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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Reproductive Biology and Endocrinology 1/2018

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

Reproductive Biology and Endocrinology > Ausgabe 1/2018
Virginie Maillard, Alice Desmarchais, Maeva Durcin, Svetlana Uzbekova, Sebastien Elis
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12958-018-0357-7) contains supplementary material, which is available to authorized users.



Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers.


The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4).


DHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion.


These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.
Additional file 5: Figure S4. Expression and localization of free fatty acid receptor 4 (FFAR4) in bovine granulosa cells by immunofluorescence with a commercial antibody against human FFAR4. Immunofluorescence was performed on granulosa cells (GC) after in vitro culture. Briefly, recovered GCs after follicle puncture and GC washing were incubated in serum-free modified McCoy’s 5A medium (2.5 × 105 viable cells/well on a 8-well chamber slide (Lab-Tek® Nunc) for 48 h. Cultures were performed in a water-saturated atmosphere containing 5% CO2 in air at 38 °C. FFAR4 (green fluorescence) was immunodetected in GC (commercial human FFAR4 rabbit antibody, Aviva Systems Biology, Clinisciences, Nanterre, France) with a similar protocol to the protocol used with the customized anti-FFAR4 antibody. The commercial anti- FFAR4 antibody was produced by using a peptide from the FFAR4 human c-terminal region, which shares 89% identity (Protein BLAST® result on NCBI website) with the Bos taurus FFAR4 (Accession number: NP_001315586.1) and no identity with other amino acid sequences of bovine proteome. Rabbit IgG was used as the control with the same secondary antibody as for FFAR4 detection. Nuclei were stained with Hoechst 33,258 (blue fluorescence). Fluorescence was observed under a Zeiss confocal microscope LSM700 (Carl Zeiss Microscopy GmbH, Munich, Germany) using an oil 63× objective and the appropriate filters. The images were captured using Zen 2012 software (black edition version 8.0, Carl Zeiss Microscopy GmbH). The picture framed in red is a magnification of the area framed in red from the original image. Bars = 10 μm. (TIF 132 kb)
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