Metabolic stress, characterized by elevated levels of free fatty acid (FFA), have been linked to reduced female fertility.
Saturated FFA (stearic acid (SA)) appears to have a dose-dependent negative effect on oocyte developmental competence
while monounsaturated oleic acid (OA) is shown to be harmless. Oocytes are protected against FFA by cumulus cells via
stearoyl-CoA desaturase 1 (SCD1) activity, which converts saturated SA into mono-unsaturated OA. To study FFA effects on
early embryonic development, embryos were cultured in the prese nce of SA and OA and SCD activity was analyzed.
Cumulus-oocyte-complexes (COCs), collected from 2-8 mm sized follicles of bovine slaughterhouse ovaries, were in vitro
matured (n=400/run) and fertilized (Aardema et al., Biol Reprod; 85: 62-69, 2011). During the first five days of embryo culture
(day 1-5; i.e. oviductal period), embryos were cultured in SOF without (control) or with FFA (FFA conc.= 25 and 50 μM OA; 25
and 50 μM SA; 25 or 50 μM OA + 25 or 50 μM SA). Fatty acids were complexed to fatty acid free BSA (10 mM) (FFA:BSA ratio
of 5:1). FFA was conjugated to albumin, likewise the transport of FFA in vivo, to solubilize FFA in an aqueous solution. At day 8
the number of blastocysts was counted. With RT-qPCR the mRNA expression of SCD1 was measured in all the conditions. The
general linear model was used for statistical analysis with SPSS 27.0. The day 8 embryos, COCs (positive control) and oocytes
(negative control) were fixated in 4% PFA and incubated with a primary antibody, SCD1, diluted 1:100 in PBST overnight at 4°C
for immunostaining. Thereafter, embryos were washed 3 times in PBST for 15 min and incubated with the second antibody,
goat anti-rabbit AlexaTM fluor 647, diluted 1:100 in PBST for 1h in the dark at RT. Confocal microscopy was performed using an
inverted Nikon A1R confocal microscope to determine the presenc e of SCD1 protein.
Exposure to 25 and 50 μM SA from day 1-5 resulted in a significantly lower blastocyst rate of respectively 18.9 ± 1.6% and 2.6
± 4.6%, compared to the control condition 25.9 ± 3.1% (n=3; p<0.05). Interestingly, exposure to 25 and 50 μM OA resulted in
a significantly higher blastocyst rate, 36.4 ± 6.3% and 34.6 ± 7.3% respectively (n=3; p<0.05). Exposure to 25 μM OA + 25 μM
SA resulted in a blastocyst rate of 26.0 ± 3.7% comparable to the control condition and was not significantly different from
exposure to 50 μM OA + 50 μM SA (25.6 ± 5.5%) (n=3; p>0.05). Interestingly, exposure to 50 μM OA + 25 μM SA resulted in a
blastocyst rate of 33.7 ± 9.3% comparable to the 50 μM OA condition (n=3; p<0.05). SCD1 was faintly expressed at RNA and
protein levels in day 8 embryos. Nevertheless, SCD1 was stronge r detected in embryos compared to oocytes.
Previously, our group demonstrated no detectable SCD1 protein levels in oocytes, but solely in cumulus cells. We here showed
that day 8 embryos express SCD1, which may protect embryos against saturated FFA. The current culture data show that OA
counteracted the negative effect of SA on embryos. Future studie s should investigate the role of OA and SCD1 in embryos.