94 NUCLEAR-CYTOPLASMIC INTERACTION AND DEVELOPMENT OF CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 147
Author(s):  
H. J. Song ◽  
E. J. Kang ◽  
B. G. Jeon ◽  
S. L. Lee ◽  
H. H. Seong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Neuronal Cell ◽  
Total Cell ◽  
Control Group ◽  
Mrna Levels ◽  
Canine Genome ◽  
Osteogenic Cell ◽  
Fetal Fibroblasts ◽  
Cytoplasmic Interaction

Although cloned canine offspring have been produced, the efficiency of nuclear transfer (NT) is still far from expectations. The most important factor interfering with the success of NT is the shortage of canine matured oocytes. In the present study, the nuclear-cytoplasmic interaction and in vitro development of porcine oocytes cloned with canine fetal fibroblasts (cFF) and canine mesenchymal stem cells (cMSC) were assessed by examining the developmental rate, embryonic morphology, total cell number, canine genome expression, and in vivo development following the transfer of iNT embryos into the oviducts of surrogate mother. Isolated cMSC from bone marrow were characterized by early transcription factors (Oct3/4, Sox2, and Nanog) and by differentiation into adipogenic, osteogenic, and neuronal cells under controlled in vitro conditions. Porcine cumulus–oocyte complexes (COC) were matured in vitro as described by Kim YS et al. (2005 Mol. Reprod. Dev. 70, 308–313). Porcine NT embryos were used for the control group. Canine MSC showed their characteristic property of attaching to plastic culture flasks and forming a monolayer of spindle-shaped cells. Adherent cells showed moderate expression of Oct3/4, Sox2, and Nanog protein by immunostaining. Following osteogenic cell induction, cMSC transformed into mineralized nodules by von Kossa staining. Oil red O staining revealed that MSC produced lipid droplets after incubation in adipogenic media. Neuronal-like cells with multipolar, round cell bodies organized into a network-like structure were confirmed by immunostaining of neuronal markers (NFM). The tissue-specific mRNA levels were confirmed after differentiation by RT-PCR (osteogenic cell, Osteocalcin; adipogenic cell, PPAR γ; neuronal cell, NFM). The control group blastocysts (porcine oocytes-pMSC) exhibited significantly (P < 0.05) greater development and total cell numbers of than blastocysts of interspecies groups (porcine oocytes-cFF and cMSC). The canine genome GAPDH was detected in cFF, cMSC, and interspecies NT embryos using PCR, whereas there was no detection in porcine oocytes and the negative control. Most of the embryos arrested at the 8- to 16-cell stage and only 5% of embryos developed to blastocyst stage. The interspecies-NT embryos (average 35 embryos/recipient dog) were surgically transferred into the oviducts of 3 recipient dogs. Two of them showed exterior signs of implantation; i.e. a slight thickening of the uterus and small pea-sized lumps. No pregnancies were detected in the recipients after ovariohysterectomy surgery. These results demonstrate the potential of cMSC isolated from bone marrow to differentiate into mesoderm (osteogenic and adipogenic cell) and ectoderm (neuronal cell)-like cells under specific induction conditions. Although interspecies NT embryos could not implant in the canine uterine wall, the results provided valuable information on canine MSC and interspecies NT. This work was supported by Grant No. 20070301034040 from Bio-Organ, Republic of Korea.

YAP1 Overexpression Is Associated with Kidney Dysfunction in Lupus Nephritis

Pathobiology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Ying Xie ◽  
Yuanyuan Ruan ◽  
Huimei Zou ◽  
Yixin Wang ◽  
Xin Wu ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Epithelial Mesenchymal Transition ◽  
Kidney Tissue ◽  
Mouse Kidney ◽  
Experimental Comparison ◽  
Control Group ◽  
Mrna Levels ◽  
Mesenchymal Transition ◽  
Protein Levels

