Early embryonic development after intracytoplasmic sperm injection of swamp buffalo (bubalus bubalis) oocytes

Abstract

Physiology of early embryonic development is required for a further application of reproductive biotechnology in swamp buffalo. However nowadays a few studies were reported and basic knowledge is limited. This thesis composes of three parts as follows: EXP.1aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. Swamp buffalo oocytes were matured and fertilized in vitro, presumptive zygotes and embryos were fixed at various time points post-IVF. Microtubules, microfilaments and chromatin were fluorescently labeled using monoclonal-α-tubulin, Phalloidin and DAPI, respectively. The redistribution pattern of cell cytoskeleton and chromosome of the zygotes and embryos was examined under an epifluorescent microscope. The results indicated that a dense network of microtubules or sperm aster in which radiating from the base of the decondensing sperm head plays a crucial role in the fertilization events about migration and apposition of male and female pronuclei in a normal fertilization process whereas microfilaments are considerably required for contractile ring formation during cleavage. Fertilization failure, at least in our current culture system, is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate. EXP. 2 aimed to : 1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; 2) compare the subsequent development of embryos following activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group); and 3)clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally-expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using RT-PCR. Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P<0.05). However, since 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation and IVF groups. Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. EXP. 3 aimed to improve sperm head decondensation by pretreating spermwith various chemicals before ICSI. Sperm were treated with the following protocols; (1) 0.1% Triton-X 100 (TX), (2) 10 µM calcium ionophore (CaI), (3) freezing and thawing (FT) without any cryoprotectant, and (4) untreated control. In each treatmentsperm were then either treated or not with 5 mMdithiothreitol (DTT). Acrosome integrity and DNA fragmentation were evaluated in sperm before ICSI by staining of sperm with fluorescein isothiocyanate–labeled peanut agglutinin and TUNEL, respectively. Then in vitro matured oocytes were subjected to ICSI using sperm pretreated as described above. The results revealed significantly increased rates of acrosome-lost sperm cells after TX and CaI treatments, whereas FT treatment and no-treatment (control) significantly increased the proportion of acrosome-reacted sperm. DTT treatment had no significant effect on acrosome configuration of sperm. DNA fragmentation was not significant difference among treatments. At 18 h post-ICSI, female pronucleus (PN) formation was found only in activated oocytes. However, among all the activated ICSI oocytes, the majority of them contained intact sperm heads. Normal fertilization characterized by two PNs without intact sperm head was only observed in CaI and FT treatment and control groups when sperm were treated with DTT before ICSI. In conclusion, these results indicated that DTT treatment of sperm with reacted acrosome before ICSI together with an additional activation of the resultant ICSI oocytes are important for successful sperm head decondensation resulting in male pronuclear formation. This study is the first report to examine the redistribution of cytoskeleton (microtubules, actin microfilaments) and indicates the dynamic of early embryo development during the first cell cycle in swamp buffalo. The fundamental knowledge and techniques from our study can be used as a tool for further investigating the embryonic development. In addition, this study confirms for the first time about the failure of traditional ICSI technique in swamp buffalo oocytes. Sperm treatment before ICSI is nescessory for successful production of normally fertilized embryos.