THE EFFECT OF HSP60 ON FERTILIZATION AND PRE-IMPLANTATION EMBRYO DEVELOPMENT IN MICE: AN IN VITRO STUDY.

CONTEXT: Heat Shock Protein 60 (HSP60) is a chaperone protein which is involved in proteins transfer and re-folding of proteins. OBJECTIVE: Importance of HSP60 in sperm capacitation and facility of sperm-oocyte membrane binding was confirmed, therefore in this study the effect of HSP60 on the rate of in vitro fertilization and the cleavage rate in mouse embryo was investigated. DESIGN: Ten male mice and twenty five female mice were involved to collect sperms and oocytes required for this study. SUBJECTS AND METHODS: Sperms were collected from the epididymis of male mouse and oocytes were collected from the oviduct of female mouse following ovarian hyperstimulation. Then, capacitated sperms and oocytes were placed together in fertilization medium in four groups in the presence of different concentrations of HSP60 (10, 50 and 100 ng/mL) and in the absence of HSP60. After calculation of the fertilization rate, zygotes were transformed into the other medium for development and the cleavage rate was monitored to blastocyst stage. RESULTS: There was not a significant difference in the rate of fertilization between 10 ng/mL HSP60 group and the control group. The rate of fertilization and two-cell embryo development decreased significantly (P≤0.05) in 100 ng/mL HSP60 compared to other experimental and control groups. Further, the rate of two-cell embryo development increased significantly (P≤0.05) in 10 ng/mL HSP60 compared to other experimental and control groups. CONCLUSIONS: The present study demonstrated that HSP60 in low dose had a positive effect on two-cell embryo development, however it did not have any significant effect on the fertilization rate. Conversely, HSP60 had adverse effects on the fertilization and cleavage rates at higher doses.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy.

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.

Proteomic analysis of the sheep caruncular and intercaruncular endometrium reveals changes in functional proteins crucial for the establishment of pregnancy.

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.