162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
A. Harvey ◽  
M. Lane ◽  
J. Thompson
Keyword(s):  
Calf Serum ◽  
Cell Number ◽  
Tunel Staining ◽  
Embryo Survival ◽  
Improved Outcomes ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).

74 EFFECT OF SIZE AND CULTURE PERIOD OF FROZEN–THAWED BOVINE TROPHOBLASTIC VESICLES ON INTERFERON-τ SECRETION

2014 ◽  
Vol 26 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
D. Yamaguchi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Significant Positive Correlation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Culture Period ◽  
Fetal Bovine ◽  
Student’S T ◽  
Student’S T Test

Frozen–thawed bovine trophoblastic vesicles (bTV) derived in vivo could secrete interferon-τ (IFN-τ) at the same level as fresh bTV on Days 4 to 6 after thawing. However, amounts of IFN-τ decreased following continuous in vitro culture (Hashiyada et al. 2012 38th IETS). Co-transfer of frozen–thawed bTV improved pregnancy rate of embryos due to the effects of IFN-τ secreted by bTV (Hashiyada et al. 2008 41th SSR). However, the relation between bTV size and IFN-τ secretion level during culture has not been well documented. The objective of present study was to characterise the concentration of IFN-τ related bTV volume and culture period after thawing of cryopreserved bTV. The bTV were prepared from Day 16 elongating blastocysts recovered nonsurgically. The dissected trophoblastic fragment, 1 to 1.5 mm in width, was cultured using TCM-199 supplemented with 20% (vol/vol) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum and 1.8 M ethylene glycol. After thawing, bTV were cultured individually with 100 μL/well/day until Day 2 (i.e. the day of thawing was defined as Day 0), and thereafter changed to 200 μL/well/day to termination at Day 10. Collection of culture media and measurement of bTV diameter were performed before cryopreservation and after thawing for every day. Interferon-τ in collected media was measured by radioimmunoassay. The estimated bTV volume was calculated based on the diameter. Data were analysed by Student's t-test. Nine fresh bTV before cryopreservation were used to assess the IFN-τ secretion for 24 h in relation to bTV volume. A significant positive correlation was observed between secreted IFN-τ (mean ± s.e.M, 19.9 ± 3.1 ng mL–1) and bTV volume (1.49 ± 0.6 mm3, r = 0.91; P < 0.01). Initial IFN-τ secretion from bTV cultured for 24 h after thawing was significantly decreased compared with that before cryopreservation (29.1 ± 2.1 ng mL–1 and 58.4 ± 4.8 ng mL–1; P < 0.01, n = 27). In continuous culture of bTV (n = 8), IFN-τ secretion increased gradually from Day 2 (23.1 ± 9.0 ng mL–1) to Day 4 (32.2 ± 8.4 ng mL–1), and then maintained this level until Day 7 (33.4 ± 14.9 ng mL–1). However, this amount of IFN-τ tended to decrease on Day 8 (24.8 ± 5.0 ng mL–1), 9 (16.5 ± 4.4 ng mL–1), and 10 (12.0 ± 1.7 ng mL–1). Interferon-τ secretion from bTV on Day 9 and 10 was lower than that on Day 3, 4, 5, 6, and 8, respectively (P < 0.05). Volume of bTV increased also from Day 2 (0.2 ± 0.1 mm3) to Day 5, 6 (0.8 ± 0.3 mm3) and 7 (0.7 ± 0.2 mm3). However, bTV volumes shrank drastically on Day 8 (0.3 ± 0.1 mm3), 9, and 10 (0.2 ± 0.1 mm3). In comparison with bTV during culture, volumes on Day 4, 5, and 7 were greater than those on Day 2 and 3, and volumes on Day 6 and 7 were greater than on Day 8, 9, and 10 (P < 0.05). These results indicate that the dynamics of IFN-τ secretion reflected the expansion or reduction of bTV in continued culture after thawing. Interferon-τ secretion might be related to bTV volume. Moreover, we reconfirmed that cryopreserved bTV highly express IFN-τ during 4 to 7 days after thawing.

