• Journal of Embryo Transfer
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• 제16권3호
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• pp.203-211
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• 2001

This study was carried out to determine sex of porcine embryos produced by in vitro fertilization. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cystein (0.1 mg/ml) and hormonal supplement (10 IU eCG and 10 IU hCG per ml) for 20~22 hrs. They were then cultured in the same medium but without hormonal supplement for additional 20~22 hrs. After culture, cumulus cells were removed and oocytes were co-incubated for 6 hrs with four different concentrations (5$\times$10$^4$, 2.5$\times$ 10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) of porcine sperm. After fertilization, oocytes were transferred into NCSU 23 with 0.4% BSA medium. The cleavage and blastocyst formation rates were evaluated at 48 and 144 hrs, respectively. In this study, the polymerase chain reaction (PCR) was used to determine the sex of porcine embryos in the stage of blastocyst. The PCR was performed using a set of oligonucleotide primers (5‘-TCATGGACCAGGTAGGGAAT-3', 5’-GAAAGACACGTCCTTGGA GA-3') for 491 bp fragment of porcine male-specific DNA sequence. In the flour different sperm concentration (5$\times$10$^4$, 2.5$\times$10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) for fertilization condition, the cleavage rate was 55.95, 67.88, 60.18 and 47.60%, respectivety, and the development rate of blastocysts was 16.03, 20.40, 21.41 and 12.37%, respectively. At 5.0$\times$10$^4$and 2.5$\times$10$^{5}$ of sperm concentrations per ml cleavage rate and development rate of blastocyst were higher than those of 5.0$\times$10$^4$and l0$\times$10$^{5}$ of sperm concentration (P<0.01). The male of porcine embryos was detected at 491 bp by PCR, and 18 of the 31 porcine blastocysts were the male (58.1%) and the rest 13 were the female(41.9%).

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.169-174
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• 2005

This study was comducted to examine the effects of culture medium, and the osmolarity and osmotic change of the culture medium on in vitro maturation of porcine oocytes and developement of porcine parthenogenetic embryos. In Experiment 1, cumulus-oocyte complexes were matured in NCSU-23, mWM and mKRB, respectively, There was no difference in maturation rate($62.1\~71.3\%$) among groups. In Experiment 2, matured oocytes in each medium were activated and cultured for 6 days in the same media. Blastocyst formation rate was higher in NCSU-23($22.9\%$) than those of others($0\~0.6\%$, P<0.05). In Experiment 3, parthenogenetic embryos were cultured for 6 days in NCSU-23 with different osmolarity(300, 280 and 256 mOsmols) adjusted by NaCl. There were no differences in development rates to the blastocyst stage($11.0\~14.4\%$) among groups. In Experiment 4, activated oocytes were cultured for 2 days in NCSU-23 with 300, 280 and 256 mOsmols and then transferred to increased or decreased osmotic condition. Blastocyst formation rate was higher in a group which was transferred from the higher osmotic condition to the lowe. osmotic condition($21.0\%$) than a contrary group( $11.8\%$, (P<0.05). This result shows that the culture medium and the osmolarity of the culture medium affect the development of porcine parthenogenetic embryos, and the change of osmolarity from the higher condition to the lower condition at a certain developmental stage can enhance the development of porcine parthenogenetic embryos.

• Current Research on Agriculture and Life Sciences
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• 제8권
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• pp.45-50
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• 1990

