Expression of enhanced green fluorescent protein in porcine- and bovine-cloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector

Uhm, Sang Jun,Gupta, Mukesh Kumar,Kim, Teoan,Lee, Hoon Taek JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.12

<P>Interspecies somatic cell nuclear transfer (iSCNT) has emerged as an important tool for studying nucleo-cytoplasmic interactions and cloning of animals whose oocytes are difficult to obtain. This study was designed to explore the feasibility of employing transgenic fibroblasts as donor cells for iSCNT. The study examined the chromatin morphology, in vitro development, and expression of an enhanced green fluorescent protein (EGFP) gene in porcine- and bovine-cloned embryos produced by iSCNT of fetal fibroblast transfected with a pLNβ-EGFP retroviral vector. Parthenogenetic and transfected or nontransfected intraspecies SCNT embryos were used as controls for comparison. Analysis of data revealed that xenogenic oocyte was able to reprogram somatic cells of different genus and supports their in vitro development to the blastocyst stage. However, the developmental rates of transgenic iSCNT embryos to the blastocyst stage were significantly lower than those of intraspecies SCNT embryos. The reduction in development rates was however, not due to integration of the transgene as the lower (P < 0.05) development rates of the intraspecies SCNT porcine or bovine embryos did not differ between transgenic and nontransgenic groups. Expression of EGFP was observed in 100% of blastocysts and mosaicism was not observed. Furthermore, after iSCNT of porcine or bovine donor nuclei into xenogenic ooplasm, patterns of nuclear remodeling in reconstructed embryos were similar. In conclusion, our data demonstrated the feasibility of producing transgenic iSCNT embryos. To our knowledge, this is the first report of transgenic cloned embryo production by iSCNT approach. In the future, this may provide a powerful research tool for studying developmental events in domestic animals and provide marked cell lines for other genetic manipulations. Mol. Reprod. Dev. 74: 1538–1547, 2007. © 2007 Wiley-Liss, Inc.</P>

Effects of calcitonin and parathyroid hormone on the regulation of cabindin-D_9k in the uterus, placenta, and fetal membrane of rats related to blood calcium level during late gestation

Hong, Eui-Ju,Choi, Kyung-Chul,Hyun, Sang-Hwan,Jeung, Eui-Bae JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.9

<P>Calbindin-D<SUB>9k</SUB> (CaBP-9k) gene is expressed in the uterus of pregnant rats, which is regulated by steroid hormones during estrous cycle or gestation. We hypothesized that there is a positive correlation between altered CaBP-9k expression and change in one or more of the hormones to provide a clue to the mechanism responsible for the altered calcium levels in the uterus, placenta, and fetal membrane during late gestation. Thus, in the present study, we investigated the effects of the hormones including estradiol (E2), calcitonin (CT), and parathyroid hormone (PTH) on the regulation of CaBP-9k in these tissues. There was an increase in the level of CaBP-9k in the uterus, placenta, and extra-embryonic membrane at late gestation, as blood calcium level increased. The protein level of CaBP-9k remained lower in the uterus at two-thirds of pregnancy, and then it rebounded abruptly during late pregnancy. During late gestation, E2 is postulated to be a dominant factor in the regulation of uterine CaBP-9k gene expression. Furthermore, we assumed that there is a positive correlation between altered expression of CaBP-9k and blood calcium level during pregnancy. The present study demonstrated the regulation of CaBP-9k mRNA in the uterus, placenta, and fetal membrane of rats, implying a role for CaBP-9k gene in the control of blood calcium in placenta and the calcium passing from maternal blood to fetal circulation. Taken together, these results suggest that major alterations in calcium metabolism caused by maternal thyroparathyroidectomy (TPTX), are sufficient to affect the changes in reproductive tissues during late pregnancy. In addition, an increase of blood calcium level is one of the most significant factors in the regulation of CaBP-9k at the transcriptional and/or translational levels in the reproductive tissues during late pregnancy. Mol. Reprod. Dev. 74: 1188–1197, 2007. © 2007 Wiley-Liss, Inc.</P>

Ectopic expression of Tollo/Toll-8 antagonizes Dpp signaling and induces cell sorting in the Drosophila wing

Kim, Sangjoon,Chung, SeYeon,Yoon, Jeongsook,Choi, Kwang-Wook,Yim, Jeongbin John Wiley & Sons 2006 Genesis Vol.44 No.11