78 INTERFERON-τ SECRETION OF CRYOPRESERVED BOVINE TROPHOBLASTIC VESICLES DERIVED FROM IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Continuous Culture ◽  
Culture Media ◽  
Calf Serum ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

The co-transfer of bovine trophoblastic vesicles (bTVs) prepared from in vivo recovered conceptuses is known to promote the successful implantation of embryos, which expected lower viability, through the effects of interferon-τ (IFN-τ) secreted by bTVs. We have reported that the pregnancy rate was improved for co-transferred embryos with frozen-thawed bTVs using the direct-transfer technique (Hashiyada et al. 2008, 41st SSR). However, the IFN-τ secretion level from cryopreserved bTVs is not well known. The objective of the present study was to measure concentration of IFN-τ released from frozen-thawed bTVs individually cultured in vitro. bTVs were prepared from elongating blastocysts 3 to 20 mm in length, following superstimulatory treatment and recovered on Day 16 post-AI, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of a 96-well plate using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 or 48 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum, 1.5 M ethylene glycol (EG) and 0.1 M sucrose or 1.8 M EG. After thawing, each bTVs was cultured for 2 days to compare IFN-τ secretion between the 2 cryoprotectants. Furthermore, transition of IFN-τ level was assessed in continuous culture until Day 10 (the day of thawing was defined as Day 0). The volume of culture medium was 100 μL well–1 day–1 until Day 2 and thereafter changed to 200 μL well–1 day–1 until termination. Exchange and collection of culture media were performed on Day 1, 2, 4, 6, 8 and 10. Collected culture media were stored at –30°C until use. IFN-τ was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analysed by Student's t-test. Initial IFN-τ secretion from bTVs before cryopreservation did not differ between 24 and 48 h of culture period to form vesicles, 44.0 ± 2.9 (mean ± standard error of the mean, n = 64) and 52.8 ± 6.4 ng mL–1 (n = 27), respectively. IFN-τ secretion was no difference between the 1.5 M EG group and the 1.8 M EG group on Day 1 (41.2 ± 4.9 ng mL–1, n = 42 and 30.4 ± 2.2 ng mL–1, n = 31) and on Day 2 (38.0 ± 5.4 and 38.2 ± 4.5 ng mL–1), respectively. In the continuous culture group (n = 28), IFN-τ secretion tended to increase from Day 2 (25.2 ± 3.4 ng mL–1) to Day 4 (51.8 ± 12.3 ng mL–1) and 6 (55.4 ± 13.3 ng mL–1) (P < 0.05). However, this amount of IFN-τ on Day 6 significantly decreased on Day 8 (25.6 ± 2.7 ng mL–1; P < 0.05) and Day 10 (15.5 ± 2.2 ng mL–1; P < 0.01), gradually. These results indicate that cryopreserved bTVs could secrete IFN-τ at the same level as fresh bTVs on Day 4 to 6 after thawing and then these amounts of IFN-τ significantly decrease in vitro.

In Vitro and in Vivo Targeting of Chronic Lymphocytic Leukemia Using CX-4945, a Clinical-Stage CK2-Specific Inhibitor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2897-2897 ◽  
Author(s):  
Leila R. Martins ◽  
Paulo Lúcio ◽  
Alice Melao ◽  
Bruno A. Cardoso ◽  
Ryan Stansfield ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Abstract Abstract 2897 Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma. We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P<0.0001 for each dose, 2-tailed paired Student's t test), as determined by Annexin V-APC/7-AAD staining. Moreover, the effect of CX-4945 was time- and dose-dependent in 4 out of 4 cases that were more thoroughly analyzed. Similar results were obtained using MEC1, MEC2, WaC3CD5, JVM3 and MO1043 cell lines whose IC50 ranged between 3.1 and 5.8μM. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival, it did not prevent CX-4945-mediated apoptosis of CLL cells. Most importantly, CX-4945 induced a stronger decrease in the viability of CLL cells from patients with higher percentage of malignant cells in the blood (R2=0.4176, P=0.0317, n=11, Pearson correlation), Binet stage B/C (P=0.0424, n=10, 2-tailed unpaired Student's t test) or higher plasma β2 microglobulin levels (P=0.0239, n=9). Furthermore, CLL cells with a higher proliferation rate (LDT < 12 months) were also more sensitive to CX-4945 (P=0.0007, n=11). In accordance, the need for treatment positively correlated with the sensitivity to CX-4945 (R2=0.4504, P = 0.0238). These observations suggest that treatment with CK2 inhibitors may be especially beneficial to patients with more advanced or aggressive disease. The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P<0.0001, 2-way ANOVA). Notably, CX-4945 was as effective as fludarabine when used as a single agent, and the combination of the two drugs was significantly more effective than fludarabine alone (P=0.0375). All treatments were well tolerated as evidenced by the maintenance of body weight and the inexistence of signs of overt toxicity. Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures: Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

