bluuebunny/crossref_metadata_2025 huggingface dataset:
271 INDUCTION OF OVULATION IN RABBIT DOES USING PURIFIED NERVE GROWTH FACTOR AND CAMEL SEMINAL PLASMA
Authors: Masdeu M. , Garcia-Garcia R. M. , Cardinali R. , Millan P. , Arias Alvarez M. , Castellini C. , Lorenzo P. L. , Rebollar P. G.
Published: December 2014

The presence of an ovulation-inducing factor (OIF) in the seminal plasma (SP) of several species with spontaneous and induced ovulation, including the rabbit, has been documented. Recent studies have demonstrated that the OIF in the SP of camels (SPCAM) is a nerve growth factor (β-NGF). The aim of this study was to determine if purified β-NGF from mouse submandibular glands or SPCAM could provoke ovulation induction in the rabbit doe. A total of 35 females were synchronized with 25 IU of equine chorionic gonadotropin (Serigan, Laboratorios Ovejero, Spain) and allocated into 4 groups. Forty-eight hours later (Day 0), does were given a single dose (IM) of 1 mL of saline solution (SS; n = 8); 1 mL of gonadorelin (GnRH; Inducel, Laboratorios Ovejero, Spain; n = 9); 24 µg of β-NGF (2.5S-NGF; Promega, USA; n = 10); or 1 mL of centrifuged raw camel SP (SPCAM; 127 pg mL–1 NGF; n = 8). After treatment, an empty catheter was introduced through the vagina to simulate the nervous/mechanical stimulus of coitus (4 animals per group). Plasma LH concentrations were determined in blood samples taken 30 min before treatment and at 0, 30, 60, 90, and 120 min after injection. Progesterone concentrations were assessed at 0 and 120 min and every 2 days until Day 6 after treatment. Concentrations of β-NGF in camel SP and hormone determinations were made by enzyme immunoassay. Ovulation rate (OR) was determined after euthanasia on Day 7. Statistical analyses using CATMOD and MIXED procedures of the SAS program to compare OR data and hormone concentrations, respectively, were performed. Ovulation occurred in 100% of GnRH (9/9), 33% (3/10) of NGF, 25% (2/8) of SS, and 0% (0/8) of SPCAM groups. Both NGF and SS ovulated females had significantly lower LH concentration than GnRH group throughout all preovulatory surge (P < 0.001). When does were not stimulated with catheter introduction, only those from the GnRH (5/5) and NGF (1/6) groups ovulated. Total number of corpora lutea in ovulated does was similar (15.9 ± 1.9, 17.0 ± 4.2, and 14.3 ± 3.1 CL in GnRH, SS, and NGF groups, respectively). Plasma P4 concentrations were normally increased from Day 2 to 6 in ovulated rabbits of all groups, but were lower at 120 min (P < 0.001) for the NGF and SS does, reaching similar levels than GnRH does at 6 days post-treatment. In the present study, β-NGF from mouse submandibular glands, but not from raw camel SP, induced ovulation in rabbit females, independently of nervous stimulus. Nonetheless, the possible low dose of β-NGF used and the origin could have been responsible for the lack of a more acute effect.We acknowledge CM, FSE, and AGL2011-23822 for funding.
28 EFFECTS OF RECLONING ON THE PRODUCTION OF PIGS OVEREXPRESSING 11 β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 (11β-HSD1)
Authors: Jeong Y. I. , Jeon Y. , Park C. H. , Ko K. H. , Jeong Y. W. , Kim Y. W. , Hyun S. H. , Yang I. S. , Hwang W. S.
Published: December 2014

