Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem (ES) cells. Differentiation capacity of the parthenogentic ES cells was rather lower than that of fertilized embryos derived ES cells, which might be the result of the absence of male genome. However, parthenogenetic ES cells might be useful research tool for genetic engineering and generating SCNT embryo derived ES cells. In our previous study, we reported that establishment of several bovine ES cell lines from in vitro fertilized (IVF) embryos named JNU-ibES. Based on this data, the objective of this study is to generate parthenogenetic ES cells and to examine their stem cell characteristics. Total 107 parthenogenetic embryos produced at day 8 or 9 were classified into their developmental stages (full expanded x 40, hatched x 67). For producing ES cells, ICM and trophetoderm-rich clumps were mechanically dissociated and were cultured on mitomycin- C treated mouse embryonic fibroblast feeder cell drop and covered with mineral oil in DMEM medium containing 20% FBS, 5 ng/ml basic FGF, 1% nonessential amino acids, and 0.55 mM b-mercaptoethanol. We obtained 20 primary parthenogenetic bovine ES (pbES)-like cell colonies. And pbES colony formation was higher in hatched blastocyst (25.4%, 16/67) than expanded blastocysts (10%, 4/40). Among those colonies, 5 pbES cell lines were successfully established and they were named as a series of JNU-pbES. These pbES cells were positively expresssed pluripotency markers such as Oct4, Nanog, TRA-1-81, SSEA-1 and alkaline phosphatase. This result demonstrated that the establishment efficiency and characteristics of pbES cell line was very similar to those of ibES cell line.

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.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt- 1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5’-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in ‒ 640 bp to ‒ 30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the in vivo or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the in vivo was increased up to 84.38% in the SCNT embryo, moreover, de novo methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the in vivo or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

The prediction of male fertility is of paramount importance for breeding animal herds when artificial insemination is applied. While the male fertility assays provide valuable quantitative data, they yield limited information concerning the functional competence of the spermatozoa. The objective of this study was to standardize a method for predicting in vivo fertility in bulls using the capacitation status that was assessed by chlortetracycline (CTC) staining. To optimize the capacitation process, sperm were treated with various concentrations of heparin (0, 10, 20, 50, and 100 μg/mL) and incubated for 10, 20, and 30 min each at 39℃ in 5% CO2. We found that maximum capacitation condition obtained from 10 μg /mL heparin treated sperm cells for 20 min (p<0.05). Optimized methods were used to determine the fertility of 17 batches of frozen bull semen representing a wide range of field fertility levels as indicated by non-return rates (NRR) (35.29% 93.18%). There was no significant correlation between NRR and the percentage of capacitated spermatozoa (B type) and non-capacitated spermatozoa (F type). However, acrosome reacted spermatozoa (AR type) was significantly correlated with NRR (p<0.01). To determine the normal range for the AR type, lower limits of the AR (%) were established as 23% for low fertility (NRR < 75%) using receiver operating characteristic curve. The overall accuracy of the assay was 88.24% for low fertility, sensitivity and specificity were 81.82 and 100%, respectively. These results indicate that capacitation status as measure by CTC staining is a useful predictor of male fertility. Therefore, low and high fertility bulls can be identified primarily by the functional capacitation status.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300 X g and 700 X g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