scholarly journals Bioinformatic and experimental findings to indicate anti-cancer targets and mechanisms of calycosin against nasopharyngeal carcinoma

2020 ◽  
Author(s):  
Fangxian Liu ◽  
Qijin Pan ◽  
Liangliang Wang ◽  
Shijiang Yi ◽  
Peng Liu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Network Pharmacology ◽  
Tunel Staining ◽  
Pharmacological Targets ◽  
Experimental Findings

Abstract Background: Calycosin is a naturally-occurring phytoestrogen that reportedly exerts anti- nasopharyngeal carcinoma (NPC) effects. Nevertheless, the molecular mechanisms for anti-NPC using calycosin remain unrevealed. Methods: Thus, a network pharmacology was used to uncover anti-NPC pharmacological targets and mechanisms of calycosin. Additionally, validated experiments were conducted to validate the bioinformatic findings of calycosin for treating NPC. Results: As results, bioinformatic assays showed that the predictive pharmacological targets of calycosin against NPC were TP53, MAPK14, CASP8, MAPK3, CASP3, RIPK1, JUN, ESR1, respectively. And the top 20 biological processes and pharmacological mechanisms of calycosin against NPC were identified accordingly. In clinical data, NPC samples showed positive expression of MAPK14, reduced TP53, CASP8 expressions. In studies in vitro and in vivo, calycosin-dosed NPC cells resulted in reduced cell proliferation, promoted cell apoptosis. In TUNEL staining, calycosin exhibited elevated apoptotic cell number. And immunostaining assays resulted in increased TP53, CASP8 positive cells, and reduced MAPK14 expressions in calycosin-dosed NPC cells and tumor-bearing nude mice. Conclusion: Altogether, these bioinformatic findings reveal optimal pharmacological targets and mechanisms of calycosin against NPC, following with representative identification of human and preclinical experiments. Notably, some of original biotargets may be potentially used to treat NPC.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 601 ◽  
Author(s):  
PA Batt ◽  
DK Gardner ◽  
AW Cameron
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Oxygen Concentration ◽  
Bovine Serum ◽  
Calf Serum ◽  
Cell Number ◽  
The Mean ◽  
Number Of Cells

The effect of oxygen concentration and the source of protein in culture medium on the development of 2- to 4-cell goat embryos in vitro was investigated. Embryos were collected from superovulated Angora-Cashmere-cross goats 48 h after ovulation and cultured for 6 days in synthetic oviduct fluid (SOF) medium under one of two oxygen concentrations (20% or 7%) and in the presence of one of five protein sources; Miles bovine serum albumin (Miles BSA), Commonwealth Serum Laboratory bovine serum albumin (CSL BSA), goat serum (GS), fetal calf serum (FCS) and human serum (HS). In the presence of 20% oxygen the percentage of embryos reaching the expanded and/or hatched blastocyst stage in SOF medium containing Miles BSA was 29%, with a mean cell number per embryo of 28.1 +/- 6.0 (+/- s.e.m.). Use of an oxygen concentration of 7% significantly increased the percentage of embryos reaching this stage (80%, P less than 0.01) and the mean number of cells per embryo (65.3 +/- 8.2, P less than 0.01). The mean number of cells of the early-cleavage-stage embryos was significantly lower when the medium contained CSL BSA, GS or FCS (42.7 +/- 5.6, 29.0 +/- 6.1 and 21.3 +/- 3.2, respectively) than with Miles BSA (92.8 +/- 6.4) or HS (104.8 +/- 17.2) (P less than 0.01). Under 7% oxygen and with Miles BSA or HS, embryos were morphologically comparable to those developed in vivo, but the mean cell numbers in vitro were only approximately half those obtained in vivo.

