Elevated free fatty acids affect bovine granulosa cell function: a molecular cue for compromised reproduction during negative energy balance

Abstract

High-yielding dairy cows postpartum face the challenge of negative energy balance leading to elevated free fatty acids levels in the serum and follicular fluid thus affecting the ovarian function. Here, we investigated effects of physiological concentrations of palmitic acid (PA), stearic acid (SA) and oleic acid (OA) on the viability, steroid production and gene expression in a bovine granulosa cell (GC) culture model. Treatment with individual and combined fatty acids increased the CD36 gene expression, while no significant apoptotic effects were observed. Both PA and SA significantly upregulated the expression of FSHR, LHCGR, CYP19A1, HSD3B1, CCND2 and increased 17β-estradiol (E2) production, while OA downregulated the expression of these genes and reduced E2. Interestingly, STAR was equally downregulated by all fatty acids and combination treatment. E2 was significantly reduced after combination treatment. To validate the effects of OA, in vivo growing dominant follicles (10–19 mm) were injected with bovine serum albumin (BSA) with/without conjugated OA. The follicular fluid was recovered 48 h post injection. As in our in vitro model, OA significantly reduced intrafollicular E2 concentrations. In addition, expression of CD36 was significantly up- and that of CYP19A1 and STAR significantly downregulated in antral GC recovered from aspirated follicles. The ovulation rates of OA-injected follicles tended to be reduced. Our results indicate that elevated free fatty acid concentrations specifically target functional key genes in GC both in vitro and in vivo. Suggestively, this could be a possible mechanism through which elevated free fatty acids affect folliculogenesis in dairy cows postpartum.

Effects of Dietary Fatty Acids on Bovine Oocyte Competence and Granulosa Cells

“…Together, these results suggest that high levels of ALA or cis-9, trans-11 CLA in FF can adversely affect GC by reducing both E2 and P4 production, which in turn could result in compromised ovarian cyclicity and impaired fertility in lactating cows. This is also in line with our recent study, where we could show that increased concentrations of OA in the follicular fluid can actually impair the functionality of GC in situ, thus obviously suppressing ovulation (21).…”

“…Primary GC culture and bovine serum albumin (BSA)-FA conjugate preparations were done as described earlier (21). For testing the effects of actual follicular concentration of ALA (Sigma-Aldrich,L2376) and cis-9, trans-11CLA (Sigma-Aldrich,16413) the media were replaced every 48 h with supplemented α-MEM media containing different concentrations of ALA (20,40, and 80 µM) or cis-9, trans-11 CLA (15, 30, and 60 µM) as BSA conjugates or non-conjugated BSA as vehicle control (Sigma-Aldrich A7030).…”

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

“…For HIF1A knockdown, GCs were transfected with 50 nM of antisense LNA negative control gapmer (A*A*C*A*C*-G*T*C*T*A*T*A*C* G*C), and anti HIF1A gapmer (A*C*T*G*A*T*C*G*A*A*G*G*A*-A*C*G) using TransIT-X2 ® transfection reagent (Mirus Bio, USA) as per the manufacturer's recommendations in a 24 well culture dishes for 6 days (from day 2 to day 8 of the culture). As established before, all cultured cells were subjected to different analyses on day 8 [20][21][22][23] .…”

“…Hence, analyzing the role of HIF1 under normoxic and hypoxic environments in the presence of FSH and IGF1 would offer important cues regarding GC physiology. Accordingly, the present investigation was carried out to identify HIF1 dependent transcriptional activity both under normoxic and hypoxic conditions using our renowned estrogen active culture model of bovine primary GC [20][21][22][23] .…”

Non-esterified fatty acids in the ovary: friends or foes?

“…Valckx et al 2014 [29] showed that in vitro exposure of murine ovarian follicles to elevated levels of NEFAs resulted in the impairment of ovarian steroidogenesis and oocyte competence for fertilization. To further understand the physiological responses under in situ conditions, Sharma et al 2019 [30] took advantage of the ultrasound-guided injection approach to inject NEFAs (oleic acid; C18:1) into the dominant follicles of heifers in situ. Interestingly, the NEFA injected animals showed reduced ovulation rates and reduced production of E2 hormone compared to that of the control group of animals ( Fig.…”