Carryover effect of atrazine and its metabolite—from treated bovine spermatozoa to the embryo’s transcriptome†

2021 ◽  
Author(s):  
A Komsky-Elbaz ◽  
D Kalo ◽  
Z Roth
Keyword(s):  
Dna Fragmentation ◽  
Annexin V ◽  
Dna Integrity ◽  
Blastocyst Formation ◽  
Transcriptome Profile ◽  
Carryover Effect ◽  
Vitro Fertilization ◽  
Cellular Processes ◽  
Bovine Spermatozoa

Abstract Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV−) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV−) and DACT (AV+, AV−) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV− vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.

scholarly journals Effect of aflatoxin B1 on bovine spermatozoa’s proteome and embryo’s transcriptome

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 709-723
Author(s):  
Alisa Komsky-Elbaz ◽  
Dorit Kalo ◽  
Zvi Roth
Keyword(s):  
Differential Expression ◽  
Dna Fragmentation ◽  
Aflatoxin B1 ◽  
Deleterious Effect ◽  
Annexin V ◽  
Dna Integrity ◽  
Biological Processes ◽  
Cleavage Rate ◽  
Bovine Spermatozoa ◽  
Cellular Pathways

This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV−) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV− spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa’s DNA integrity, additional damage mechanisms are involved.

219 SORTING OF EQUINE SPERM USING A MICROFLUIDIC DEVICE AS A METHOD OF SPERM SELECTION FOR IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 241 ◽  
Author(s):  
R. Gonzalez ◽  
E. Carnevale
Keyword(s):  
Dna Fragmentation ◽  
Microfluidic Device ◽  
Membrane Integrity ◽  
Sperm Parameters ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Normal Morphology ◽  
Vitro Fertilization ◽  
Sperm Sorting

Microfluidic technology can be used for sperm separation. Microfluidic devices generate a fluid flow to sort sperm from a media reservoir into a collection chamber. In the human and mouse, the use of microfluidic devices resulted in the selection of sperm with improved sperm motility, normal morphology, and DNA integrity for in vitro fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI). With the use of microfluidic sperm separation, centrifugation can be eliminated, diminishing the risk of reactive oxygen species exposure and DNA damage. We hypothesised that equine sperm can be separated using a microfluidic sorting device (Fertile PlusTM Sperm Sorting Chip; DxNow, Worcester, MA, USA) to improve the quality of sperm for ICSI. The aim of our research was to evaluate sperm parameters, including motility, morphology, membrane integrity, and DNA integrity, in frozen-thawed samples of equine semen before and after sorting using the Fertile Plus Sperm Sorting Chip. Two experiments were performed. In Experiment 1, the microfluidic device was used to separate frozen-thawed semen samples (n = 10) from research stallions (n = 3) with good quality frozen semen; all semen was frozen by one method in our laboratory. In Experiment 2, clinical samples of frozen-thawed semen (n = 11) from 7 stallions were evaluated. The semen was of variable quality and frozen at different facilities. Sperm analyses included (1) motility, (2) morphology (Hancock stain, Animal Reproduction Systems, Chino, CA, USA), (3) live-dead sperm (Hancock stain), (4) membrane integrity (HOS, hypo-osmotic swelling test), and (5) DNA fragmentation (SCD, sperm chromatin dispersion). Two sample t-tests were used to compare sperm parameters. In Experiment 1, use of the Fertile Plus Sperm Sorting Chip improved sperm parameters between the original and sorted samples, respectively: sperm motility (37.2 ± 13.0% and 62.2 ± 15.6%; P = 0.002), normal morphology (60.1 ± 12.2% and 75.5 ± 9.7%; P = 0.006), percentage live sperm (55.8 ± 16.0% and 73.6 ± 12.9%; P = 0.03), HOS (33.7 ± 7.2% and 48 ± 9.7%; P = 0.001) and sperm DNA fragmentation (12.3 ± 4.4% and 5.6 ± 4.4%; P = 0.004). When the Fertile Plus Sperm Sorting Chip was used in Experiment 2 to separate frozen-thawed semen from various sources, improvements were noted between the original and sorted samples, respectively, with increased motility (22.0 ± 13.0% and 57.0 ± 11.6%; P = 0.0009), normal morphology (58.4 ± 9.6% and 74.0 ± 10.3%; P = 0.005), a higher percentage of live sperm (55.5 ± 11.2% and 68.3 ± 14.2%; P = 0.04), and decreased sperm DNA fragmentation (22.3 ± 14.7% and 8.2 ± 8.3%; P = 0.004); no effect was observed on HOS (21.2 ± 6.0% and 24.9 ± 11.5%; P = 0.19). Our results demonstrate that use of the Fertile Plus Sperm Sorting Chip resulted in a subpopulation of sperm with improved quality parameters. Separation of sperm using a microfluidic device has the potential to select sperm with desirable characteristics for equine assisted reproductive techniques.

