Abstract
BackgroundCryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes.MethodsImmature oocytes (germinal vesicles) isolated from ovaries of normal bitches (> 6 months of age) were either vitrified in open pulled straw (OPS) using 20% ethylene glycol (EG) and 20% dimethyl sulfoxide (DMSO) as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were investigated for nuclear maturation following in vitro maturation (IVM), ultrastructural changes using transmission electron microscopy (TEM) and gene expression using RT-PCR. Fresh immature oocytes were used as the control group.ResultsThe rate of resumption of meiosis in vitrified-warmed oocytes (53.4%) was significantly (P < 0.05) lower than those of control (93.8%) and exposure (91.4%) groups. However, there were no statistically significant differences among groups in the rates of GV oocytes reaching the maturation stage (metaphase II, MII). The ultrastructural alterations revealed by TEM showed that cortical granules, mitochondria, lipid droplets and smooth endoplasmic reticulum (SER) were affected by vitrification procedures. RT-PCR analysis for gene expression revealed no differences in HSP70, Dnmt1, SOD1 and BAX genes among groups, whereas Bcl2 was strongly expressed in vitrified-warmed group when compared to the control.ConclusionImmature canine oocytes were successfully cryopreserved, resumed meiosis and developed to the MII stage. The information obtained in this study is crucial for the development of an effective method to cryopreserve canine oocytes for establishment of genetic banks of endangered canid species.
Highlights
Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology
In contrast to most of other mammals that oocytes are at the MII stage when ovulated, canine oocytes released from ovaries are at the prophase I stage of the first meiotic division and they subsequently completed nuclear maturation within 6072 h in the oviduct [1]
Experiment 1: The effect of vitrification on nuclear maturation of vitrified-warmed canine oocytes A total of 284 oocytes were vitrified
Summary
Cryopreservation of oocytes, which is an interesting procedure to conserve female gametes, is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. A major obstacle for the development of assisted reproductive technologies in canines is the low percentage of oocytes reaching the maturation stage (i.e., metaphase II, MII) following IVM. Ultrastructural studies on vitrified bovine oocytes have revealed that intercellular communication between the cumulus cells and oocyte might have been interrupted and that the zona pellucida might have been modified by premature cortical granule release [10]. The aim of this study was to investigate the effects of vitrification on nuclear maturation, ultrastructural changes and gene expression on vitrified-warmed immature canine oocytes
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