The Sertoli cell tight junction (TJ) of the seminiferous epithelium is important for the developmental process of spermatogenesis as it separates germ cells in the seminiferous tubules from the general circulation in the testicular interstitium. Absence of the TJ leads to spermatogenic arrest and infertility. TJs form at puberty as circulating gonadotrophins luteinising hormone/testosterone and follicle stimulating hormone increase. Several studies have demonstrated hormonal regulation of the two major TJ proteins, claudin-11 and occludin, and also of TJ function in vitro and in vivo. Men with low levels of circulating gonadotrophins exhibit an immature and dysfunctional TJ phenotype, which is reversed upon the exogenous application of gonadotrophins. This thesis hypothesises that claudin-11 and occludin are the major contributors to TJ function, and that gonadotrophins regulate TJ function and structure via these two proteins in several species including humans. This PhD was divided into four separate studies to address these hypotheses. The first study selectively silenced the genetic expression of claudin-11 and occludin with small interfering RNA (siRNA) in cultured immature rat Sertoli cells to determine their contribution to Sertoli cell TJ function in vitro. siRNA treatment against either protein significantly (p less than 0.01) reduced TJ function by ~50% as assessed by transepithelial electrical resistance. Immunocytochemistry displayed marked reductions in the localisation of these proteins to the TJ after siRNA treatment. It was concluded that both proteins significantly contributed to TJ function in vitro. The second and third studies then aimed to study hormonal regulation of the TJ in vivo. Weekly injections of the gonadotrophin releasing hormone antagonist acyline were used to suppress circulating gonadotrophins and spermatogenesis in adult rats. Acyline treatment disrupted i) the localisation of occludin to the TJ and ii) TJ function as shown by permeability to a biotin tracer, which was impermeable to TJs in controls. Short-term hormone replacement partially restored the effects of gonadotrophin suppression. It was concluded that gonadotrophins regulate the maintenance of the TJ in rats in vivo. The third study used the hypogonadal (hpg) mouse, which is a naturally occurring model of gonadotrophin deficiency with inactive spermatogenesis. Claudin-11 in hpg mice was not localised at the TJs, and these were dysfunctional as shown by permeability to biotin. Following hormone treatment, TJs were structurally and functionally competent, demonstrating that gonadotrophins also regulate the formation of TJs in vivo. The fourth study subsequently analysed TJs in gonadotrophin suppressed men, and it was found that claudin-11 staining was reduced from continuous bands in control men, to punctate staining in gonadotrophin-suppressed men, demonstrating that gonadotrophins also regulate the localisation of claudin-11 to the TJ in men in vivo. In summary, it is concluded that the Sertoli cell TJ is hormonally regulated, and that the major contributors to TJ function in vivo and in vitro are claudin-11 and occludin. It is hypothesised that the reduction of claudin-11 localisation to the TJ in men may also result in a loss of human Sertoli cell TJ function, suggesting that the TJ may be a potential target of hormonal contraception in men.

Thesis advisor: Daniel A. Kirschner / The myelin sheath of the central nervous system contains a network of interlamellar tight junctions known as the radial component. Ablation of claudin-11, a tight junction protein, results in the absence of the radial component and compromises the passive electrical properties of the myelin sheath. Although tight junctions are known to regulate paracellular diffusion, this barrier function has not been directly demonstrated for the radial component, and some evidence suggests that the radial component may also, or instead, mediate adhesion between myelin membranes. To investigate the physical properties of claudin-11 tight junctions, we first compared fresh, unfixed Claudin 11-null and control nerves using X-ray diffraction. In Claudin 11-null tissue, we detected no changes in myelin structure, stability, or membrane interactions, which argues against the notion that myelin tight junctions exhibit significant adhesive properties. To examine myelin permeability in the absence of the radial component, we measured the kinetics of osmotic compaction and recovery in knockout and control myelin. We found that myelin lacking claudin-11 responded more rapidly to osmotic stress, indicating an increase in permeability to water and small osmolytes. To further test this hypothesis, we explored the possibility of measuring the diffusion of water through myelin using neutron diffraction, a technique that had been pioneered in myelin decades ago but was largely unused because of previous limitations in neutron technology. After establishing that present-day neutron instruments were capable of measuring diffusion in myelin, we applied this technique to samples from mice lacking claudin-11. Consistent with our X-ray diffraction studies, we found that H2O-D2O exchange was more rapid in Claudin 11-null mice compared to controls. Thus, our data indicate that the radial component serves primarily as a diffusion barrier and elucidate the mechanism by which tight junctions govern myelin function. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.