- L-lactate induces specific genome wide alterations of gene expression in cultured bovine granulosa cells. - Background: Previously, we could show that L-lactate affects cultured bovine granulosa cells (GC) in a specific manner driving the cells into an early pre-ovulatory phenotype. - Conclusions: Our data clearly show that L-lactate alters gene expression in cultured bovine GC in a broad, but obviously specific manner. - Besides the release of a fertilizable oocyte in particular in the bovine this transi- tion involves a deep transformation of somatic cells of the follicular wall into luteal cells. - In the bovine, cells of the granulosa and theca layers migrate and largely intermingle with each other during CL for- mation [1]. - This remodeling of the follicle is preceded and accompanied by a profound and meticulous regula- tion of gene expression in particular in the granulosa cell layer. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - From this knowledge, typical markers for LH-dependent differentiation in the bovine could be established [3], whereby in particular the role of various growth-factors as those of the TGFbeta superfamiliy or EGF and their role during fol- licle differentiation were analyzed in detail [12–15]. - Additionally, it was shown that the L-lactate levels in rats increase at the expected time of the LH-surge implicating a regulatory role that forces GC differentiation [19]. - During the present study we ana- lyzed the effects of L-lactate in a genome wide mRNA microarray approach with subsequent bioinformatic evaluation of the datasets to elucidate the underlying pathways and biological processes.. - Additionally, a small portion of the freshly aspirated sample-pool was preserved as “pre-cultured sample” for qPCR analysis in liquid nitrogen. - For seed- ing, the GC were thawed and immediately transferred into α-MEM and centrifuged at 500 x g for 3 min to ensure fast removing of the freezing media. - GC were cultured for 8 days at 37 °C and 5% CO 2 and two-third of the media with or without L-lactate or ve- hicle was exchanged every other day.. - Validation of the microarray data was accomplished with quantitative real-Time PCR (qPCR). - At the end of each run, the melting point was analysed to check for amplification of the right products. - Therefore, five different di- lutions of the respective standards g DNA/reaction) were freshly prepared and co-amplified.. - The two most stable reference genes were obtained by using the geNORM algorithm imple- mented in the NormqPCR package for R [20], revealing TBP and B2M as the most stable genes. - Hybridization was done overnight in the GeneChipR Hybridization Oven (Affymetrix) and visualized with the Affymetrix Gene- Chip Scanner 3000. - Results of the array have been submitted to the GEO database (GSE121408). - Bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA, Qiagen) using a modified list of the treat- ments L-lactate vs. - Out of the modified list 2193 genes could be mapped in IPA, settings for the pathway analysis were restricted to genes with FC. - All genes having a FC be- tween |1.2| and |1.5| were characterized as abundant in the dataset but did not influence the pathway analysis.. - Raw data from the microarray analysis were initially ana- lyzed by principal component analysis (PCA) to reduce the multidimensionality of the dataset. - Individual samples of the dataset were plotted and revealed the greatest vari- ability between the different groups of culture conditions on the x-axis with a variation of 30.9% (Fig. - Individual samples of the same culture condition clustered tightly together. - The higher number (788) of differentially regulated clus- ters (representing 735 annotated genes) of the L-lactate treatment compared to the untreated cells is in line with the PCA, demonstrating strongest effects between. - 1 Principal component analysis (PCA) and Venn diagram of the microarray datasets. - Principal Component Analysis of the qPCR dataset revealed the highest vari- ation of 71.7% between the L-lactate treatment and both other culture conditions “NaCl” and “untreated” controls (Fig. - Comparing the effects of lactate treatment with the NaCl vehicle control 487 transcript clusters. - Further analysis identified TNF, beta-estradiol, progesterone and Vegf as major up- stream regulators, which might be involved as activating factors and thus responsible for the observed changes of the expression profile (Table 4 and Additional file 1:. - Interestingly, the Regulator Effects analysis of IPA identified a putative activation of the functions “pro- liferation”, “vascularization”, “angiogenesis” or “cell movement” whereas the biological function “organismal death” was predicted to be inhibited (Fig. - Our data clearly show that any treatment with either lac- tate or the NaCl vehicle control significantly changed the global gene expression profiles of cultured GC when compared to untreated controls. - The effect of NaCl alone is clearly visible in the PCA of the microarray dataset Table 1 Comparison of qPCR and microarray data by Pearson. - that separates the vehicle control and untreated cells, re- vealing the sensibility of the cell culture model to the composition of media. - However, focusing on markers of GC differ- entiation specific effects of L-lactate are even more clearly visible without any unspecific changes in the gene expression profile. - This limits the extrapolation of the data obtained in vitro to in vivo conditions.. - 2 Validation of the microarray data from selected marker genes of the folliculo-luteal transition with quantitative real-time PCR. - a PCA of the qPCR data showed highest variation between L-lactate treated and both control groups. - NaCl treated cells revealed similar fold changes in the microarray and qPCR dataset. - NaCl vehicle control). - HAS2 hyaluronan synthase E-07 2.70E-05. - EREG Epiregulin 5.05 3.57E-09 2.00E-06. - VNN1 vanin E-07 1.20E-05. - SLC1A5 solute carrier family 1 (neutral amino acid transporter), member E-07 2.70E-05. - LOC508666 C-C motif chemokine E-07 1.70E-05. - 2.31 2.62E-07 1.90E-05. - But for most of the pathways no prediction regarding activation or inactivation