Hypothesis regarding the effects of gonadotropins on the level of free fatty acids and phospholipids in serum and follicular fluid during controlled ovarian stimulation

Abstract Background: Granulosa cells (GCs) in cumulus oophorus highly express follicle stimulating hormone receptor (FSHR), which is the most important mediator of both estradiol synthesis and oocyte maturation. Obese women have elevated free fatty acids (FFAs) levels in their follicular fluids and decreased FSHR expression in GCs, which is related to an altered protein kinase B/glycogen synthase kinase 3β (Akt/GSK3β) signaling pathway. Such FFA increases accompany 3-fold rises in pseudokinase 3 (TRIB3) expression and reduce the Akt phosphorylation status in both the human liver and in insulinoma cell lines. Therefore, in a high FFA environment, we determined if TRIB3 mediates regulation of FSHR via the Akt/GSK3β signaling pathway in human GCs. Methods: GCs from women undergoing in vitro fertilization were collected and designated as high and low FFAs cohorts based on their follicular fluid FFA content. GCs with low FFA levels and a human granulosa-like tumor (KGN) cell line were exposed to palmitic acid (PA), which is a dominate FFA follicular fluid constituent. The effects were assessed of this substitution on the Akt/GSK3β signaling pathway activity as well as the expressions of TRIB3 and FSHR at both the gene and protein levels by qPCR, Western blot and immunofluorescence staining analyses. Meanwhile, the individual effects of TRIB3 knockdown in KGN cells and p-AKT inhibitors were compared to determine the mechanisms of FFA-induced FSHR downregulation.Results: The average FSH dose consuming per oocyte (FSH dose/oocyte) was elevated and Top embryo quality ratio was decreased in women with high levels of FFAs in their follicular fluid. In these women, the GC TRIB3 and ATF4 protein expression levels were upregulated which was accompanied by FSHR downregulation. Such upregulation was confirmed based on corresponding increases in their gene expression levels. On the other hand, the levels of p-Akt decreased while p-GSK3β increased in the GCs. Moreover, TRIB3 knockdown reversed declines in FSHR expression and estradiol (E2) production in KGN cells treated with PA, which also resulted in increased p-Akt levels and declines in the p-GSK3β level. In contrast, treatment of TRIB3-knockdown cells with an inhibitor of p-Akt (Ser473) resulted in rises in the levels of both p-GSK3β as well as FSHR expression whereas E2 synthesis fell. Conclusions: During exposure to a high FFA content, TRIB3 can reduce FSHR expression through stimulation of the Akt/GSK3β pathway in human GCs. This response may contribute to inducing oocyte maturation.

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

Endocrine Connections ◽

10.1530/ec-19-0011 ◽

2019 ◽

Vol 8 (5) ◽

pp. 493-505 ◽

Cited By ~ 5

Author(s):

Arpna Sharma ◽

Vijay Simha Baddela ◽

Frank Becker ◽

Dirk Dannenberger ◽

Torsten Viergutz ◽

...

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Energy Balance ◽

Free Fatty Acids ◽

Dairy Cows ◽

Combination Treatment ◽

Follicular Fluid ◽

Negative Energy

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.

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1 CUMULUS CELLS PROTECT OOCYTES AGAINST POTENTIAL LIPOTOXICITY FROM ELEVATED FREE FATTY ACID CONCENTRATIONS IN FOLLICULAR FLUID

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab1 ◽

2013 ◽

Vol 25 (1) ◽

pp. 148

Author(s):

H. Aardema ◽

F. Lolicato ◽

B. A. J. Roelen ◽

P. L. A. M. Vos ◽

J. B. Helms ◽

...

Keyword(s):

Fatty Acids ◽

Oleic Acid ◽

Fatty Acid ◽

Free Fatty Acid ◽

Free Fatty Acids ◽

Follicular Fluid ◽

Animal Health ◽

Cumulus Cells ◽

The Impact

Metabolic conditions characterized by elevated free fatty acid levels in the blood are often associated with reduced fertility performance. Increased concentrations of saturated free fatty acids can induce lipotoxicity in cumulus–oocyte-complexes in vitro, while unsaturated fatty acids like oleic acid are mostly harmless and able to counteract the impact of saturated fatty acids (Aardema et al. 2011 Biol. Reprod. 85, 62–69). This study investigates the impact of elevated free fatty acids in the blood on the follicular fluid and the lipid of cumulus and oocytes derived from these follicles. Furthermore, in vitro maturing oocytes were exposed to free fatty acid concentrations measured in follicles of control and metabolically stressed animals from this study to determine the impact on oocyte developmental competence. Cyclic heifers (n = 12) were synchronized (7 days CIDR®) and superstimulated from Day 10 of the synchronized cycle [4 days of Folltropin-V® (Bioniche Animal Health Inc., Belleville, ON, Canada) in decreasing doses; in total 200 mg]. Heifers received ad libitum grass silage, apart from the experimental group (n = 6), which was metabolically stressed during the period of superstimulation. Ovaries were collected by ovariectomy at final maturation, 22 h after the induced LH peak. Follicular fluids and cumulus–oocyte complexes (COC) were collected from follicles of ≥8 mm. To determine the free fatty acid and lipid composition, blood, follicular fluid, cumulus cells, and oocytes were analyzed with mass spectrometry. The COC (4 runs, 400 per group) derived from slaughterhouse ovaries were in vitro matured in a standard medium without or with the dominating free fatty acids, saturated palmitic and stearic and unsaturated oleic acid, in concentrations measured in follicular fluid of control (80, 70, and 100 µM) and experimental heifers (150, 100, and 200 µM) and fertilized and cultured until the blastocyst stage. Culture data were analyzed by one-way ANOVA and lipid data by two-sample t-test (P ≤ 0.05 considered significant). Procedures were approved by the Institutional Animal Care and Use Committee. Metabolic stress resulted in elevated free fatty acid levels in blood (from 430 ± 70 to 1048 ± 190 µM) and follicular fluid (from 357 ± 72 to 670 ± 133 µM), with relatively high oleic acid concentrations in follicular fluid (+10%). The increased levels of free fatty acids in follicular fluid resulted in a massive increase of fatty acids in the cumulus cells, but oocytes did only show marginal changes. In line with this, maturation in the presence of elevated palmitic, stearic, and oleic acid did not impair oocyte developmental competence and resulted in comparable blastocyst rates for the standard medium and the free fatty acid control and metabolic stress medium (31 ± 8.7, 34 ± 7.8, and 28 ± 1.7%). Thus, cumulus cells appear to protect oocytes against potential lipotoxicity from elevated free fatty acid concentrations by the accumulation of these fatty acids. This work was funded by Pfizer Animal Health.

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