This study was carried out ot investigate the effects of fetal calf serum (FCS) and gonadotropins supplemented to the medium on maturation and fertilization in vitro of porcine follcular oocytes. Ovaries were obtained from gilts at local slaughter-house. Oocyte-cumulus complexes were recovered by puncturing the ovarian follicles(3~5 mm in diameter). The complexes from individual ovaries were pooled in a $0.4m{\ell}$ droplet of medium covered with paraffin oil, then washed twice in fresh droplet and cultured for 36hrs in culture media according to experimental conditions. Boar epididymal spermatozoa were capacitated by preincubation for 4hrs in m-KRB medium and the preincubated spermatozoa were insemenated in the fertilization medium containing the cultured oocytes. The results obtained in this study are summarized as follows: 1. The maturation rates of oocytes cultured in m-KRB and m-KRB supplemented to 10% FCS were 82 and 37%, respectively. When PMSG, hCG. and PMSGt hcG($10Iu/m{\ell}$) were added to the media supplemented to 10% FCS, the maturation rates were 66, 58 and 68%, respectively. 2. Expansion of cumulus cells was not occured in m-KRB and m-KRB supplemented to 10% FCS. However, when PMSG, hCG and PMSG+hCG($10Iu/m{\ell}$) were added to m-KRB supplemented to 10% FCS, the expansion rates of cumulus cell layers were 92, 13 and 91%, respectively. 3. When oocytes were mltured in m-KRB, the rates of penetration and formation of male pronucle: were 93 and 7%, respectively. By adding FCS and gonadotropin to m-KRB, the penetration and formation of male pronuclei were 100 80%, respectively.

• Journal of Embryo Transfer
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• 제22권1호
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Development and Reproduction
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• 제13권4호
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• pp.329-339
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• 2009

Cortisol is present in high concentration in the ovary and its receptor is expressed in the ovarian cells. Moreover, cortisol is known to have a role in steroid synthesis and cell metabolism in human granulosa and lutein cells. However, little is known of the role of cortisol presenting in high concentration in the follicles after LH surge on the granulosa-lutein cells. Therefore, the this study we evaluated the apoptosis and the production of progesterone $(P_4)$ and estradiol $(E_2)$ in the granulosa-lutein cells that are obtained during oocyte-retrieval after treatment with 5, 50, and $500{\mu}g/m\ell$ cortisol and 1 IU/$m\ell$ FSH. Results of DNA fragment analysis and TUNEL assay demonstrated that DNA fragmentation and the rate of apoptotic cells were increased in a dose-dependent manner showing a significant increase in 50 and $500{\mu}g/m\ell$ cortisol treated cells. We found, however, that FSH did not suppress the apoptosis of the cells induced by cortisol. In the results of chemiluminescence assay for $P_4$ and $E_2$, $P_4$ production was decreased by cortisol treatment, whereas $E_2$ was not changed. We also demonstrated that FSH did not inhibit the suppressive effect of GnRH on $P_4$ production as the result of apoptosis. The present study suggests that cortisol of high concentration could cause the apoptosis of human granulosa-lutein cells by suppressing the production of $P_4$. However, we need more studies to elucidate the mechanism by which cortisol induces apoptosis in human granulosa-lutein cells in view of the fact that our results are inconsistent with previous reported data.

• Korean Journal of Veterinary Research
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• 제37권1호
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• pp.71-78
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• 1997

This study was designed to investigate the number of the growing and mature follicles following gonadotrophin treatments for superovulation in mature rats. Eighteen mature rats (Sprague-Duwely, initially 190~230gm) were randomly alloted into 3 groups. One group was control group, another FSH-treated group was injected intramuscularly with 0.5 units of follicular stimulating hormone (FSH) / rat, and third PMS and HCG-treated group was intramuscularly injected with 20~25IU of pregnant mare serum (PMS) / rat and then at the 48 hrs later, with 20~25IU of human chorionic gonadotrophin (HCG) / rat. The uteri and ovaries of rats were collected and then were observed grossly and serial sections of paraffin embedding ovaries were stained with H-E. Number of ovarian follicles by following 3 grades of large, middle and small follicles from secondary and tertiary follicles were investigated by LM photography of preparations. Small follicles were classified as secondary follicles of preantral follicles with more than 2 layers of granulosa cells surrounding the oocyte and middle follicles were classified as secondary follicles with early signs of antral cavity or with more than one small cavity on either side of the oocytes and large follicles were classified as tertiary follicles with a single medium sized antral cavity or large well-formed antral cavity. In gross findings, the uteri were slightly swelling in FSH-treated group and markedly swelling or filled with fluid in the uterine lumen in PMS and HCG-treated group. In histological findings, the shape and size of the follicles were diverse in middle and large follicles of FSH-treated group and PMS and HCG-treated group, and proportion of atretic follicles was increased in FSH-treated group and PMS and HCG-treated group than those in control group. The uteri of FSH-treated group and PMS and HCG-treated group were hypertropied or filled with fluid in the lumens and walls of uteri. The wall tissue layers were flattened and their blood and lymph vessels were dilated. The mean number of follicle per ovary in control group were appeared to be $17.1{\pm}5.6$($14.0%{\pm}4.6%$), $37.8{\pm}9.1$($30.9{\pm}7.4%$) and $67.6{\pm}30.1$($55.2{\pm}24.6%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $122.5{\pm}40.0$. The mean number of follicle per ovary in FSH-treated group were appeared to be $22.8{\pm}7.0$($17.4%{\pm}5.3%$), $43.4{\pm}6.6$($33.2{\pm}5.1%$) and $64.5{\pm}13.0$($49.3{\pm}9.9%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $130.7{\pm}16.6$. The mean number of follicle per ovary in PMS and HCG-treated group were appeared to be $29.7{\pm}11.0$($16.3%{\pm}6.0%$), $61.9{\pm}17.2$($33.9{\pm}9.4%$) and $91.1{\pm}28.2$($49.9{\pm}15.4%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $182.6{\pm}32.7$. The above findings reveal that large follicles were increased 29.8% in FSH-treated group and 73.7% in PMS and HCG-treated group than those in control group and in histologic findings, proportion of atretic follicles were more increased in ovaries with more number of more developing follicles.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.311-318
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• 1997