could be made. - In this study we investigated the global change of gene expression by comparing cells treated with L-lactate or. - NaCl vehicle control. - TXNIP , coding for the thioredoxin interacting protein, was remarkably up-regulated (FC 21.97) in L-lactate treated GC indicating a role in the L-lactate induced differentiation process. - The strongly reduced expression in high density GC culture model might therefore mirror the need of the cells for increased glucose uptake under these “glucose-deficient” conditions, whereas increased expression in the present L-lactate supplementation model could be a consequence of the ample supply with an alternative energy source thus reducing the need for glucose uptake.. - Also H19 gene expression was observed to be signifi- cantly up-regulated in L-lactate treated GC (FC 12.4).. - As a regulator of the tissue response to oxidative stress VNN1 modulates the gluta- thione store [33]. - MYC was shown to be down-regulated in the L-lactate treated GC compared to the vehicle control (FC − 2.4).. - Earl- ier it was proposed that both PKA and Epac are involved in the process of luteinization activated by LH [40–43].. - In our former study of density-driven differentiation in bovine GC we could also highlight the involvement of the “cAMP-mediated Signaling” pathway [26]. - Thus, the results of the present study reflect a LH induced like differentiation of bovine GC upon L-lactate treatment.. - NTN1 was down-regulated with a fold change of − 2.1 in L-lactate treated GC compared to the control, which would suggest that vascular regulation or angiogenic processes are not induced. - Interestingly, netrin-1 was found to be present in follicular fluid as well as in the theca and granulosa cell layer of swine antral follicles and was proposed to have anti-angiogenic functions [51]. - It seems that the final answer of NTN1 function regarding angiogenesis is not that straightforward in granulosa cells and needs further investigations.. - SLIT and ROBO were present in the microarray dataset but no differential expression of these could be ob- served. - Nonetheless, SLIT and ROBO expression could be detected in human luteinized GC or in the CL and are regulated by steroid hormones [54]. - In the microarray dataset we could identify the expression of several subunits of NMDARs in bovine GC, however without any differential regulation of these.. - However, if L-lactate signaling might be mediated via NMDA recep- tors in bovine granulosa cells still has to be elucidated.. - GnRH controls the release of the gonadotropins FSH and LH by diverging pulse frequencies. - Progesterone, on the other hand, is a critical parameter for the establishment of an active corpus luteum indicating a transition of the GC’s phenotype to- wards luteinization. - However, whereas expression of the key gene of progesterone synthesis HSD3B1 is very high in fully luteinized GC (i.e. - It is commonly known that angiogenic processes contribute to ovulation and the later formation of the corpus luteum [60, 61]. - The function “organismal death ” could be identified as inactivated, thus indicating that L-lactate treatment does not affect the viability of the cultured GC. - The func- tion “cell movement” could be identified as activated, which is in line with the forthcoming breakup of follicu- lar cell layers and necessary migratory processes during the formation of the corpus luteum.. - Taken together our data provide novel insights into a possible regulatory role of increased concentrations of L-lactate on cells of the granulosa in large follicles dur- ing the folliculo-luteal transition. - Our data suggest that the biological function of L-lactate in the granulosa cell layer of growing follicle is complex and by far exceeding its role as a product of hypoxic metabolism and energy source. - GC: Granulosa cells. - DK did the mRNA microarray analysis and JV contributed to the experimental design and preparation of the manuscript. - The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.. - Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG. - Down-regulation of genes encoding steroidogenic enzymes and hormone receptors in late preovulatory follicles of the cow coincides with an accumulation of intrafollicular steroids. - Transcriptome analysis of granulosa cells after conventional vs long FSH-induced superstimulation in cattle. - PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. - Regulation of LH receptor and PGF2alpha receptor signaling by the regulator of G protein signaling 2 (RGS2) in human and mouse granulosa cells. - Expression and regulation of regulator of G- protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells. - Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle. - Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post- LH stage. - A tissue culture model of estrogen-producing primary bovine granulosa cells. - Low oxygen levels induce early Luteinization associated changes in bovine granulosa cells.. - Increasing cell plating density mimics an early post- LH stage in cultured bovine granulosa cells. - Cultured bovine granulosa cells rapidly lose important features of their identity and functionality but partially recover under long-term culture conditions. - Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density. - The expression profile of the H19 gene in cattle.. - Steroid hormones modulate H19 gene expression in both mammary gland and uterus. - Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.. - Global gene expression in granulosa cells of growing, plateau and atretic dominant follicles in cattle.. - Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation. - In-vitro study of gonadotrophin signaling pathways in human granulosa cells in relation to progesterone receptor expression.. - Intracellular signaling in the gonads. - Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes. - The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early luteal phase of the bovine estrous cycle. - Meta-analysis of gene expression profiles in granulosa cells during folliculogenesis.. - Angiogenesis in the corpus luteum
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