The incidence of metabolic syndrome is increasing globally, as the prevalence of obesity continues to rise. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, animal disease models are required for the study of metabolic syndrome. The overexpression of 11 β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome; thus, we attempted to produce pigs with overexpression of 11β-HSD1 gene by somatic cell nuclear transfer (SCNT). However, low transgenic (TG) efficiency has been an obstacle to the production of TG pigs. A SCNT method in which somatic cells derived from TG pig are used as the nuclear donor (re-cloning method) is an effective technique for TG pig production. In this study, we attempted to increase TG efficiency by the re-cloning method. Pregnancy efficiency, production efficiency, and TG efficiency were compared with sources of donor cells (transfected TG fetal fibroblast v. TG fibroblast derived from newborn TG cloned pig). A total of 1382 and 881 TG SCNT embryos were produced from fetal fibroblast v. cloned fibroblast, and then transferred to 13 and 10 recipients. The pregnancy rate was not significantly different (30.8% v. 20.0%). Seventeen live piglets and 5 stillborn piglets were born from 4 recipients in the fetal fibroblast group, and 8 live piglets, 2 stillborn piglets, and 3 mummies were born from 2 recipients in the cloned fibroblast group. There were no significant differences in the production efficiency (3.7% v. 5.0%). All of the 13 re-cloned piglets showed reporter and target gene integration. But, of 22 fetal fibroblast-cloned piglets, reporter gene integration was confirmed in 9, but only 3 clone piglets showed reporter gene integration. Efficiency of TG was significantly increased in re-cloning group (13.6% v. 100.0%). In this study, TG efficiency of 11β-HSD1 overexpressed pigs was improved by re-cloning method. These results indicate that re-cloning is an efficient method for production of TG cloned pigs.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.
287 EFFECT OF FOLLICULAR AGING ON THE ATP CONTENT AND DISTRIBUTION OF MITOCHONDRIA IN BOVINE OOCYTES
Authors: Dadarwal D. , Dias F. , Adams G. , Singh J.
Published: December 2014

Our objective was to determine how follicular aging affects the distribution and content of mitochondrial population and ATP in in vivo-matured bovine oocytes. We hypothesised that in vivo-matured bovine oocytes obtained from aged follicles (84 h of gonadotropin starvation) have altered mitochondrial distribution and decreased cytoplasmic ATP content compared to those obtained immediately at the end of a superstimulatory protocol (no starvation). Follicular waves were synchronized by ablation 5 to 8 d after ovulation and a CIDR device was given. Starting on the day of wave emergence (Day 0), short FSH and FSH starvation groups (n = 5 heifers each) were given 8 doses of FSH im over 4 d and the long FSH group (n = 4) was given 14 doses over 7 d. Two doses of PGF were given on Day 4 (short FSH) or Day 7 (FSH starvation and long FSH groups), the CIDR was removed, and LH was given 24 h after second PGF treatment. The ovaries were removed 24 h later by colpotomy and cumulus-oocyte-complexes (COC) were collected from follicles ≥8 mm. Denuded oocytes were either stained with Mitotracker Deep Red FM and imaged by confocal microscopy or processed for ATP assay. Mitochondria numbers were assessed by segmentation of 3D datasets. Proportions of COC within each grade were compared using Fischer's exact test, and ATP and mitochondrial data were compared by analysis of variance. Short and long FSH groups had a greater proportion of Grade 1 expanded COC than the FSH starvation group (P = 0.02). The ATP content of oocytes (from expanded COC) tended to be higher in the long FSH group than short FSH (P = 0.09), and the FSH starvation group was intermediate. The ATP content of oocytes from compact COC did not differ among groups (P = 0.49). The proportion of mitochondrial clusters was highest (P = 0.01) and the proportion of individual mitochondria was lowest (P = 0.01) in the FSH starvation group compared to short and long FSH groups. Mitochondria from the long FSH and FSH starvation groups had twice the relative intensity compared to the short FSH group (P < 0.01). In conclusion, follicular aging (FSH starvation) was associated with a decrease in oocyte morphologic grade and marked clustering of mitochondria, which may be a reflection of oxidative stress and atresia.
292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES
Authors: Souza E. D. , Rabelo N. C. , Araujo T. D. , Assunção C. M. , Quintão C. C. R. , Viana J. H. M. , Louro I. D. , Camargo L. S. A.
Published: December 2014

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes.

Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments

Financial support comes from CNPq, FAPEMIG, and FAPES.
293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING
Authors: Lekola K. P. M. , Ng'ambi J. W. , Nkadimeng N. , Mphaphathi M. L. , Nedambale T. L.
Published: December 2014

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.
295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS
Authors: Jeon Y. , Baasanjav B. , Jeong Y. I. , Jeong Y. W. , Kim Y. W. , Hyun S. H. , Yang I. S. , Hwang W. S.
Published: December 2014

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P < 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P < 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P < 0.05). Total cell numbers of BL were significantly higher (P < 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.
322 SPERM PRESERVATION BY FREEZE-DRYING IN ENDANGERED ANIMALS
Authors: Kaneko T.
Published: December 2014

