Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression, resulting in low fertility of pig oocytes

Abstract

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm-penetrated oocytes were observed to some extent (30-40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two-cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP3 R1), the channel responsible for Ca(2+) release during IVF in porcine oocytes. Localization of IP3 R1 close to the plasma membrane and total expression level of IP3 R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified-warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP3 R1 by the vitrification procedure.

Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

“…Accordingly, an increased frequency of non-penetrated parthenogenetic oocytes was observed in the matured-vitrified group in the present study. A plausible explanation for this phenomenon may be alteration or damage of Ca 2+ regulating organelles such as mitochondria and the endoplasmic reticulum, as reported previously in mouse, porcine and human oocytes [8][9][10]15].…”

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

“…Vitrification and warming by the Cryotop method was performed as describe [28] with some modification. PN embryos were selected and exposed for 10 min to PB1, i.e., phosphate-buffered saline (PBS) supplemented with 20% (v/v) fetal calf serum (FCS), 15% (v/v) ethylene glycol (EG) and COOH-PLL.…”

The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

“…Immunostaining was carried out as previously described (Hirose et al . ). Following three washes in phosphate‐buffered saline (PBS) (Nissui, Tokyo, Japan) containing 0.1% polyvinyl alcohol (PBS‐PVA), oocytes were fixed for 60 min in PBS‐PVA containing freshly prepared 2% (w/v) paraformaldehyde and 0.2% (v/v) Triton X at 24°C.…”

“…Our previous study confirmed that the CT1 antibody can be used for detection of IP 3 R1 in pig oocytes (Hirose et al . ). Following primary antibody incubation, oocytes were washed with PBS‐BSA (three times for 15 min each) and incubated for 1 h in a secondary antibody (Alexa 488 anti‐rabbit immunoglobulin G (IgG)) diluted 1:100 in PBS‐BSA at 24°C.…”

“…) and that the decrease in IP 3 R1 expression may be involved in the low fertility of cryopreserved oocytes (Hirose et al . ). However, no one has yet reported on the IP 3 R1 distribution in oocytes of domestic species.…”