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Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and nontargeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either western blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein–related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were “less distinct” than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin-related junctions (ESs). Summary sentence Cortactin knockdown disrupts basal TBCs and perturbs basal ES turnover. Graphical Abstract

The release of late spermatids from the seminiferous epithelium requires the internalization of intercellular junctions by Sertoli cell specific structures called “tubulobulbar complexes” (TBCs). These large, endocytic devices likely evolved from classic clathrin-mediated-endocytosis (CME) machinery, but have several important morphological differences to CME vesicles. Most notable among these differences is that extensive endoplasmic reticulum (ER) membrane contact sites (MCSs) occur with TBCs and not with clathrin-coated pits. One of the well-established functions of ER MCSs is lipid exchange. Previously, we have established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. Here we use an in vivo knockdown approach to probe function. The testes of Sprague–Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution stimulated emission depletion, and electron microscopy. The knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3 day intervals. Compared to controls, sections from ORP9 siRNA-injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release.

Dominant and recessive mutations in podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for podocalyxin in all tissues (Podxl+/−). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl−/−), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/− mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/− mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/− mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.

Separation of respiratory and digestive tracts in the mammalian pharynx is critical for survival. Food must be kept out of the respiratory tract, and air must be directed into the respiratory tract when breathing.

International audience; Ulcerative colitis (UC) is a chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal pathogenic E. coli. These "pathobionts" exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of relevant animal models. Here, we established a mouse model using a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. Strain p19A was found to adhere to the cecal mucosa of Sigirr-/-mice, causing modest inflammation. Moreover, it dramatically worsened dextran sodium sulfate-induced colitis. This potentiation was attenuated using a p19A strain lacking α-hemolysin genes, or when we targeted pathobiont adherence using a p19A strain lacking the adhesin FimH, or following treatment with FimH antagonists. Thus, UC pathobionts adhere to the intestinal mucosa, and worsen the course of colitis in susceptible hosts.

Effective 'valving' in the upper aerodigestive tract (UAT) is essential to temporarily separate the digestive and respiratory pathways. Marine mammals are largely dedicated to feeding underwater, and in many cases swallowing prey whole. In seals, little work has been done to explore the anatomy and function of the UAT in the context of valving mechanisms that function to separate food and air pathways. Here we use videofluoroscopy, gross dissection, histology and computed tomography (CT) renderings to explore the anatomy of the larynx and soft palate in the harbour seal (

Tubulobulbar complexes (TBCs) internalize intercellular junctions during sperm release. One of the characteristic features of TBCs is that they form “bulbs” or swollen regions that have well-defined membrane contact sites (MCS) with adjacent cisternae of endoplasmic reticulum. Previously, we have localized the IP3R calcium channel to the TBC bulb-ER contacts and have hypothesized that fluctuations in local calcium levels may facilitate the maturation of TBC bulbs into putative endosomes, or alter local actin networks that cuff adjacent tubular regions of the TBCs. To test this, we injected the testes of Sprague Dawley rats with small interfering RNAs (siRNAs) against IP3R1 and processed the tissues for either western blot, immunofluorescence, or electron microscopy. When compared to control testes injected with nontargeting siRNAs, Sertoli cells in knocked-down testes showed significant morphological alterations to the actin networks including a loss of TBC actin and the appearance of ectopic para-crystalline actin bundles in Sertoli cell stalks. There also was a change in the abundance and distribution of TBC-ER contact sites and large internalized endosomes. This disruption of TBCs resulted in delay of the withdrawal of apical processes away from spermatids and in spermiation. Together, these findings are consistent with the hypothesis that calcium exchange at TBC-ER contacts is involved both in regulating actin dynamics at TBCs and in the maturing of TBC bulbs into endosomes. The results are also consistent with the hypothesis that TBCs are part of the sperm release mechanism.

Listeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1–based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1–based internalization event can exceed the theoretical size limit for this endocytic pathway., Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.

