Browsing by Subject "Testis"

The mouse gonad begins its development as a bipotential primordia, capable of developing into a testis or ovary depending on the presence of the sex-determining gene, Sry. In the XY gonad, opposing pro-testis and pro-ovary pathways compete in gonadal supporting cells. While the individual cellular decision process is well understood, the higher-level process of coordination of cell fates across the gonad remains to be explained. The testis and ovary exhibit distinct patterns of differentiation, suggesting that either development of each organ requires a particular organizing principle or bipotentiality requires regional separation for fate specification or stabilization. The overall goal of this work is to improve characterizations of the spatiotemporal features of sex determination and gonadogenesis, including cell fate organization, morphogenic processes, and system context.Though several hypotheses have connected gonad morphogenesis to sex determination, the morphogenic processes that occur in the gonad have not been sufficiently characterized for formulating testable hypotheses. To capture and analyze the complexity of genital ridge morphogenesis, we generated a 3-dimensional time course of gonad development in native form and context using whole embryo tissue clearing and light sheet microscopy. Analysis revealed that the early gonad exhibits anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain. In extending characterization to the neighboring nephric ducts, we found a close alignment of gonad and mesonephric duct movements as well as delayed duct development in Cbx2 mutants, which undergo XY sex reversal and gonad dysgenesis. These data support mechanical integration of gonad and mesonephric duct morphogenesis. In investigating the mechanisms underlying the center-to-pole pattern of testis differentiation, we performed anteroposterior axis analyses and ex vivo gonad reconstruction cultures. These experiments allowed us to rule out two commonly accepted theories in the field: paracrine relay and center-first Sry expression. After searching for patterns in other cellular processes during gonadogenesis, including cell cycle arrest and coelomic epithelium proliferation, we uncovered a center-biased pattern of supporting cell precursor ingression. The updated model indicates that differences between the patterns of differentiation in the testis and ovary are due to features of their respective regulatory networks connecting their fate dynamics to different general gonadal organizing principles acting upstream of supporting cell differentiation. Following recent work on the rete testis and rete ovarii suggesting these structures contribute to gonadal supporting cell populations, we characterized early development of the rete and adjacent tissues in both sexes. Comparison of the GATA4+/PAX8+ presumptive rete with mesonephric and gonadal cells led to the identification of undescribed patterns in mesonephros development which may play a role in sexual dimorphism of the rete. Cells of the rete may derive from mesonephric condensates in a process similar to kidney nephron development. Cell cycle analysis revealed the mesonephric tubules and early rete to be a largely non-proliferating population of cells, suggesting expansion through recruitment of new cells. These results were used to establish preliminary theories for lineage relationships in early urogenital development. Initial attempts at lineage tracing to test the theory were unsuccessful. The findings presented here contribute to a more comprehensive and systems level understanding of sex determination and gonad development. In particular, the incorporation of high-resolution spatial information into theories of sex determination serves to connect individual cell fate decisions to organ level patterns of differentiation in space and time. These results will be useful for novel hypothesis generation as well as for designing more detailed models and simulations of sex determination and gonadogenesis.

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.