Dynamic Regulation of HIF1α and Oxygen-Sensing Factors in Cyclic Bovine Corpus Luteum and During LPS Challenge

Abstract

Understanding the corpus luteum (CL) and its role in cattle reproduction is crucial, particularly as it is a progesterone source for the establishment and maintenance of pregnancy. Reduced oxygen levels significantly impact these processes. This study investigated the effects of the luteal stage on the spatio-temporal gene expression patterns of HIF1α and oxygen-sensing factors, as well as the impact of lipopolysaccharide (LPS)-induced inflammation on these factors. Endothelial inflammatory responses were also addressed. The samples included CL collected at the early, mid, and late stages, as well as biopsies from mid-luteal stage cows treated either with saline (controls) or LPS. Samples collected in subsequent cycles assessed potential carryover effects. RT-PCR revealed upregulation of HIF1α, PHD1, PHD3, FIH, and VHL encoding genes in the mid-luteal stage. In situ hybridization revealed the compartmentalization of HIF1α and its regulators within the luteal and endothelial cells, suggesting their cell-specific roles. LPS treatment affected PHD1 and PHD3 expression, while increasing endothelial pro-inflammatory factors ICAM1 and NFκB, suggesting vascular inflammation and modulated oxygen sensing. These findings reveal new insights into the spatio-temporal expression of HIF1α-regulating factors in the CL, highlighting their potential role in controlling luteal function, detailing their cellular compartmentalization, and the effects of LPS-mediated inflammatory responses.

Keywords: HIF1α; LPS; bovine corpus luteum; hypoxia.

Figure 1

Relative mRNA expression of HIF1A (HIF1α) and HIF1α-regulating factors, i.e., EGLN2 (PHD1), EGLN1 (PHD2), EGLN3 (PHD3), HIF1AN (FIH), and VHL in bovine CL during early, mid-, and late luteal stages. Relative gene expression as determined by semi-quantitative real-time (TaqMan) PCR is presented as mean ± standard error of mean (±SEM). Bars with asterisks differ at * p < 0.05 and ** p < 0.01.

Figure 2

Luteal localization of transcripts (mRNA) encoding for HIF1A (HIF1α) (A,B), EGLN2 (PHD1) (C,D), EGLN1 (PHD2) (E), EGLN3 (PHD3) (F,G), and VHL (H,I) in bovine CL, shown at mid-luteal stage. Black arrow: large luteal cells; grey arrow: small luteal cells; arrowhead: vessel. Explanations in text. The sense probes were employed as negative controls (smaller inserts at the same magnification to (B,D,E,G,I)).

Figure 3

Relative mRNA expression of HIF1A (HIF1α) and its regulatory factors, EGLN2 (PHD1), EGLN1 (PHD2), EGLN3 (PHD3), HIF1AN (FIH), and VHL during and after E. coli-LPS treatment. Relative gene expression as determined by semi-quantitative real-time (TaqMan) PCR is presented as mean ± standard error of mean (±SEM). Bars with asterisks differ at * p < 0.05 and ** p < 0.01.

Figure 4

Relative mRNA expression of ICAM1 and NFKB2 during and after E. coli-LPS treatment. Relative gene expression as determined by semi-quantitative real-time (TaqMan) PCR is presented as mean ± standard error of mean (±SEM). Bars with asterisks differ at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

Schematic representation of the involvement of HIF1α and oxygen-sensing factors in the cyclic bovine corpus luteum, as well as their expression in the CL of cows treated with lipopolysaccharide (LPS; gram-negative bacterial endotoxin). The data regarding STAR derive from [32]. This figure was created with BioRender.com.