Phosphorylation of proteins is a post-translational modification process which plays a significant role in a wide range of cellular processes. Addition or removal of phosphate groups result in conformational changes in proteins leading either to their activation or inactivation. Tyrosine phosphorylation of protein is associated with sperm function in several mammalian species. The control of this process may via the changes in cyclic adenosine monophosphate (cAMP); the changes in cAMP levels that occur in the spermatozoa regulate protein kinase A (PKA) activity which, in turn, leads to the tyrosine phosphorylation of protein substrates by either the activation of sperm tyrosine kinases and/or the inhibition of phosphoprotein phosphatases. Cyclic nucleotides, in particular, cAMP, are important regulators of various maturation events in sperm including capacitation and motility. Interestingly, some environmental chemicals (ECs) may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems. Otherwise, because the mature spermatozoa are transcriptionally inactive, therefore the study of sperm proteins phosphorylation may permit more information about the agents and conditions affects on sperm function. In the present study, to examine the effect of ECs on human sperm function, human spermatozoa were incubated with a group of ECs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo- pdioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, western blot analysis was carried out using extracted sperm proteins. Antiphosphorylation antibody (pY20) was used to determine sperm tyrosine phosphorylation after EDs treatment. The pY20 antibody labeled three common bands of approximately 90, 110, and 150 KDa. There were no significant differences between negative and positive control groups in regard to the tyrosine phosphorylated proteins except at the band with molecular weight 110 KDa. However, except Diaz treatment group, the other treatment groups showed decreasing (TCDD, Gen, NP, BPA, and DBCP) or increasing (Atraz) in the tyrosine phosphorylated proteins at least in one band from the three common bands studied. Therefore, it sug-gests that ECs effectively alters human sperm function and this effect may detect via their effect on tyrosine phosphorylation pattern.

In mammals, the meiosis division in testes produces equal numbers of two different types of gametes: X chromosome-bearing sperm (X-spermatozoa) and Y chromosomebearing sperm (Y-spermatozoa), which have equal potential to fertilize the oocytes. Therefore, the expected 1: 1 sex ratio is observed. However, under some conditions like endocrine disruptors (EDs) exposure the sex ratio is deviated than the expected with more males or more females. And recently many hypotheses have been postulated to explain the mechanism of sex ratio deviation; however none of them introduced a proven experimental explanation. To solve this enigma, we hypothesized that the differences between X- and Y-spermatozoa survivability under specific conditions due to differences in their chromosome contents are the key leading to the sex ratio alteration. To examine our hypothesis, we combined two techniques; first, hypo-osmotic swelling (HOS) test that was applied to test viability of spermatozoa and second, fluorescence in situ hybridization that was applied on HOS-treated spermatozoa to define sex chromosome composition. In the present study, human spermatozoa were incubated with a group of EDs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, the viability of spermatozoa and their sex chromosome contents were evaluated simultaneously. Among seven chemicals studied only four chemicals (Atraz, DBCP, TCDD, and Diaz) significantly decreased Y-sperm viability when compared to those of X-spermatozoa in the same treatment group and viability of Y-spermatozoa when compared to those in the negative and positive (DMSO) control groups (p<0.05). Also, in these four treatment groups the sex ratio of live sperm population was significantly lowered compared to the control groups (p<0.05). Otherwise, Gen, BPA, and NP did not show any significant effect on viability of Yspermatozoa or decreasing sex ratio in live sperm population as compared to the control groups. It has been proven that TCDD, DBCP, and the pesticides decrease the sex ratio, but the same effect was not observed in case of Gen, BPA, and NP. From the present findings, there is no doubt that the EDs may alter sex ratio via decreasing Y-spermatozoa viability.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

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.

