and 8.1, and the ovalation rate was 60.1%. 2. Of 220-ova recovered, 75(34.1%) were morula and 91(41.4%) were blastocyst, and the morphological normal and abnormal rate of ova recovered were 75.5% and 24.5%, respectively. 3. Of 39 frozen/thawed embryos, the scores of normal morula and blastocyst, after thawing were 79.2%(19/24) and 73.3%(11/15). The average rate of frozen/thawed embryos which appeared morphologically normal post thawing was 76.9%(30/39). 4. The sex ratio was measured using the embryos treated with immunofluorescence assay to examine the relationship between embryo developmental stage, sex ratio of morula stage embryo was 42.2%(19/45) fluorescing and 57.8%(26/45) non-fluorescing, on the other hand, the ratio switched to 46.8%(29/62) fluorescing and 53.2%(33/62) non-fluorescing embryo in blastocyst stage. The sex ratio was also measured between fresh and frozen/thawed embryos, fresh and frozen/thawed treated embryos were indicated 45.8%(38/83) fluorescing, 54.2%(45/83) non-fluorescing and 41.7%(10/24) fluorescing, 58.3%(14/24) non-fluorescing. This trend indicated the approximal sex ratio was 1 : 1. 5. The result of karyotype test showed the successful rate of sexing embryo is fluorescing and non-fluorescing was 21.2%(7/33) and 29.6%(8/27). The female to male ratio within 33 fluorescing was 28.6 : 71.4, and the ratio of 27 non-fluorescing embryos was 87.7 : 12.5. 6. Of the embryo transferred after assignment of H-Y phenotype, five of the fluorescing embryos survived to term, all was males. Whereas six non-fluorescing embryos also survived to term and the sexes of the calves were 1 male 5 female.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1023-1029
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• 2007

Inhibins participate in the regulation of pituitary follicle-stimulating hormone synthesis and secretion, follicular maturation and steroidogenesis in the female. Inhibin ${\beta}_A$ gene (INHBA) was studied as a candidate gene for the prolificacy of sheep. Single nucleotide polymorphisms of the entire coding region and partial 3' untranslated region of INHBA were detected by PCR-SSCP in two high fecundity breeds (Small Tail Han and Hu sheep) and six low fecundity breeds (Dorset, Texel, German Mutton Merino, South African Mutton Merino, Chinese Merino and Corriedale sheep). Only the PCR products amplified by primers 3, 4 and 5 displayed polymorphisms. For primer 3, genotype CC was only detected in Chinese Merino sheep, genotype AA was detected in the other seven sheep breeds. Genotype BB was only detected in Hu sheep. Only Hu sheep displayed polymorphism. Eight or four nucleotide mutations were revealed between BB or CC and AA, respectively, and these mutations did not result in any amino acid change. For primer 4, genotypes EE, EG and GG were detected in Dorset and German Mutton Merino sheep, genotypes EE, EF and FF were detected in Chinese Merino sheep, only genotype EE was detected in the other five sheep breeds. Only Dorset, German Mutton Merino and Chinese Merino sheep displayed polymorphism. Sequencing revealed one nucleotide mutation ($114G{\rightarrow}A$) of exon 2 of INHBA gene between genotype FF and genotype EE, and this mutation did not cause any amino acid change. Another nucleotide change ($143C{\rightarrow}T$) was identified between genotype GG and genotype EE, and this mutation resulted in an amino acid change of $serine{\rightarrow}leucine$. For primer 5, genotypes KK and KL were detected in German Mutton Merino and Corriedale sheep, genotypes KK, LL and KL were detected in the other six sheep breeds. Genotype MM was only detected in Hu sheep. All of these eight sheep breeds displayed polymorphism. Sequencing revealed one nucleotide mutation ($218A{\rightarrow}G$) of exon 2 of the INHBA gene between genotype LL and genotype KK, and nine nucleotide mutations between genotype MM and genotype KK. These mutations did not alter amino acid sequence. The partial sequence (395 bp for exon 1 and 933 bp for exon 2) of the INHBA gene in Small Tail Han sheep (with genotype KK for primer 5) was submitted into GenBank (accession number EF192431). Small Tail Han sheep displayed polymorphisms only in the fragment amplified by primer 5. The Small Tail Han ewes with genotype LL had 0.53 (p<0.05) or 0.63 (p<0.05) more lambs than those with genotype KL or KK, respectively. The Small Tail Han ewes with genotype KL had 0.10 (p>0.05) more lambs than those with genotype KK.