Sperm preservation is a useful tool for conservation of endangered animals. Freeze-drying sperm have been studied as new preservation method in various mammals as samples can be preserved in a refrigerator at 4°C or ambient temperature. Sperm preservation by freeze-drying is the ultimate method by which sperm can be stored that neither required specialised cryoprotectants nor constant supply of liquid nitrogen. We established the freeze-drying method that mouse and rat sperm could be preserved long-term at 4°C after freeze-drying using a simple solution containing 10 mM Tris and 1 mM EDTA (TE buffer; 2012 PLoS ONE 7, e35043; 2012 Cryobiology 64, 211–214). Using this method, the fertility of the chimpanzee, giraffe, and jaguar sperm after freeze-drying were estimated. Ejaculated chimpanzee and giraffe and cauda epididymal jaguar sperm were freeze-dried using TE buffer. Sperm were rehydrated with sterile distilled water after storage at 4°C for 1 month. Sperm with normal shape were injected into mouse oocytes in CZB medium with HEPES, and oocytes were then cultured in vitro for 6 to 8 h in the same media. In all animals, pronuclei and sperm tail were observed into oocytes without artificial activation after injection of freeze-dried sperm. When chimpanzee, giraffe, and jaguar sperm were injected into oocytes, 86% (12/14), 100% (12/12), and 96% (22/23) of oocytes formed 2 distinct pronuclei. This study demonstrated that the sperm of various animals could be decondensed into the mouse oocytes after freeze-drying using the same protocol. A further advantage is that freeze-dried sperm can be transported oversea at ambient temperature. Freeze-drying preservation without using liquid nitrogen can be protected strongly valuable gametes of endangered animals even in the event of unexpected accidents and disaster such as earthquakes and typhoons. Freeze-drying of sperm has been applied as a “freeze-drying zoo” for conservation of endangered animals (http://www.anim.med.kyoto-u.ac.jp/reproduction/home.aspx).
333 GENERATION OF OOCYTE-LIKE STRUCTURE FROM OVARIAN SURFACE EPITHELIAL STEM CELLS OF GOAT
Authors: Fatima S. , Sharma V. , Saini S. , Saugandhika S. , Malik H. N. , Kumar S. , Malakar D.
Published: December 2014

Stem cells have potential for therapeutic application. Continuous repair of ovarian surface epithelium following folliculogenesis and ovarian carcinoma suggests the presence of stem cells in ovarian epithelial cells. In vitro gametogenesis in livestock will result in large numbers of oocytes production from a single ovary, resulting in faster multiplication of superior germplasm of livestock species, treatment of infertile animals, and conservation of endangered species. The present study was conducted with the objective of in vitro differentiation of putative ovarian surface epithelial stem cells into oocyte-like structures in goat model. Ovary samples of 1- to 2-year-old goats were collected from slaughterhouse. The surface of the ovary was gently scraped using sterile blunt scraper to isolate ovarian surface epithelial stem cells. These scraped cells were cultured in DMEM/F12 supplemented with 20% FBS for 3 weeks in 5% CO2 at 37°C with maximum humidity. The cultured stem cells were characterised for stemness by RT-PCR and immunostaining for Oct4, Sox2, and Nanog genes after 3 weeks. These putative stem cells were in vitro differentiated spontaneously to oocyte-like structures in DMEM/F12 medium and characterised for premeiotic markers by RT-PCR and immunostaining for VASA, DAZL, and STELLA genes. Results of this study provide evidence for the presence of putative stem cells with pluripotent characteristics in the ovarian surface epithelium. The cultured cells were found to be round in shape, with a high nucleus to cytoplasm ratio under inverted microscope, and found positive for stem cell markers of Oct4, Sox2, and Nanog genes. A total of 66 oocyte-like structures were produced from 12 ovaries. These oocyte-like structures were nearly similar to oocytes produced in vivo, both morphologically and in molecular gene expression. The oocyte-like structures were also found positive for premeiotic markers of VASA, DAZL, and STELLA genes by RT-PCR and immunostaining. From this study, we concluded that the ovarian surface epithelial cells have putative stem cells which can be in vitro differentiated into oocyte-like structures in goat. These oocyte-like structures need further characterisation of their surface membrane, more molecular markers, and following their developmental potential. These oocytes can help for multiplication of elite germplasm, curing infertile animals, and saving endangered species.
334 FACTORS AFFECTING HEMATOPOIETIC ENGRAFTMENT OF MONKEY EMBRYONIC STEM CELLS IN SHEEP FETUSES
Authors: Nagao Y. , Abe T. , Hara A. , Sarentonglaga B. , Yamaguchi M. , Ogata K. , Fukumori R. , Hanazono Y.
Published: December 2014