scholarly journals Preparation of cyclodextrin nanoparticles and evaluation of its effect on the capacitation of bovine spermatozoa used in the in vitro fertilization

2018 ◽  
Vol 9 (4) ◽  
pp. 112
Author(s):  
Heba F Salem ◽  
Mai Raslan ◽  
Hanaa Suliman ◽  
Tamer Essam ◽  
Saber Abd-Allah
Keyword(s):  
Data Analysis ◽  
In Vitro Fertilization ◽  
Interval Data ◽  
Fertilization Rate ◽  
Vitro Fertilization ◽  
Bovine Spermatozoa ◽  
The Media ◽  
Biomedical Interventions ◽  
Positive Effect

<p>This study was conducted to produce nanosized cyclodextrin (NCD) and assess its effect on bovine spermatozoa during In vitro fertilization (IVF) to optimize the capacitation media for successful IVF. Therefore, Four cyclodextrin formulations were prepared and characterized. Data analysis revealed the best formula (F2) showed a smallest particle size (15 nm), zeta potential (-37 mv), and higher yield percentages (95%) was selected for spem capacitation. Motile spermatozoa were separated from frozen-thawed semen by a swim-up procedure and capacitated in IVF-TALP medium with different formulae of NCD or CD or without treatments (control) and incubated for 3hours(hr) at 38°C and evaluated every one (hr) interval. Data analysis revealed that the formulation of cyclodextrin nanoparticles (F2<strong>)</strong> after (2hr) incubation in the media gave best effect on sperm capacitation and acrosme reaction (AR) and effect of sperm treated with NCD on fertilization rate was evaluated. The results showed that the proportion of Oocytes fertilized was increased significantly in F2 (60%) than in the control (35%), and cyclodextrin group (50%) groups (<em>p</em>&lt;0.05). It could be inferred from this investigation that cyclodextrin nanoparticles can be used for biomedical interventions in bovine spermatozoa. NCD improve sperm motility, viability, and (AR), also fertilization rate of sperm treated with NCD increase. So NCD gave positive effect on sperm functions during IVF. </p>

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

2017 ◽  
Vol 29 (1) ◽  
pp. 185
Author(s):  
L. R. Madzhie ◽  
M. A. Raseona ◽  
L. P. Nethenzheni ◽  
O. Ajao ◽  
M. L. Mphaphathi ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Culture Media ◽  
Uv Light ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Murine Embryos

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

scholarly journals Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization

2020 ◽  
Vol 21 (10) ◽  
pp. 3692 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Sanghoon Lee ◽  
Soo-Hyun Park ◽  
Min Ju Kim ◽  
Hyo-Gu Kang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Cumulus Cell ◽  
Annexin V ◽  
Ros Generation ◽  
Vitro Fertilization ◽  
Porcine Oocyte ◽  
Mitochondrial Distribution

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 μM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 μM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 177-185 ◽  
Author(s):  
A. Nader Fatehi ◽  
Bernard A.J. Roelen ◽  
Ben Colenbrander ◽  
Eric J. Schoevers ◽  
Bart M. Gadella ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Physical Contact ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization

The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

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