1. Objective Conclusion Materials & Methods Results
19th Congress on Reproductive Biomedicine & 14th Congress on Stem Cell Biology and Technology
29-31 August, 2018
Maturational genes upregulation and mitochondrial activity enhancement in mouse
in-vitro matured oocytes applying granulosa cells conditioned medium
Elnaz Zand1, Mohammad Jafari Atrabi2, Vahid Akbarinejad3, Rouhollah Fathi4
1,2,4Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
3Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran, Iran
* Corresponding address:
Objective
Conclusion
Results
In vitro maturation (IVM) is a promising technique in the case of women with normal and intricate cycles. The miscarriage rate after transfer of embryos derived from IVM is about 20-60%. It se ems improving the IVM method leads to better results in post fertilization stages. The purpose of this study was to improve both cytoplasmic and nuclear maturation of immature oocytes using granulosa cells conditioned medium.
The results of present study indicated that Granulosa Cells Conditioned Medium 50% (GCCM50) will improve MII rate, viability and mitochondrial potential activity rather than the other control and experimental groups.
Materials & Methods
Fig. 3 : JC-1 staining for mitochondrial membrane potential activity; red to green: increasing of membrane potential activity (MPA). Green: JC-1 monomeric form (low intensity), Red: JC1-aggregated form (high intensity).
Graph; *significant with other groups: P < 0.05.
Germinal Vesicles from NMRI mice were collected and cultured in 3 conditions: Base Medium (control), Granulosa Cells Conditioned Medium100% (GCCM100) and 50 % (GCCM50), After 24 hours, the mitochondrial activity potential and viability of MII were evaluated by JC1 and Trypan blue stainings, respectively. Gene Expression of Cyclin B1, Cdk1 and Gdf9 were analyzed by the means of Real Time PCR just in control, GCCM100 and GCCM50 because of their interesting results.
Fig. 1: Percentage of polar body released oocytes (M II rate) in all control (BM) and Experimental groups (GCCM50 and 100). *significant with other groups: P<0.05.
Results
Relative gene expression level to 18S
Fig. 2: Trypan blue staining to select the viable germinal vesicles for IVM and viable in vitro matured oocytes (M II oocytes). Viable GV (A) and dead GV (B) before IVM, C) viable MII oocyte after maturation in base medium (control group), viable MII oocytes after maturation in GCCM100 (D) and GCCM50 (E). Graph indicates different viability rate after IVM; A with a: P =
The results revealed reaching to MII stage was greater in GCCM50 among the groups (p<0.05)(Fig. 1).
The viability rate of the in vitro matured oocytes was higher in GCCM50 between all groups (Fig. 2).
Also, Jc-1 staining demonstrated that GCCM50 composition is more beneficial for maintaining the mitochondrial activity potential as compared to the other groups (Fig. 3).
The analysis of gene expression due to GCCM50 treatment, concerned with Cdk1 and Gdf9, was higher than control and GCCM100 groups, while the expression of Cyclin B1 was not different among the groups (Fig. 4).
Fig. 4: Gene expression level of CdK1, CycB1 and Gdf9 in in vitro matured oocytes (M II) of all control (BM) and experimental (GCCM50 and 100) groups (*significant with BM)(**significant with BM and GCCM50)(P<0.05).