Morphology and Mechanics of the Fin Whale Esophagus: The Key to Fast Processing of Large Food Volumes by Rorquals.

Lunge feeding rorqual whales feed by engulfing a volume of prey laden water that can be as large as their own body. Multiple feeding lunges occur during a single foraging dive and the time between each lunge can be as short as 30 s (Goldbogen et al. 2013). During this short inter-lunge time, water is filtered out through baleen to concentrate prey in the oral cavity, and then the prey is swallowed prior to initiating the next lunge. Prey density in the ocean varies greatly, and despite the potential of swallowing a massive volume of concentrated prey as a slurry, the esophagus of rorqual whales has been anecdotally described as unexpectedly narrow with a limited capacity to expand. How rorquals swallow large quantities of food down a narrow esophagus during a limited inter-lunge time remains unknown. Here, we show that the small diameter muscular esophagus in the fin whale is optimized to transport a slurry of food to the stomach. A thick wall of striated muscle occurs at the pharyngeal end of the esophagus which, together with the muscular wall of the pharynx, may generate a pressure head for transporting the food down the esophagus to the stomach as a continuous stream rather than separating the food into individual boluses swallowed separately. This simple model is consistent with estimates of prey density and stomach capacity. Rorquals may be the only animals that capture a volume of food too large to swallow as a single intact bolus without oral processing, so the adaptations of the esophagus are imperative for transporting these large volumes of concentrated food to the stomach during a time-limited dive involving multiple lunges.

Retia mirabilia: Protecting the cetacean brain from locomotion-generated blood pressure pulses.

Cetaceans have massive vascular plexuses (retia mirabilia) whose function is unknown. All cerebral blood flow passes through these retia, and we hypothesize that they protect cetacean brains from locomotion-generated pulsatile blood pressures. We propose that cetaceans have evolved a pulse-transfer mechanism that minimizes pulsatility in cerebral arterial-to-venous pressure differentials without dampening the pressure pulses themselves. We tested this hypothesis using a computational model based on morphology from 11 species and found that the large arterial capacitance in the retia, coupled with the small extravascular capacitance in the cranium and vertebral canal, could protect the cerebral vasculature from 97% of systemic pulsatility. Evolution of the retial complex in cetaceans-likely linked to the development of dorsoventral fluking-offers a distinctive solution to adverse locomotion-generated vascular pulsatility.

Mechanical contribution of lamellar and interlamellar elastin along the mouse aorta.

The mechanical properties of aortic elastin vary regionally, but the microstructural basis for this variation is unknown. This study was designed to identify the relative contributions of lamellar and interlamellar elastin to circumferential load bearing in the mouse thoracic and abdominal aortas. Forces developed in uniaxial tests of samples of fresh and autoclaved aorta were correlated with elastin content and morphology obtained from histology and multiphoton laser scanning microscopy. Autoclaving should render much of the interlamellar elastin mechanically incompetent. In autoclaved tissue force per unit sample width correlated with lamellar elastin content (P≪0.001) but not total elastin content. In fresh tissue at low strain where elastin dominates the mechanical response, forces were higher than in the autoclaved tissue, but force did not correlate with total elastin content. Therefore although interlamellar elastin likely contributed to the stiffness in the fresh aorta, its contribution appeared not in proportion to its quantity. In both fresh and autoclaved tissue, elastin stiffness consistently decreased along the abdominal aorta, a key area for aneurysm development, and this difference could not be fully accounted for on the basis of either lamellar or total elastin content. These findings are relevant to the development of mathematical models of arterial mechanics, particularly for mouse models of arterial diseases involving elastic tissue. In microstructural based models the quantity of each mural constituent determines its contribution to the total response. This study shows elastin's mechanical response cannot necessarily be accounted for on the basis of fibre quantity, orientation, and modulus.

Cardiovascular design in fin whales: high-stiffness arteries protect against adverse pressure gradients at depth.

Fin whales have an incompliant aorta, which, we hypothesize, represents an adaptation to large, depth-induced variations in arterial transmural pressures. We hypothesize these variations arise from a limited ability of tissues to respond to rapid changes in ambient ocean pressures during a dive. We tested this hypothesis by measuring arterial mechanics experimentally and modelling arterial transmural pressures mathematically. The mechanical properties of mammalian arteries reflect the physiological loads they experience, so we examined a wide range of fin whale arteries. All arteries had abundant adventitial collagen that was usually recruited at very low stretches and inflation pressures (2-3 kPa), making arterial diameter largely independent of transmural pressure. Arteries withstood significant negative transmural pressures (-7 to -50 kPa) before collapsing. Collapse was resisted by recruitment of adventitial collagen at very low stretches. These findings are compatible with the hypothesis of depth-induced variation of arterial transmural pressure. Because transmural pressures depend on thoracic pressures, we modelled the thorax of a diving fin whale to assess the likelihood of significant variation in transmural pressures. The model predicted that deformation of the thorax body wall and diaphragm could not always equalize thoracic and ambient pressures because of asymmetrical conditions on dive descent and ascent. Redistribution of blood could partially compensate for asymmetrical conditions, but inertial and viscoelastic lag necessarily limits tissue response rates. Without pressure equilibrium, particularly when ambient pressures change rapidly, internal pressure gradients will develop and expose arteries to transient pressure fluctuations, but with minimal hemodynamic consequence due to their low compliance.