The upper respiratory tract of rorquals, lunge-feeding baleen whales, must be protected against water incursion and against the risk of barotrauma at depth, where air-filled spaces like the bony nasal cavities may experience high adverse pressure gradients. We hypothesize these two disparate tasks are accomplished by paired cylindrical nasal plugs that attach on the rostrum and deep inside the nasal cavity. Here we present evidence that the large size and deep attachment of the plugs is a compromise allowing them to block the nasal cavities to prevent water entry while also facilitating pressure equilibration between the nasal cavities and ambient hydrostatic pressure (Pamb) at depth. We investigated nasal plug behaviour using videos of rorquals surfacing, plug morphology from dissections, histology, and MRI scans, and plug function by mathematically modelling nasal pressures at depth. We found each nasal plug has three structurally distinct regions: a muscular rostral region, a predominantly fatty mid-section, and an elastic tendon that attaches the plug caudally. We propose muscle contraction while surfacing pulls the fatty sections rostrally, opening the nasal cavities to air, while the elastic tendons snap the plugs back into place sealing the cavities after breathing. At depth, we propose Pamb pushes the fatty region deeper into the nasal cavities, decreasing air volume by about half and equilibrating nasal cavity to Pamb, preventing barotrauma. The nasal plugs are a unique innovation in rorquals that demonstrate their importance and novelty during diving, where pressure becomes as important an issue as the danger of water entry.

Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal crypts. Unlike intestinal goblet cells, which secrete the mucin Muc2, Paneth cells are best known for producing an array of antimicrobial factors. We unexpectedly identified Muc2 staining localized around Paneth cell granules. Electron microscopy (EM) confirmed an electron lucent halo around these granules, which was lost in Paneth cells from Muc2-deficient (−/−) mice. EM and immunostaining for lysozyme revealed that Muc2−/− Paneth cells contained larger, more densely packed granules within their cytoplasm, and we detected defects in the transcription of key antimicrobial genes in the ileal tissues of Muc2−/− mice. Enteroids derived from the small intestine of wild-type and Muc2−/− mice revealed phenotypic differences in Paneth cells similar to those seen in vivo. Moreover, lysozyme-containing granule release from Muc2−/− enteroid Paneth cells was shown to be impaired. Surprisingly, Paneth cells within human ileal and duodenal tissues were found to be Muc2 negative. Thus Muc2 plays an important role in murine Paneth cells, suggesting links in function with goblet cells; however human Paneth cells lack Muc2, highlighting that caution should be applied when linking murine to human Paneth cell functions. NEW & NOTEWORTHY We demonstrate for the first time that murine Paneth cell granules possess a halo comprised of the mucin Muc2. The presence of Muc2 exerts an impact on Paneth cell granule size and number and facilitates the release and dispersal of antimicrobials into the mucus layer. Interestingly, despite the importance of Muc2 in murine Paneth cell function, our analysis of Muc2 in human intestinal tissues revealed no trace of Muc2 expression by human Paneth cells.

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+podocin+ZO-1+) and microvillus-rich apical membranes (podocalyxin+), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration.

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility., IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.

Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.

A sphincter on the inferior vena cava can protect the heart of a diving mammal from overload when elevated abdominal pressures increase venous return, yet sphincters are reported incompetent or absent in some cetacean species. We previously hypothesized that abdominal pressures are elevated and pulsatile in fluking cetaceans, and that collagen is deposited on the diaphragm according to pressure levels to resist deformation. Here we tested the hypothesis that cetaceans generating high abdominal pressures need a more robust sphincter than those generating low pressures. We examined diaphragm morphology in seven cetacean and five pinniped species. All odontocetes had morphologically similar sphincters despite large differences in collagen content, and mysticetes had muscle that could modulate caval flow. These findings do not support the hypothesis that sphincter structure correlates with abdominal pressures. To understand why a sphincter is needed, we simulated the impact of oscillating abdominal pressures on caval flow. Under low abdominal pressures, simulated flow oscillated with each downstroke. Under elevated pressures, a vascular waterfall formed, greatly smoothing flow. We hypothesize cetaceans maintain high abdominal pressures to moderate venous return and protect the heart while fluking, and use their sphincters only during low-fluking periods when abdominal pressures are low. We suggest pinnipeds, which do not fluke, maintain low abdominal pressures. Simulations also showed retrograde oscillations could be transmitted upstream from the cetacean abdomen and into the extradural veins, with potentially adverse repercussions for the cerebral circulation. We propose that locomotion-generated pressures have influenced multiple aspects of the cetacean vascular system.