• The Korean Journal of Zoology
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• v.32 no.3
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• pp.281-289
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• 1989

The present study was disigned to determine the concentration of bioavailable steroid hormones in the atretic follicular fluid (FF). The concentradons of progesterone (P), testosterone (T), estradiol (E), androstenedione (A), and 5-$\alpha$ dihydrotestosterone (DIlT) were determined by the established methods of luminescent immunoassay (LIA) or radioimmunoassay (RIA). Concentrations of T, A and Diff in human FF from smail (< 6 mm). medium (8-15 mm), and large (> 15 mm) atretic follicles were significandy higher than those of normal ones (p < 0.01). However, the levels of T, A and DHT in smail atretic foflicle were significandy lower than those found in normal one. The concentrations of P in atretic FF from porcine small (< 3 mm), medium (4-6 mm), and large (> 7 mm) follicles were not different from that of normal ones. However, the concentration of E in atretic forncles of each group was significantly lower than that of normal group (p < 0.001 in each group). On the other hand, the percentages of bioavailable T (BI) in human FF were significandy (p <0.001) higher than those in normal groups. The BT in normal or atretic FF was more than 90 % of total T. The present result demonstrates that the bioavailable androgen, but not E levels in atretic follicles is higher than that of normal one, and that the atretic mechanism might be dependent on the ovarian forncle size in the developmental stage and on the animal model system. Moreover, the present study suggests that the steroids found in the FF are the bioavailable forms and the concentration of BT in FF could be used as one of the valuable criteria classifying the ovarian atretic follicle.

• The Korean Journal of Malacology
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• v.27 no.3
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• pp.261-271
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• 2011

The gametogenic cycle, the spawning season and the biological minimum sizes in female and male Protothaca (Notochione) jedoensis were investigated by quantitative statistical analysis. In females, monthly changes in the percents of the follicle areas to the ovarian tissue areas and the percents of the oocyte areas to the ovarian tissue areas increased in February and reached the maximum in April, and then gradually decreased from May to July, with the spawning peak between June and July. In males, monthly changes in the percents of the testicular tissue areas to total tissue areas and the percents of the spermatogenic stage areas to the testicular tissue areas increased in February and reached the maximum in April, and then showed a rapid decrease from May to July. From these data, it is apparent that the number of spawning seasons in female and male P. (N.) jedoensis occurred once a year, from May to July. Therefore, P. (N.) jedoensis in both sexes showed a unimodal gametogenic cycle during the year. Compared the gametogenic cycle by quantitative statistical analysis in 2007 with the previous qualitative results of this species, the results of the gametogenic cycle calculated by quantitative statistical analysis showed some differentiations in the spawning seasons evaluated by the gonad index by qualitative histological analysis. The intervals of the beginning of two spawning seasons showed one month between the results of quantitative and qualitative analyses. The biological minimum sizes (considering to 50% of group sexual maturity) in female and male clams by quantitative analysis of this species are 32.01 mm in shell length in females and 30.58 mm in males, respectively. According to the mean shell length fitted to von Bertalanffy's equation, 30.58 and 32.01 mm in shell length were considered to be two years old. Therefore, we assume that both sexes of this population begin reproduction from two years of age.

• The Korean Journal of Malacology
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• v.25 no.2
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• pp.145-151
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• 2009

The ultrastructural changes in germ cells during oogenesis of the melania snail, Semisulcospira libertina libertina have been investigated by light and electron microscopy. The ovary is located on the surface of the hepatopancreas in the spiral posterior region. The ovary exhibited greenish color in the gonadal mature season. The ovary was composed of a number of oogenic follicles. Oogenesis was divided into five stages with histological features: (1) oogonia, (2) previtellogenic, (3) initial vitellogenic, (4) active vitellogenic, and (5) mature stages. Oogonia were oval in shape, $4-6\;{\mu}m$ in diameter, and had a large nucleus. Previtellogenic oocytes were about $20\;{\mu}m$ in diameter and the cytoplasm reacted with hematoxylin in H-E satin. Initial vitellogenic stage, oocytes were $60-80\;{\mu}m$ in diameter, and small yolk granules of low electron density are scattered in the cytoplasm. Oocytes in the initial vitellogenic stage were connected with ovarian follicle by egg stalk. Active vitellogenic oocyte were $100-120\;{\mu}m$ in diameter. Electron density, size and quantity of yolk granules that are distributed in the cytoplasm have increased from the previous stage. Result of TEM observations, the oocyte contains well-developed Golgi complex, endoplasmic reticula and tubular mitochondria in the cytoplasm. Cytoplasm of mature oocyte was filled with proteinaceous yolk globules of high electron density. In this stage, the length of microvilli in the egg envelope was approximately $1.1\;{\mu}m$.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.