The role of dynamin 3 in the testis.

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.

Non-muscle cofilin is a component of tubulobulbar complexes in the testis.

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus).

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.

Rat seminiferous epithelium contains a unique junction (Ectoplasmic specialization) with signaling properties both of cell/cell and cell/matrix junctions.

The seminiferous epithelium contains unique actin related cell-cell junctions, termed ectoplasmic specializations (ESs). Turnover of these junctions is fundamental to sperm release and to movement of spermatocytes from basal to adluminal compartments of the epithelium during spermatogenesis. In this study we report several novel observations related to the spatial and temporal distribution of integrin-related signaling molecules at ESs. We confirm the presence of beta(1)-integrin at these sites and further demonstrate co-localization of integrin linked kinase (ILK). beta(1)-Integrin and ILK were shown by immunoprecipitation to associate in whole cell lysates of seminiferous epithelium. This observation provides the first evidence for a direct beta(1)-integrin/ILK interaction in noncultured epithelium. Pan-cadherin and beta-catenin antibodies did not react at ESs. Rather, antibodies reacted with desmosome-like junctions that are present both at basal junctional complexes between Sertoli cells and at sites of attachment to spermatogenic cells. Focal adhesion kinase (FAK), a known integrin-associated molecule, did not codistribute with beta(1)-integrins and did not associate with these adhesion molecules in immunoprecipitation studies. Although FAK was expressed in the epithelium, it appeared to be limited to the cytoplasm of early spermatogenic cells. Significantly, polyclonal antibodies against phosphotyrosine-containing residues reacted strongly at ESs, with highest levels detected during sperm release and turnover of basal junction complexes. Our observations indicate that ESs share cell signaling features both of cell-cell junctions and of cell-extracellular matrix junctions.

Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo.

We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt.

NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization.

Teleost fishes, living in fresh water, engage in active ion uptake to maintain ion homeostasis. Current models for NaCl uptake involve Na(+) uptake via an apical amiloride-sensitive epithelial Na(+) channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or alternatively by an amiloride-sensitive Na(+)/H(+) exchange (NHE) protein, and apical Cl(-) uptake mediated by an electroneutral, SITS-sensitive Cl(-)/HCO(3-) anion-exchange protein. Using non-homologous antibodies, we have determined the cellular distributions of these ion-transport proteins to test the predicted models. Na(+)/K(+)-ATPase was used as a cellular marker for differentiating branchial epithelium mitochondria-rich (MR) cells from pavement cells. In both the freshwater tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), V-ATPase and ENaC-like immunoreactivity co-localized to pavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical anion-exchanger-like immunoreactivity is found in the MR cells. Thus, a freshwater-type MR chloride cell exists in teleost fishes. The NHE-like immunoreactivity is associated with the accessory cell type and with a small population of pavement cells in tilapia.

Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri).

The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.

Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques (ectoplasmic specializations).

The mechanism responsible for spermatid translocation in the mammalian seminiferous epithelium was proposed to be the microtubule-based transport of specialized junction plaques (ectoplasmic specializations) that occur in Sertoli cell regions attached to spermatid heads. These plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments and the adjacent plasma membrane. It is predicted that motor proteins function to move the junction plaques, and hence the attached spermatids, first towards the base and then back to the apex of the epithelium, along microtubules. If this hypothesis is true, motor proteins should be associated with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. In addition, isolated junction plaques should support microtubule movement both in the plus and minus directions to account for the bidirectional translocation of spermatids in vivo. To determine if cytoplasmic dynein is localized to the endoplasmic reticulum of the plaques, perfusion-fixed rat testes were immunologically probed, at the ultrastructural level, for the intermediate chain of cytoplasmic dynein (IC74). In addition, testicular fractions enriched for spermatid/junction complexes were incubated with and without gelsolin, centrifuged and the supernatants compared, by western blot analysis, for Glucose Regulated Protein 94 (a marker for endoplasmic reticulum) and IC74. At the ultrastructural level, the probe for IC74 clearly labelled material associated with the cytoplasmic face of the endoplasmic reticulum component of the junction plaques. In the gelsolin experiments, both probes reacted more strongly with appropriate bands from the gelsolin-treated supernatants than with corresponding bands from controls. To determine if the junction plaques support microtubule transport in both directions, polarity-labelled microtubules were bound to isolated spermatid/junction complexes and then assessed for motility in the presence of ATP and testicular cytosol (2 mg/ml). Of 25 recorded motility events, 17 were in a direction consistent with a plus-end directed motor being present, and 8 were in the minus-end direction. The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions and that both a kinesin-type and a dynein-type motor may be associated with the junction plaques. The data also indicate that cytoplasmic dynein is localized to the cytoplasmic face of the endoplasmic reticulum component of the plaques.

