Expression pattern of endothelin system components and localization of smooth muscle cells in the human pre-ovulatory follicle

Abstract

Background: Whether ovarian follicular rupture involves contractile activity or not has been debated for decades. Recently, study in the rodents has indicated that an endogenously produced potent vasoconstrictive peptide, endothelin-2 (EDN2), may induce follicular constriction immediately prior to ovulation. This study was aimed to systematically characterize the human ovarian endothelin system and localize smooth muscle cells to assess the possible involvement of contractile activity in human ovulation.

Methods: This is a prospective experimental study. Study subjects were 20 women aged 20-38 years who underwent IVF owing to tubal or male factors. Expression patterns of messenger RNAs (mRNAs) for EDN1, EDN2, EDN3, endothelin-converting enzyme-1 (ECE1 and ECE2), endothelin receptor A (ET(A)) and ET(B) in the granulosa cells (GCs) and cumulus cells and endothelin peptide concentration in the pre-ovulatory follicles were measured at 36 h after hCG injection. In addition, localization of ovarian smooth muscle cells and endothelin receptor expression were determined in normal (non-IVF patient) ovaries.

Results: Pre-ovulatory follicles express mRNA for EDN1 and EDN2, ECE1, ECE2, ET(A) and ET(B), but not EDN3, contain highly concentrated endothelin peptides (105.9 pg/ml) and are surrounded by theca externa that are made mostly of multicell layer non-vascular smooth muscle cells. ET(A) expression is localized in the smooth muscle cells of theca externa, theca interna and GC, whereas ET(B) expression is confined to theca interna.

Conclusions: Pre-ovulatory follicles contain highly concentrated endothelins and are surrounded by non-vascular smooth muscle cells that express endothelin receptor, suggesting involvement of endothelin-induced contractile action in ovulation in the human ovary.

Figure 1

Expression of endothelin system components in the GCs of a human pre-ovulatory follicle. (A) mRNA expression profiles of endothelin components in GCs obtained from a pre-ovulatory follicle. After 40 cycles of PCR amplifications, the PCR products were separated by electrophoresis on 1.2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The real-time amplification profiles (B) and melting curves (C) of endothelin component mRNA. Purple, EDN1; yellow, EDN2; red, EDN3; yellowish green, ET_A; green, ET_B; sky blue, ECE1; blue, ECE2; pink, GAPDH. (D) EDN3 mRNA expression profiles in GC of eight different patients and BE2 cell line that constitutively expresses EDN3 mRNA. β-Actin was used as an internal control.

Figure 2

Relative mRNA levels of endothelin subtypes in the GC and endothelin peptide levels of human pre-ovulatory follicles. (A) Endothelin peptide concentrations in the pre-ovulatory follicles (n = 6). The serum endothelin level was measured for reference. (B) Relative mRNA levels of endothelin subtypes calculated by using C_T values from the real-time PCR (n = 8). The level of each mRNA was normalized with the corresponding β-actin transcript level. Data are the mean ± SEM, and different letters indicate significance at P < 0.05. The C_T values of the EDN1 and EDN2 are 27.97 ± 0.58 and 24.38 ± 0.57, respectively.

Figure 3

Comparison of the endothelin component mRNA levels between human GCs and CCs. After real-time PCR, expression levels of each target gene in GCs and CCs were calculated from C_T values, and the fold difference in the relative transcript level was determined against that of GC (n = 8). Data are the mean ± SEM, and different letters indicate significance at P < 0.05. Note a higher expression of ET_B in the CC compared with GC.

Figure 4

Comparison of endothelin system component expression between human pre-ovulatory follicles with immature oocyte versus mature oocytes. Equal amounts of mRNAs isolated from human immature or mature CCs that were isolated from the same ovary were reverse transcribed and amplified using real-time PCR. The expression level was calculated from C_T values, and the mRNA ratio was determined relative to that of mature CC (n = 7). Data are the mean ± SEM. Note that there was no difference in the mRNA level for any of the endothelin components.

Figure 5

Localization of αSMA, ET_A and ET_B protein in large antral follicle. Specific antibodies for αSMA (A and B), ET_B (C) and ET_A (D) proteins were used to stain adjacent sections of a normal human ovary followed by counter staining by Mayer's hematoxylin. The high magnification images (×400; B–D) were taken from the marked (arrow) area of (A) (×12.5). inTH, theca interna; exTH, theca externa; BL, blood vessel; AT, antrum. Note that ET_B expression is specific to theca interna (C), whereas ET_A expression is seen in the GC, theca interna and theca externa (D) where αSMA staining is most intense (B). No positive signal was seen in the negative controls (no primary antibody; not shown). These are representative images from one of the seven ovaries that had at least one healthy large follicle in each ovary.