The Role of Aquaporin 3 in the Movement of Water and Cryoprotectants in Mouse Morulae1

Tan

et al. 2013

Cell Physiol Biochem

Background/Aims: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

Section: Discussionsupporting

confidence: 64%

“…Down-regulation of AQP3 has been shown to be involved in impaired function of several cell types including oocytes, embryos and somatic cells. Knockout of AQP3 in mouse zygotes significantly decreased water and cryoprotectant permeability [21]. Impaired proliferation of epidermal cells was observed in AQP3 null mice [22].…”

Section: Discussionmentioning

confidence: 99%

Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

Tan

et al. 2013

Cell Physiol Biochem

“…[27] for mouse oocytes consists of ethylene glycol (EG), acetamide, sucrose, and Ficoll dissolved in isotonic phosphate buffered saline (PB1). The oocyte is highly permeable to EG and acetamide [5,28], but is impermeable to the last three. This composition is typical of vitrification solutions in that it consists of permeable and non-permeable solutes, low and high molecular weight solutes, and has a very high total molality.…”

Section: Resultsmentioning

confidence: 99%

“…Then the left end of the Cryotop holder [3] is pressed manually, tipping the sample out of the LN 2 in the Styrofoam well, releasing the LN 2 cover slide to cover the well, and firing the laser as the slide slips into position. The operational details are as follows: The LN 2 well [5] in the Styrofoam block is filled with LN 2. Then,…”

Section: The Laser the Laser Pulse The Cryo Jig And The Resultant mentioning

confidence: 99%

Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse

2014

Self Cite

Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below − 100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.

J Animal Sci Biotechnol

“…Numerous research articles have focused on CPA permeability and the rate at which it enters cells [5,6]. Other studies have investigated incubation times in both the pretreatment and vitrification solutions and found that the temperature used during the handling procedure is also important for successful vitrification [7-9].…”

Section: Introductionmentioning

confidence: 99%

An efficient method for the sanitary vitrification of bovine oocytes in straws

Zhou

et al. 2014

BackgroundAt present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study.ResultsWhen in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation.ConclusionsThese results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.