Unique and multifunctional adhesion junctions in the testis: ectoplasmic specializations.

In this paper, we review the structure and function of a unique type of actin-related intercellular adhesion junctions in the testis. Based on their ultrastructure, the junctions are divided into five distinct domains. The currently identified molecular components of each domain are summarized. In addition, the architecture of the mammalian system is compared with that of non-mammalian vertebrates. Functionally, the junctions are related to the turnover of adhesion between Sertoli cells, to the attachment of spermatids to the seminiferous epithelium, and to sperm release. They also are part of the mechanism by which spermatids are moved through the epithelium. Evidence consistent with adhesion and motility related functions is discussed. Control, both of junction turnover and of microtubule-based transport, is identified as an important avenue for future research.

Spermatid translocation in the rat seminiferous epithelium: coupling membrane trafficking machinery to a junction plaque.

In this study, we demonstrate that specialized junction plaques that occur between Sertoli cells and spermatids in the rat testis support microtubule translocation in vitro. During spermatogenesis, Sertoli cells are attached to spermatids by specialized adhesion junctions termed ectoplasmic specializations (ESs). These structures consist of regions of the plasma membrane adherent to the spermatid head, a submembrane layer of tightly packed actin filaments, and an attached cistern of endoplasmic reticulum. It has been proposed that motor proteins on the endoplasmic reticulum interact with adjacent microtubules to translocate the junction plaques, and hence the attached spermatids, within the epithelium. If this hypothesis is true, then isolated junctions should support microtubule transport. To verify this prediction, we have mechanically isolated rat spermatids, together with their attached ESs, and tested them for their ability to transport microtubules in vitro. Most assays were done in the presence of 2 mg/ml testicular cytosol and at room temperature. ESs attached to spermatids supported microtubule translocation. In some cases in which motility events were detected, microtubules moved smoothly over the junction site. In others, the movement was slow but progressive, saltatory and "inch-worm-like." No motility was detected in the absence of exogenous ATP or in the presence of apyrase (an enzyme that catalyses the breakdown of ATP). Our results are consistent with the microtubule-based motility hypothesis of spermatid translocation.

Rat testis motor proteins associated with spermatid translocation (dynein) and spermatid flagella (kinesin-II).

In this study, we report sites in the seminiferous epithelium of the rat testis that are immunoreactive with antibodies to the intermediate chain of cytoplasmic dynein and kinesin II. The study was done to determine whether or not microtubule-dependent motor proteins are present in Sertoli cell regions involved with spermatid translocation. Sections and epithelial fragments of perfusion-fixed rat testis were probed with an antibody (clone 74.1) to the intermediate chain of cytoplasmic dynein (IC74) and to kinesin-II. Labeling with the antibody to cytoplasmic dynein was dramatically evident in Sertoli cell regions surrounding apical crypts containing attached spermatids and known to contain unique intercellular attachment plaques. The antibody to kinesin II reacted only with spermatid tails. The levels of cytoplasmic dynein visible on immunoblots of supernatants collected from spermatid/junction complexes treated with an actin-severing enzyme (gelsolin) were greater than those of controls, indicating that at least some of the dynein may have been associated with Sertoli cell junction plaques attached to spermatids. Results are consistent with the conclusion that an isoform of cytoplasmic dynein may be responsible for the apical translocation of elongate spermatids that occurs before sperm release. Also, this is the first report of kinesin-II in mammalian spermatid tails.

Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells.

Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules. Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed for plectin and vimentin using antibodies. Tissue was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to the nucleus and peripherally to intercellular attachment sites.

Immunolocalization of proton-ATPase in the gills of the elasmobranch, Squalus acanthias.

Proton-ATPase was localized to mitochondria-rich cells in the interlamellar region of the gills of the elasmobranch, Squalus acanthias. Localization was accomplished using a polyclonal antibody specific for the 70 kDa subunit of the (V-type) proton-ATPase as confirmed by Western blot analysis. In addition, significant levels of N-ethymaleimide sensitive ATPase activity (0.116 +/- 0.026 mumol Pi.mg-1 protein.h-1) were also measured in crude gill membrane preparations. These data provide, for the first time, direct evidence of the localization of elements possibly involved in branchial acid-base (or ionic) regulation in elasmobranchs.