91 PRELIMINARY RESULTS OF SURGICAL EQUINE EMBRYO TRANSFER AFTER OPEN PULLED STRAW VITRIFICATION

2006 ◽  
Vol 18 (2) ◽  
pp. 154 ◽  
Author(s):  
G. Duchamp ◽  
F. Guignot ◽  
J. Grizelj ◽  
M. Vidament ◽  
P. Mermillod
Keyword(s):  
Calf Serum ◽  
Embryo Cryopreservation ◽  
Success Rates ◽  
Ringer Lactate ◽  
Embryo Survival ◽  
Preliminary Study ◽  
Higher Sensitivity ◽  
Conventional Freezing

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after open pulled straw (OPS) vitrification (Moussa et al. 2005 Theriogenology 64, 1619–1632). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB), and blastocysts (B) ranging from 140 to 320 μm in diameter were collected (n = 20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon and Squires 1988 J. Am. Vet. Med. Assoc. 192, 401–406). Then, embryos were vitrified using the OPS method described by Berthelot et al. (2001 Reprod. Nutr. Dev. 41, 267–272). Briefly, embryos were washed twice in HEMES-TCM-199 + 20% newborn calf serum (NBCS) for 1 min, equilibrated in HEPES-TCM-199 + 20% NBCS with 7.5% dimethyl sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min, and then with 18% DMSO + 18% EG + 0.4 M sucrose for 45 s. One embryo was then loaded per straw. For transfer, four straws were quickly thawed (5 s in air) and the narrow end of the straw containing the embryo was immersed in HEPES-TCM-199 + 20% NBCS + PBS + 0.2 M sucrose. Five to 8 min after thawing, four embryos were surgically transferred into the cranial portion of the uterine horn in each of five pony mare recipients. Five days after transfer, embryos recovered by transcervical flushing of the uterus were classified as viable if morphology was normal, no dark inner cells were present, the capsule was intact, and the diameter was at least 1000 μm. The results are shown in the table. One recipient of vitrified embryos had an endometritis and no embryo was recovered. From the four other recipients, nine embryos were recovered out of 16 (56%) transferred, seven of which were viable (44%). The results of the present preliminary study demonstrating survival of equine embryos transferred after OPS vitrification is very encouraging. However, the results should be confirmed by birth of foals after transfer of OPS-vitrified embryos to recipients. Table

Over Expression of MN1 Confers Resistance to Chemotherapy In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 969-969
Author(s):  
Timothy Pardee ◽  
Teresa Mascenik ◽  
Britt H. Bolemon ◽  
Guerry J Cook
Keyword(s):  
Bone Marrow ◽  
The United States ◽  
Genetic Alterations ◽  
T Test ◽  
Over Expression ◽  
Student’S T ◽  
Worse Prognosis ◽  
Student’S T Test

