A spatial model showing differences between juxtacrine and paracrine mutual oocyte-granulosa cells interactions.

The bidirectional communication between oocytes and granulosa cells are mediated by several factors via a local feedback loop(s). The current model was carried out to study the spatial mutual interaction of porcine denuded oocytes and granulosa cells either in direct contact (juxtacrine) or paracrine co-culture using transwell system. Transwell 0.4 µm polyester membrane inserts were used to permit oocytes-granulosa cells paracrine communication with a distance of 2 mm between them in co-culture. Oocytes were cultured with granulosa cells in a defined basic maturation medium for 44 h. In results, oocyte secreted factors (OSFs; GDF9 and BMP15) temporal expression showed progressive decrement by the end of culture in case of direct contact with granulosa cells while it was increased progressively in the paracrine co-culture groups. However, oocytes that were cultured in direct contact showed a significant increase in blastocyst development after parthenogenetic activation than the paracrine co-cultured ones (20% vs. 11.5%, respectively). By the end of culture, granulosa cell count in direct contact showed a significant decrease than the indirect co-culture group (1.2 × 105 cell/mL vs. 2.1 × 105 cell/mL, respectively). Steroids (P4 and E2) and steriodogenesis enzymes mRNA levels showed significant temporal alterations either after 22 h and 44 h of IVM in both juxtacrine and paracrine co-culture systems (P ≤ 0.05). CX43 was much more highly expressed in the granulosa of the direct contact group than the indirect co-culture group. These results indicate the difference in mutual communication between oocytes and granulosa cells that were cocultured either in direct contact (juxtacrine) or with a short distance (paracrine) and propose a new paradigm to study different ovarian follicular cells interaction.

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.