<b><i>Objective:</i></b> The goal of the present study was to determine the expression of yes-associated protein 1 (YAP1) in renal tissues of mice with lupus nephritis (LN) and elucidate its role in the progression of renal fibrosis. <b><i>Methods:</i></b> C57BL/6 mice and MRL/lpr mice were selected for experimental comparison. Mouse kidney tissues were removed and sectioned for hematoxylin and eosin staining, Masson’s trichome staining, Sirius staining, and immunohistochemistry. The mRNA and protein levels of YAP1 in mouse kidney tissues were detected, and the correlation between YAP1 and fibronectin (FN) mRNA levels was analyzed. Mouse renal epithelial cells were used for in vitro experiments. After transfection and stimulation, the cells were divided into 4 groups, namely the C57BL/6 serum group (group 1), the MRL/lpr serum group (group 2), the MRL/lpr serum + siRNA-negative control group (group 3), and the MRL/lpr serum + siRNA-YAP1 group (group 4). Epithelial-mesenchymal transition (EMT) markers in each group were detected by Western blotting and immunofluorescence staining. Serum creatinine, blood urea nitrogen, and urinary protein levels were detected and assessed for their correlation with YAP1 mRNA levels by Spearman’s analysis. <b><i>Results:</i></b> Compared to C57BL/6 mice, MRL/lpr mice exhibited obvious changes in fibrosis in renal tissues. In addition, YAP1 expression was significantly higher in the renal tissues of MRL/lpr mice than in those of C57BL/6 mice, and YAP1 mRNA levels were positively correlated with those of FN. YAP1 silencing in lupus serum-stimulated cells could effectively relieve serum-induced EMT. Finally, we observed that YAP1 mRNA levels in mouse kidney tissue were significantly and positively correlated with the degree of renal function injury. <b><i>Conclusion:</i></b> YAP1 expression in the kidney tissues of LN mice was higher than that observed in normal mice, indicating that YAP1 may play an important role in the occurrence and development of LN.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

scholarly journals A Prospective Controlled Trial to Evaluate Safety and Efficacy of in vitro Expanded Recipient Regulatory T Cell Therapy and Tocilizumab Together With Donor Bone Marrow Infusion in HLA-Mismatched Living Donor Kidney Transplant Recipients (Trex001)

2021 ◽  
Vol 7 ◽  
Author(s):  
Rainer Oberbauer ◽  
Matthias Edinger ◽  
Gabriela Berlakovich ◽  
Peter Kalhs ◽  
Nina Worel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Kidney Transplant ◽  
Graft Function ◽  
Control Group ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Post Transplant ◽  
Donor Bone Marrow ◽  
Living Donor Kidney Transplant

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR &lt;35 mL/min/1.73 m2], graft-vs.-host disease or patient death by 12 months) and efficacy (total leukocyte donor chimerism within 28 days post-transplant) are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

scholarly journals Cytogenetic studies and in vitro colony growth in patients with mastocytosis

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1928-1932 ◽  
Author(s):  
B Swolin ◽  
S Rodjer ◽  
G Roupe
Keyword(s):  
Bone Marrow ◽  
Growth Pattern ◽  
Bone Marrow Cells ◽  
Systemic Mastocytosis ◽  
Colony Growth ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Normal Peripheral Blood ◽  
Abnormal Growth

Abstract Cytogenetic analysis of bone marrow cells and in vitro growth for bone marrow granulocytic-macrophage stem cells have been performed in 13 patients with mastocytosis, six with systemic mastocytosis, and seven with urticaria pigmentosa. Clones with chromosome abnormalities were found in five patients. The number of clusters and/or colonies after seven days in culture was increased in seven patients, compared with the growth in a control group. Three patients with chromosome abnormalities showed an abnormal growth pattern, yet exhibited normal peripheral blood values. Two patients with systemic mastocytosis had clones with chromosome abnormalities and some abnormal hematological values. The proportion of patients with chromosome abnormalities and an abnormal growth pattern was higher among these patients with mastocytosis than in healthy control subjects. These results may be of interest when discussing the origin of mast cell disorders and indicate an association with the myeloproliferative disorders.

41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

2014 ◽  
Vol 26 (1) ◽  
pp. 134
Author(s):  
L. N. Moro ◽  
M. I. Hiriart ◽  
J. Jarazo ◽  
C. Buemo ◽  
A. Sestelo ◽  
...  
Keyword(s):  
Species Conservation ◽  
Total Cell ◽  
Domestic Cat ◽  
Control Group ◽  
Felis Silvestris ◽  
Blastocyst Formation ◽  
Acinonyx Jubatus ◽  
Cell Numbers ◽  
Number Of Cells

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

2009 ◽  
Vol 21 (1) ◽  
pp. 148
Author(s):  
D. N. Q. Thanh ◽  
K. Matsukawa ◽  
M. Kaneda ◽  
S. Akagi ◽  
Y. Kanai ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Monozygotic Twins ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
First Polar Body ◽  
Blastocyst Quality ◽  
Single Blastomeres

