Reproductive cytotoxicity is predicted by magnetic resonance microscopy and confirmed by ubiquitin-proteasome immunohistochemistry in a theophylline-induced model of rat testicular and epididymal toxicity.

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques. In vivo T1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring. In vivo MRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting that in vivo MRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use of in vivo MRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

LPS-sensory peptide communication in experimental cystitis.

Stimulation of sensory nerves can lead to release of peptides such as substance P (SP) and consequently to neurogenic inflammation. We studied the role of bacterial lipopolysaccharide (LPS) in regulating SP-induced inflammation. Experimental cystitis was induced in female mice by intravesical instillation of SP, LPS, or fluorescein-labeled LPS. Uptake of fluorescein-labeled LPS was determined by confocal analysis, and bladder inflammation was determined by morphological analysis. SP was infused into the bladders of some mice 24 h after exposure to LPS. In vitro studies determined the capacity of LPS and SP to induce histamine and cytokine release by the bladder. LPS was taken up by urothelial cells and distributed systemically. Twenty-four hours after instillation of LPS or SP, bladder inflammation was characterized by edema and leukocytic infiltration of the bladder wall. LPS pretreatment enhanced neutrophil infiltration induced by SP, increased in vitro release of histamine, tumor necrosis factor-alpha, and interferon-gamma, and significantly reduced transforming growth factor-beta1 release. These findings suggest that LPS amplifies neurogenic inflammation, thereby playing a role in the pathogenesis of neurogenic cystitis.

Subcellular localization of beta1,4-galactosyltransferase on bull sperm and its function during sperm-egg interactions.

The process of sperm-oocyte recognition is a complex interaction between the plasma membrane of sperm and the extracellular matrix of the oocyte. The best studied mammalian system is the mouse, in which sperm plasma membrane receptors recognize specific oligosaccharides on the egg coat glycoprotein ZP3. A well-defined ZP3 receptor on mouse sperm is beta1,4-galactosyltransferase (GalT). In this study, we investigated the possibility that GalT is present on bull sperm, and that it may participate during bovine sperm-oocyte binding. Using Western immunoblotting, bull sperm were found to have a protein of molecular weight similar to mouse GalT at approximately 60 kDa. Immunogold low voltage scanning electron microscopy reveals that GalT epitopes are confined to the anterior cap of fresh or capacitated bull sperm. To investigate the function of bovine sperm GalT, fresh bull sperm were pretreated with either preimmune or anti-GalT antibody and added to in vitro-matured bovine oocytes. Sperm exposed to preimmune serum fertilized 82.7% (153 of 185) of the oocytes, whereas sperm exposed to anti-GalT antiserum fertilized only 42.3% (202 of 478) of the oocytes. We determined whether the inhibition of fertilization resulted from a direct inhibition of sperm-oocyte binding. The number of sperm bound to eggs was determined by low voltage scanning electron microscopy following pretreatment with preimmune or anti-GalT antibody. An average of 25.3+/-2.2 (mean +/- SEM) sperm bound per half-oocyte when treated with preimmune serum. In contrast, exposure of sperm to anti-GalT antiserum significantly lowered (P<0.001) the frequency of sperm binding to 9.9+/-0.8 bound per half-oocyte. These results show that GalT is present on the anterior cap of the bovine sperm head, where it participates in fertilization by facilitating sperm-oocyte binding. The function of GalT in both the murine and bovine systems suggests that it may serve as a generalized gamete receptor in mammals.

Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction.

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe MitoTracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 nM MitoTracker. Exposure of bull sperm to 0.1-10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization.

Use of gold-labeled ovalbumin to correlate antigen deposition and localization with tissue response.

This study was performed to evaluate a technique for correlating the location of antigen within sensitized tissues with physiological response. Guinea pigs actively sensitized by intraperitoneal injection of ovalbumin (OVA) were euthanized, and urinary bladders were removed. Gold beads (18 nM diameter) were conjugated to OVA (OVA-Au) and bovine serum albumin (BSA-Au). Bladder tissue was suspended in tissue baths, exposed to OVA, OVA-Au, BSA, and BSA-AU, and tissue contraction and histamine release were determined. Bladder tissues were examined by electron microscopy to determine distribution of gold-labeled antigen at 1 and 5 min after exposure. Exposure of bladder tissue from sensitized guinea pigs to OVA stimulated concomitant contraction and histamine release which reached maximal levels within 3 min; bladder tissue from control, nonsensitized guinea pigs did not respond to OVA. BSA failed to stimulate response from OVA-sensitized or control bladder tissue. Labeled antigen was adhered to mucosa of sensitized bladder tissue 1 min after exposure to OVA-Au. OVA-Au was present within the mucosa and submucosa of sensitized tissues within 5 min. OVA-Au did not adhere to, or become internalized by, control tissues, and BSA-Au did not adhere to, or become internalized by, any tissues. Labeling of antigen with gold allowed the location of antigen within tissues to be determined and did not affect the response of sensitized tissues to antigen exposure.

Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures.

