The reverse cholesterol transport system as a potential mediator of luteolysis in the primate corpus luteum

Abstract

The cessation of progesterone (P(4)) production (i.e. functional regression), arguably the key event in luteolysis of the primate corpus luteum (CL), is poorly understood. Previously, we found that genes encoding proteins involved in cholesterol uptake decreased, while those involved in cholesterol efflux (reverse cholesterol transport, RCT) increased in expression during spontaneous functional regression of the rhesus macaque CL, thereby potentially depleting the cholesterol reserves needed for steroidogenesis. Therefore, a comprehensive analysis of the components necessary for RCT was performed. RCT components were expressed (mRNA and/or protein) in the macaque CL including cholesterol sensors (liver X receptors alpha or NR1H3; and beta or NR1H2), efflux proteins (ATP-binding cassette subfamilies A1 (ABCA1) and G1), acceptors (apolipoproteins A1 or APOA1; and E or APOE), and plasma proteins facilitating high-density lipoprotein formation (lecithin:cholesterol acyltransferase or LCAT; phospholipid transfer protein or PLTP). ABCA1, APOE, PLTP, and NR1H3 increased, while lipoprotein receptors decreased, in expression (mRNA and/or protein) through the period of functional regression. The expression of APOA1 and APOE, as well as NR1H3, was greatest in the CL and tissues involved in regulating cholesterol homeostasis. Immunolocalization studies revealed that RCT proteins and lipoprotein receptors were expressed in large luteal cells, which possess intracellular cholesterol reserves during periods of P(4) synthesis. Lipid staining revealed changes in luteal cholesterol ester/lipid distribution that occurred following functional regression. These results indicate that decreased cholesterol uptake and increased RCT may be critical for the initiation of primate luteolysis by limiting intracellular cholesterol pools required for steroidogenesis.

Figure 1. Lipoprotein receptor and ABC transporter protein levels throughout the period of spontaneous functional regression in the rhesus macaque CL

Panel A contains representative Western blots for SCARB1, LDLR, ABCA1, ABCG1, and TUBB using samples pooled from CL collected at either the mid-late (ML), functional late (FL), functionally-regressed late (FRL), or very-late (VL) stages of the luteal phase. The results of densitometry analyses of individual luteal homogenates (n = 4 CL/group) for the lipoprotein receptors SCARB1 and LDLR, as well as the cholesterol efflux proteins ABCA1 and ABCG1, are presented in panels B and C, respectively. Error bars indicate one standard error of the mean (SEM). Columns with different letters of the same case are significantly different (p< 0.05).

Figure 2. Localization of lipoprotein receptors and ABC transporters in the rhesus macaque CL throughout the period of spontaneous functional regression

Representative photomicrographs of SCARB1, LDLR, ABCA1, and ABCG1 from the mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) luteal stages are shown. The insets in the upper left corner of the ML samples for SCARB1 and LDLR, as well as in the FRL samples for ABCA1 and ABCG1, are sections that were processed with primary antibody preabsorbed with immunizing peptide. The approximate locations of various cell types are indicated in the FL panel for each protein and include: large luteal cells (L); small luteal or stromal cells (S); and blood vessels (V). The scale bar in the lower right hand corner of each image is 50 µm. Arrows indicate the appearance of plasma membrane localization of ABCA1 and ABCG1 in FRL stage CL.

Figure 3. Apolipoprotein expression in the rhesus macaque CL

Panel A contains microarray and Q-PCR expression data for APOE during the early (E), mid (M), mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) stages. Error bars indicate one SEM (n = 4 CL/group). Upper case letters denote significant differences (p< 0.05) for microarray data and lower case letters denote significant differences for Q-PCR data. Panel B contains representative photomicrographs for APOE IHC performed using CL obtained from the E through VL stages of the luteal phase. The inset in the upper left corner of the VL sample is from a control section that was probed with primary antibody preabsorbed with its immunizing peptide. The approximate locations of various cell types are indicated in the ML panel and include: large luteal cells (L); small luteal or stromal cells (S); and blood vessels (V). The scale bar in the lower right hand corner of each image is 100 µm. Panel C contains microarray and Q-PCR expression data for APOA1 that was analyzed in the same manner as for Panel A. Panel D contains representative photomicrographs from APOA1 IHC analysis. The details are similar to the description for panel B except the negative control inset is in the FL sample and consists of a control section probed with primary antibody preabsorbed with purified full-length human APOA1.

