• 대한생식의학회:학술대회논문집
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• 2005.06a
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• pp.126-126
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• 2005

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.93-102
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• 1998

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptides, which may mediate or potentiate gonadotropin action. Inhibin also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia. Therefore, we studied the differences of serum inhibin, gonadotropin and androgen levels in the women with only the ultrasound findings and no disorder, and polycystic ovary (PCO) with ovulatory disturbance. We prospectively analysed forty-three women with PCO. The diagnosis of PCO was based on typical appearance of the ovaries on TVS. Twelve women with regular menstrual cycle and normal ovarian morphology were selected as control. Basal levels of inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol $(E_2)$, testosterone (T), androstenedione (ADD), dehydroepiandrosterone-sulfate (DS), prolactin and TSH in serum were determined. There were significant differences in basal LH levels and LH/FSH ratio between the control and the women with PCO. The basal levels of inhibin and $E_2$ in the oligo-amenorrheic PCO (N=34) were significantly higher than those in the control. There was higher negative correlation between the inhibin and T levels in the oligo-amenorrheic PCO, but, not in the regular cycling PCO. Also, there was higher positive correlation between the LH and T levels in the oligo-amenorrheic PCO, but not in the regular cycling PCO. These data presume that the initial event of PCO is elevated pituitary LH secretion. Elevated levels of LH may down-regulate LH receptors on granulosa cells and also cause hypertrophy of the thecal layer. High level of androgen secreted by the hypertrophied thecal layer may stimulate inhibin secretion from granulosa cells and can be converted to estrogen by extraovarian tissues and could serve to augment pituitary sensitivity to GnRH with a resultant secretion of more LH than FSH. Inhibin may inhibit FSH action on granulosa cell in the PCO follicle, impairing follicular development and dominant follicle selection resulted in ovulatory disturbance.

• Journal of Veterinary Clinics
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• v.9 no.1
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• Development and Reproduction
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• v.1 no.1
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• pp.67-77
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• 1997

Mammalian ovary consists of various growing stages of follicles. Ovarian follicular growth and differentiation, however, can be distinguished into recruitment, growth, selectiona nd ovulation. while only minute of the selected follicles ovulate their oocytes, all the rest follicles disappear by atresia. this atresia is an important event of which physiological mechanism must be resolved. The present study was carried out to investigate the effects of various doses of pregnant mare's serum gonadotropin (PMSG) on the oocyte quality, ovulation rate, and the early embryonic development in immature mice. Immature mice were administrated with 5, 20, or 40 IU PMSG. At every 12 hour up to 72 hour after treatment, body and ovary weights were measured. Oocytes were flushed from the oviducts under the dissecting microscope and observed under the inverted microscope. Late 2-cell embryos were collected from the mice which were superovulated by the same dosage of PMSG followed by 5 IU hCG 47 hours after PMSG-treatment. The percentage of abnormal oocytes was higher in 20 or 40 IU PMSG-treated animals than 5 IU PMSG-treated ones. Ovulation occured at 12 hours afger PMSG injection in all experimental groups. The percentage of retrieved abnormal oocytes increased in the 20 or 40 IU PMSG-treated goups but not in 5 IU PMSG-treated group. There was no significant difference in the mating rate among the groups [52.6% (10/19), 66.7% (10/15), 44.0% (11/25) : 5, 20, 40 IU group respectively] ; however, ther was a significant (p<0.01) increase of embryo retrieval rates in 5 and 20 IU-treated groups compared with that in 40 IU-treated group [89.2% (239-268), 85.5% (224/262), 40.0% (18/45)]. There was significant (p<0.01) increase of embryo development rates in 5 IU-treated group compared with that in 20 and 40 IU-treated group [231/239(96.7), 179/224(79.9), 77.8(14/18)]. In conclusion, higher doses of PMSG injection increased the occurrence of abnormal oocytes ovulation in immature mice. The most of oocytes collected from 5 or 20 IU-PMSG-treated group has fertilizabioity. But in mice injected iwth higher doses of PMSG, their oocytes exhibit less fertilizability and, even fertilized, all oocytes are not fully capable of development.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.71-81
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• 1994

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.171-178
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• 1999

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.249-254
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• 2012

This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p<0.05 or p<0.01). The proliferation of the cells cultured in the SS group was significantly lower than that of the other treatment groups (p<0.01). The population of apoptotic and necrotic cells in the SS group was significantly higher than that of either the control or the pFF group (p<0.01). The number of embryos that cleaved (p<0.05) and developed into blastocysts (p<0.01) in the SS group was significantly lower than that of either the control or the pFF group. Compared to other groups, the blastocysts produced from the donor cells in the pFF group had higher total cells and lower apoptotic cells (p<0.05). It can be concluded that pFF supplementation in the donor cell culture medium positively affects cell death, cell cycle and quality of the cloned blastocyst.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.341-348
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• 2011

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.