scholarly journals Transcriptional, Mitochondrial Activity, and Viability of Egyptian Buffalo’s Granulosa Cells In Vitro Cultured under Heat Elevation

2021 ◽  
Vol 11 (2) ◽  
pp. 193-201
Author(s):  
Nasser Ghanem ◽  
Marwa Said Faheem ◽  
Romysa Samy ◽  
Ashraf Hesham Barkawi
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Transcriptional Activity ◽  
The Other ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Fluorescent Intensity ◽  
Stressed Group ◽  
Other Hand

It is documented that heat stress caused impairment on the reproductive performance of dairy animals. However, there are few reports that have focused on the molecular and intracellular responses of in vitro cultured buffalo granulosa cells during heat elevation. The present study was conducted to investigate the effect of heat elevation during in vitro culture of buffalo granulosa cells on their viability, quality, mitochondrial activity, and transcriptional activity. Granulosa cells were harvested after aspiration of cumulus-oocytes complexes that were collected from abattoir ovaries. The granulosa cells were cultured in vitro either at a normal physiological temperature suitable for oocyte maturation and embryo development (38.5°C) or exposed to the elevated temperature of 40.5°C on day 3 of culture (the first two days were for confluence) for two hours of culture then continued at 38.5°C up to day 7 of culture. The viability of granulosa cells was measured using trypan blue and quality was estimated by measuring the level of intracellular reactive oxygen species (ROS) on day 7. Moreover, metabolic activity was performed by measuring the fluorescent intensity of mitochondria. Moreover, transcriptional activity was done by profiling four selected candidate genes using quantitative real-time PCR. The results indicated that the granulosa cells viability rate significantly decreased in the heat stress group (25.1 ± 3.7), compared to the control group (36.6 ± 5.3) on confluence day (day 3). In addition, the viability rate on the last day of culture (day 7) decreased in heat stress, compared to control (83.7 ± 4.5 and 97.4 ± 0.4, respectively). On the other hand, there was a nonsignificant difference in ROS profile between the control (21.7*104 ± 1.3) and the heat-stressed group (15.7 ± 0.7) on day 7 of culture. However, the mitochondrial fluorescent intensity was higher in the control (21.9 ± 1.9) than in the heat-stressed group (15.4 ± 0.8) on day 7 of culture. The expression of cellular defense (HSF1) and apoptosis-inducing gene (P53) were significantly up-regulated in granulosa cells exposed to heat elevation, compared to the control group. On the other hand, the steroidogenesis-regulating gene (StAR) was down-regulated in granulosa cells cultured under heat shock, compared to the control group. In conclusion, heat stress reduced the viability of granulosa cells by inducing the expression of an apoptosis-related gene (P53) and compromised expression of genes regulating the steroid biosynthesis, which resulted in up-regulation of cell defense gene (HSF1) in an attempt to ameliorate the deleterious effect of heat stress on the biological activity of the granulosa cells.

404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
T. S. Kim ◽  
Y. Cao ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
C. K. Park
Keyword(s):  
Gene Transfer ◽  
Transfection Efficiency ◽  
The Other ◽  
Control Group ◽  
Dna Uptake ◽  
Alkaline Lysis ◽  
Exogenous Dna ◽  
Other Hand ◽  
Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

Cellular and Transcriptional Adaptation of Bovine Granulosa Cells Under Ethanol-Induced Stress In Vitro

2020 ◽  
Author(s):  
Md Mahmodul Hasan Sohel ◽  
Mostafa Abdulkareem Salman ◽  
Abdurrahman Ayvaz
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Ethanol Exposure ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Antioxidant Genes ◽  
Nrf2 Pathway ◽  
Transcriptional Adaptation ◽  
Time Point

Abstract Aims Granulosa cells (GCs) are the major cellular component in a follicular microenvironment and play an indispensable role in ovarian function. This study was conducted to investigate the effects of ethanol exposure on the cellular and transcriptional changes of ovarian GCs. Methods For this purpose, bovine GCs were exposed to different concentrations of ethanol (0, 50, 100, 200, 500 and 1000) to mimic the effects of alcohol in in vitro. Subsequently, 100 and 1000 mM concentrations were discarded from further experiments, as 100 mM was not different from 50 mM, and 1000 mM was supertoxic to the cells. Results The results showed that there was a gradual loss of cell viability with the increase of the ethanol concentration, i.e. lowest viability was observed at the highest concentration (1000 mM), which is further supported by cell proliferation assay. Mitochondrial activity decreased significantly at higher concentrations. The expression of NRF2 decreased significantly (P < 0.05) in ethanol-exposed cells compared with the cells in the control group at the 6-h time point, whereas the expression was increased in 500 mM concentration at the 24-h time point. The expression of antioxidant genes, downstream to Nrf2-pathway activation, showed that overall expression pattern similar to NRF2. Conclusion The result of this study prompted us to postulate that ethanol exposure decreases the ability of GCs to handle stress by downregulating the expression of genes involved in Nrf2-pathway.