<P>The wing imaginal disc of Drosophila consists of the primordia for the adult wing and the body wall. The zinc-finger transcription factor Teashirt (Tsh) is expressed in the region proximal to the wing primordium and regulates the formation of the wing-body wall boundary. Here, we report that Tollo/Toll-8, a member of Toll family transmembrane proteins, is also expressed proximal to the wing domain. Ectopic expression of Decapentaplegic (Dpp), a morphogen for wing development, represses tollo expression in the proximal domain. Likewise, misexpression of Tollo in the presumptive wing strongly antagonizes the effects of Dpp signaling. The extracellular domain of Tollo containing the Leucine-Rich Repeats (LRR) is required for the inhibition of Dpp signaling in the wing. Furthermore, clones of cells with Tollo overexpression are sorted out from the surrounding wild-type cells, resulting in the formation of epithelial folds around the clone boundaries. Tsh is ectopically induced at the border of Tollo-expressing clones. Despite the strong effects of Tollo overexpression on Dpp signaling and cell sorting, loss-of-function tollo mutants are viable with normal external morphology. Our data suggest that Tollo function might be redundant but is sufficient to antagonize Dpp signaling and induce sorting of Tollo expressing cells from the wing cells to develop proximal cell fate. genesis 44:541–549, 2006. © 2006 Wiley-Liss, Inc.</P>

Enhanced two-photon luminescence from nanoporous gold capped with microcontact-printed salts

Wi, J. S.,Park, J. H.,Tominaka, S.,Lee, J. Y. John Wiley Sons, Ltd 2014 Physica Status Solidi. Rapid Research Letters Vol.8 No.1

We report that a nanoporous Au film capped with a dielectric surface layer enables the effective absorption of near-infrared light and intense emission of two-photon luminescence. In this approach, nanoscale pores in Au are incorporated by chemical dissolution and microscale surface patterns are added on the nanoporous Au by microcontact print lithography. Electromagnetic simulation shows that the strong local electric fields are concentrated in the vicinity of nanopores in the Au film and are further increased by the surface dielectric patterns, which leads to a 40-fold increase in the intensity of the two-photon luminescence, as verified by photoemission measurement. ((c) 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Influence of ovarian hyperstimulation and ovulation induction on the cytoskeletal dynamics and developmental competence of oocytes

Lee, Seung Tae,Han, Ho Jae,Oh, Seo Jin,Lee, Eun Ju,Han, Jae Yong,Lim, Jeong M. JOHN WILEY & SONS LTD 2006 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.73 No.8

<P>This study was undertaken to determine the effects of gonadotrophin on cytoskeletal dynamics and embryo development and its role in improving the retrieval of developmentally competent oocytes. Female golden hamsters were injected with human chorionic gonadotrophin (hCG; 5-, 7.5- or 15-IU) on the day 4 of estrus, pregnant mare serum gonadotrophin (PMSG; 5-, 7.5- or 15-IU) on the day 1 of estrus, or 15-IU hCG at 56 hr post-15-IU PMSG injection in any cycle except estrus. Increasing the hCG dose decreased not only retrieval rate of 2-cell embryo but development to blastocyst after subsequent in vitro culture. Whereas, although increasing the PMSG dose induced increasing the number of 2-cell embryo and blastocyst, 15-IU PMSG injection caused retardation of development to blastocyst. No 2-cell embryos were retrieved by injecting both PMSG and hCG. The injections of 15-IU hCG and 7.5- or 15-IU PMSG inhibited the proliferation of trophectodermal and inner cell mass cells, respectively. Gonadotrophin injection didn't influence microtubular spindle formation, but 5- or 15-IU hCG, 15-IU PMSG, or PMSG and hCG injections induced aberrant cortical granule (CG) and microfilament distribution. After 15-IU hCG or PMSG and hCG injections, fewer oocytes had enriched cortical actin domains, and the expression of α-, β- and γ-actin genes was greatly increased. In conclusion, a high dose of gonadotrophins alters the microfilament and CG distribution, which in turn reduces the developmental competence of oocytes. Injecting a reduced dose of PMSG to initiate ovarian hyperstimulation without triggering ovulation contributes to the efficient retrieval of developmentally competent oocytes. Mol. Reprod. Dev. 1022–1033, 2006. © 2006 Wiley-Liss, Inc.</P>

Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain

Ji, Kyung-Ae,Yang, Myung-Soon,Jeong, Hey-Kyeong,Min, Kyoung-Jin,Kang, Seung-Hee,Jou, Ilo,Joe, Eun-Hye John Wiley & Sons, Inc. 2007 Glia Vol.55 No.15

<P>Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)-injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule-1, Iba-1), as well as a general marker for these inflammatory cells (CD11b). CD11b-immunopositive (CD11b<SUP>+</SUP>) cells and Iba-1<SUP>+</SUP> cells displayed similar behavior in intact and LPS-injected brain at 6 h after the injection. Interestingly, however, CD11b<SUP>+</SUP> cells and Iba-1<SUP>+</SUP> cells displayed significantly different behavior at 12 h: Iba-1<SUP>+</SUP> cells disappeared while CD11b<SUP>+</SUP> cells became round in shape. We found that CD11b/Iba-1-double positive (CD11b<SUP>+</SUP>/Iba-1<SUP>+</SUP>) ramified microglia died within 6 h after LPS injection. The round CD11b<SUP>+</SUP> cells detected at 12 h were MPO<SUP>+</SUP>. These CD11b<SUP>+</SUP>/MPO<SUP>+</SUP> cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO<SUP>+</SUP> neutrophils expressed inducible nitric oxide synthase, interleukin-1β, cyclooxygenase-2, and monocyte chemoattractant protein-1, but died within 18 h. CD11b<SUP>+</SUP> cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba-1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS-injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain. © 2007 Wiley-Liss, Inc.</P>

