The Influence of Estrogen Signaling on Male Reproduction in Medaka (Oryzias latipes)

Abstract

<p>Endocrine disrupting chemicals (EDCs) are ubiquitous and often act as xenoestrogens with the ability to disrupt estrogen signaling through differential binding to the various estrogen receptors. Exposure to these xenoestrogens has led to detrimental effects on male reproduction. In fish, observed effects include sex reversal, presence of testicular oocytes, altered courting behavior, vitellogenin synthesis in males, altered fertility and gonadal histopathology. Understanding how xenoestrogens exert their effects is complicated by the existence of multiple estrogen receptors (ESR1, ESR2a, ESR2b, and GPER), coupled with their ability for crosstalk and differential binding capability of selective estrogen receptor modulators (SERMS). Additionally, estrogen can signal through both classic genomic signaling and nongenomic signaling. Furthermore, the importance of estrogen signaling in normal male reproduction is just beginning to be understood. The primary goal of this dissertation was to assess the implications of aberrant estrogen signaling on male reproductive capacity, testicular morphology and gene expression changes in the small aquarium model fish, medaka, by investigating effects of a general estrogen receptor agonist, ethinylestradiol (EE2), and those of a G-protein estrogen receptor (GPER) specific agonist, G-1. This was assessed through breeding experiments, histological assessment of testicular morphology and microarray assessment of testicular gene expression changes following exposure to EE2 and G-1. Finally, a comparison of altered testicular morphology between EE2 and G-1 induced changes was further assessed using a variety of histological techniques. The findings demonstrate that a 14-day exposure to EE2 impaired male reproductive capacity and altered testicular morphology and gene expression in a time- and dose-dependent manner. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. These morphologic changes were highly associated with gene expression changes. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell proliferation, collagen production/extracellular matrix organization, and protein ubiquitination among others. Comparatively, a 14-day exposure to G-1 did not affect male reproductive capacity but did alter testicular morphology and gene expression. The histological analysis found an increased cellularity of the interstitium leading to thickened interstitium but no change in germinal epithelium. The microarray data indicate differential expression in genes most commonly involved in cell cycle, cell proliferation, apoptosis, transcription, translation, and ubiquitination. Finally, an assessment of the testicular histological phenotypes following EE2 and G-1 exposure indicate different morphologic changes led to thickened interstitium observed in the two exposures. In EE2 exposed fish, thickening of interstitium was associated with increased collagen deposition on the periphery of the organ while the interior thickening was primarily due to the collapse of intralobular space associated with decreased germinal epithelium. In the G-1 exposed fish, the thickened interstitium was due to increased cellularity. A modest increase in cell proliferation was observed contributing to the increase in interstitial cells, however, it is also possible that there is a decrease in normal apoptosis and cell turnover as well. These findings highlight the importance of anchoring gene expression changes with morphology and ultimately proper tissue/organ function as well as the potential differences in effects that may occur with EDCs and SERMs.</p>