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Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

ABSTRACT: Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca²⁺]i). However, little information about the mechanism by which vitrification increases [Ca²⁺]i levels or a procedure to regulate [Ca²⁺]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca²⁺]i, endoplasmic reticulum Ca²⁺ (ER Ca²⁺), and mitochondrial Ca²⁺ (mCa²⁺) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca²⁺ release into the cytoplasm, consequently increasing the [Ca²⁺]i and mCa²⁺ levels. Supplementing the cells with 10??M 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca²⁺]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1??M ruthenium red (RR) significantly inhibited the Ca²⁺ flux from the cytoplasm into mitochondria; maintained normal mCa²⁺ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

SUBMITTER: Wang N

PROVIDER: S-EPMC5587528 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Wang Na N Hao Hai-Sheng HS Li Chong-Yang CY Zhao Ya-Han YH Wang Hao-Yu HY Yan Chang-Liang CL Du Wei-Hua WH Wang Dong D Liu Yan Y Pang Yun-Wei YW Zhu Hua-Bin HB Zhao Xue-Ming XM

Scientific reports 20170906 1

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, ...[more]

PMID: 28878377

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Project description:BackgroundIn livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Dataset Information

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Publications

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

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