• Development and Reproduction
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• v.8 no.1
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• pp.27-33
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• 2004

To investigate the mechanism of germ cell death in postnatal stage of mouse, the involvement of apoptotic executioners, caspase-3 and caspase-activated DNase(CAD), and apoptotic initiators, Bax Fas and Fas ligand, in the germ cell death has been studied. Immune-labels of active caspase-3 and CAD were located in TUNEL-positive, apoptotic, oocytes as well as normal oocytes of primary or secondary follicles. CAD immune-labels were also detected in the nucleus of TUNEL-positive oocytes. Most of oocytes showing positive immune-labeling of active caspase-3 or CAD had vacuoles in their cytoplasm, which is the morphological characteristic of oocyte during folliclar atresia. Bax immune-stains were detected in the atretic oocytes which showed the vacuole in their cytoplasm. Positive immune-labels for Fas ligand was localized in TUNEL-positive or atretic oocytes. Presence of immunoreactivity of active caspase-3 and CAD in TUNEL-positive germ cells implicate that active raspase-3 and CAD might play a role in germ cell apoptosis during early development of mouse ovarian follicle. Immunohistochemical localization of Bax and Fas ligand in TUNEL-positive oocytes suggests that these might be the most plausible modulator of oocyte apoptosis.

• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.67-67
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• 2003

Early development of porcine oocytes fertilized in vitro was examined in different culture conditions. Porcine ovaries were collected from local slaughter-house. Cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cystein, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for 42~44 hrs. The frozen-thawed spermatozoa were washed by centrifigation 2 times at 1, 500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, 1$\mu\textrm{g}$/ml streptomycin and 1ng/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 2.5$\times$10$^{6}$ cells/ml motile sperm during fertilization in vitro. At 8hrs h after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine and 4 mg/ml BSA and cultured for 7 days. In first experiment, the mean numbers of oocytes collected from 20 ovaries were 674.4 oocytes, and 4.1(27.6), 12.5(84.0), 25.4(171.6) and 57.9%(390.8) for A, B, C and D grade in morphological classification. In the second experiment, when culture medium was supplemented with various concentrations of EGF, the proportions of oocytes cleaved were 56.9, 55.7, 61.9 and 54.7% for 0, 5, 10 and 20ng/ml EGF. The higher proportions(15.1%) of oocytes developed to morular stage were obtained at concentration of 10ng/ml than 0 and 5ng/ml EGF (P<0.05). However, the proportions of embryos developed to blastocyst stage were not significantly different among concentrations of EGF. In another experiment, when the medium supplemented with catalase was used, the proportions of oocytes cleaved were higher in the concentration of 0 unit (56.5%, 61/108) than 100 and 1, 000 unit/ml of catalase (P<0.05). Although the developmental capacity of embryos was improved by medium with 0 unit/ml compared with 100, 500 and 1, 000 units/ml of catalase in oocytes developed to morula and blastocyst stages, were not significantly different among concentrations of catalase.

• The Korean Journal of Pesticide Science
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• v.5 no.4
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• pp.11-19
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• 2001

This review discusses the effects and roles of insect hormones and insect growth regulators (IGRs) on vitellogenesis in adult insects. Insect vitellogenesis is regulated by hormones such as juvenile hormone (JH), ecdysteroids, and neurosecretory hormones (ovaryecdysteroidogenic hormone : OEH) released by neurosecretory cells, diet, and other elements(male specific protein of sperm fluid). In the fat bodies, the vitellogenins are synthesized by the stimulation of JH released by corpus allatum (CA) and ecdysteroids produced by follicle cells with the ovary in most insects. Furthermore, vitellogenins are released into the hemolymph, transported to the ovarioles by carrier protein, and incorporated into oocytes for the developing ovary. Of IGRs, juvenile hormone and its mimics such as methoprene and pyriproxifen appear to have pharmacological effects such as membrane lysis, destruction of salivary grand and midgut epithlial cells, fat body cells, and ovarian tissue, and also anti-juvenile hormone such as precocenes I and II appear to have specific cytotoxicity such as inhibition of corpus allatum and oocytes development. These results suggest that IGRs may be useful as agents for integrated pest management.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.11
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• pp.1573-1582
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• 2015

This study was to investigate the effect of soyabean isoflavones (SIF) on onset of puberty, serum hormone concentration, and gene expression in hypothalamus, pituitary and ovary of female Bama miniature pigs. Fifty five, 35-days old pigs were randomly assigned into 5 treatment groups consisting of 11 pigs per treatment. Results showed that dietary supplementation of varying dosage (0, 250, 500, and 1,250 mg/kg) of SIF induced puberty delay of the pigs with the age of puberty of pigs fed basal diet supplemented with 1,250 mg/kg SIF was significantly higher (p<0.05) compared to control. Supplementation of SIF or estradiol valerate (EV) reduced (p<0.05) serum gonadotrophin releasing hormone and luteinizing hormone concentration, but increased follicle-stimulating hormone concentration in pigs at 4 months of age. The expression of KiSS-1 metastasis-suppressor (KISS1), steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase ($3{\beta}-HSD$) was reduced (p<0.01) in SIF-supplemented groups. Expression of gonadotropin-releasing hormone receptor in the pituitary of miniature pigs was reduced (p<0.05) compared to the control when exposed to 250, 1,250 mg/kg SIF and EV. Pigs on 250 mg/kg SIF and EV also showed reduced (p<0.05) expression of cytochrome P450 19A1 compared to the control. Our results indicated that dietary supplementation of SIF induced puberty delay, which may be due to down-regulation of key genes that play vital roles in the synthesis of steroid hormones.

