Ovarian superovulation was achieved using pregnant mare serum gonadotropin (PMSG, Cat. Nor-272-a, Prospec, East Brunswick, NJ), and human chorionic gonadotropin (HCG, Cat. C1063-1VL, MilliporeSigma, Burlington, MA). Materials for in-vitro culture, including potassium simplex optimized medium (KSOM, Cat. MR-121-D) and hyaluronidase, were purchased from MilliporeSigma (Burlington, MA) and sterile mineral oil (Cat. ART-4008-5P) from CooperSurgical (Trumbull, CT). OMOPS handling medium consisted of MgSO4 (1.2 mM); Glucose (0.5 mM); L-Lactate (6.0 mM); GlutaGRO (1.0 mM); Tarine (0.1 mM); NEAA (1x); EDTA (0.01 mM); Alpha Lipoic Acid (10 uM); Gentamicin (10 ug/ml); Hyaluronan (0.125 mg/mL); NaHCO3 (5.0 mM); MOPS (20.0 mM); pyruvate (0.2 mM); Citrate (0.5 mM); FAF BSA (4.0 mg/ml). A pure phthalate mixture was made by calculating and combining the appropriate amount of each phthalate according to the following percentages 35% DEP, 21% DEHP, 15% DBP, 15% DiNP, 8% DiBP, and 5% BBzP in 0.05% DMSO. Phthalates were purchased from Sigma. In previous, preliminary studies, incubation of mouse embryos in this phthalate mixture, at 1 µg/mL, reduced blastocyst formation by 50%. This concentration was used to produce the sick batch of embryos.
Phthalates
Manufactured by Merck Group
Sourced in United States, France
Phthalates are a class of chemical compounds commonly used in the manufacturing of laboratory equipment. They serve as plasticizers, enhancing the flexibility and durability of various materials used in the production of lab instruments and apparatus.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
12 o tetradecanoylphorbol 13 acetate tpa, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Neutral red solution, by Merck Group (1 mentions)
Alamar blue solution, by Thermo Fisher Scientific (1 mentions)
4 protocols using phthalates
1
Ovarian Superovulation and Phthalate Exposure
2
Biochemical Reagents for Cell Assays
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All ingredients for phosphate-buffered saline (PBS, 10 mM, pH 7.4), formaldehyde, methanol, phthalates (purity: >97%), lucifer yellow dye dilithium salt, 12-O-tetradecanoylphorbol 13-acetate (TPA) and neutral red solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Additionally, 5-carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM) and Alamar Blue® solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
3
Phthalate Mixture Preparation and Dosing
4
Phthalate Exposure Protocol in Mice
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Exposure to phthalates (Sigma Aldrich, Saint-Quentin Fallavier, France) was performed as recently described (Adam et al., 2021; (link)Ahmadpour et al., 2021) (link). The phthalates were first dissolved in absolute ethanol, then incorporated into food as previously detailed (Ahmadpour et al., 2021 (link) studies showing an external co-exposure to these molecules and the presence of their metabolites in urinary samples (Dewalque et al., 2014; (link)Martine et al., 2013) (link). Mice were weighed weekly for the duration of the exposure and phthalate doses were adjusted to their body weights and calculated for a daily food intake of 5 g per animal (Cheema et al., 2019; (link)Dombret et al., 2017) (link). It has already been shown that such a phthalate exposure protocol had no effect on the body weight of individuals between the first and the last day of treatment (Ahmadpour et al., 2021) (link).
Analyses were performed on 4 cohorts each comprising animals distributed equally between the 4 treatment groups.
Briefly, two cohorts were used for immunohistochemistry, immunocytochemistry and microscopy studies. One was used for the capillary-enriched fraction procedure for western blot analysis and one for in situ zymography and Fluoro-Jade® C fluorescent investigation.
Analyses were performed on 4 cohorts each comprising animals distributed equally between the 4 treatment groups.
Briefly, two cohorts were used for immunohistochemistry, immunocytochemistry and microscopy studies. One was used for the capillary-enriched fraction procedure for western blot analysis and one for in situ zymography and Fluoro-Jade® C fluorescent investigation.
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