Additional file 1: of Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

Figure S1. Comparison of the expression of marker genes in GC cultured immediately after isolation vs. GC cultured after cryo-preservation. No difference between GC cultured either directly after isolation or after cryo-preservation could be observed. Transcript abundance is shown as absolute expression (copy number per μg RNA) of n = 3, student’s t-test revealed no significant differences. (TIFF 226 kb

Affected “Diseases and Functions” in granulosa cells under acute pre-ovulatory heat stress conditions.

<p>Only significantly affected functions with p < 0.05 (indicated threshold line) according to bioinformatics IPA analysis were shown.</p

Size of dominant follicles as determined by ultrasonography at the times of treatment with PGF_2α and GnRH, and intra-follicular steroid contents.

<p>Size of dominant follicles as determined by ultrasonography at the times of treatment with PGF_2α and GnRH, and intra-follicular steroid contents.</p

Hierarchical cluster analysis of heat affected genes.

<p>Based on the abundance of 88 differentially expressed granulosa cell transcripts (│FC│ >2 and unpaired One-Way ANOVA P<0.05) different samples were clustered using the respective tool of the Transcriptome Analysis Console software.</p

Animal data of heat-stressed (HS) and pair-fed (PF) cows determined in the respiration chamber.

<p>Animal data of heat-stressed (HS) and pair-fed (PF) cows determined in the respiration chamber.</p

Data_Sheet_9_Low Oxygen Levels Induce Early Luteinization Associated Changes in Bovine Granulosa Cells.xlsx

<p>During follicle maturation, oxygen levels continuously decrease in the follicular fluid and reach lowest levels in the preovulatory follicle. The current study was designed to comprehensively understand effects of low oxygen levels on bovine granulosa cells (GC) using our established estrogen active GC culture model. As evident from flow cytometry analysis the viability of GC was not found to be affected at severely low oxygen condition (1% O₂) compared to normal (atmospheric) oxygen condition (21% O₂). Estimations of hormone concentrations using competitive radioimmunoassay revealed that the production of estradiol and progesterone was significantly reduced at low oxygen condition. To understand the genome-wide changes of gene expression, mRNA microarray analysis was performed using Affymetrix’s Bovine Gene 1.0 ST Arrays. This resulted in the identification of 1104 differentially regulated genes of which 505 were up- and 599 down-regulated under low oxygen conditions. Pathway analysis using Ingenuity pathway analyzer (IPA) identified 36 significantly affected (p < 0.05) canonical pathways. Importantly, pathways like “Estrogen-mediated S-phase Entry” and “Cyclins and Cell Cycle Regulation” were found to be greatly down-regulated at low oxygen levels. This was experimentally validated using flow cytometry based cell cycle analysis. Up-regulation of critical genes associated with angiogenesis, inflammation, and glucose metabolism, and down-regulation of FSH signaling, steroidogenesis and cell proliferation indicated that low oxygen levels induced early luteinization associated changes in granulosa cells. Identification of unmethylated CpG sites in the CYP19A1 promoter region suggests that granulosa cells were not completely transformed into luteal cells under the present low oxygen in vitro condition. In addition, the comparison with earlier published in vivo microarray data indicated that 1107 genes showed a similar expression pattern in granulosa cells at low oxygen levels (in vitro) as found in preovulatory follicles after the LH surge (in vivo). Overall, our findings demonstrate for the first time that low oxygen levels in preovulatory follicles may play an important role in supporting early events of luteinization in granulosa cells.</p

Top 20 regulated genes according to microarray analysis.

<p>Mean hybridization signals and fold change (FC) differences are shown.</p

qPCR re-evaluation of selected, differentially expressed transcripts according to microarray analysis.

<p>qPCR re-evaluation of selected, differentially expressed transcripts according to microarray analysis.</p

Experimental design of the present heat stress model.

<p>Non-pregnant German Holstein cows in established 2^nd lactation were randomly allocated to two groups: a PF control (n = 4) with a proportional feed restriction like it was measured in the HS group before, and an experimental HS group (n = 4). During the experimental period of 96h in a climate/respiration chamber at 28°C or 15°C the cows were synchronized with PGF_2α (Prostaglandin F2α) and subsequent GnRH injections prior to slaughter and sampling. Concentrations of O₂, CO₂ and CH₄ in the chamber were measured during the last 24hrs before slaughter.</p

Presentation_2_Low Oxygen Levels Induce Early Luteinization Associated Changes in Bovine Granulosa Cells.ZIP

<p>During follicle maturation, oxygen levels continuously decrease in the follicular fluid and reach lowest levels in the preovulatory follicle. The current study was designed to comprehensively understand effects of low oxygen levels on bovine granulosa cells (GC) using our established estrogen active GC culture model. As evident from flow cytometry analysis the viability of GC was not found to be affected at severely low oxygen condition (1% O₂) compared to normal (atmospheric) oxygen condition (21% O₂). Estimations of hormone concentrations using competitive radioimmunoassay revealed that the production of estradiol and progesterone was significantly reduced at low oxygen condition. To understand the genome-wide changes of gene expression, mRNA microarray analysis was performed using Affymetrix’s Bovine Gene 1.0 ST Arrays. This resulted in the identification of 1104 differentially regulated genes of which 505 were up- and 599 down-regulated under low oxygen conditions. Pathway analysis using Ingenuity pathway analyzer (IPA) identified 36 significantly affected (p < 0.05) canonical pathways. Importantly, pathways like “Estrogen-mediated S-phase Entry” and “Cyclins and Cell Cycle Regulation” were found to be greatly down-regulated at low oxygen levels. This was experimentally validated using flow cytometry based cell cycle analysis. Up-regulation of critical genes associated with angiogenesis, inflammation, and glucose metabolism, and down-regulation of FSH signaling, steroidogenesis and cell proliferation indicated that low oxygen levels induced early luteinization associated changes in granulosa cells. Identification of unmethylated CpG sites in the CYP19A1 promoter region suggests that granulosa cells were not completely transformed into luteal cells under the present low oxygen in vitro condition. In addition, the comparison with earlier published in vivo microarray data indicated that 1107 genes showed a similar expression pattern in granulosa cells at low oxygen levels (in vitro) as found in preovulatory follicles after the LH surge (in vivo). Overall, our findings demonstrate for the first time that low oxygen levels in preovulatory follicles may play an important role in supporting early events of luteinization in granulosa cells.</p