Oleic acid induces down-regulation of the granulosa cell identity marker FOXL2, and up-regulation of the Sertoli cell marker SOX9 in bovine granulosa cells

High-yielding dairy cows suffer from negative energy balance after parturition [1, 2]. To meet their energy requirements in spite of low blood glucose levels lactating dairy cows mobilize fat from adipose tissue. Consequently, levels of free fatty acids like palmitic acid (PA, 16:0), stearic acid (SA, 18:0), oleic acid (OA, 18:1) and of β-hydroxybutyric acid increase in the plasma and follicular fluid [3, 4], which can cause decreased milk yield and increased vulnerability to infections, metabolic diseases and sub-fertility. Short-term fasting of cattle also increases the levels of different fatty acids in the follicular fluid and especially of OA [5]. In our previous study, we showed that OA at physiological concentrations remarkably affected the cell morphology, reduced the abundance of functionally important transcripts as FSHR, LHCGR, STAR, CYP11A1, HSD3B1 and CYP19A, and decreased the production of 17-beta estradiol (E2) in cultured bovine granulosa cells (GC). In contrast, the expression of CD36 and SLC27A1, encoding fatty-acid transporters was increased [6]. Sahmi et al. [7] showed that the transcription factor and GC identity marker FOXL2 is vital for higher activity of the CYP19A1 promoter P2 in bovine GC. So we hypothesized that OA may affect the expression of FOXL2 and thus the GC identity. FOXL2 is involved in the regulation of the Sertoli cell marker SOX9 in the ovary at early developmental and adult stages [8‐12]. In adult mice the deletion of FOXL2 in GC increases the expression of SOX9 and development of seminiferous tubule-like structures in the ovary [11]. Also the deletion of estrogen receptors or low levels of estrogen resulted in a similar phenotype of GC [12‐14], thus demonstrating that the expression of SOX9 in ovaries is normally suppressed via ESR2 (estrogen receptor 2) and E2 along with FOXL2. FOXL2 also positively regulates the expression of ESR2, PPARG and FST, whereas it negatively regulates the expression of PTGS2 [12, 15, 16]. These genes are essentially involved in normal GC function [17‐21]. SOX9 is a very early and permanently expressed marker of Sertoli cells [22]. Loss- and gain-of-function studies revealed that SOX9 is required for Sertoli cell differentiation. Deletion of SOX9 in XY gonads leads to male-to-female sex reversal and misexpression in XX mice to female-to-male sex reversal [23, 24], thus demonstrating that SOX9 plays an essential role during testis differentiation processes [25‐28]. On the other hand, the loss of the SRY-dependent SOX9 inducibility in bipotential gonadal cells is the earliest sign of pre-granulosa cell differentiation in XX gonads [29].

During the present study we analysed effects of OA in cultured bovine GC on FOXL2 and SOX9, which are essentially involved in the maintenance of granulosa and Sertoli cell identity, respectively. In addition, we also studied effects of OA on other functionally important genes that are regulated by FOXL2 like ESR2, PPARG, FST and PTGS2.

The results showed that OA clearly down regulated the transcript abundance and protein levels of FOXL2, but up regulated those of SOX9 (Fig. 1). As OA inhibited FOXL2 mRNA and protein levels, we analyzed the effects of OA on the expression of genes that are involved in different functions of GC and are regulated by FOXL2 as FST, ESR2, PTGS2 and PPARG [12]. The results showed that OA significantly down regulated the expression of ESR2, PPARG and FST as compared with vehicle treated controls, whereas PTGS2 was increased (Fig. 2).

In the present study OA markedly reduced the levels of the transcription factors FOXL2 and ESR2, and increased the expression of SOX9. It is a very well established fact that FOXL2 is involved in maintaining the identity of GC [12]. The deletion of FOXL2 in GC of adult mice resulted in the development of seminiferous tubule-like structures along with the expression of Sertoli-cell marker genes in the ovary [11]. Georges et al. [12] showed that knockdown of FOXL2 or ESR2 leads to a rise of SOX9 levels and in case of a knockdown of both genes, SOX9 levels became even more effectively up-regulated. The same study also showed that knockdown of ESR1 has no clear effect on SOX9 repression following E2 treatment, but ESR2 knockdown resulted in an increase of SOX9 expression, whereas SOX9 expression was markedly increased in the absence of FOXL2. Thus, FOXL2 and ESR2/E2 repress SOX9 expression in follicular GC independent of each other [12]. In addition to this, it has been shown that FOXL2 regulates many genes like FST, PTGES2 and PPARG [12]. Our results showed that OA down regulated the expression of FST and PPARG along with that of FOXL2. The expression of FST in the ovary and GCs is regulated by FOXL2 [8, 16]. Knockdown of FOXL2 leads to decreased expression of PTGS2 and PPARG in murine primary GC [12]. Our results showed that OA induced down-regulation of PPARG along with FOXL2 but not of PTGS2, which was increased by OA treatment. Kim et al. [15] showed that FOXL2 represses the expression of PTGS2, which would be in line with our data. This suggests that OA may up-regulate SOX9 expression by reducing the levels of FOXL2, ESR2 and of E2. The data of our present study demonstrate that the expression of FOXL2, ESR2, PPARG, and FST was clearly decreased. In contrast, the expression of PTGS2 and in particular of SOX9 was increased by OA. Accordingly, we propose that OA induces the Sertoli cell marker SOX9 in GC eventually leading to a partial loss of GC identity, which may severely compromise GC’s functionality.

Our data suggest that (i) OA treatment reduced the expression of FOXL2. (ii) The inhibition of FOXL2 expression in turn may lead to the down-regulation of ESR2, FST and PPARG. (iii) The low levels of FOXL2, ESR2 and of E2 may overcome transcriptional suppression of the Sertoli cell marker SOX9 and thus induce trans-differentiation-like processes, which would severely compromise GC functionality. This novel observation could partly explain the negative effects of increased OA concentrations on post-partum fertility in dairy cows.