In the last few decades with the industrial revolution many environmental contaminants have estrogenic activity (endocrine disruptors, EDs) are released into the environment affecting the male reproductive system and male fertility. Sperm motility is one of the initial tests performed to assess sperm function; only motile sperm can achieve fertilization in vivo. The present study aimed to investigate the possible effects of a group of EDs that represent a widespread chemicals in the environment genistein (Gen), is a naturally occurring isoflavone (100 μM), bisphenol A (BPA), that is used in the manufacture of plastics and other products and released largely into the environment (100 μM), nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical (10 μg/ml), TCDD, that is formed as an unwanted by-product in the manufacture of chlorinated hydrocarbons (2.5 μg/ml), atrazine (Atraz) is a herbicides (500 μM), dibromochloropropane (DBCP) is a pesticide (10 μg/ml), and diazinone (Diaz) is a insecticide (500 μM) on human sperm motility and kinematic characteristics. Human spermatozoa were incubated in Ham's F10 media with/without the tested chemicals or DMSO as positive control for 6 hr at 37℃ in 5% CO2. Then, sperm motility was assessed using computer assisted semen analyzer. Interestingly, all the chemicals tested significantly decreased sperm motility as compared to the control groups. However, only Diaz significantly decreased sperm kinematic characteristics namely, VCL, VSL, STR, VAP, and ALH. We suggest that the environmental chemicals may have an effect on male fertility via decreasing sperm motility.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane. Na+/K+-ATPase consists of α, β, and γ subunits, but only α and β subunits are needed for basic functions. Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca2+ exchanger involved in intracellular calcium extrusion. Our previous study showed that calcium regulatory molecules including Na+/Ca2+ exchanger are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs, however, expression of Na+/K+-ATPase in the uterine endometrium has not been determined. Thus, we examined expression of α1 (ATP1A1) and β1 (ATP1- B1) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of ATP1A1 m- RNA in the uterine endometrium during the estrous cycle and early pregnancy were higher than those during mid and term pregnancy, and that levels of ATP1B1 mRNA were highest on day (D) 12 of the estrous cycle. In situ hybridization analysis revealed that ATP1A1 and ATP1B1 mRNAs were localized to luminal (LE) and glandular epithelia (GE) in the endometrium. During mid to term pregnancy, localization of ATP1A1 mRNA was confined to LE, GE, and chorionic membrane (CM) of areolae and ATP1- B1 mRNA was localized to LE, GE and CM with the strongest intensity in LE of areolae. Signal intensity of ATP1B1 mRNA in LE was slightly stronger than that in GE. RT-PCR analysis showed that ATP1A1 and ATP1B1 mRNAs were expressed in conceptuses on D12 and D15 of pregnancy. These results showed that ATP1A1 and ATP1B1 were expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase may play a key role in the establishment and maintenance of pregnancy by regulating intracellular concentrations of various ions including calcium at the maternal-fetal interface in pigs.

Proteases and their inhibitors are involved in the process of pregnancy by remodeling uterine endometrium and placenta in many mammals. During placentation, proteases and their inhibitors contribute to formation of epitheliochorial type placentation in pigs. Our previous study showed that LGMN and CST6 were expressed in the uterine endometrium and localized mainly to glandular epithelial cells (GE) and chorionic membrane (CM) during mid to late pregnancy. In this study, we investigated expression of LGMN and CST6 in the uterine endometrium and fetal membrane during pregnancy in pigs. Uterine endometrial tissue samples and fetal membrane samples were collected from D30, D60, D90, and D114 of pregnancy. Real-time RT-PCR analysis showed that both LGMN and CST6 mRNAs were detected in the uterine endometrium and fetal membrane in all samples with higher levels during mid to late stage of pregnancy. Analysis by immunoblotting revealed that LGMN protein was present in the porcine uterine endometrium and fetal membrane. Based on the placental and endometrial distribution of proteases and their inhibitors, we examined LGMN mRNA and LGMN protein expression in the neonatal pigs. In situ hybridization analysis using the intestine from D90 of piglet revealed that LGMN mRNA was highly expressed in the absorptive epithelium of the intestinal villi. Immunohistochemical experiments demonstrated that LGMN protein was localized to epithelial villi. These results suggest a possible role of LGMN in modification of proteins that are transported through the fetal membrane from the uterine for successful transport and utilization in the fetus.

Prostaglandins (PGs) are critical lipid mediators involved in many reproductive processes including luteolysis, maternal recognition of pregnancy, and implantation in domestic animals. In pigs, PGs, especially PGE2 and PGF2α, are produced in the uterine endometrium. The actions of PGE2 and PGF2α are mediated by signaling receptors, PTGERs and PTGFR, respectively, but their expression in the uterine endometrium is not well elucidated. In this study, we determined expression of PTGERs and PTGFR in the uterine endometrium during the estrous cycle and pregnancy in pigs. Uterine endometrial tissue samples were collected from Day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90, and D114 of pregnancy. Temporal expression of all genes studied was analyzed by real-time RT-PCR. PTGERs except for PTGER1 were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of PTGER2 and PTGER3 mRNA increased during early pregnancy and late pregnancy, respectively, and levels of PTGER4 mRNA were not changed during pregnancy. Levels of PTGFR mRNA were highest on D90 of pregnancy. Results of this study showed that expression of PG receptors was dynamically regulated in the uterine endometrium during pregnancy in pigs. These results indicate that actions of PGs are dependent on types of receptors and is critical to support the establishment and maintenance of pregnancy at the maternal-fetal interface in pigs.