Abstract Abstract 969 Acute myeloid leukemia (AML) is an accumulation of immature myeloid precursors that leads to progressive marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of active research the overall 5 year survival remains a dismal 30–40%. The backbone of initial therapy for the last 30 years is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is characterized by heterogeneous genetic alterations that can be used to delineate prognosis. Using standard karyotyping techniques patients can be divided into good, intermediate and poor prognostic categories. There is a clear link between these chromosomal aberrations and response to chemotherapy as complete remission rates are significantly different between groups. Patients with no detectable cytogenetic abnormality fall into an intermediate prognostic group with a very heterogeneous outcome. Recent work has begun to uncover submicroscopic genetic alterations that effect prognosis for these patients. These alterations can be mutations, over or under expression of a particular gene. The MN1 gene encodes a transcription co-factor first identified by its involvement in a balanced translocation in a patient with a meningioma. Since its initial description it has been found over-expressed in multiple AML patient samples. There are several reports that over-expression of MN1 confers a worse prognosis in AML. High MN1 expressers were less likely to achieve a remission and had a lower 3 year survival rate. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. However, the effect of MN1 over expression on response to standard chemotherapy is currently unknown. To answer this question we used a murine model of AML driven by MLL-ENL. AML blasts were infected with retroviral vectors that contained MN1 and a GFP reporter. Partially infected blast populations were then exposed to various concentrations of either Ara-C or doxorubicin and the ratio of GFP positive and negative cells was compared to untreated controls. When blasts were exposed to 150 nM Ara-C the GFP+ percentage went from 21.10 (+/− 0.5302) in the control samples to 35.68 (+/−1.230) in the treated samples. This result was even more profound when cells were treated with 15 ng/ml doxorubicin where the percentage went from 21.10 (+/− 0.5302) to 80.27 (+/−1.615). Both results were highly statistically significant by two tailed student's t test with p values of 0.004 and <0.0001 respectively. Consistent results were obtained in multiple different infections and with separately derived MLL-ENL lines. These data demonstrate that blasts expressing MN1 had an advantage when exposed to either Ara-C or doxorubicin although the effect was far more pronounced with doxorubicin exposure. MN1 expressing blasts were also resistant to the combination of Ara-C and doxorubicin. In order to determine if MN1 conferred resistance to Ara-C and doxorubicin in vivo we injected sublethally irradiated, Ly5.1+ C57Bl6 recipients with a partially infected population of blasts. Ly5.1+ animals do not express the Ly5.2 allele; thus, staining cells for Ly5.2 allows differentiation of leukemic cells from endogenous marrow. Eight days after injection of blasts animals were treated with 100 mg/kg Ara-C plus 3 mg/kg doxorubicin daily for 5 days or observed. On day 6 animals were sacrificed and bone marrow from bilateral femurs was harvested, stained for Ly5.2 and analyzed by flow cytometery. Animals treated with Ara-C plus doxorubicin had 90.58% (+/−0.6638) Ly5.2+, GFP+ blasts compared to 55.38% (+/−5.245) in control animals. This result was highly statistically significant with a p value of <0.0001 by two tailed student's t test. This observation was reproducible in a separately derived MLL-ENL driven cell line. These data suggest that over expression of MN1 in this murine AML model confers resistance to both Ara-C and doxorubicin in vitro and in vivo and provides a biological explanation for the clinical observation that it confers a worse prognosis. The mechanisms involved in this resistance are currently under study. Disclosures: No relevant conflicts of interest to declare.

Immunosurgical studies on Inner cell mass development in rat and mouse blastocysts before and during implantation in vitro

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 255-269
Author(s):  
Horst Spielmann ◽  
Ursula Jacob-Müller ◽  
Werner Beckord
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Cell Number ◽  
C57bl Mouse ◽  
Before And After ◽  
Rabbit Complement

Eighty per cent of rat blastocysts (Wistar, SW72) cultured for 96 h in NCTC-109 supplemented with fetal calf serum (FCS) hatched from the zona pellucida and developed a trophoblast giant cell layer. Thirty seven per cent of the rat blastocysts developed an inner cell mass (ICM) which, in about 7 %, consisted of two germ layers (ectoderm and endoderm), compared to 84% in NMRI mice. A significantly better ICM development was obtained with cultured rat blastocysts that had hatched in vivo. Similar to the in vivo situation LDH-5 was present in rat blastocysts after implantation in NCTC-109-FCS. Differentiation of C57BL mouse blastocysts in NCTC-109-FCS proceeded as poorly as in the rat. ICM development of rat and mouse blastocysts in NCTC-109-FCS was studied in detail. ICMs of the two species were isolated immunosurgically using complement from different species, e.g. human, rat and rabbit complement, since guinea-pig complement did not lyse trophectoderm cells of rat blastocysts. All immunosurgically isolated rat ICMs degenerated within 48 h, but mouse ICMs isolated with rat or rabbit complement developed significantly better than mouse ICMs isolated with guinea-pig complement. Determinations of theblastocyst total cell number (BTCN) and of the cell number of immunosurgically isolated ICMs were performed in rat and mouse blastocysts to investigate growth kinetics of the ICM before implantation in vitro. In the mouse an exponential increase in both BTCN and cell number of the ICM was observed during the 48 h before implantation in NCTC-109-FCS and also during the 16-24 h before implantation in vivo. In the rat, doubling of the BTCN was found only during the first 24 h in NCTC-109-FCS and there was hardly any increase in the cell number of the ICM during the first 48 h in culture. ICM growth of blastocysts in NCTC-109-FCS is, therefore, stimulated in the mouse before and after implantation and. in the rat it is inhibited already before implantation.

