In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during In Vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

Although in vitro production (IVP) techniques of porcine follicular oocytes have progressed and are well studied, the developmental potential of porcine oocytes matured in vitro remains low compared with those matured in vivo. It is well known that one of the reason occurred impair in vitro maturation (IVM) of porcine oocytes is the oxidative stress. Oxidative stress is mainly caused by reactive oxygen species (ROS) generation formed during cellular metabolism. β-cryptoxanthin (BCX) is one of the carotenoid pigment and possesses strong anti-oxidative and free radical scavenging activities and suppresses lipid peroxidation and nitrogen oxide production. The objective of this study was to examine the effects of BCX treatment on porcine oocyte during IVM and their in vitro developmental potential. The follicular oocytes were cultured in IVM medium supplemented with 0, 0.1, 1, 10 and 100 μM BCX (control, 0.1 B, 1 B, 10 B and 100 B). In analysis of intracellular ROS expression level after IVM, 1 B group was the lowest among all groups (p<0.05), while other BCX treated groups are similar to control group. Also, 1 B group was significantly decreased during the classified oocyte maturation stage (GVBD, MⅠ and MⅡ) than control (p<0.05). In addition, the relative mRNA expression level of antioxidant gene (superoxide dismutase-2 and peroxiredoxin-5) was significantly higher in 1 B group than control (p<0.05). After parthenogenetic activation, there was no different in the cleavage rate between two groups, however, the blastocyst formation rate was significantly higher in 1 B group than in control (p<0.05). In embryo quality, the total cell number and DNA fragmentation of blastocysts were no different between two groups. These results demonstrate that BCX is helpful for decreasing ROS level of porcine follicular oocytes and improves their developmental potential.

The follicular lymphoid hyperplasia is a very rare nonneoplastic lymphoproliferative disease in the oral mucosa. Clinically, it usually presents as asymptomatic, non-ulcerated, normal-colored, soft, sessile, unilateral and slow growing mass in the posterior hard palate. Therefore, soft-tissue mass of the hard palate can be a difficult diagnostic challenge for the clinician. Herein, we report a case of follicular lymphoid hyperplasia located in the left posterior hard palate. A 40-year-old male presented a soft, non-ulcerated, painless mass of 1.5 X 1.0cm in size. Radiographically, it was no bony involvement. Histology revealed germinal center surrounding mantle zone of small lymphocytes. Immunohistochemistry showed CD3,CD20 were postive but Bcl-2 and cyclin D1 were negative. The patient underwent surgical excision and 3-year follow-up has not shown any evidence of recurrence.

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3 hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot (20 mg × 6 / 300 IU) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ± SEM) observed which developed to larger than 2 mm in diameter were 1.6 ± 2.5, 4.3 ± 3.1, 5.5 ± 4.2, 6.6 ± 2.1 and 8.8 ± 7.8, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + 10 μg/ml FSH + 1 μg/ml 17β-estradiol for 27 h at 39℃ in 5% CO2 in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6- DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

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.

In the present study, we investigated the effect of porcine follicular fluid (PFF) concentration (10% vs. 1%) and protein-free media (PFF 0%) on maturation of porcine oocytes in vitro and analysed difference in gene expression in resulting blastocysts following parthenogenetic activation. Three groups were tested; 1) 10% PFF: Tissue culture medium (TCM) 199+10% PFF; 2) 1% PFF: TCM 199+1% PFF; and 3) 0.1% PVA: TCM 199+0.1 PVA. Cumulus-oocyte-complexes were cultured in the respective media containing gonadotrophin (1 ug/ml), epidermal growth factor (10 ng/ml), cystein (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 ug/ml), 9-cis retinoic acid (5 nM) for 20～22 h and then without hormonal supplements for an additional 20-22 h. Data was analyzed using statistical analysis system(SAS) program. There was no significant difference in oocyte maturation rate. However, significantly higher (p<0.05) proportions of embryos developed to the blastocyst stage when oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 vs. 54.6±3.1 vs. 54.4±2.5, respectively). The relative abundance (ratio to beta-actin mRNA) of gene transcripts related to apoptosis in blastocysts was measured by real- time PCR. The expression of anti-apoptotic gene (BclxL) was up-regulated and the expression of pro-apoptotic gene (Bax) was down-regulated in 10% PFF group than in the other groups. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by reduction of apoptosis. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), MKE (#10033839-2011-13), Institute for Veterinary Science, the BK21 program and TS Corporation.

The objective of this study was to examine the effect of thymidine treatment during in vitro maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group (p<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group (p<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group (p<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups (p<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

The event that occur in sperm during chemotaxis are only partly known. As a essential step of fertilization, sperm cells should undergo capacitation process inside female genital tracts. To understand the molecular event of calcium signals on sperm cells, Fluo 4 loaded spermatozoa was treated with follicular fluid. The motility of sperm was reduced by follicular fluids. Simultaneously, level of calcium in head and tail was also reduced for 5 10 second. The inhibition of sperm motility was believed as a reversible event, so the follicular fluid in graffiaan follicles in vivo could act as a selector on active spermatozoa that recover motility and calcium signals during ovulation. This suggested that the normal levels of calcium in sperm was also critical for active state of sperm cells and the follicular fluids during ovulation could inhibit the motility of sperm cell via calcium signaling.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.

In this study, we analyzed expression patterns of apoptotic and autophagic gene products in culture follicular cells of normal and miniature pigs to assess the effect of hormones on the choice for programmed cell death. Autophagic activity progressively increased from control cultures to luteinizing hormone (LH)-treated cultures of follicular cells of normal pigs, but decreased from the LH to follicle stimulating hormone (FHS) +LH-treated cultures. Expression of Casp-3 protein in follicular cells was highest in LHtreated cultures, but the activity of Casp-3 decreased in the control, FSH-treated, and FSH+LH-treated cultures. The activity of the apoptosis protein was highly expressed in the control, LH-treated, and FSH+LH-treated follicular cells of miniature pigs, but autophagy- associated proteins were expressed at low levels in all treatments groups of the miniature pig. The expression of autophagy and apoptosis proteins appeared similar in control and rapamycin-treated cells. In addition, stimulation with FSH triggered the activation of autophagy in the follicular cells of normal pigs, but induced apoptosis in the follicular cells of miniature pigs. A similar effect was obtained when LH was applied. These results suggest that the autophagy process and FSH stimulation is more effective for stable and innovative follicular cell development.

It was conducted the experiment, divided into three groups as normal, poor and polycystic ovary syndrome, to detect the change of protein patterns in follicular fluid on ovarian response following controlled ovarian hyperstimulation for human IVF outcome. In the normal group, it was confirmed reproducible 57 spots in the detected total 81 spots. Then 1 spot was not found in the other groups. In the poor responder group, it was found reproducible 53 spots in the detected total 98 spots. 6 spots were down-regulation and 7 spots were up-regulation comparable with normal group. There were not 5 spots in poor responder group comparable with other groups. In the polycystic ovary syndrome group, it was expressed reproducible 53 spots in the detected total 80 spots and 3 spots were just expressed in this group. However, 4 spots were not found in polycystic ovary syndrome. 9 spots were up-regulation comparable with normal group. Significant up and down-regulation spots among the each groups were identified. The results were a cytosolic carboxypeptidase, a signal-induced proliferation-associated protein 1, a ceruloplasmin, a keratin(type Ⅱ cytoskeletal 1), a polypeptide N-acetylgalactosaminyltransferase 2, a serine/threonine-protein phosphatase 4 regulatory subunit 4. It was identified that 8 spots, 6 kinds of protein are corresponded with NCBInr database research, but 10 spots were failed in the identification. In conclusion, it has been confirmed change and expression of protein on the ovarian response following COH of human.

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of MⅡ stage.

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1～2 mm, 2～6 mm and 6～10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2～6 mm follicle and 5 spots from follicle fluid of 6～10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2～6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6～10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.