Successful pregnancy requires suppression of maternal immune response to the implanting conceptus, which acts as a semiallograft. During the implantation period in humans and rodents, various immune modulators are produced at the maternal-fetal interface and regulate functions of cytotoxic T cells and NK cells for protection of conceptuses from the maternal immune system. However, maternal immune responses to the conceptuses during the establishment and maintenance of pregnancy are not much understood in pigs which show true epitheliochorial type placentation. Previously, we reported that SLA-DQ molecule, a type of MHC class II molecules, is expressed in the uterine endometrium during pregnancy in a stage- and cell type specific manner, and that SLA-DQ expression is essential for the maintenance of pregnancy. Thus, to understand the role of SLA-DQ and maternal-fetal immune interaction, we examined expression of CD80 and CD86, co-stimulators for T cell activation, in the uterine endometrium during pregnancy. We also measured levels of CD80 and CD86 mRNAs in the uterine endometrium of pigs carrying conceptuses derived from somatic cell nuclear transfer (SCNT) and those from natural mating on Day 12 of pregnancy. Expression of endometrial CD80 mRNA was affected by day of pregnancy, and levels of CD80 mRNA were significantly higher on Day 15 of pregnancy than those of the estrous cycle. Expression of CD86 mRNA did not change during pregnancy. Levels of CD80 and CD86 mRNAs were not different in the uterine endometrium of pigs carrying SCNT derived conceptuses on D12 of pregnancy compared to those with conceptuses derived from natural mating. These findings suggest that CD80 and CD86 are involved in immune interactions at the maternal-fetal interface during pregnancy for the establishment and maintenance of pregnancy in pigs.

Immunological rejection of the organ grafted onto a primate arises from two antibody mediated processes, hyperacute rejection (HAR) and acute humoral rejection (AHR). Functional ablation of α1,3-galactosyltransferase (GalT) and concurrently overexpression of complement regulatory proteins are known to inhibit HAR and AHR. In previous study, we reported that production of porcine male fibroblasts harboring a MCP expression cassette targeted to GalT locus. In this study, we constructed a different MCP expression cassette, in which the EF1α promoter regulates MCP expression and internal ribosome entry site-mediated neomycin resistance gene expression. Subsequently, this cassette was inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Female fibroblasts were isolated from ear skin of 10 days old miniature pig, and used for nucelofection of the the construct for MCP expression at GalT locus. PCR analysis showed that four clones of forty neomycin resistant clones carry MCP expression cassette at exon 9 of the GalT gene. Two clones analyzed downregulated GalT expression, as determined by quantitative reverse transcriptase polymerase chain reaction. Flow cytometry analysis showed that MCP was efficiently expressed at the cell surface.

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.

Although the National Institute of Health (NIH, USA) miniature pigs were developed specifically for xenotransplantation, the cloning efficiency is still very low. To increase the efficiency, an advanced somatic cell nuclear transfer (SCNT) method may need. In the present study, we report the productions of genetically modified cloned pigs using the frozen-thawed donor cells without culture before SCNT. Fibroblasts were isolated from an ear skin of a 10-day-old NIH miniature pig. The fibroblast cells were genetically modified with the human CD73 (hCD73). For SCNT, somatic cells transfected with hCD73 were used as donor cells. The survival rate of the somatic cells was significantly higher in 0 h (95%) compared with 1 h (81%) after thawing (p<0.05). We obtained the pregnancy (38.9%, 7/18) and delivery (11.1%, 2/18) rate, respectively. Totally 7 genetically modified cloned piglets were delivered. Among them, 2 piglets were survived and 5 piglets were born stillbirth. The healthy 2 piglets are still survived (≥6 months).