403. Differential effect of hexoses on sperm metabolism and function in culture

2008 ◽  
Vol 20 (9) ◽  
pp. 83
Author(s):  
H. W. Bakos ◽  
M. Lane
Keyword(s):  
Lipid Peroxidation ◽  
Dna Damage ◽  
Carbohydrate Metabolism ◽  
Culture Media ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Function ◽  
Ros Production ◽  
Sperm Dna Damage ◽  
The Media

Currently there is lack information regarding how human spermatozoa regulate their energy metabolism. This is surprising considering that carbohydrate metabolism is a vital point for the understanding of sperm function. This coupled with the increased use of assisted reproductive technology and the importance of a well balanced culture media has led us to hypothesise that an imbalance of carbohydrate presence in the media may alter sperm function, particularly in relation to oxidative stress, DNA damage and lipid peroxidation. Sperm samples were obtained from three healthy normospermic donors for this study. Motile sperm were separated from semen samples using density gradient separation. Samples were incubated at different media conditions with varying glucose or fructose concentrations (0, 2.5, 25 mM) for 6–24hrs. Reactive oxygen species (ROS) were measured using 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (DCFDA). Sperm DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). Lipid peroxidation was assessed using the probe BODIPY (581/591) C11. Carbohydrate uptake from the media was measured using a fluorometric procedure. Statistical differences between treatments were assessed by ANOVA and Bonferroni post-hoc test. No significant motility differences were found following treatments. Results showed an increased level of ROS production as glucose concentration increased (P < 0.05). This was accompanied by an increased number of TUNEL positive cells (P < 0.05). Furthermore, lipid peroxidation of spermatozoa was significantly increased when incubated under high glucose concentrations (P < 0.01). In contrast, increases in fructose concentrations did not alter ROS levels or the number of TUNEL positive cells. Sperm metabolised both glucose and fructose in vitro and the removal of one carbohydrate resulted in a compensatory increase in the metabolism of the other. To our knowledge, this is the first report providing evidence that altered carbohydrate metabolism may induce ROS production, lipid peroxidation and increase the number of sperm exhibiting DNA damage.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 ◽  
Author(s):  
Gabriela de Oliveira Fernandes ◽  
Marcella Pecora Milazzotto ◽  
Andrei Antonioni Guedes Fidelis ◽  
Taynan Stonoga Kawamoto ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Markers ◽  
Culture Medium ◽  
Culture Media ◽  
Ionization Mass ◽  
Metabolic Profiles ◽  
Bovine Embryos ◽  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

scholarly journals Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oils from AgNPs and AuNPs Elicited Lavandula angustifolia In Vitro Cultures

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 606 ◽  
Author(s):  
Aneta Wesołowska ◽  
Paula Jadczak ◽  
Danuta Kulpa ◽  
Włodzimierz Przewodowski
Keyword(s):  
Molecular Weight ◽  
Silver Nanoparticles ◽  
Essential Oils ◽  
Culture Media ◽  
In Vitro Cultures ◽  
Gas Chromatography Mass Spectrometry ◽  
Lavandula Angustifolia ◽  
Gold And Silver Nanoparticles ◽  
The Media

The aim of this study was to determine how the addition of gold and silver nanoparticles to culture media affects the composition of essential oils extracted from Lavandula angustifolia propagated on MS media with the addition of 10 and 50 mg·dm−3 of gold (24.2 ± 2.4 nm) and silver (27.5 ± 4.8 nm) nanocolloids. The oil extracted from the lavender tissues propagated on the medium with 10 mg·dm−3 AgNPs (silver nanoparticles) differed the most with respect to the control; oil-10 compounds were not found at all, and 13 others were detected which were not present in the control oil. The addition of AuNPs (gold nanoparticles) and AgNPs to the media resulted in a decrease of lower molecular weight compounds (e.g., α- and β-pinene, camphene, δ-3-carene, p-cymene, 1,8-cineole, trans-pinocarveol, camphoriborneol), which were replaced by those of a higher molecular weight (τ- and α-cadinol 9-cedranone, cadalene, α-bisabolol, cis-14-nor-muurol-5-en-4-one, (E,E)-farnesol).

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Culture Media ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Ros Production ◽  
Oxygen Species

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P &lt; 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P &lt; 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

99 DIFFERENTIAL GENE REGULATION OF STEROIDOGENIC TRANSCRIPTS AND ESTRADIOL PRODUCTION FOLLOWING IN VITRO PIG EMBRYO ELONGATION IN ALGINATE HYDROGEL THREE-DIMENSIONAL MATRIX

2012 ◽  
Vol 24 (1) ◽  
pp. 162
Author(s):  
J. R. Miles ◽  
C. N. Sargus ◽  
S. A. Plautz ◽  
J. L. Vallet ◽  
A. K. Pannier
Keyword(s):  
Equal Opportunity ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Differential Gene Regulation ◽  
Alginate Hydrogels ◽  
The Media ◽  
And Control

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production. †USDA is an equal opportunity provider and employer.

Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation

2019 ◽  
Vol 26 (12) ◽  
pp. 1575-1581 ◽  
Author(s):  
Senay Cankut ◽  
Turgay Dinc ◽  
Mehmet Cincik ◽  
Guler Ozturk ◽  
Belgin Selam
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Tunel Assay ◽  
University Hospital ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Dna Fragmentation ◽  
Liquid Form ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Before And After

Aim: Human sperm DNA fragmentation is one of the factors suggested for male infertility. The ratio of sperm DNA damage in semen may adversely affect both the fertilization rate and the embryo development of in vitro fertilization/ intracytoplasmic sperm injection cycles. Sperm cryopreservation both increases the success rates in assisted reproductive techniques (ARTs) and contributes to the preservation of fertility before testis surgery, chemotherapy, and radiotherapy. The aim of the current study is to determine sperm DNA fragmentation, following cryopreservation. Methods: A cross-sectional, observational study was conducted at a university hospital infertility clinic. One hundred (n = 100) volunteer fertile men (ages between 21 and 39 years) with normozoospermic sperm parameters were involved in the current study. Sperm DNA damage was evaluated with the Halosperm technique and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fresh samples were studied in liquid form. The remaining samples were kept frozen and then thawed after 1 month and reevaluated with the Halosperm technique and TUNEL assay. Results were then compared between the fresh and frozen samples. Results: Sperm DNA fragmentation results with the Halosperm technique both before and after cryopreservation were 25% (5%-65%) and 40% (6%-89%), respectively, with a statistically significant increase (15%; P < .001). Sperm DNA fragmentation results by TUNEL assay before and after cryopreservation were 17% (3%-43%) and 36% (7%-94%), respectively, with a statistically significant increase (19%; P <.001). Conclusion: The current data demonstrate increased sperm DNA damage after cryopreservation. Further studies may contribute to development of less harmful techniques and cryoprotectants in order to improve the results of ART.

scholarly journals Lycopene Modulates THP1 and Caco2 Cells Inflammatory State through Transcriptional and Nontranscriptional Processes

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Njock Makon-Sébastien ◽  
Fouchier Francis ◽  
Seree Eric ◽  
Villard Pierre Henri ◽  
Landrier Jean François ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Culture Media ◽  
Cell Types ◽  
Ros Production ◽  
M Cell ◽  
Low Concentration ◽  
Inflammatory State ◽  
Proinflammatory Gene ◽  
Inflammatory Effect

We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS) production, and metalloprotease (MMP9) activity. THP1 and Caco2 cell lines were used asin vitromodels for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA), lipopolysaccharide (LPS) or tumor necrosis factor (TNF). In THP1 cells, short term pretreatment (2 h) with a low concentration (2 μM) of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF) used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h) with lycopene. At low concentration (2 μM or less), lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM–20 μM), an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals.

Effects of parathyroid hormone on bone acid hydrolases in tissue culture

1968 ◽  
Vol 46 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Susan Tolnai
Keyword(s):  
Parathyroid Hormone ◽  
Acid Phosphatase ◽  
Bone Cells ◽  
Culture Media ◽  
Enzyme Synthesis ◽  
Maximum Activity ◽  
Acid Protease ◽  
Acid Hydrolases ◽  
The Media

Experiments were carried out to assess the activity of acid hydrolases of bone cells under in vitro conditions. Rat and mouse skull bones were kept in vitro for 2 to 6 days in the presence and absence of added parathyroid hormone. The activities of acid protease (cathepsin D), acid phosphatase, and β-glucuronidase were measured in the culture media and in homogenates prepared from the cultured bones. All three enzymes showed increased activity in various degrees in the media when parathyroid hormone (PTE) was present, but increased enzyme synthesis in the homogenates could be detected only in the case of acid protease and acid phosphatase. The acid protease showed maximum activity at pH 3.0–3.2, and the PTE-stimulated increase was time-dependent. Lysozyme incorporated into the culture medium stimulated acid protease activity, while histone had an opposite effect. Cultures kept in incomplete media for a limited period of time also responded to PTE treatment with an increased release of acid hydrolases into the medium.

scholarly journals Sperm DNA damage has a negative effect on early embryonic development following in vitro fertilization

2018 ◽  
Vol 20 (1) ◽  
pp. 75 ◽  
Author(s):  
Wei-Wei Zheng ◽  
Ge Song ◽  
Qi-Ling Wang ◽  
Shan-Wen Liu ◽  
Xiao-Li Zhu ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Early Embryonic Development ◽  
Vitro Fertilization ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Negative Effect
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