Previously, we generated monkey/sheep haematopoietic chimeras by in utero transplantation (IUT) of monkey embryonic stem (ES); however, the factors that control how the ES cells successfully engraft and differentiate into haematopoietic tissue in sheep fetuses remain uncertain. Here, we examined factors that might influence donor cells and recipient sheep and affect successful ES cell engraftment. We transplanted either undifferentiated monkey ES cells or ES-derived cells at an early haematopoietic differentiation stage into sheep fetuses. The latter cells were allowed to differentiate by culturing on OP9 cell layers for 6 days. Cells were transplanted into the liver or subcutaneous tissue of recipient sheep fetuses at 43 to 50 or 51 to 67 days of gestation (full term = 147 days) using ultrasound to identify the site for transplantation. After birth, monkey haematopoietic engraftment in the bone marrow was analysed in 40 lambs using colony-PCR with cells grown in methylcellulose in the presence of defined cytokines; teratoma formation was analysed by biopsy and immunohistochemistry. We found that haematopoietic engraftment was only observed when ES-derived cells at the early differentiation stage were transplanted into fetal livers at 51 to 67 days of gestation (6/9). However, teratoma formation with mature monkey tissue structures was only observed following transplantation of undifferentiated ES cells into fetal subcutaneous tissues at 43 to 50 days of gestation (4/6), but that was not observed when both types of cells were transplanted into the liver (0/18) or at 51 to 67 days of gestation (0/24). These results demonstrate that the differentiation status of the donor cells, the transplantation site, and the age of the fetus at transplantation are important factors in engraftment and differentiation into haematopoietic tissue or teratoma formation in sheep fetuses.
335 GENE THERAPEUTIC EFFECT OF ENGINEERED STEM CELLS EXPRESSING CYTOSINE DEAMINASE AND INTERFERON-B AGAINST ENDOMETRIAL CANCER IN A XENOGRAFT MOUSE MODEL
Authors: Yi B.-R. , Choi K.-C.
Published: December 2014

To generate an animal model of endometrial cancer, Ishikawa cells (2 × 106 cells) were implanted via subcutaneous injection into SCID mice. In order to evaluate the therapeutic effect on tumour growth, human neural stem cells (HB1.F3), transduced with genes expressing cytosine deaminase (CD) and interferon-β (IFN-b), were labelled with the membrane dye stained with CM-DiI and were injected nearby into the tumour masses (250 mm3) in these SCID mice (n = 24). Two days after stem cell injections, 5-fluorocytosine (5-FC) was intraperitoneally injected (500 mg kg–1 per day) for 14 consecutive days. Mice were sacrificed at 48 h after the last treatment with 5-FC delivered by intraperitoneal injection for 14 days. Results demonstrated that tumour mass growth in mice treated with stem cells and 5-FC was significantly inhibited (450 mm3 ± 52) in comparison to that of nontreated mice (1400 mm3 ± 124; P < 0.01). We confirmed that injected stem cells migrated to the tumour masses by visualising the red-coloured cells in the endometrial cancer masses. The RT-PCR analysis showed that the VEGF gene was highly expressed in endometrial cancer cells, while migration-induced uPA, SDF-1, SCF, MCP-1, IL-6, and IL-8 genes were moderately expressed in these tumour cells. Histological analysis of tumour masses showed that the aggressive nature of endometrial tumour masses, determined by labelled stem cells, was inhibited in treated mice. A further analysis showed that expression of proliferating nuclear antigen (PCNA) was significantly decreased in tumour masses from treated mice. To evaluate the effect of 5-FU on Ishikawa cells, 5-FU (1.0 mg mL–1) was added to in vitro-cultured cells and the levels of IFN-a/b receptor 2 (IFNAR2) and BAX, a proapoptotic genes, were detected by RT-PCR. In these cells, IFNAR2 and BAX genes were significantly increased by the addition of 5-FU. Taken together, these results indicate that co-expression of CD and IFN-b significantly inhibited the growth of endometrial tumour masses in the presence of 5-FC, and 5-FU and IFN-b inhibits tumour growth by inhibition of DNA synthesis and enhancing the apoptotic cascade, respectively. We provide evidence to support the efficacy of therapeutic stem cell-based immuno-therapy involving the targeted expression of CD and IFN-b genes at endometrial cancer sites.This work was supported by a National Research Foundation of Korea (NRF) grant (2013R1A1A2059092) funded by the Ministry of Education, Science and Technology (MEST) of the Republic of Korea government.
336 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING SF1 INTO THE STEROID-PRODUCING CELLS
Authors: Kang H. Y. , Choi Y.-K. , Jeung E.-B.
Published: December 2014

