Determination of atresia condition in hake ovary samples from the Bay of Biscay: histological, immunohistochemical and western blot analysis.

Supervisors: Maren Ortiz-Zarragoitia, Oihane Diaz de Cerio (PiE-UPV/EHU).
Atresia in hake (Merluccius merluccius) ovary was investigated using histological, immunohistochemical and molecular approaches during the reproductive season of 2016 to 2017. A total of 153 female were analyzed and atretic follices were detected in 57 % of females. Alpha atresia was detected in most of the females with atretic follicles 36%, beta atresia was detected in 14% of female and only 7% of atretic females showed ovaries containing both alpha and beta atresia follicles. The 83% of females showing atresia was at previtellogenic stage while the 17% was at vitellogenic ovarian stage. Both previtellogenic and vitellogenic atretic follicle were immunoreactive with MDM2, RPL5 and RPL11, although MDM2 reaction observed in non atretic follicle. For the three proteins both alpha and beta atretic follicle showed strong staining in follicular theca cell layer, ooplasm and nuclear periphery. Advanced beta atretic stages were immunoreactive with MDM2 and RPl11 as indicated by mild chromatin in follicle cell layer and in nuclear area respectively. Western blot analysis gave positive immunoreactivity for previtellogenic, vitellogenic, atretic and non atretic follicle with MDM2, RPL5 and RPL11. Atretic and non atretic follicle in vitellogenic and previtellogenic follicle produced a single band of 55kDa for MDM2, 34kDa for RPl5 and 22KDa for RPl11. In all cases atretic follicles produced moderately strong band compared to that of non atretic follicles, although densitometry analysis did’t produce any significant difference between immunoreactive band of atretic and non atretic follicle for MDM2 and RPL11. Densitometry of RPL5 exhibits significant variation between atretic and non atretic follicles for both gametogenic stages. The positive reaction of antibodies in immunohistochemlistry and western blot analysis indicated the presence of MDM2, RPl5 and RPL11 protein during the follicular regression of hake ovary. Positive labeling also suggested that, the pathway modulating P53 activity is modified in atretic follicles in comparison to nonatretic follicles. Immunolabelling with RPL11 and RPl5 of atretic follicles (both previtellogenic and vitellogenic) but not in non-atretic ones and higher RPl11 and RPL5 levels in atretic follicles determined by western blot demonstrated that P53 pathway could be activated, but we need further analysis involving P53 to prove it.

Keywords: atresia, hake ovary, apoptosis, autophagy, histology, immunohistochemistry, western blotting