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P < 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
H. T. Lee ◽  
J. M. Jang ◽  
S. H. Lee ◽  
M. K. Gupta
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Cell Number ◽  
Control Group ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle&apos;s non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 &plusmn; 0.3 vs. 27.5 &plusmn; 0.3&percnt;, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 &plusmn; 0.9 vs. 33.4 &plusmn; 0.3&percnt;, respectively). In the SCNT group, however, both cleavage (73.6 &plusmn; 0.2 vs. 64.2 &plusmn; 0.4&percnt;) and blastocyst rate (18.7 &plusmn; 0.2 vs. 13.8 &plusmn; 0.3&percnt;) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 &plusmn; 1.8 vs. 14.6 &plusmn; 4.9&percnt;) than those of control group (P &lt; 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 &plusmn; 4.9 vs. 66.5 &plusmn; 3.3) as well as SCNT-derived (43.1 &plusmn; 2.6 vs. 31.8 &plusmn; 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

182 In Vitro Derivation of Male Germ Cells from Bovine Bone Marrow-Derived Mesenchymal Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
J. Cortez ◽  
J. Bahamonde ◽  
J. Palomino ◽  
M. De los Reyes ◽  
C. Torres ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Mrna Expression ◽  
Male Germ Cell ◽  
Bovine Bone ◽  
Mrna Levels ◽  
Germ Cell Differentiation

During the last few years, the in vitro derivation of germ cell lineages from stem cells has emerged as an exciting new strategy for obtaining mature gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Germ cell differentiation and gametogenesis is a complex process and potential of different stem cell donors (i.e. SSC, ESC, iPSC) for in vitro male germ cell derivation has been inconsistent. Mesenchymal stem cells (MSC) may be suitable candidates for in vitro gamete derivation considering their (1) plasticity that is not limited to mesodermal derivatives, (2) availability of abundant tissues sources for isolation, (3) high proliferative potential, (4) simple and inexpensive isolation, and (5) high potential for cell therapy, including autologous or allogenic transplantation. The present study aimed to induce differentiation of MSC isolated from bone marrow derived from bovine male fetuses (bfMSC) into the germ cell lineage using an in vitro approach based on the exogenous effect of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4). Differentiation media consisted in control media (DMEM with high glucose plus 10% fetal bovine serum, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1amphotericin B) supplemented with RA (0.01, 0.1, or 1 µM) or BMP4 (10, 50, or 100 ng mL−1). Cell samples were obtained from differentiating and control bfMSC cultures and analysed for expression of housekeeping genes β-ACTIN and GAPDH, pluripotent genes OCT4 and NANOG, germ cell genes FRAGILLIS, STELLA, and VASA, male germ cell genes DAZL, PIWIl2, and STRA8, and meiotic biomarker SCP3 by quantitative-PCR (Q-PCR). OCT4, NANOG, and DAZL were immunodetected in undifferentiated and differentiated bfMSC using flow-cytometry analysis. The mRNA expression of DAZL was activated by RA or BMP4 supplementation, although no differences (P > 0.05) were detected among different concentrations. DAZL and NANOG mRNA levels increased (P < 0.05) from Day 7 to Day 21 during supplementation of RA (0.1 μM). In comparison, DAZL mRNA levels increased (P < 0.05) at Day 14 during supplementation of BMP4 (100 ng). OCT4 and SCP3 mRNA levels were not affected by RA or BMP4 treatments. Transcripts of FRAGILLIS, STELLA, VASA, PIWIl2, and STRA8 were not detected in control or differentiated bfMSC. Higher (P < 0.05) percentages of undifferentiated bfMSC were positive for NANOG (80.6%) and OCT4 (83.4%). DAZL- and NANOG-positive cells were 2.1% and 2.9%, and 95.9% and 97.8% at Days 0 and 21 of RA treatment, respectively. Data indicated that expression of germ cell biomarker DAZL in bfMSC is activated and increased after in vitro supplementation of RA and BMP4. Moreover, NANOG mRNA levels were regulated by RA treatment. Similar levels of SCP3 mRNA expression suggest that differentiated bfMSC were not induced into meiosis. Thus, exposure of bfMSC to RA or BMP4 under in vitro conditions might induce an early stage of premeiotic germinal differentiation.

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