Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations.
Abstract: Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.
Publication Date: 2013-01-23 PubMed ID: 23340077DOI: 10.1017/S0967199412000627Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper investigates different drug combinations for the artificial activation of egg cells (oocytes) in horses and cows, with the goal of improving techniques for cloning (nuclear transfer) and sperm injection (Intracytoplasmic Sperm Injection). The authors found that all drug combinations successfully activated bovine oocytes, particularly with high calcium concentrations, whereas for equine oocytes, high calcium levels were not beneficial.
Research Objectives
- The study aimed to enhance the efficiency of nuclear transfer (cloning) and Intracytoplasmic Sperm Injection by artificially activating oocytes in bovine and equine species.
- The authors experimented different combinations of drugs – calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP) – in varying calcium concentrations to test their effectiveness.
Methodology
- The oocytes were collected and matured in vitro for 24 to 30 hours before being artificially activated.
- The artificial activation was conducted in the presence of low or high concentrations of calcium with the various drug combinations.
- The activated oocytes were assessed using the Hoechst 33342 stain and observed under a microscope.
Findings
- All combinations of drugs were effective in activating bovine oocytes, with the most successful results occurring when high concentrations of calcium were used.
- For equine oocytes, high concentrations of calcium did not exhibit a beneficial effect for the parthenogenetic (asexual reproduction) activation.
- The combination of ionomycin with either 6-DMAP or roscovitine effectively induced the artificial activation of equine oocytes.
Significance
- The findings of this preliminary study provide vital insights for the improvement of nuclear transfer and Intra-Cytoplasmic Sperm Injection techniques in bovines and equines.
- These results may also contribute to the development of better protocols for the artificial activation of oocytes in these species.
Cite This Article
APA
Fernandes CB, Devito LG, Martins LR, Blanco ID, de Lima Neto JF, Tsuribe PM, Gonçalves CG, da Cruz Landim-Alvarenga F.
(2013).
Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations.
Zygote, 22(3), 387-394.
https://doi.org/10.1017/S0967199412000627 Publication
Researcher Affiliations
- Department of Animal Reproduction,School of Veterinary Medicine and Animal Science,University of São Paulo (USP),Prof. Dr Orlando Marques de Paiva,87 Postal code: 05508 270-São Paulo/SP-Brazil.
- Department of Animal Reproduction and Veterinary Radiology,School of Veterinary Medicine and Animal Science,São Paulo State University (UNESP),Rubião Júnior District s/n.-Postal code: 18618-970-Botucatu-SP,Brazil.
- Department of Animal Reproduction and Veterinary Radiology,School of Veterinary Medicine and Animal Science,São Paulo State University (UNESP),Rubião Júnior District s/n.-Postal code: 18618-970-Botucatu-SP,Brazil.
- Department of Animal Reproduction and Veterinary Radiology,School of Veterinary Medicine and Animal Science,São Paulo State University (UNESP),Rubião Júnior District s/n.-Postal code: 18618-970-Botucatu-SP,Brazil.
- Department of Animal Reproduction and Veterinary Radiology,School of Veterinary Medicine and Animal Science,São Paulo State University (UNESP),Rubião Júnior District s/n.-Postal code: 18618-970-Botucatu-SP,Brazil.
- Endogin,Comandante João Ribeiro de Barros,Km 226,Bauru-SP,Brazil.
- Department of Animal Reproduction,School of Veterinary Medicine and Animal Science,University of São Paulo (USP),Prof. Dr Orlando Marques de Paiva,87 Postal code: 05508 270-São Paulo/SP-Brazil.
- Department of Animal Reproduction and Veterinary Radiology,School of Veterinary Medicine and Animal Science,São Paulo State University (UNESP),Rubião Júnior District s/n.-Postal code: 18618-970-Botucatu-SP,Brazil.
MeSH Terms
- Adenine / analogs & derivatives
- Adenine / pharmacology
- Animals
- Calcium / pharmacology
- Cattle
- Cells, Cultured
- Cycloheximide / pharmacology
- Female
- Horses
- In Vitro Oocyte Maturation Techniques
- Ionomycin / pharmacology
- Oocytes / drug effects
- Oocytes / physiology
- Parthenogenesis / drug effects
- Purines / pharmacology
- Roscovitine
- Strontium / pharmacology
Citations
This article has been cited 7 times.- Chen C, Huang Z, Dong S, Ding M, Li J, Wang M, Zeng X, Zhang X, Sun X. Calcium signaling in oocyte quality and functionality and its application. Front Endocrinol (Lausanne) 2024;15:1411000.
- Wang M, Zhu L, Liu C, He H, Wang C, Xing C, Liu J, Yang L, Xi Q, Jin L. A Novel Assisted Oocyte Activation Method Improves Fertilization in Patients With Recurrent Fertilization Failure. Front Cell Dev Biol 2021;9:672081.
- Unnikrishnan V, Kastelic J, Thundathil J. Intracytoplasmic Sperm Injection in Cattle. Genes (Basel) 2021 Jan 29;12(2).
- Stein P, Savy V, Williams AM, Williams CJ. Modulators of calcium signalling at fertilization. Open Biol 2020 Jul;10(7):200118.
- Tibary A. Grand Challenge Animal Reproduction-Theriogenology: From the Bench to Application to Animal Production and Reproductive Medicine. Front Vet Sci 2017;4:114.
- Hernández-Pichardo JE, Ducolomb Y, Romo S, Kjelland ME, Fierro R, Casillas F, Betancourt M. Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm. J Anim Sci Biotechnol 2016;7:65.
- Haverfield J, Nakagawa S, Love D, Tsichlaki E, Nomikos M, Lai FA, Swann K, FitzHarris G. Ca(2+) dynamics in oocytes from naturally-aged mice. Sci Rep 2016 Jan 20;6:19357.
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