Figure 4. Expression of proteins that promote HDL formation in the rhesus macaque CL

Panel A contains microarray expression data for LCAT and PLTP during the early (E), mid (M), mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) stages of the luteal phase. Error bars indicate one SEM (n = 4 CL/group). Columns with different letters of the same case are significantly different (p< 0.05). Panel B contains representative photomicrographs for LCAT and PLTP IHC analysis from throughout the luteal phase. The inset in the upper right corner of the M image (LCAT), or VL image (PLTP), are control sections where the primary antibody was excluded. The approximate locations of various cell types are indicated in the FL panel and include: large luteal cells (L); small luteal or stomal cells (S); and blood vessels (V). The scale bar in the lower right hand corner of each image is 50 µm.

Figure 5. NR1H2 and NR1H3 expression in the rhesus macaque CL

Panel A contains microarray and Q-PCR expression data for NR1H2 and NR1H3 during the early (E), mid (M), mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) stages of the luteal phase. Panel B contains Western blot data for NR1H2 and NR1H3. Images are from pooled CL collected at the ML, FL, FRL, or VL stages. The graph presents densitometry results from analysis of individual CL (n = 4 CL/group). Error bars indicate one SEM. Columns with different letters of the same case are significantly different (p< 0.05).

Figure 6. Localization of NR1H2 and NR1H3 expression in the rhesus macaque CL throughout the period of spontaneous functional regression

Representative photomicrographs of NR1H2 and NR1H3 from the mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) stages are shown. The insets in the upper left corner of the FL 200× panel for NR1H2, and FRL panel for NR1H3, are control sections incubated with primary antibody preabsorbed with its immunizing peptide (NR1H2), or where the primary antibody was excluded (NR1H3). The approximate locations of various cell types are indicated in the FL panel for each protein and include: large luteal cells (L); small luteal or stromal cells (S); and blood vessels (V). The scale bar in the lower right hand corner of each image is 50 µm.

Figure 7. Tissue distribution of APOA1, APOE, NR1H2, and NR1H3 gene expression in the rhesus macaque

Agarose gel images of PCR amplification products are shown. Gene-specific primers were used to amplify cDNA collected from various rhesus macaque tissues. The number of cycles used in each image is: APOA1 = 25, APOE = 24, NR1H2 = 34, NR1H3 = 26; and PPIA = 26. These cycle numbers correspond approximately with the mid-linear range of amplification for each gene as determined from pooled luteal cDNA. A 100 base-pair (BP) ruler is included with each image, and the 500 BP band is marked. Expected product size for each gene is indicated.

Figure 8. Rhesus macaque luteal cholesterol levels and cholesterol ester/lipid droplet localization prior to and following spontaneous functional regression

Panel A contains normalized cholesterol concentrations in CL extracts from the early (E), mid (M), mid-late (ML), functional late (FL), functionally-regressed late (FRL), and very-late (VL) stages of the luteal phase. Error bars indicate one SEM. Columns with different letters of the same case are significantly different (p< 0.05). Representative lipid droplet staining and localization results, as determined by confocal microscopy, are presented in panel B for the mid and functionally-regressed late stages. Actin is represented by blue fluorescence, nuclei are red, and neutral lipids including cholesterol esters are green. The inset in the mid 400× lipid overlay is a parallel section that was extracted with methanol to remove all lipids (background control). The approximate locations of various cell types are indicated in the first image for each stage and include: large luteal cells (L); small luteal or stromal cells (S); and blood vessels (V). The scale bar in the lower right hand corner of each image is 50 µm.