Intracytoplasmic sperm injection (ICSI) was performed on rhesus monkey oocytes, and the resultant microtubule and DNA configurations were imaged by laser-scanning confocal microscopy. In addition, polyspermic oocytes fertilized by ICSI were examined by transmission electron microscopy (TEM). Successful rhesus fertilization by ICSI revealed microtubule and DNA configurations similar to those observed during in vitro fertilization of human and rhesus monkey oocytes, including sperm aster formation, pronuclei decondensation, spindle formation, and cell division. Several abnormalities, however, were also observed: 1) inability to complete meiosis; 2) inability to undergo male or female pronucleus formation; 3) separation of the sperm tail from the sperm nucleus; 4) premature chromosome condensation with the formation of a paternal meiotic spindle; and 5) formation of multiple female pronuclei (karyomeres) during chromosome decondensation. TEM analysis revealed that sperm can undergo decondensation in the presence of an intact acrosome at least 18 h after sperm injection. These results demonstrate the utility of rhesus ICSI in pre-clinical applications as well as with endangered species. However, the different types of fertilization failures observed here indicate that although ICSI may be a readily accepted means of fertilization of human oocytes in many clinics, we should further characterize the cellular and genetic abnormalities associated with ICSI in both human and nonhuman primates.

Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly.

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction.

Differential release of prostaglandins and leukotrienes by sensitized guinea pig urinary bladder layers upon antigen challenge.

The relative contributions of mucosal/submucosal and detrusor layers to the release of inflammatory mediators were investigated in isolated, ovalbumin (OVA) sensitized guinea pig urinary bladders. Ovalbumin challenge of sensitized mucosa induced release of prostaglandins (PG): PGE2, PGD2 and PGF2 alpha, in that order of magnitude. The total release of PGs was significantly higher from the mucosa/submucosa than from the detrusor/serosa. Under the same conditions, net release of leukotriene (LT) was observed predominantly from the detrusor. The total amount of histamine released from the mucosa was greater than that from the muscle layer. These results indicate differential production and release of inflammatory mediators from the mucosal/submucosal and detrusor smooth muscle layers. These results may have serious implications in disorders, such as interstitial cystitis, involving bladder mucosal damage. The cytoprotective effect of PGE2 is likely to be lost when the mucosa is damaged, and LT release from deeper layers may contribute significantly to symptoms of bladder inflammation.

Alteration of reactivity of native arteries induced by venous graft placement.

Occlusion of aortocoronary venous grafts can be due to thrombosis, atherosclerosis, or vasospasm. Investigations have focused on properties of the graft itself, and little is known about the vascular reactivity and function of the native arteries proximal and distal to the vein graft, although spasm of the native artery distal to the graft site has been observed in patients. We hypothesized that the function of the endothelium of the native arteries may be altered after surgery. Autogenous venous grafts were placed in femoral arteries of rabbits to study the reactivity of the native arteries after grafting. Four weeks after graft implantation, the vein graft, ipsilateral vein, and native artery proximal and distal to the graft were removed for in vitro studies. Morphological evaluation by scanning electron microscopy and fluorescence microscopy after labeling with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate indicated the presence of an intact, metabolically active endothelial layer. There was no alteration in the contractile responses to phenylephrine of the arteries, vein grafts, or veins. Precontracted vein grafts, veins, and arterial segments proximal to the grafts relaxed when exposed to endothelium-dependent vasodilators (acetylcholine, arachidonic acid, and substance P), but the native arteries distal to the grafts did not. In bioassay cascade experiments, the distal artery did not release any measurable relaxing factor when exposed to acetylcholine. We conclude that the endothelium of the distal artery did not function normally. The extent and reversibility of altered endothelial function remain to be determined. This observation may help to explain the occurrence of myocardial infarction after aortocoronary bypass grafting in some patients.

Removal of venous endothelium with air.

Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or enzymatic processes. It has been observed that the technique used for the removal of arterial endothelium may profoundly affect smooth muscle function and release of prostanoids by the vessel wall. The function and patterns of regeneration of arterial endothelium have been extensively described, but there is a relative paucity of information about the venous endothelium, due in part to the difficulty of its removal. We developed a technique for removal of the endothelium of rabbit femoral veins by passing a stream of air through the lumen of the vessel to dry and remove the endothelium. The effectiveness of endothelium removal was verified by the lack of in vitro reactivity to endothelium-dependent relaxing substances, examination of frozen sections of vessels, labeled with fluorescent-tagged acetylated low-density lipoprotein, with fluorescent light microscopy and scanning electron microscopy of vessel segments. Air drying effectively removed the endothelium and abolished mechanical responses to endothelium-dependent vasodilators but did not affect the function of the smooth muscle. We propose the use of air to remove endothelium from veins to be used to study endothelium-derived factors since this method achieves complete removal of endothelium without causing detectable damage (morphological or functional) to the remainder of the vessel wall.

Phagocytosis of opsonized Treponema pallidum subsp. pallidum proceeds slowly.

Macrophages were found to phagocytize Treponema pallidum subsp. pallidum attached to polycarbonate filters. This environment simulated the in vivo interaction of surface-adherent treponemes with macrophages. The phagocytosis of T. pallidum subsp. pallidum was found to proceed slowly. Heat-killed T. pallidum subsp. pallidum were susceptible to opsonization with 2% immune serum, whereas live treponemes were resistant to this concentration of antibody. High concentrations of immune serum were found to increase phagocytosis of the spirochetes. Live T. pallidum subsp. pallidum had bound limited quantities of immunoglobulin G in vivo, and only opsonization with 20% immune serum resulted in a detectable increase in surface-bound immunoglobulin in vitro. Kinetic studies suggested a steady rate of phagocytosis that is considerably slower than with other bacteria. Scanning electron microscopy studies of the phagocytizing macrophages showed that the treponemes were detached from the membrane filters and scooped onto the ruffled portion of the macrophage surface. This lengthy physical process, along with the lack of a dramatic increase in ingestion after opsonization, may account for the slow rate of phagocytosis.