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we obtained SF1 cDNA from mRNA of murine pituitary glands and constructed the SF1-expressing vector controlled by the CMV promoter. The SF1 transgenic mouse embryonic stem cells (SF1-mES cells) were established through transfection using the nucleofector (Lonza) and selection process using G418 at 250 μg mL–1. The SF1-mES cells were aggregated in hanging drops for 2 days and were transferred to suspension culture for an additional 1 day in mouse basal differentiation medium. Three-day-old SF1-mESC-derived EB were attached onto 6 well culture plates and differentiated into granulosa-like cells. Differentiated SF1-mES cells were analysed by expression of steroidogenesis-related genes and gonadal lineage-markers to the level of mRNA via real-time PCR method. To test the phenotype for granulosa-like cells, we confirmed transcripts of specific forkhead transcription factor FOXL2 and the follicle-stimulating hormone receptor (FSHR). On the other hand, we monitored some specific genes related to differentiation into testicular tissue. We observed the progress to primitive streak-mesendodermby gene expression analyses. In addition, we observed that differentiated SF1-mES cells express steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, and CYP19A1. Using the advanced approach, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic and gonadal lineages. We also induced granulosa-like cells. We established the effective protocol to generate ovarian cells. The derivation of these cells explores new avenues for the further study and potential application of these cells in steroidogenesis.
358 EXCESS EXPRESSION OF 11β-HSD1 CONTRIBUTES TO LETHALITY THROUGH DYSFUNCTION IN ENERGY BALANCE BETWEEN ANABOLIC PROCESS AND ENERGY RECOVERY PROCESS
Authors: Kang H. Y. , Jeung E.-B.
Published: December 2014

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) effectively amplifies glucocorticoid action in liver, adipose tissue, and brain. This enzyme converts the inactive 11-keto form into a glucocorticoid (e.g. cortisol). Glucocorticoids (GC) are steroid hormones found in the body and produced by the adrenal cortex, the outer layer of the adrenal glands. Glucocorticoids are lipophilic and readily access their intracellular receptors. Glucocorticoids regulate carbohydrate, fat, and protein metabolism. In a previous study, we constructed a vector composed of 2 parts: the 11β-HSD1 expression cassette and the selection cassette containing EGFP and Neo resistant genes. Porcine fibroblasts overexpressing 11β-HSD1 under the control of adipose tissue-specific aP2 promoter were established and used in somatic cell nuclear transfer (SCNT). Somatic cells from the resulting stillborn transgenic piglets were used in a second round of SCNT. Non-obese, transgenic piglets overexpressing 11β-HSD1 were obtained and were identified through PCR-based methods using specific primers for the targeting cassettes from the genomic DNA of piglets. Six live piglets, 1 stillborn piglet, and 3 mummies were born. Integration of target gene into the genomic DNA was confirmed for all of them. However, all 6 live piglets died within 1 month. All of the piglets had displayed hypoglycemia. Increased expression of 11β-HSD1 in metabolic tissues induced up-regulation of gluconeogenesis-related genes (G6PT, G6Pase, PEPCK, HNF4a, FOXO1) in liver and kidney, and showed up-regulation of lipogenesis-related genes (SREBP1c, FASN, DGAT, ACC, SCD) in muscle. The AMPK and SIRT signalling, which controls energy balance and mitochondrial biogenesis, was also stimulated. We propose that overexpression of 11β-HSD1 evokes the excess production and action of glucocorticoid or its receptors, and activates gluconeogenic and lipogenic pathways. For this reason, AMPK and SIRT1 signalling was induced. Also, in compensation of energy loss by anabolic processes, the expression of mitochondrial biogenesis-related genes was increased. Finally, the constitutive expression of 11β-HSD1 might continuously activate complementary energy-gaining processes, and these problems could develop into more fatal diseases that resulted in the piglets' death.
44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Kondo S. , Imai K. , Dochi O.
Published: December 2014

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium.

Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos
52 EFFECT OF CARBOXYLATED POLY-L-LYSINE ON THE POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE BLASTOCYST
Authors: Ha A. N. , Lee K. L. , Fakruzzaman Md. , Kim S. S. , Park P. R. , Jin J. I. , Hyon S. H. , Kong I. K.
Published: December 2014

Recently, there has been development of an antifreeze polyamino acid (carboxylated poly-l-lysine: PLL) as new cryoprotectants (CPA). This compound counts as amphoteric macromolecular cationic and anionic substituents (polyampholyte) by chemically modifying poly-lysine. In addition, PLL is highly safe and frequently used as a food additive in substitution for other CPA. Other common CPA have high toxicity and caused physiological damage. In vitro-produced blastocysts were randomly assigned into 3 groups: (1) vitrified embryos with PLL vitrification solution [PLL-vit-1: 15% PLL + 15% ethylene glycol (EG); PLL-vit-2: 30% PLL + 30% EG + 0.5 M sucrose], (2) vitrified embryos with Vajta et al. solution (Conv-vit-1: 7.5% dimethyl sulfoxide + 7.5% EG; Conv-vit-2: 16.5% dimethyl sulfoxide + 16.5% EG + 0.5 M sucrose), and (3) nonvitrified blastocysts (control). All embryos were frozen by droplet vitrification method. First, the PLL-vitrified embryos were exposed to 5, 10, and 15 min in the PLL-vit-1 and then putted for 30 to about 60 s in the PLL-vit-2. Then, we compared with each group regarding exposure time of Vit-1. Post-thaw survival rate of each exposure time did not significantly differ among the 3 groups (100 v. 100 v. 100%). However, hatching rate of the 10-min exposure group was higher than that of 5- and 15-min groups (75.0 v. 25.0 v. 66.7; P < 0.05). Therefore, we confirmed that exposure time of Vit-1 was exposed for a minimum of 10 min. The post-thawed survival rate of each vitrification method was not significantly different between PLL-vit and Conv-vit groups (97.7 v. 86.4%). The total cell numbers of blastocyst did not significantly differ among groups. However, the apoptotic cell numbers of blastocyst was significantly different between the control and Conv-vit groups (0.4 ± 0.6 v. 4.4 ± 3.9; P < 0.05) but was not different in control v. PLL-vit (0.4 ± 0.6 v. 2.1 ± 2.4) and Conv-vit v. PLL-vit (4.4 ± 3.9 v. 2.1 ± 2.4). In conclusion, PLL-vit for bovine blastocyst could reduce toxicity and osmotic shock and showed high efficiency on the quality of post-thawed bovine blastocysts compared with that of Conv-vit.This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009587022014), IPET (Grant No. 112020-3), and a scholarship from the BK21 plus program. A-Na Ha, Kyeong-Lim Lee, and Md. Fakruzzaman were supported by BK21 plus fellowships at Gyeongsang National University, Republic of Korea.
54 CRYOPRESERVATION OF BOVINE IN VITRO-PRODUCED EMBRYOS: INTRINSIC FACTORS DETERMINING VITRIFICATION OUTCOMES
Authors: Saucedo M. N. , Kurome M. , Reichenbach M. , Wolf E. , Reichenbach H.-D.
Published: December 2014

As a part of a study on bovine embryo genomic evaluation, effects of an intact (iZP) or opened zonae pellucidae (oZP) of in vitro-produced embryos on vitrification outcomes were assessed. In the first experiment, only iZP embryos were subjected to vitrification, using either the hollow fibre vitrification method (HFV; Matsunari et al. 2012 J. Reprod. Dev.) or the cryologic vitrification method (CVM; CryoLogic®, Blackburn, Victoria, Australia). Developmental stage (morula = M; early blastocyst = EaB; blastocyst = B) and quality (good = 1; fair = 2) before vitrification were evaluated. In a first set of experiments, quality 1 and 2 iZP embryos were vitrified either by the HFV or the CVM method. A significant difference between the 2 methods was found when comparing overall survival rates (24–48 h post-thaw; HFV 59.32% v. CVM 78.9%; P < 0.001; unpaired t-test, GraphPad Prism, GraphPad Software Inc., La Jolla, CA, USA). Concerning hatching rates, no significant difference was found (HFV 48.7% v. CVM 57.8%; P > 0.1). In order to evaluate influence of embryo stage and quality on HFV or CVM outcomes, iZP embryos vitrified by the HFV method were classified regarding quality (1: n = 207; 2: n = 66; total n = 273) and embryo stage (M: n = 78; EaB: n = 74; B: n = 121). Concerning survival rates, no significant difference was found between M 1 (57.4%) and M 2 (44.6%), or EaB 1 (80%) and EaB 2 (68%). However, a significant difference was found when comparing B 1 (77%) and B 2 (29%; P < 0.05). With regard to hatching rate, no significant difference was found between M 1 (39.9%) and M 2 (30%), or EaB 1 (66.6%) and EaB 2 (57%). However, significant difference was found between B 1 (60.9%) and B 2 (22%; P < 0.05). With regard to the CVM (1: n = 172; 2: n = 82; total n = 254), no significant difference was found when analysing survival rates of EaB 1 (82%) and EaB 2 (80%), or B 1 (87%) and B 2 (74%), whereas the survival rates were significantly different between M 1 (80%) and M 2 (53%; P < 0.05). Significant differences regarding hatching rates were not found between M 1 (51%) and M 2 (28%) or EaB 1 (55%) and EaB 2 (65%), whereas the hatching rate of B 1 (76%) was not significantly higher than that of B 2 (48.8%; P < 0.05). In a second set of experiments, oZP EaB (1: n = 14) and oZP B (1: n = 30) were vitrified by the CVM method. No significant difference regarding survival rates concerning stage (95 and 72%, EaB and B, respectively), or between treatments (EaB iZP v. oZP, and B iZP v. oZP; P > 0.1) was found. Effect of ZP, either intact or opened, does not seem to affect survival rates (judged by their re-expansion 24–48 h post-thaw) of good-quality embryos.The authors thank the Bavarian Research Foundation for the financial support (AZ-1031-1; DOK-153-12).
63 STEPWISE THAWING PRESERVES MITOCHONDRIA MEMBRANE POTENTIAL OF BOAR SPERMATOZOA EXTENDED IN GLYCEROL-FREE MEDIUM CONTAINING TREHALOSE
Authors: Athurupana R. , Funahashi H.
Published: December 2014

Motility and penetrability of spermatozoa are significantly affected by the energy produced in mitochondria. The survival of cryopreserved spermatozoa is influenced by the warming rate as well. The objective in the present study was to evaluate the effect of stepwise thawing on post-thaw motility and mitochondria activity of boar spermatozoa. The sperm samples collected from a Berkshire boar with high fertility at an AI centre were diluted in egg yolk-based and glycerol-free freezing extender containing 100 mM trehalose and 0.25% Equex STM™. The samples were cryopreserved using the straw freezing procedure. Best thawing durations in high temperatures were examined in a previous study. Straws were immersed in 80, 70, and 60°C water for 6, 8, and 10 s and continued to thaw in 39°C for 54, 52, and 50 s, respectively. Sperms thawed at 39°C for 60 s were considered as the control. Frozen-thawed spermatozoa were analysed for motility with computer-assisted semen analysis and mitochondrial membrane potential (MMP) with JC-1/PI staining (Table 1). Data were analysed with ANOVA and Pearson correlation. Motility and MMP of frozen-thawed sperm were significantly lower than fresh samples. Motility was fairly high compared with frozen-thawed controls, when thawed rapidly in 2-steps, though the difference was not significant. The MMP was also significantly higher when thawed in 2-steps from 80 or 70°C, then to 39°C, as compared with frozen-thawed controls, and it was positively correlated with the thawing temperature (r = 0.64; P < 0.01). There was a positive trend between thawing temperature and motility, but it was not significant (r = 0.37; P = 0.11). In conclusion, our results suggest that stepwise thawing with a rapid thawing at the beginning preserves post-thaw motility and MMP of boar spermatozoa.

Table 1.Effect of thawing temperature on post-thaw motility and mitochondrial membrane potential (MMP)1
87 OVARIAN RESERVE, EMBRYO PRODUCTION, AND THEIR CORRELATION WITH ANTI-MÜLLERIAN HORMONE (AMH) IN HOLSTEIN COWS
Authors: Verstegen J. , Rozner A.
Published: December 2014

