Volume 12, Issue 2, Pages 109-120 (September 2005) Gonadal steroid modulation of the diabetes (db/db) mutation-induced hyperlipometabolic, hypogonadal syndrome: Restoration of female reproductive tract cytochemical and structural indices  David R. Garris  Pathophysiology  Volume 12, Issue 2, Pages 109-120 (September 2005) DOI: 10.1016/j.pathophys.2005.03.001 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Photomicrographic (HRLM) and DCSP cytochemical lipodeposition comparisons of representative control (A and B: ×400) and littermate 8-week-old diabetes (db/db) mutant (C and D: ×400) uterine endometrial epithelial (UEE) cells demonstrating the hypercytolipidemia (*) associated with expression of the diabetes hypogonadal syndrome. Ovarian follicular (secondary) granulosa cell (GC) layers from +/? (E and F: ×400) and db/db mutants (G and H: ×400) demonstrating elevated interstitial (i) and GC cytolipidemia. Normal UEE and follicular GC compartments presented a viable cytoarchitecture with minimal cytoplasmic lipid deposits as denoted by DCSP cytochemical analysis (B and F). In contrast, the suppressed follicular maturation and enhanced atresia rates, as well as utero-atrophy, that characterize the hypogonadal db/db mutants were associated with pronounced hypercytolipidemia (D and H) in FRT samples as indicated by lipid (yellow fluorescent depositions) cytochemical analysis. Expansion of UEE and GC cytochemical lipid (triglyceride) pools in db/db (D and H) groups was recognized to be accentuated relative to the cytoplasmic volume and distribution patterns of lipid vacuoles in +/? genotype controls (B and F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Representative TEM photomicrographs from db/db C57BL/KsJ mice following chronic, low-dose oil- (A: ×6400), P- (B: ×6400) and E2- (C: ×6000) HRx treatments between 3 and 8 weeks of age. In oil-HRx groups (A), hypercytolipidemia dominated the UEE cellular profiles, characterized by expanded basal, perinuclear and apical lipid (*) pools adjacent to nuclear pycnotic convolutions (arrows). Following P-HRx (B), cytoplasmic lipidemia was reduced, and organelle profiles enhanced, in db/db mice; however, nuclear membranes convolutions persisted. In contrast, E2-HRx (C) in db/db mutants stimulated smooth nuclear membranes, well defined cytoplasmic organelles with expanded golgi cisternal (gc) channels and limited cytolipidemia. Ovarian follicular (secondary antral) GC compartments (D: ×4300) from oil-HRx db/db groups presented dense interstitial and cellular lipidemia characteristic of premature hypogonadal atrophy. Following P-HRx (E: ×4700), GC cytoplasmic profiles indicated reduced cytolipidemia associated with prominent organelle compartment expansion, but the retention of perinuclear lipid (*) pools present in dispersed GC populations. In contrast, E2-HRx (F: ×4300) promoted well defined GC cytoarchitecture profiles, including minimal cytolipidemia, expanded organelle compartments with dense mitochondrial (m), endoplasmic reticulum (er) and golgi expansions. Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 The influences of P-HRx (A–D: ×1000) on UEE (A and B) and follicular GC (C and D) cytoarchitecture and cytolipid deposition analysis in db/db mutants at 8 weeks of age. P-HRx restricted the mutation-induced UEE hypercytolipidemia (*) to basopolar compartments in affected cells (A and B), in contrast to the dispersed lipid pools observed in oil-HRx groups (Fig. 1). Follicular GC hypercytolipidemia remained prominent within the ovarian interstitium, but was reduced in the GC compartments relative to oil-HRx groups (Fig. 1). In contrast, E2-HRx diminished the cytolipid profiles in db/db UEE (E and F) and ovarian follicular GC (G and H) compartments to comparable oil-HRx +/? indices (Fig. 1). Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Electron photomicrographic analysis of db/db C57BL/KsJ mutant UEE cellular ultrastructure following oil- (A: ×6200), P- (C: ×6700) and E2- (E: ×7400) HRx treatments, and comparable digital-color enhancement TEM micrographic depiction (B, D and F) following computerized, color-scale imaging of intracellular lipid depositions. In comparison to the oil-HRx db/db-associated hypercytolipidemia (*) and nuclear membrane pycnotic convolution profiles (A and B), P-HRx groups (C and D) demonstrated reduced intracellular and perinuclear lipid (*) depositions, but remained sensitive to intranuclear lipid (Ln) invasion. In contrast, E2-HRx groups (E and F) demonstrated minimal cytolipidemia (*), smooth nuclear (n) contours, dispersed (active) chromatin profiles and prominent organelle compartmentalization with expanded golgi cisternal (gc) volumes. Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Representative photomicrographs of db/db-associated nuclear apoptosis (T: TUNEL-labeled 3′-DNA fragmentation) within follicular GC compartments by HRLM (A, D and G: ×1000) analysis, sequential section cytochemical analysis of hyperlipidemia (B, E and H: ×1200) and co-localization of nuclear lipoapoptosis (i.e., lipid infiltration of TUNEL-labeled nuclei) and cytolipid (*) depositions (C, F and I: ×1200) following oil-HRx (A–C), P-HRx (D–F) or E2-HRx (G–I) between 3 and 8 weeks of age. In oil-HRx db/db mutant groups (A–C), TUNEL-labeled (T) cellular apoptosis, hypercytolipidemia (*) and nuclear lipidemia (Ln) characterized the follicular GC compartment profiles associated with depressed ovarian biomass (Table 1). In P-HRx db/db groups (D–F), TUNEL-indexed apoptosis and cytoplasmic lipidemia (*) were presented in selected GC cell populations, which demonstrated nuclear lipoapoptosis (Ln). In contrast, E2-HRx db/db groups (G–I) demonstrated GC compartments composed of cells exhibiting viable cytoarchitectures (with minimal TUNEL-labeled (T) apoptosis and cytolipid (*) accumulation) in the absence of nuclear lipodepositions, which typically characterized oil-HRx (C) mutant groups. Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 High magnification (×2500) cytolipidemia (A) and TUNEL (B) imaging of db/db UEE tissue and stromal (s) compartments accentuating the intercellular hyperlipidemia (*: fluorescent yellow lipid) and co-incident lipoapoptotic nuclear dissolution (arrow) that characterizes the uterine organoinvolution process by 8 weeks of age in oil-HRx (A and B) C57BL/KsJ mice. Following P-HRx (C and D), db/db UEE samples demonstrated a moderate reduction in hypercytolipidemia profiles, but TUNEL-labeled (white label within nuclear envelope) nuclear lipoinvasion (apoptosis) remained a consistent cytocharacteristic (B and D: arrows). In contrast, E2-HRx (E and F) of db/db C57BL/KsJ mice minimized UEE cytolipidemia (E), restraining lipid vacuole accumulations to the normal basopolar cytoplasmic compartment typical of oil-HRx +/? groups (Fig. 1), and prevented the occurrence of nuclear lipid invasion associated with apoptosis-induced cytolipoatrophy (F), under increased tissue mass and normoglycemic systemic (Table 1) indices. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Pathophysiology 2005 12, 109-120DOI: (10.1016/j.pathophys.2005.03.001) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions