Gene Expression of ACTH, Glucocorticoid Receptors, 11βHSD Enzymes, LH‐, FSH‐, GH Receptors and Aromatase in Equine Epididymal and Testicular Tissue

Abstract

Glucocorticoids (GCs) are important mediators of the stress response and have been implicated in the function and regulation of testicular functions in different species. In many tissues, intracellular glucocorticoid activity is controlled by either or both of the two known isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 and 2, which interconvert active and inactive GCs. Little is known about the effects of stress on fertility in the equine species. The main objective of the present study was to investigate the expression of receptors for GCs and adrenocorticotropic hormone [ACTH, melanocortin 2 receptor (MC2R)] as well 11βHSD1 and 11βHSD2 in male equine epididymal and testicular tissue. In addition, expression of aromatase P-450 and receptors for luteinizing hormone (LHR), follicle stimulating hormone (FSHR) and growth hormone (GHR) was studied. Reverse transcriptase PCR and quantitative real-time PCR were performed in tissue from the epididymis (caput and cauda) and testes collected from nine healthy mature stallions (age 4-10 years). mRNA for ACTH and GC receptors as well as 11βHSD1 and -2 were found in epididymal and testicular tissue. Expression of the genes studied was always positive in testicular tissue, while it was inconsistent in epididymal tissue. Quantitative gene expression in relation to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was significantly correlated (R = 0.403, p < 0.001). Quantitative PCR in relation to β-actin revealed significant differences in the gene expression of 11βHSD1, 11βHSD2, LHR, FSHR, MC2R and aromatase between tissue collected from caput epididymidis, cauda epididymidis and testicular parenchyma (p < 0.05). With GAPDH, differences between tissues were significant for 11βHSD1, 11βHSD2 and MC2R (p < 0.05) In addition, high concentrations of mRNA of aromatase and receptors of LH and FSH were found in testicular tissue, while a pronounced expression of GH receptor was present in epididymal tissue. The results support the hypothesis of an interaction between the pituitary-adrenal axis and testicular function in the stallion.

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes

“…This study identified very high expression of the NR3C1 gene, which encodes the GR protein, in the testes of adult stallions. It agrees with previous work that localized the GR protein primarily in the Leydig cells of stallion testes . These data indicate that the steroidogenic Leydig cells are poised to respond to glucocorticoids such as dexamethasone.…”

“…Leydig cells were cultured from one testis from each of the three Quarter Horse stallions using a previously optimized protocol with modifications . Numerous cultures can be set up from one stallion because each testis weighs an average of 165 g and contains 13% Leydig cells by volume . Briefly, fresh testis parenchyma (10 g) was minced and incubated in 12 mL of HBSS containing 1 mg/mL collagenase, 0.25 mg/mL soy bean trypsin inhibitor and 1 mg/mL BSA in a 37°C water bath with shaking at 100 rpm for 30 minutes.…”

“…However, glucocorticoids also affect the Leydig cells in the testes directly. Leydig cells express very high levels of the NR3C1 gene encoding the glucocorticoid receptor (GR) protein . In the testes of rats injected with radioactive dexamethasone, 86% of the radioactivity in the testis parenchyma was specifically bound to GR proteins within Leydig cells, which compose only 3% of the volume of the rat testis .…”

Global Gene Expression Analysis in Human Uterine Epithelial Cells Defines New Targets of Glucocorticoid and Estradiol Antagonism1

“…Remarkably, the folliclestimulating hormone receptor (FSHR) gene is one of the genes regulated only in the presence of Dex and E 2 in ECC1 cells. Expression levels were 3.4-fold lower than those of the vehicle, which is supported by reports of glucocorticoid repression of FSHR in the male reproductive tract (epididymis and testicular tissue) [49]. FSHR is required for fertility and mRNA expression increases in the human endometrium following ovulation [50,51].…”

A comparison between the equine and bovine hypothalamus-pituitary-adrenocortical axis

“…On the other hand, the enzyme 11bHSD type 2 interconverts active cortisol into inactive cortisone. For instance, 11bHSD1 and 2 were found in epididymal and testicular tissue from healthy mature stallions [96].…”