Anti-Müllerian hormone (AMH) is a small peptide hormone that has been associated with ovarian follicular reserve in humans and in some animal species including bovine. Profiles of AMH, as well as the relationship between serum AMH to oocyte number and in vivo embryo production, were evaluated in Holstein cows. AMH levels were determined in 15 unstimulated cows at monthly intervals for 4 months and in 394 male and 399 female developing Holstein animals from birth to adulthood. Also, AMH was measured in 41 heifers at the time of ovum pick-up (OPU) and 125 heifers at the time of embryo flushing. Superovulation was induced before OPU or embryo flushing using a modified Ovsynch protocol with 4 days of decreasing FSH (Pluset H®, MOFA Global, Verona, WI, USA). Blood samples were collected using serum tubes and spun within 2 h. The samples were stored at –20°C until evaluated for AMH using the AMH-Bovine specific immunoassay® (MOFA Global). AMH levels in males and females peaked at 2 months of age and then decrease as they reached adulthood. The average AMH level of adult cows was stable for each of the 4 monthly measurements, with a high correlation between all values per animal (r2 = 0.9077; P < 0.01), suggesting that AMH levels are consistent for at least 4 consecutive months. However, AMH levels were lowest during the summer months, suggesting a seasonal change in AMH secretion. Animals repeatedly ovarian stimulated showed decreasing AMH levels (509 ± 295, 299 ± 210, 211 ± 119) with subsequent stimulations. There was also a significant decrease in the number of embryos recovered (5.7 ± 4, 2.2 ± 1.9; P = 0.02); however, the number of oocytes was not altered by multiple stimulations (9.9 ± 9.8, 8.1 ± 6.2; P = 0.57). Because AMH and embryo numbers decreased after multiple stimulations, the first AMH value and results of the first OPU or embryo flush were used for the correlation of AMH to the number of oocytes or embryos. Animals were separated into 3 AMH categories: low (<100), normal (100–400), and high (>400 pg mL–1). High AMH OPU animals had significantly higher numbers of oocytes than the normal or low AMH groups (13.8 ± 9.2, 9.2 ± 5.3, 5.6 ± 3.9; P = 0.001). High AMH flushed animals had significantly higher numbers of embryos than animals with low AMH (10.9 ± 8.0, 5.7 ± 5; P = 0.002). Statistical analyses were performed using Statview 5. Differences were determined using Student's t-test; P < 0.05 was considered significant. In conclusion, AMH serum concentrations are consistent over multiple months; however, blood should not be taken for animal selection by AMH after ovarian stimulations have begun and should be interpreted with caution during the summer months. AMH is highly associated with superovulation response and oocyte and embryo production and should improve efficiency of multiple-ovulation embryo transfer.
Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in
Drosophila
oenocytes
Authors: Chatterjee Debamita , Katewa Subhash D. , Qi Yanyan , Jackson Susan A. , Kapahi Pankaj , Jasper Heinrich
Published: December 2014

Significance

This study characterizes the transcriptional response to fasting in adult flies in a tissue-specific manner, highlighting a central role for adult oenocytes in the regulation of lipid mobilization and supporting the proposed analogy between oenocytes and mammalian hepatocytes. A surprising and critical role for insulin signaling activity in the oenocyte fasting response is identified, and it is shown that the
Drosophila
insulin-like peptide 6 (dILP6), which is induced in the fat body in response to starvation, mediates this response. A new paracrine role for insulin signaling in regulating the interaction between adipose tissue and hepatocyte-like cells in the metabolic adaptation to fasting is thus identified.
Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP–sugar synthesis to support viral protein glycosylation
Authors: DeVito Stefanie Renee , Ortiz-Riaño Emilio , Martínez-Sobrido Luis , Munger Joshua
Published: December 2014

Significance

Viruses use the host cell to provide the energy and molecular subunits to assemble viral progeny. The progeny of a variety of viral families possess envelope glycoproteins that are essential for viral infection. The production of these functional glycoproteins requires an ample supply of UDP–sugar subunits that serve as the substrates for glycosylation reactions. Our results indicate that human cytomegalovirus induces a viral metabolic program that activates pyrimidine biosynthesis to drive UDP–sugar biosynthesis. This metabolic activation is important for viral protein glycosylation and high-titer viral replication. Further, our results suggest that this metabolic link between pyrimidine and UDP–sugar biosynthesis is shared between evolutionarily diverse viral families, which may provide novel avenues for antiviral therapeutic intervention.
Inhibition of malaria parasite growth by quinomycin A and its derivatives through DNA-intercalating activity
Authors: Hayase Hiroki , Watanabe Nobumoto , Lim Chung Liang , Nogawa Toshihiko , Komatsuya Keisuke , Kita Kiyoshi , Osada Hiroyuki
Published: December 2014

Quinomycin A and its derivatives were identified as potent antimalarial (Plasmodium falciparum) agents in a screen of the RIKEN NPDepo chemical library. IC50 values of quinomycin A and UK-63,598 were approximately 100 times lower than that of the antimalarial drug chloroquine. This activity was mitigated by the addition of plasmid DNA, suggesting that these compounds act against parasites by intercalating into their DNA.
A general form of Green’s Formula and the Cauchy Integral Theorem
Authors: Cufí Julià , Verdera Joan
Published: December 2014

We prove a general form of Green’s Formula and the Cauchy Integral Theorem for arbitrary closed rectifiable curves in the plane. We use Vituśkin’s localization of singularities method and a decomposition of a rectifiable curve in terms of a sequence of Jordan rectifiable sub-curves due to Carmona and Cufí.