scholarly journals Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress Under In Vitro Condition

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 794
Author(s):  
Marwa S. Faheem ◽  
Nasser Ghanem ◽  
Ahmed Gad ◽  
Radek Procházka ◽  
Sherif M. Dessouki
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Molecular Mechanisms ◽  
Control Group ◽  
Adaptive Responses ◽  
Biological Responses ◽  
Heat Treated ◽  
Integration Mechanism ◽  
Total Antioxidant

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

scholarly journals Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium

2021 ◽  
Vol 8 ◽  
Author(s):  
Martha Valdivia ◽  
Zezé Bravo ◽  
Jhakelin Reyes ◽  
Gustavo Gonzales
Keyword(s):  
Cell Viability ◽  
Antioxidant Properties ◽  
Testicular Tissue ◽  
The Other ◽  
Mitochondrial Activity ◽  
Isolated Cells ◽  
Male Adult ◽  
Testicular Cells ◽  
Other Hand

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

295 RELATIONSHIP BETWEEN PLASMINOGEN ACTIVATOR/PLASMIN SYSTEM AND IN VITRO FERTILIZATION ABILITY IN THE PIG

2006 ◽  
Vol 18 (2) ◽  
pp. 255
Author(s):  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
C.-K. Park
Keyword(s):  
Plasminogen Activator ◽  
Formation Rate ◽  
Sodium Dodecyl ◽  
The Other ◽  
Tissue Type ◽  
Bromophenol Blue ◽  
Control Group ◽  
Fertilization In Vitro ◽  
Other Hand

Plasminogen activators (PAs) are serine proteases, known to be secreted by a large number of cell types, that convert plasminogen into plasmin. The present study was undertaken to identify PAs in porcine gametes and to investigate a possible role of plasminogen in fertilization in vitro in the pig. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG). To determine the changes of PA activities in porcine oocytes during maturation, the cumulus-oocyte complexes (COCs) were incubated in NCSU-33 in an atmosphere of 5% CO2 in air at 39�C for 0, 24, or 48 h. On the other hand, to investigate the release of PAs by boar spermatozoa, fresh spermatozoa were pre-incubated in fertilization medium (mTBM) for 0, 2, 4, or 6 h. After culture, 40 COCs, 40 cumulus-free oocytes, and 40 � 106 spermatozoa were separately put into microtubes containing 20 �L of sample buffer [5.0% (w:v) sodium dodecyl sulfate (SDS), 20% (v:v) glycerol, and 0.0025% (w:v) bromophenol blue in 0.125 M Tris-HCl buffer] and frozen at -80�C until used for analysis. PA activities in porcine oocytes and spermatozoa were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). In the COCs cultured for 24-18 h, tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed. Also, PA activities increased as duration of culture increased. However, no uPA activity was detected in cumulus-free oocytes. In procine fresh spermatozoa, tPA, uPA, and tPA-PAI were observed. When spermatozoa were incubated for 2, 4, or 6 h in fertilization medium, the rate of acrosome reaction (AR) in spermatozoa increased as the duration of culture increased, but PA activities decreased gradually. However, PA activities in sperm-conditioned medium increased as duration of culture increased. On the other hand, to determine the effect of plasminogen on fertilization ability of porcine oocyte and spermatozoa, plasminogen (50 �g/mL) was added in fertilization medium. Addition of plasminogen to fertilization medium increased (P < 0.05) AR in spermatozoa and sperm binding to the zona pellucida (ZP), compared with control group. The ZP solubility (zona digestion time) was higher in medium with than that without plasminogen. When porcine oocytes and spermatozoa were co-incubated in fertilization medium with plasminogen, the polyspermic rate was lower in medium with than that without plasminogen. Also, plasminogen significantly (P < 0.05) increased formation rate of the male pronucleus in oocytes penetrated by spermatozoa. These results suggest that supplementing of plasminogen in fertilization medium may play a positive role in improving of fertilization ability in vitro in the pig.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

scholarly journals Fetal erythropoiesis in steel mutant mice. III. Defect in differentiation from BFU-E to CFU-E during early development

Blood ◽  
1978 ◽  
Vol 51 (3) ◽  
pp. 539-547 ◽  
Author(s):  
DH Chui ◽  
SK Liao ◽  
K Walker
Keyword(s):  
Progenitor Cells ◽  
Early Development ◽  
The Other ◽  
Mutant Mice ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Other Hand ◽  
Colony Forming Units ◽  
Fetal Erythropoiesis

Abstract Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

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