Francisella novicida is a surrogate pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. One of the primary sites of Francisella infections is the liver where >90 % of infected cells are hepatocytes. It is known that once Francisella enter cells it occupies a membrane-bound compartment, the Francisella-containing vacuole (FCV), from which it rapidly escapes to replicate in the cytosol. Recent work examining the Francisella disulfide bond formation (Dsb) proteins, FipA and FipB, have demonstrated that these proteins are important during the Francisella infection process; however, details as to how the infections are altered in epithelial cells have remained elusive. To identify the stage of the infections where these Dsbs might act during epithelial infections, we exploited a hepatocyte F. novicida infection model that we recently developed. We found that F. novicida ΔfipA-infected hepatocytes contained bacteria clustered within lysosome-associated membrane protein 1-positive FCVs, suggesting that FipA is involved in the escape of F. novicida from its vacuole. Our morphological evidence provides a tangible link as to how Dsb FipA can influence Francisella infections.

The endoplasmic reticulum (ER) forms a continuous network throughout morphologically differentiated Sertoli cells. It is an integral component of intercellular adhesion junctions in this cell type, as well as forming membrane contact sites with the plasma membrane and intracellular organelles. One of the major functions of the ER in cells generally is maintaining calcium homeostasis and generating calcium signals. In this review, we discuss what is currently known about the overall pattern of distribution of the ER in Sertoli cells and the location of calcium regulatory machinery in the various subdomains of the organelle. Current data is consistent with the hypothesis that calcium signaling by the ER of Sertoli cells may play a significant role in events related to junction remodeling that occur in the seminiferous epithelium during spermatogenesis.

Internal pressures change throughout a cetacean's body during swimming or diving, and uneven pressures between the thoracic and abdominal compartments can affect the cardiovascular system. Pressure differentials could arise from ventral compression on each fluke downstroke or by a faster equilibration of the abdominal compartment with changing ambient ocean pressures compared with the thoracic compartment. If significant pressure differentials do develop, we would expect the morphology of the diaphragm to adapt to its in vivo loading. Here, we tested the hypothesis that significant pressure differentials develop between the thoracic and abdominal cavities in diving cetaceans by examining diaphragms from several cetacean and pinniped species. We found that: (1) regions of cetacean diaphragms possess subserosal collagen fibres that would stabilize the diaphragm against craniocaudal stretch; (2) subserosal collagen covers 5–60% of the thoracic diaphragm surface, and area correlates strongly with published values for swimming speed of each cetacean species (P

Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection.

The tongue of rorqual (balaenopterid) whales slides far down the throat into the expanded oral pouch as an enormous mouthful of water is engulfed during gulp feeding. As the tongue and adjacent oral floor expands and slides caudoventrally, it glides along a more superficial (outer) layer of ventral body wall musculature, just deep to the accordion-like ventral throat pleats. We hypothesize that this sliding movement of adjacent musculature is facilitated by a slick, stretchy layer of loose areolar connective tissue that binds the muscle fibers and reduces friction: fascia. Gross anatomical examination of the gular region of adult minke, fin, and humpback whales confirms the presence of a discrete, three-layered sublingual fascia interposed between adhering fasciae of the tongue and body wall. Histological analysis of this sublingual fascia reveals collagen and elastin fibers loosely organized in a random feltwork along with numerous fibroblasts in a watery extracellular matrix. Biomechanical testing of tissue samples in the field and laboratory, via machine-controlled or manual stretching, demonstrates expansion of the sublingual fascia and its three layers up to 250% beyond resting dimensions, with slightly more extension observed in anteroposterior (rather than mediolateral or oblique) stretching, and with the most superficial of the fascia's three layers. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:735-744, 2019. © 2018 Wiley Periodicals, Inc.