Bypassing acrosome reaction and fusion process in intracytoplasmic sperm injection(ICSI), most of injected spermatozoa still contain intact acrosome contents and plasma membrane. It Is not known yet what acrosome contents and plasma membrane of spermatozoa have effect on the development of embryo. For further understanding of fertilization process after ICSI, we studied the time of pronucleus formation, disappearance and first cleavage in human zygote, and pregnancy rate in relation to acrosome reaction rate of spermatozoa after ICSI. Seventy cycles undergoing ICSI program were randomly selected. Sperm suspension from 38 cycles were treated 50% human follicular fluid(hFF) for 3 hours in order to induce acrosome reaction, others were not treated as control. Acrosome reaction in hFF treated and non-treated group was assessed by fluorescein isothiocyanate(FITC)-conjugated Arachis hypogea(PNA) and Pisum sativum agglutinin(PSA). Oocytes were classified into 'good' and 'poor' according to their morphology. After ICSI, fertilization of oocytes were assessed by detection of two pronuclei at 16 hours. The pronuclei disappearance and first cleavage of zygotes were observed at 24 hours, and then embryos were transferred to uterus after culture for 72 hours. The rate of acrosome reaction of spermatozoa in hFF treated group was significantly higher than that in control(p<0.01). Fertilization rates of good oocytes were not different both control and hFF treated group(81.3%(174/206) vs. 72.1%(102/130)). But, in poor oocytes, the fertilization rates in hFF treated group(72.1%(149/183)) were increased compared than those of control group (63.6%(98/140), p<0.01). In either good or poor oocytes, the rates of pronuclei disappearance in hFF treated-spermatozoa injected oocytes were higher than control (59.1%(103/174), 56.4%(84/149) vs. 32.4%(33/102), 37.8%(37/98), p<0.01). Also, the rates of thirst cleavage were increased in hFF treated group (31%(54/174), 24.1%(36/149)) compared than those of control group (10.8%(11/102), 13.2%(13/98), p<0.01). The pregnancy rates of hFF treated group (42.1%(16/38)) were slightly higher than control group (28.1%(9/32), p>0.05). But, the pregnancy rate of group which possessed more than one cleavaged zygote at 24 hours was higher than group which did not (45.2%(19/42) vs. 21.4%(6/28), p<0.05). From these results, the development of zygotes were faster in higher acrosome reacted sperm group than lower acrosome reacted sperm group after ICSI. Our results may be explained that acrosomal membrane and plasma membrane are easily detached from spermatozoa in acrosome reacted spermatozoa compared with acrosome intact sperm in the cytoplasm of oocyte during pronuclear formation. We conclude that the injection of acrosome reacted spermatozoa will increase the pregnancy rate as they can induce fast embryonic development in ICSI.