195 Nuclear progesterone receptor and mitochondria profiling during bovine oocyte growth

Previous data from our group described the localization of the nuclear progesterone receptor (nPR) in growing oocytes, showing foci arranged in a clustered pattern in oocytes 60–80 μm in diameter and a more dispersed distribution in larger oocytes (i.e. >90 μm in diameter). The initial step in progesterone (P₄) synthesis involves the transport of cholesterol from the cytoplasm into the inner mitochondrial membrane. Once inside the mitochondria, cholesterol is converted into the P₄ precursor, pregnenolone. Since the distribution of nPR foci in oocytes <80 μm was similar to the arrangement of mitochondria in growing oocytes, we hypothesised that the nPR associates with mitochondria during this stage of oocyte growth. To this end, the objective of the study was to determine the extent to which nPR colocalizes with mitochondria during the bovine oocyte phase. Bovine cumulus–oocyte complexes (COC) were recovered from ovaries collected at a local abattoir, by ovarian slicing. The COCs were measured, allocated to the following diameter groups: 60–<100 μm, 100–110 μm, and 120–130 μm, and incubated 30 minutes in Stroebech IVM media containing 5 μM cilostamide and 200 nM MitoTracker Deep Red (Invitrogen, M22426) at 39°C in 5% CO₂. The oocytes were denuded by repeated pipetting and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Expression of nPR was investigated by whole-mount immunofluorescent analysis of the fixed oocytes. Immunolabelled oocytes were visualised using a Carl Zeiss LSM 800 Airy confocal system. The resulting images were analysed in ImageJ to determine the number of nPR foci and mitochondria, and the volume of each oocyte. Data were analysed by Poisson regression to determine the correlation between the number of nPR foci and oocyte diameter, and by analysis of variance to evaluate nPR foci/mitochondria colocalization/μm³ and oocyte diameter. Expression of nPR isoform A (nPR-A) was detected in the cytoplasm and nucleus of oocytes in all. The number of nPR-A foci was positively correlated with oocyte diameter and nuclear volume (P < 0.001). In addition, oocytes from the smallest diameter group presented higher variability of nPR foci/μm³ compared with oocytes in the largest group (182 ± 19.08 vs 48.8 ± 84.70, P < 0.05). Oocytes from 60–100 μm were characterised by an even distribution of mitochondria throughout the cytoplasm, specifically surrounding the nucleus. In contrast, oocytes >100–130 μm showed a mitochondrial dispersion around the oocyte cortex and small aggregations in the periphery of the oocyte. The colocalized number of mitochondria and nPR-A foci/μm³ was significantly higher in the smaller oocytes (60–100 μm) compared with the oocytes 100–130 μm (517 ± 75.44 vs 160 ± 76.14, P < 0.01). The distribution of nPR foci and mitochondria is similar during bovine oocyte growth, suggesting an interaction during this process. However, the exact nature of this interaction and its significance are yet to be fully understood. Further functional studies are required to gain more insights into the role and variability of this association.

The work was funded by an EU Marie Sklodowska-Curie Innovation Training Network Action EUROVA_ETN#860960.