The G-Protein-Coupled Membrane Estrogen Receptor Is Present in Horse Cryptorchid Testes and Mediates Downstream Pathways

Abstract

Cryptorchidism in horses is a commonly occurring malformation. The molecular basis of this pathology is not fully known. In addition, the origins of high intratesticular estrogen levels in horses remain obscure. In order to investigate the role of the G-protein-coupled membrane estrogen receptor (GPER) and establish histological and biochemical cryptorchid testis status, healthy and cryptorchid horse testes were subjected to scanning electron microscopy analysis, histochemical staining for total protein (with naphthol blue black; NBB), acid content (with toluidine blue O; TBO), and polysaccharide content (with periodic acid-Schiff; PAS). The expression of GPER was analyzed by immunohistochemistry and Western blot. GPER-mediated intracellular cAMP and calcium (Ca2+) signaling were measured immunoenzymatically or colorimetrically. Our data revealed changes in the distribution of polysaccharide content but not the protein and acid content in the cryptorchid testis. Polysaccharides seemed to be partially translocated from the interstitial compartment to the seminiferous tubule compartment. Moreover, the markedly decreased expression of GPER and GPER downstream molecules, cAMP and Ca2+, suggests their potential role in testis pathology. Increased estrogen levels in cryptorchid conditions may be linked to disturbed GPER signaling. We postulate that GPER is a prominent key player in testis development and function and may be used as a new biomarker of horse testis in health and disease.

Keywords: G protein-coupled receptor; cryptorchidism; estrogens; horse.

Figure 1

The general structure of healthy (A,A’,C,E,G,I) and cryptorchid (B,D,F,H,J) horse testicular tissues, respectively, which were used for analysis via scanning electron microscopy. Bars represent 1 µm. Histochemical staining of healthy and cryptorchid horse testes for the presence and localization of total protein content by naphthol blue black (NBB) (E,F), acidic content by toluidine blue O (TBO) (G,H), and polysaccharide content by PAS (I,J). Bar 20 µm. Histochemical staining was performed on three serial sections from each subject’s testis. IT—interstitial tissue, ST—seminiferous tubules.

Figure 2

Left panel. Qualitative expression and relative expression (arbitrary units) of GPER in healthy (control) and cryptorchid horse testes. The relative amount of GPER normalized to β-actin. The relative intensity of bands from three separate analyses is expressed as means ± SD. Asterisks show significant differences between healthy and cryptorchid testes. Significant difference is denoted *** p < 0.001. Right panel. Representative microphotographs of immunohistochemical localization of GPER (A,B) and INSL3 (C,D and higher magnification C’, D’ with positively immunostained individual Leydig cells) in healthy (A,C,C’) and cryptorchid (B,D,D’) horse testes. a,b—negative controls representative for GPER or INSL3 staining. Staining with DAB and counterstaining with hematoxylin. Bar 45 µm. Staining was performed at three serial sections from each animal.

Figure 3

Upper panel: cAMP (A) and Ca2+ (B) levels; lower panel: representative microphotographs of immunohistochemical localization of PKA (C,C’), IP3 I (D,D’), and quantitative analysis of PKA or IP3 I immunosignals in the seminiferous tubules (ST) and interstitial tissues (IT) (E) of healthy (control) and cryptorchid horse testes. Data are expressed as means ± S.D. Significant differences between healthy and cryptorchid testes are denoted as ** p < 0.01 and *** p < 0.001. Analyses were performed in triplicate.

Figure 4

Schematic representation of the biochemical and molecular status of healthy and cryptorchid horse testes demonstrating the possible effects of GPER downstream pathways via cAMP and Ca2+ on the testes from development to maturity under both normal in healthy testes and the increased estrogen levels in cryptorchid testes.