Nerves that supply the floor of the oral cavity in rorqual whales are extensible to accommodate the dramatic changes in tissue dimensions that occur during "lunge feeding" in this group. We report here that the large nerves innervating the muscle component of the ventral grooved blubber (VGB) in fin whales are branches of cranial nerve VII (facial nerve). Therefore, the muscles of the VGB are homologous to second branchial arch derived muscles, which in humans include the muscles of "facial expression." We speculate, based on the presence of numerous foramina on the dorsolateral surface of the mandibular bones, that general sensation from the VGB likely is carried by branches of the mandibular division (V3) of cranial nerve V (trigeminal nerve), and that these small branches travel in the lipid-rich layer directly underlying the skin. We show that intercostal and phrenic nerves, which are not extensible, have a different wall and nerve core morphology than the large VGB nerves that are branches of VII. Although these VGB nerves are known to have two levels of waviness, the intercostal and phrenic nerves have only one in which the nerve fascicles in the nerve core are moderately wavy. In addition, the VGB nerves have inner and outer parts to their walls with numerous large elastin fibers in the outer part, whereas intercostal and phrenic nerves have single walls formed predominantly of collagen. Our results illustrate that overall nerve morphology depends greatly on location and the forces to which the structures are exposed. Anat Rec, 300:1963-1972, 2017. © 2017 Wiley Periodicals, Inc.

Tubulobulbar complexes are elaborate clathrin/actin related structures that form at sites of intercellular attachment in the seminiferous epithelium of the mammalian testis. Here we summarize what is currently known about the morphology and molecular composition of these structures and review evidence that the structures internalize intercellular junctions both at apical sites of Sertoli cell attachment to spermatids, and at basal sites where Sertoli cells form the blood–testis barrier. We present updated models of the sperm release and spermatocyte translocation mechanisms that incorporate tubulobulbar complexes into their designs.

The mouse protein phosphatase genePpp1ccis essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in thePpp1ccmutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in thePpp1ccmutant phenotype. A comparison ofPpp1ccandTssk1/2knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.

Background During late prenatal and early postnatal life, the reproductive system in males undergoes an extensive series of physiological and morphological changes. Prenatal ethanol (EtOH) exposure has marked effects on the development of the reproductive system, with long-term effects on function in adulthood. The present study tested the hypothesis that prenatal EtOH exposure will delay the onset of spermatogenesis. Methods Development of the seminiferous tubules and the onset of spermatogenesis were examined utilizing a rat model of fetal alcohol spectrum disorder (FASD). Male offspring from ad libitum-fed control (C), pair-fed (PF), and EtOH-fed (prenatal alcohol exposure [PAE]) dams were terminated on postnatal (PN) days 5, 15, 18, 20, 25, 35, 45, and 55, to investigate morphological changes through morphometric analysis of the testes from early neonatal life through young adulthood. Results PAE males had lower relative (adjusted for body weight) testis weights compared with PF and/or C males from PN15 through puberty (PN45). In addition, fewer gonocytes (primordial germ cells) were located on the basal lamina on PN5, while more of those touching the basal lamina were dividing in PAE compared with PF and C males, suggesting delayed cell division and migration processes. As well, the percentage of tubules with open lumena was lower in PAE compared with PF and C males on PN18 and 20, and PAE males had fewer primary spermatocytes per tubule on PN18 and round spermatids per tubule on PN25 compared with C males. Finally, the percentage of tubules at stages VII and VIII, when mature spermatids move to the apex of the epithelium and are released, was lower in PAE compared with PF and/or C males in young adulthood (PN55). Conclusions Maternal EtOH consumption appears to delay both reproductive development and the onset of spermatogenesis in male offspring, with effects persisting at least until young adulthood.