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

Hossein, Mohammad Shamim,Kim, Min Kyu,Jang, Goo,Oh, Hyun Joo,Koo, OkJae,Kim, Jeong Joo,Kang, Sung Keun,Lee, Byeong Chun,Hwang, Woo Suk JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.9

<P>Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 µM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 µg/ml estrogen, 0.5 µg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin–streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 µM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 µM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 µM cysteamine to the maturation medium improved IVM of canine oocytes. Mol. Reprod. Dev. 74: 1213–1220, 2007. © 2007 Wiley-Liss, Inc.</P>

Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

Choi, Bok Ryul,Koo, Bon Chul,Ahn, Kwang Sung,Kwon, Mo Sun,Kim, Jin-Hoi,Cho, Seong-Keun,Kim, Kyoung Mi,Kang, Jee Hyun,Shim, Hosup,Lee, Hyuna,Uhm, Sang Jun,Lee, Hoon Taek,Kim, Teoan JOHN WILEY & SONS LTD 2006 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.73 No.10

<P>A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.</P>

In vitro development and cell allocation of porcine blastocysts derived by aggregation of in vitro fertilized embryos

Lee, Sang-Goo,Park, Chi-Hun,Choi, Don-Ho,Kim, Hye-Sun,Ka, Hak-Hyun,Lee, Chang-Kyu JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.11

<P>In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to those of their in vivo counterparts. The objective of this study was to increase developmental competence and to gain an understanding of cell allocation in blastocysts derived from the aggregation of four-cell stage porcine embryos produced in vitro. After removal of the zona pellucida, two (2×) and three (3×) four-cell stage embryos were aggregated by co-culturing them in aggregation plates. Five days after aggregation, the developmental ability and the number of cells in the aggregated embryos were determined. The percentage of blastocysts was higher (P < 0.05) in both the 2× and 3× aggregated embryos (66.6% and 72.0%, respectively) compared to that of the 1× embryos and the intact controls (43.1% and 36.4%, respectively). The total cell number of blastocysts also increased in aggregated embryos compared to that of intact controls (2.6-fold for 2× and 3.4-fold for 3×) (P < 0.05). The cells of two differentially stained embryos were started to mix at 72 hr after aggregation. In vitro-fertilized porcine aggregates (2×) were developed to blastocyst with a random distribution of cells from each embryo. The mRNA levels for the oct-4, bcl-xL and connexin 43 genes were higher (P < 0.05) and bak gene were lower (P < 0.05) in both the 2× and 3× aggregated embryos than the intact controls. Therefore, the aggregation of the four-cell stage embryos could be used to improve the quality of porcine preimplantation stage embryos produced in vitro. Mol. Reprod. Dev. 74: 1436–1445, 2007. © 2007 Wiley-Liss, Inc.</P>

Characterization of pig vasa homolog gene and specific expression in germ cell lineage

Lee, Gab Sang,Kim, Hye Soo,Lee, So Hyun,Kang, Min Soo,Kim, Dae Yong,Lee, Chang Kyu,Kang, Sung Keun,Lee, Byeong Chun,Hwang, Woo Suk JOHN WILEY & SONS LTD 2005 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.72 No.3

<P>The vasa gene is known to be an important factor for germ cell development in both invertebrates and vertebrates. In the present study, we cloned the porcine vasa homolog (Pvh, 2,172 bps) and investigated its expression at mRNA and protein levels. The isolated cDNA had deduced 724 amino acid residues with significant homology to mouse (85%) and human (91%) vasa. In adult tissues, Pvh transcript was restricted to the ovary and testis, and was undetectable in somatic tissues. During preimplantation embryo development, Pvh was transcribed in oocytes and fertilized 2-cell embryos, but not in other preimplantation embryos. In fetal stage, the transcript of Pvh gene was expressed in all fetal stage, except in day 17–18. Immunohistochemical analysis of fetal and adult gonad revealed that the Pvh protein was localized in oocytes and spermatocytes, consistent with mRNA expression. Interestingly, Pvh protein was also observed in proliferating primordial germ cells (PGCs) and freshly isolated PGCs, but not in embryonic germ cells. Our results suggest that Pvh gene can be a useful marker for germ cell development in pigs. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.</P>