Spatially dynamic intercellular adhesion junction is coupled to a microtubule-based motility system: evidence from an in vitro binding assay.

During spermatogenesis, spermatids change position in the seminiferous epithelium along an axis that is perpendicular to the seminiferous tubule wall. During this period, spermatids are attached to apical invaginations of Sertoli cells. In areas of this attachment, unique junction plaques occur in Sertoli cells. These plaques consist of regions of the plasma membrane involved with intercellular adhesion, a layer of actin filaments that are hexagonally packed, and an underlying cistern of endoplasmic reticulum (ER). It previously has been proposed that these junction plaques, and therefore the attached spermatids, are translocated, by motor proteins, along microtubule tracts in the Sertoli cell. If this is true, microtubules should bind to the junction plaque in a nucleotide dependent fashion. To verify this prediction, seminiferous epithelia of the rat were separated from tubule walls and then mechanically fragmented. These epithelial preparations were incubated, in both the presence and absence of 10 mM Mg(+)(+)ATP, with exogenous microtubules stabilized with taxol. Then unbound microtubules were separated from microtubules bound to larger epithelial components by centrifuging the samples through a step sucrose gradient. The fraction enriched for elongate spermatids was collected and processed for electron microscopy. The results indicate that the junction plaques remain attached to spermatids, the plaques are intact, and the cytoplasmic face of the ER binds microtubules in a nucleotide dependent fashion. The results are consistent with the presence of motor proteins on the ER component of the junction plaques and with the general hypothesis of microtubule-dependent spermatid translocation.

The perinuclear centriole-containing centrosome is not the major microtubule organizing center in Sertoli cells.

Microtubules are abundant in Sertoli cells and are predominantly arranged parallel to the long axis of the cell. In addition, the centrioles occur in a perinuclear position in the basal one third of the cell. In this study we investigate the importance of the perinuclear centriole-containing centrosome as a microtubule organizing center (MTOC) in Sertoli cells. In all experiments, rat testes were perfusion fixed and then processed for electron and/or fluorescence microscopy. For fluorescence microscopy, fragments or dissected pieces of seminiferous epithelium were labeled for tubulin and actin. The three-dimensional pattern of microtubules in Sertoli cells was determined using data collected with a confocal microscope and analyzed using the NIH-Image program (written by Wayne Rasband at the NIH). The detailed arrangement of microtubules around, and the number of microtubule ends associated with, the centrosome were determined from composite projections constructed from serial thin sections. The nucleating potential of the perinuclear centrosome was determined by perfusing testes for 6 h with 10 micrograms/ml nocodazole and then for up to 3 h with control buffer prior to fixation and analysis with confocal and standard fluorescence microscopy. Microtubules are not organized around a focal perinuclear site and few microtubule ends are associated with the centrosome. Moreover, in cells recovering from nocodazole treatment, microtubules first appear in apical (peripheral) processes. Our data indicate that the centriole-containing perinuclear centrosome is not a significant MTOC in Sertoli cells. Rather, microtubules are nucleated in peripheral regions and project basally. Based on the observations that microtubules appear to "cuff" the nucleus, intermediate filaments are concentrated around the nucleus, microtubules project into the perinuclear intermediate filament network, and microtubules and intermediate filaments are often coaligned, we suggest that microtubules are anchored into position at the base of the cell via linkages with the intermediate filament network. Our nucleation-anchorage model of microtubule organization in Sertoli cells may be applicable to other epithelial systems.

Lack of inter-species reactivity between antigens and antibodies is overcome by protease treatment of western blots.

The analysis of proteins by Western blotting is frequently limited by the inability of antibodies to recognize antigenic epitopes in proteins of different species. In the present study, we investigated the influence of mild protease digestion on the reactivity of nitrocellulose-blotted proteins and microscopic sections with antibodies produced against analogous proteins of different species. The proteins were partially purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations ranging from 0.5 to 80 micrograms/mL, for different time periods prior to reaction with the first antibody. After incubation with the second antibody, the bands were visualized by chemiluminescence. The following antibody/antigen pairs produced no signal by conventional methods but resulted in the appropriate bands following protease treatment: (i) monoclonal antibody (Mab) to human keratin #18/rat keratin; (ii) Mab to starfish extracellular matrix/mouse laminin; (iii) rabbit antiserum to human platelet myosin II/ratfish nonmuscle myosin II. In the latter system, protease treatment also revealed previously hidden epitopes in microscopic sections. Appropriate controls demonstrated that the antibodies retained their specificity after the protease treatment of the preparations. Optimal conditions varied and had to be defined for each protein under study.(ABSTRACT TRUNCATED AT 250 WORDS)