A new integrin-associated protein, CPNA-1, which is essential for embryonic muscle development, is characterized. CPNA-1 contains a C-terminal copine domain. PAT-6 (actopaxin, parvin) recruits CPNA-1, and CPNA-1 recruits M-line proteins, including UNC-89 (obscurin)., We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1–null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.

Summary Peripheral nerves are susceptible to stretch injury [1–4] and incorporate structural waviness at the level of the axons, fascicles, and nerve trunk to accommodate physiological increases in length [5, 6]. It is unknown whether there are limits to the amount of deformation that waviness can accommodate. In rorqual whales, a sub-group of baleen whales, nerves running through the ventral groove blubber (VGB) associated with the floor of the mouth routinely experience dramatically large deformations. In fact, some of these nerves more than double their length during lunge feeding and then recoil to a short, compressed state after each lunge [7–9]. It is unknown how these nerves have adapted to operate in both extended and recoiled states. Using micro-CT and mechanics, we have discovered that the VGB nerves from fin whales require two levels of waviness to prevent stretch damage in both extended and recoiled states. The entire nerve core itself is highly folded when recoiled and appears buckled. This folding provides slack for extension but unavoidably generates large stretches at the bends that could damage nerve fascicles within the core. The strain at the bends is minimized by the specific waveform adopted by the core [10, 11], while the existing bending strains are accommodated by a second level of waviness in the individual fascicles that avoids stretch of the fascicle itself. Structural hierarchy partitions the waviness between the two length scales, providing a mechanism to maintain total elongation while preventing the stretching of fascicles at the bends when recoiled.

Tubulobulbar complexes are clathrin/actin-based structures that internalize intercellular junctions in the testis. They resemble coated pits with extremely long necks that are cuffed by dendritic actin networks. As the structures mature, a swollen region or bulb develops near the end of each complex. The bulbs lack actin cuffs and are closely associated with cisternae of endoplasmic reticulum. The bulbs expand and are internalized and enter endocytic compartments of the Sertoli cell. Previous immunofluorescence studies have demonstrated that markers for early endosomes (Rab5 and EEA1) are associated with tubulobulbar complexes and are localized at or near the ends of the structures. Here we use a pre-embedding immunoelectron microscopic technique to accurately localize these markers to apical tubulobulbar complexes that occur at junctions between Sertoli cells and spermatids. Staining for Rab5 occurs at bulbs, identified by the presence of two plasma membranes and a close association with cisternae of endoplasmic reticulum. EEA1 is associated with large vesicles that lack an association with the endoplasmic reticulum. Labeling for nectin-3, an adhesion junction protein in the spermatid plasma membrane, occurs at junctions, TBC bulbs, and in associated double membrane vesicles. Our results suggest that Rab5 associates with junction protein containing bulbs prior to their internalization and that EEA1 associates with the structures later and after internalization. We conclude that at tubulobulbar complexes in Sertoli cells of the seminiferous epithelium, the identity of 'bulbs' as putative early endosomes begins to be established prior to their undergoing scission or budding from their parent structures. Anat Rec, 300:1160-1170, 2017. © 2017 Wiley Periodicals, Inc.

