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.

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing γ‐gtubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The γ‐gtubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas γ‐g tubulin spots in the SCNT embryos were observed only during the inter‐ganaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution Ⅰ: 11% Lactose or Triladyl + egg yolk; solution Ⅱ: solutionⅠ + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CTC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p< 0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum‐starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or 150 μM roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine‐treated groups (27.6%) than that of the roscovitine‐treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine‐treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine‐treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine‐treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine‐treatment creates a more suitable condition for nuclear reprogramming after SCNT.

The present study was performed to identify changes of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in porcine oviduct epithelial cells (POECs) during the estrous cycle. POECs obtained from ovary in pre‐ovulatory (Pre‐Ov), early to mid‐luteal stage (Early‐mid L) and post‐ovulatory stage (Post‐Ov). For the examine of PA activity, 1×105 fresh cells of POECs were cultured in DMEM/Ham F‐12 containing 10% FBS and 0.2% amphotericin under humidified atmosphere of 5% CO2 in air and 38℃. The urokinase‐type PA (uPA) was observed at 7 days of POECs culture. PA activity was measured with culture prolonged of 0, 3, 6, 12 and 24 hafter culture of 7 days. The PA activity were high significantly (p<0.05) at 12 h of culture, but PA activity were decreased with culture periods increased. The PA activity in POECs of Post‐Ov stage were higher significantly (p<0.05) than that of Early‐mid L and Pre‐Ov stage. When PAI‐1 and PAI‐2 were added during the POECs culture, the PA were observed significant low activity (p<0.05). The PA activity and protein expression were decreased by PA inhibitor. This results suggest that PAI‐1 and PAI‐2 have a suppressive action on change of PA activity uring the estrous cycle of pigs. Specifically, this study using PA inhibitor was effect the PA activity and PAI expression in oviduct epithelial cells in pigs.

Changing Proteins in Granulosa Cells during Follicular Development in Pig In-Soon Chae1, Dong-Min Jang3, Hee-Tae Cheong2, Boo-Keun Yang1 and Choon-Keun Park1,† 1College of Animal Life Sciences, 2School of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, Korea 3CHA Stem Cell Institute, CHA Hospital, Seoul 463-712, Korea ABSTRACT This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is 2～4 mm and 6～10 mm, were collected from ovary of slaughtered pig that each follicle of diameter 1～4 mm and 6～10 mm. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 200 μl. Immobilized pH gradient(IPG) strip used 18 cm, 3～10 NL. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of 6～10 mm follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kDa protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development.

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed (37℃) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher(83.6±2.0 vs 59.0±4.4%) than un- separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form un-separaed sperm was significantly(p<0.05) higher(13.6±0.8 vs 8.1±0.6%) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher(20.2±0.4 vs 16.8±2.8%) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing β-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a ɤ-tubulin spot. However, ɤ-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.