• Korean Journal of Animal Reproduction
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• v.11 no.3
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• pp.223-229
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• 1987

This study was carried out to investigate effects of superovulation and time of embryo recovery on ovarian response, recovery rate and developmental stage of embryo in donor and effects of methods of synchronization, number of corpus luteum (CL), stage of embryo and time of embryo transfer on ovarian response, conception rate and number of newborn in recipients which were transferred on 2.5, 3.5 and 4.5 days after synchronization. The results obtained are as follows; 1. The ovulation point of superovulated donor on 2.5, 3.5 and 4.5 days after copulation was 23.3, 35.3 and 23.3, respectively. The number of embryos recovered from the donors on 2.5, 3.5 and 4.5 days after copulation was 23.3, 25.8 and 19.8, respectively. The ovulation point and number of embryos recovered on 3.5 days were greater than those of 2.5 and 4.5 days. Among 232 embryos recovered on 3.5 days after copulation, 84 were blastocyst and 62 were hatching blastocyst. 2. The number of CL in recipients on 2.5, 3.5 and 4.5 days after synchronization was 3.2, 2.9 and 3.8 and showed no difference among the days. 3. When the number of CL was 0, 2-3, 4-6 and more than 7 the pregnancy raet of recipients was 0, 37.5, 66.7 and 75%, respectively. The pregnancy rate of recipients increased as the number of CL increased. 4. The pregnancy rate of transferred morula, blastocyst and hatching blastocyst was 32.0, 37.2 and 24.7%, respectively. The blastocyst nhowed highest pregnancy rate. 5. When the recipients were synchronized by HCG, the number of CL, unruptured follicle, hemorrhage, pregnancy rate and number of young were 5.5, 6.4, 3.3, 72.7% and 3.3, whereas that of GnRH were 2.3, 4.4, 2.8, 25.0% and 1.2, respectively. Recipients synchronized by HCG showed better results than GnRH. 6. When the embryos were collected on 2.5 days after copulation and transferred to the synchromized recipients, the pregnancy rate and the number of young born was 62.58% and 3.1, respectively. Those of 3.5 and 4.5 days after copulation was 57.1% and 1.3, and 37.5% and 1.6. The 2.5 days showed higher pregnancy rate and number of young born than 3.5 and 4.5 days.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.79-86
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• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.239-244
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• 1998

The purpose of this study is compare IVF cycle outcome in poor responders between clomiphene citrate (CC) stimulated and controlled ovarian hyperstimulation (COH) protocol. A total of 94 patients responding poorly in previous IVF cycles (estradiol<600 pg/ml or less than 3 oocytes retrieved) subsequently underwent either COH (COH group: 122 cycles, 68 patients) or CC-stimulated cycles (CC group: 43 cycles, 26 patients). CC was administered for five consecutive days starting on cycle day 3 at a dose of 100 mg daily. Serial transvaginal ultrasound examination was done from cycle day 8. Urine was collected $3\sim4$ times before hCG injection for the detection of LH surge. The hCG was administered when serum estradiol reached greater than 150 pg/ml and mean follicle diameter>16 mm. In COH group, ovarian stimulation was done using short protocol (GnRH-a/FSH/HMG/hCG). No difference in age or number of transferred embryos was found between CC group and COH group. COH group had significantly (p<0.05) higher mean peak level of $E_2$ ($810{\pm}112$ vs $412{\pm}55$ pg/ml) and greater number of retrieved oocytes ($3.0{\pm}0.2$ vs $2.0{\pm}0.2$) than CC group. CC group had transferred embryos $(1.8{\pm}0.2)$ compared with $(2.1{\pm}0.2)$ in COH group. However, CC group had higher pregnancy rate than COH group per retrieval [26.9% (7/26) vs 6.2% (6/97)], or per transfer [31.8% (7/22) vs 7% (6/86)]. Although cycle cancellation rate in CC group (48.8%) was higher than that of COH group (21.3%), the pregnancy rate per cycle in CC group was still higher (16.3%) than COH group (4.9%). In addition, implantation rate in CC group was 17.5% (7/40), which was significantly (p<0.01) higher than 3.9% (7/180) in COH group. These data suggest that oocyte and embryo quality are lower in COH cycles of poor responders than CC cycles. We suggest that clomiphene citrate stimulated IVF cycle may be more efficient than COH IVF cycle in poor responders in terms of lower costs and higher pregnancy performance.