Basal tubulobulbar complexes (TBCs) that occur at attachment sites between neighboring Sertoli cells are subcellular machines that internalize intercellular junctions during movement of spermatocytes from basal to adluminal compartments of the seminiferous epithelium. Each complex consists of an elongate tubular extension of two attached plasma membranes, and is capped at its distal end by a clathrin-coated pit. The tubular region is surrounded by a cuff of actin arranged in a dendritic network. Near the end of the complex, a bulbous region forms that lacks the actin cuff but is closely associated with cisternae of endoplasmic reticulum. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. Previous research has shown that when the actin network is perturbed using the actin filament-disruptor, cytochalasin D, apical tubulobulbar complexes that are associated with spermatids were associated with lower levels of actin, patchy actin networks and swollen tubular regions. Here we explored the effects of actin network perturbation on the morphology of basal tubulobulbar complexes in stage V seminiferous tubules. Isolated rat testes were perfused ex vivo for one hour with oxygenated Krebs-Henseleit buffer (with BSA) containing either 40 μM cytochalasin D or control solution containing DMSO and perfusion-fixed for electron microscopy. Compared to control, actin cuffs in drug-treated TBCs appeared less uniform and patchy. In addition, the tubular regions of the complexes appeared swollen. Our results are consistent with the conclusion that intact networks of actin filaments are required for maintaining the structural integrity of basal TBCs. Anat Rec, 299:1449-1455, 2016. © 2016 Wiley Periodicals, Inc.

Top ocean predators have evolved multiple solutions to the challenges of feeding in the water. At the largest scale, rorqual whales (Balaenopteridae) engulf and filter prey-laden water by lunge feeding, a strategy that is unique among vertebrates. Lunge feeding is facilitated by several morphological specializations, including bilaterally separate jaws that loosely articulate with the skull, hyper-expandable throat pleats, or ventral groove blubber, and a rigid y-shaped fibrocartilage structure branching from the chin into the ventral groove blubber. The linkages and functional coordination among these features, however, remain poorly understood. Here we report the discovery of a sensory organ embedded within the fibrous symphysis between the unfused jaws that is present in several rorqual species, at both fetal and adult stages. Vascular and nervous tissue derived from the ancestral, anterior-most tooth socket insert into this organ, which contains connective tissue and papillae suspended in a gel-like matrix. These papillae show the hallmarks of a mechanoreceptor, containing nerves and encapsulated nerve termini. Histological, anatomical and kinematic evidence indicate that this sensory organ responds to both the dynamic rotation of the jaws during mouth opening and closure, and ventral groove blubber expansion through direct mechanical linkage with the y-shaped fibrocartilage structure. Along with vibrissae on the chin, providing tactile prey sensation, this organ provides the necessary input to the brain for coordinating the initiation, modulation and end stages of engulfment, a paradigm that is consistent with unsteady hydrodynamic models and tag data from lunge-feeding rorquals. Despite the antiquity of unfused jaws in baleen whales since the late Oligocene (∼23-28 million years ago), this organ represents an evolutionary novelty for rorquals, based on its absence in all other lineages of extant baleen whales. This innovation has a fundamental role in one of the most extreme feeding methods in aquatic vertebrates, which facilitated the evolution of the largest vertebrates ever.

Caenorhabditis elegans pxl-1 is the orthologue of vertebrate paxillin and is expressed in body wall and pharyngeal muscle. In body wall muscle PXL-1 localizes to dense bodies, M-lines, and adhesion plaques, and in pharyngeal muscle PXL-1 localizes to podosome-like actin attachment complexes. PXL-1 is required in the pharynx for muscle contraction and viability., We have identified the gene C28H8.6 (pxl-1) as the Caenorhabditis elegans orthologue of vertebrate paxillin. PXL-1 contains the four C-terminal LIM domains conserved in paxillin across all species and three of the five LD motifs found in the N-terminal half of most paxillins. In body wall muscle, PXL-1 antibodies and a full-length green fluorescent protein translational fusion localize to adhesion sites in the sarcomere, the functional repeat unit in muscle responsible for contraction. PXL-1 also localizes to ring-shaped structures near the sarcolemma in pharyngeal muscle corresponding to podosome-like sites of actin attachment. Our analysis of a loss-of-function allele of pxl-1, ok1483, shows that loss of paxillin leads to early larval arrested animals with paralyzed pharyngeal muscles and eventual lethality, presumably due to an inability to feed. We rescued the mutant phenotype by expressing paxillin solely in the pharynx and found that these animals survived and are essentially wild type in movement and body wall muscle structure. This indicates a differential requirement for paxillin in these two types of muscle. In pharyngeal muscle it is essential for contraction, whereas in body wall muscle it is dispensable for filament assembly, sarcomere stability, and ultimately movement.

