Prostaglandin synthesis, metabolism, and signaling potential in the rhesus macaque corpus luteum throughout the luteal phase of the menstrual cycle

Abstract

Prostaglandins in the corpus luteum (CL) reportedly serve as luteotropic and luteolytic agents. Based mainly on studies conducted in domesticated animals and rodents, prostaglandin E2 (PGE2) is generally considered a luteotropic factor, whereas uterine-derived prostaglandin F2alpha (PGF2alpha) initiates luteolysis. However, the role of prostaglandins in regulating primate luteal structure-function is poorly understood. Therefore, a comprehensive analysis of individual mRNA or proteins that are involved in PGE2 and PGF2alpha biosynthesis, metabolism, and signaling was performed using CL obtained at distinct stages of the luteal life span during the menstrual cycle in rhesus monkeys. Peak levels of proteins involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 2, microsomal PGE2 synthase-1) and signaling (PGE2 receptor 3) occurred during periods corresponding to development and maintenance of the primate CL. Immunohistochemistry studies indicated that large luteal cells express PGE2 synthesizing and signaling proteins. Expression of PGE2 synthesizing and signaling proteins significantly decreased preceding the period of functional regression of the CL, which also coincided with increasing levels of PGF2alpha receptor protein expression within the large luteal cells. Moreover, significant levels of mRNA expression for several aldoketo reductase family members that synthesize PGF2alpha from other prostaglandins were observed throughout the rhesus macaque luteal phase, thus supporting the possibility of intraluteal PGF2alpha production. Collectively, our results indicate that there may be intraluteal synthesis and signaling of PGE2 during development and maintenance of the primate CL, followed by a shift to intraluteal PGF2alpha synthesis and signaling as the CL nears the time of luteolysis.

Figure 1

PTGS2 and PTGES protein levels in rhesus macaque CL obtained throughout the luteal phase. A, Representative Western blots for PTGS2, PTGES, and TUBB using samples pooled from CL collected at each stage of the macaque luteal phase. The negative control is pooled midstage CL in which the primary antibody was omitted (PTGS2) or probed with preabsorbed primary antibody (PTGES). B and C Quantitative assessment of PTGS2 and PTGES expression, respectively, as determined by densitometry. Levels of PTGS2 and PTGES expression from individual CL (n = 4 CL/stage) were normalized to TUBB, and the resultant ratio was analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Error bars, 1 sem. Columns with different letters are significantly different (P < 0.05).

Figure 2

Localization of PTGS2 and PTGES proteins in rhesus macaque CL. Representative photomicrographs of PTGS2 (A) and PTGES (B) IHC. The insets in the lower left corner of the first image for A and B are adjacent sections that were processed with primary antibody preabsorbed with immunizing peptide. Approximate locations of various cell types are indicated. L, Large luteal cells; S, small luteal or stromal cells; V, blood vessel. Scale bar in the lower right hand corner of the higher-magnification images, 50 μm.

Figure 3

PTGER1, PTGER2, and PTGER4 mRNA levels in rhesus macaque CL obtained throughout the luteal phase. Levels of mRNA were determined by Q-PCR and normalized to 18s rRNA in individual CL (n = 4 CL/stage). Data were analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Error bars, 1 sem. Columns with different letters or numbers are significantly different (P < 0.05), with uppercase letters, lowercase letters, and numerals corresponding to PTGER1, PTGER2, and PTGER4, respectively.

Figure 4

PTGER3 protein levels in rhesus macaque CL obtained throughout the luteal phase. A, Representative Western blot for PTGER3 using pooled CL samples for each stage of the luteal phase. The upper image is PTGER3 and the lower corresponds to TUBB levels, which served as a loading control. The negative control is pooled early-stage CL probed with primary antibody that had been preabsorbed with its immunizing peptide. B, Mean level of expression as determined by densitometry. Levels of PTGER3 from individual CL (n = 4 CL/stage) were normalized to TUBB, and the resultant ratio was analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Error bars, 1 sem. Columns with different letters are significantly different (P < 0.05).

Figure 5

Localization of PTGER3 protein in rhesus macaque CL. The first two images are from an early-stage CL section. The inset in the top image is an adjacent section that was probed with primary antibody that had been preabsorbed with immunizing peptide. The bottom image is PTGER3 staining in very late stage CL. Approximate locations of various cell types are indicated. L, Large luteal cells; S, small luteal or stromal cells; and V, blood vessel. Note the apparent decrease in staining of vascular endothelial cells (indicated by arrows) between the early and very late stages. The scale bar in the lower right hand corner of the higher magnification images, 50 μm.

Figure 6

HPGD protein levels throughout the rhesus macaque luteal phase. A, Representative Western blot for HPGD using pooled CL samples corresponding to each stage of the macaque luteal phase. The upper image is HPGD and the lower corresponds to TUBB, whereas the negative control is the pooled late CL sample probed with primary antibody that had been preabsorbed with its immunizing peptide. B, Densitometry data for each of the detected HPGD isoforms normalized to TUBB (n = 4 CL/stage). The resultant ratio was analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Error bars, 1 sem. Columns with different letters are significantly different (P < 0.05).

Figure 7

PTGFR protein levels throughout the rhesus macaque luteal phase. A, Representative Western blot for PTGFR using pooled CL samples for each stage of the macaque luteal phase. PTGFR levels are shown in the upper image, whereas the levels of the internal control TUBB are shown in the lower image. The negative control is the pooled late CL sample probed with primary antibody that had been preabsorbed with its immunizing peptide. B, Mean level of PTGFR expression as determined by densitometry. Levels of PTGFR from individual CL (n = 4 CL/stage) were normalized to TUBB, and the resultant ratio was analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Error bars, 1 sem. Columns with different letters are significantly different (P < 0.05).

Figure 8

Localization of PTGFR protein in rhesus macaque CL. Staining for PTGFR in early-stage (top panel) and late-stage (middle panel) CL as well as a higher magnification (bottom panel) for the late-stage CL. The inset in the middle panel is the negative control from an adjacent late stage section. Approximate locations of various cell types are indicated:. L, Large luteal cells; S, small luteal or stromal cells; V, blood vessel. The scale bar in the lower right hand corner of the bottom panel, 50 μm.

Figure 9

Intraluteal AKR family member 1B1, 1C1, 1C2, and 1C3 mRNA levels throughout the rhesus macaque luteal phase. A, Microarray expression data of two genes that possess PGFS activity, AKR1B1 and AKR1C3. Microarray data were analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Columns with different letters are significantly different (P < 0.05) with upper-case letters representing AKR1B1 mRNA levels and lower-case letters representing AKR1C3 mRNA levels. B, Microarray expression data for AKR1C1 and AKR1C2. C, Q-PCR results for AKR1C1 throughout the rhesus macaque luteal phase. Q-PCR data were analyzed by ANOVA followed by comparison between groups using the Student-Newman-Keuls test. Columns with different letters are significantly different (P < 0.05). The error bars (A–C), 1 sem.