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).

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

Successful pregnancy requires well-coordinated interactions between the maternal uterus and the developing embryo in pigs. In pigs, implantation begins around Day 12 of pregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes various factors such as estrogens, interleukin-1 beta (IL1B), and interferons. Estrogens produced by conceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogen action is explained by the endocrine and exocrine theory. The uterine endometrium becomes receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells and increasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone, progesterone, but the presence of conceptus in the uterus also induces changes of endometrial functions, including most importantly maternal recognition of pregnancy. Many factors, such as hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs. However, understanding of the cellular and molecular events occurring in the endometrium is not complete. In recent studies we made some progress on understanding of expression and function of genes involved in maternal-fetal interaction for the establishment and maintenance of pregnancy in the uterine endometrium in pigs. Firstly, we found that lysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of implantation and LPA receptor 3 was uniquely expressed in the endometrium during early pregnancy. Secondly, we observed that salivary lipocalin (SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time of embryo implantation, and its expression was regulated by IL1B. Furthermore, expression of IL1B receptors are regulated by estrogen and IL1B, and IL1B functions in expression of genes related to prostaglandin synthesis and transport. Thirdly, we found that calcium regulatory molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo implantation and the maintenance of pregnancy. Finally, we observed that an MHC class II molecule, SLA-DQ, is expressed in the uterine endometrium at the time of conceptus implantation and its expression is essential for successful pregnancy, indicating that appropriate maternal-fetal immune interaction is required for the maintenance of pregnancy. Further analysis of these molecules will provide insights into the cellular and molecular basis of maternal-and fetal interaction during pregnancy in pigs.

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

Rice stripe virus (RSV), the type member of the genus Tenuivirus, causes rice stripe disease and the viral transmission is mediated through the sucking by small brown planthopper, Laodelphax striatellus. Considerations have been mainly focused on the protection of rice from RSV and/or the planthopper, rather than the interaction between RSV and the insect. To clarify the interaction, in this work, mRNA was extracted from RSV-viruliferous planthopper with non-viruliferous control, and expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing technology for comparative analysis. RSV-viruliferous planthopper had ca. 2500 isotigs, which included genes on biological process (19%), cellular component (13%), molecular function (22%) and no hits (46%) from gene ontology (GO) analysis; this structure was similar to the control. However, in the viruliferous planthopper, 109 isotigs were up-regulated and 660 isotigs were down-regulated, compared to the non-viruliferous control. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.