101 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 159 ◽  
Author(s):  
R. Li ◽  
L. Lai ◽  
D. Wax ◽  
Y. Hao ◽  
Z. Zhong ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Nuclear Transfer ◽  
Lipid Droplets ◽  
Harmful Effect ◽  
Calf Serum ◽  
Fibroblast Cell ◽  
Cell Number ◽  
Freezing And Thawing

In the production of cloned pigs, a large number of nuclear transfer (NT) embryos generally need to be transferred into a single surrogate. Thus, attempts to conduct embryo transfer can be frustrating when either a synchronized surrogate is not available, or enough NT embryos are not produced. This problem would be solved if one could cryopreserve the porcine nuclear transfer embryos. Cryopreservation of porcine embryos has been successful only for in vivo-derived embryos. In vitro-derived porcine embryos are sensitive to chilling, and this sensitivity has been attributed to the lipid droplets in the cytoplasm. In previous reports, the viability of cyropreserved embryos was improved by removal of lipid drops from the cytoplasm. Therefore we designed a procedure to cryopreserve cloned blastocysts by a combination of the open pulled straw (OPS) vitrification method with removal of lipid drops from the oocyte. In vitro-matured MII oocytes were enucleated, and centrifuged (10 000 rpm, 5 min) to polarize the lipid droplets. This was followed by removal of the polarized lipid droplets and transfer of a donor fetal-derived fibroblast cell into the perivitelline space by micromanipulation. After electrical activation and fusion, the NT embryos were cultured in PZM-3 medium with 4 mg/mL BSA. Day 5 and Day 6 blastocysts (manipulation day was Day 0) were vitrified by equilibration with 25 mM HEPES-buffered TCM-199 containing 10% ethylene glycol, 10% DMSO, and 20% fetal calf serum for 2 min, followed by exposure to 20% ethylene glycol and 20% DMSO. Embryos were loaded into an OPS straw and immediately plunged into liquid nitrogen. The process from exposure of embryos to vitrification solution to plunging was 25–30 s. Embryos were thawed by immersing the end of the OPS straw in 0.3 M sucrose in which embryos were kept for 5 min, and then in 0.2 M sucrose for 5 min. Some embryos were cultured in PZM-3 for 12 h to determine the percentage and cell number of re-expanded blastocysts. The others were transferred to the uterus of a surrogate gilt within 3 h of thawing. Lipid removal appeared to have no harmful effect on embryo development and cell number of the blastocysts. Interestingly, a higher blastocyst percentage (28.8%, 178/619) was obtained with NT embryos from which the lipid had been removed as compared to normal NT (19.6%, 44/225; P < 0.01). The cell number (31.2 ± 7.7) of re-expanded blastocysts in the delipation group was comparable with normal NT blastocysts (33.6 ± 14.1, P = 0.33). The survival rate of blastocysts after freezing and thawing was enhanced after delipation (delipation group: 66.7%, 14/21; normal NT group: 21.9%, 9/42; P < 0.01). Two hundred and fourteen delipatized NT blastocysts were transferred to four surrogates after freezing and thawing. Three of the surrogates showed a delayed estrus cycle and one is still pregnant as confirmed by ultrasound scanning. We show that the combination of the OPS vitrification method with removal of lipid drops of oocyte cytoplasm might be an efficient way to cryopreserve porcine NT blastocysts. Funding for this project was from the NIH HL51670 and RR018877 and Food for the 21st Century.

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