title: Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs.
abstract: To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.
authors.affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. Electronic address: nakai3@affrc.go.jp.
authors.forename: M
authors.initials: M
authors.lastname: Nakai
authors.affiliation: Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Ito
authors.affiliation: Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Kashiwazaki
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; Laboratory of Embryo Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam; Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: N T
authors.initials: NT
authors.lastname: Men
authors.affiliation: The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Yamaguchi, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Tanihara
authors.affiliation: Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Noguchi
authors.affiliation: Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kaneko
authors.affiliation: Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Onishi
authors.affiliation: Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Yamaguchi, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D000818:Animals; D001755:Blastocyst; D002467:Cell Nucleus / Q000502:physiology; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008297:Male; D009865:Oocytes / Q000502:physiology*; D010703:Phorbol Esters / Q000008:administration & dosage / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology; D013552:Swine / Q000196:embryology*
chemical_list: D010703:Phorbol Esters; C008874:phorbol-12,13-diacetate; D011493:Protein Kinase C
keywords: Intracytoplasmic sperm injection; Oocyte activation; Phorbol 12-myristate 13-acetate; Porcine; Protein kinase C