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The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing <jats:italic>in vitro<\/jats:italic> fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F\u2010actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early\u2010stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F\u2010actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC\u2010mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes\/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F\u2010actin), and assess expression of the DNA double\u2010strand break marker \u03b3\u2010H2AX and the DNA repair protein RAD51 in control and Cr(VI)\u2010exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. \u00a9 2024 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: <jats:italic>In vivo<\/jats:italic> EDC treatment of rats and immunostaining of treated oocytes<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: <jats:italic>In vitro<\/jats:italic> fertilization and immunostaining of early\u2010stage embryos<\/jats:p>","DOI":"10.1002\/cpz1.70060","type":"journal-article","created":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T10:49:24Z","timestamp":1734950964000},"source":"Crossref","is-referenced-by-count":3,"title":["Employment of a Newly Defined <i>In Vitro<\/i> Fertilization Protocol to Determine the Cytoskeletal Machinery, DNA Damage, and Subsequent DNA Repair Resulting from Endocrine Disruption by Hexavalent Chromium in Rat Metaphase II Oocytes"],"prefix":"10.1002","volume":"4","author":[{"given":"Liga","family":"Wuri","sequence":"first","affiliation":[{"name":"Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences Texas A&amp;M University College Station Texas"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul W.","family":"Zarutskie","sequence":"additional","affiliation":[{"name":"Department of Clinical Medicine &amp; Primary Care Sam Houston State University College of Osteopathic Medicine Conroe Texas"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joe A.","family":"Arosh","sequence":"additional","affiliation":[{"name":"Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences Texas A&amp;M University College Station Texas"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sakhila K.","family":"Banu","sequence":"additional","affiliation":[{"name":"Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences Texas A&amp;M University College Station Texas"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,12,23]]},"reference":[{"key":"e_1_2_8_2_1_1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.142711"},{"key":"e_1_2_8_2_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/humupd\/dmy019"},{"key":"e_1_2_8_2_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/humupd\/dmq036"},{"key":"e_1_2_8_2_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.taap.2011.01.011"},{"key":"e_1_2_8_2_5_1","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod.114.125708"},{"key":"e_1_2_8_2_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/toxsci\/kfx224"},{"key":"e_1_2_8_2_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/ijc.34272"},{"key":"e_1_2_8_2_8_1","doi-asserted-by":"publisher","DOI":"10.1080\/03009734.2020.1727073"},{"key":"e_1_2_8_2_9_1","doi-asserted-by":"publisher","DOI":"10.1289\/ehp.1205301"},{"key":"e_1_2_8_2_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tox.2022.153306"},{"key":"e_1_2_8_2_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xfss.2020.12.001"},{"key":"e_1_2_8_2_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ceb.2010.08.007"},{"key":"e_1_2_8_2_13_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adc9161"},{"key":"e_1_2_8_2_14_1","doi-asserted-by":"publisher","DOI":"10.1530\/jrf.0.0590329"},{"key":"e_1_2_8_2_15_1","doi-asserted-by":"publisher","DOI":"10.4103\/0971\u20106866.50867"},{"key":"e_1_2_8_2_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.reprotox.2023.108492"},{"issue":"3","key":"e_1_2_8_2_17_1","first-page":"156","article-title":"A history of developments to improve in vitro fertilization","volume":"114","author":"Eskew A. 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