Zona pellucida glycoprotein 3 (pZP3) and integrin 2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

B. Kempisty, M. Jackowska, H. Piotrowska, P. Antosik, M.Woźna, D. Bukowska, K.P. Brüssow, J.M. Jaśkowski

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells.
The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test.
Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test.
We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm.
Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocyte may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.