Human male infertility affects ∼5% of men, with one-third suffering from testicular failure, likely the result of an underlying genetic abnormality that disrupts spermatogenesis during development. Mouse models of male infertility such as thePpp1ccknockout mouse display very similar phenotypes to humans with testicular failure. MalePpp1ccmutant mice are sterile due to disruptions in spermatogenesis that begin during prepubertal testicular development, and continue into adulthood, often resulting in loss of germ cells to the point of Sertoli cell-only syndrome. The current study employs light and electron microscopy to identify new morphological abnormalities inPpp1ccmutant seminiferous epithelium. This study reveals that germ cells become delayed in their development around stages VII and VIII of spermatogenesis. Loss of these cells likely results in the reduced numbers of elongating spermatids and spermatozoa previously observed in mutant animals. Interestingly,Ppp1ccmutants also display reduced numbers of spermatogonia compared with their wild-type counterparts. Using electron microscopy, we have shown that junction complexes inPpp1ccmutants are ultrastructurally normal, and therefore do not contribute to the breakdown in tissue architecture seen in mutants. Electron microscopy revealed major acrosomal and chromatin condensation defects inPpp1ccmutants. Our observations are discussed in the context of known molecular changes inPpp1ccmutant testes.

ObjectiveThe attaching and effacing (A/E) pathogens enterohaemorrhagic Escherichia coli, enteropathogenic E coli and Citrobacter rodentium colonise intestinal tracts, attach to enterocytes, collapse infected cell microvilli and alter numerous host cell processes during infection. Enterocyte alterations result in numerous small molecules being released from host cells that likely contribute to diarrhoeal phenotypes observed during these infections. One possible route for small molecules to be released from intestinal cells may be through functional gap junction hemichannels. Here we examine the involvement of these hemichannels during the diarrhoeal disease caused by A/E pathogens in vivo.DesignMice were infected with the diarrhoea-causing murine A/E pathogen C rodentium for 7 days. Connexin43 (Cx43) protein levels and immunolocalisation in the colon were initially used to determine alterations during A/E bacterial infections in vivo. Connexin mimetic peptides and connexin permeable tracer molecules were used to gage the presence and function of unpaired connexin hemichannels. The role of Cx43 in diarrhoea generation was assessed by comparing infections of wild-type mice to Cx43 mutant mice and determining the water abundance in the colonic luminal material.ResultsWe demonstrate that Cx43 protein levels are increased in colonocytes during in vivo A/E bacterial infections, resulting in functionally open connexon hemichannels in apical membranes of infected cells. Moreover, infected Cx43 +/− mice do not suffer from diarrhoeal disease.ConclusionsThis study provides the first evidence that functional connexon hemichannels can occur in the intestine and are a novel molecular mechanism of water release during infectious diarrhoea.

White adipose tissue (fat) is the primary organ for energy storage and its regulation has serious implications on human health. Excess fat tissue causes significant morbidity, and adipose tissue dysfunction caused by excessive adipocyte hypertrophy has been proposed to play a significant role in the pathogenesis of metabolic disease. Studies in both humans and animal models show that metabolic dysfunction is more closely associated with visceral than subcutaneous fat accumulation. Here, we show that in mice fed a high-fat diet, visceral fat (VAT) grows mostly by hypertrophy and subcutaneous fat (SAT) by hyperplasia, providing a rationale for the different effects of specific adipose depots on metabolic health. To address whether depot expansion is controlled at the level of stem/progenitor cells, we developed a strategy to prospectively identify adipogenic progenitors (APs) from both depots. Clonogenic assays and in vivo bromodeoxyuridine (BrdU) studies show that APs are eightfold more abundant in SAT than VAT, and that AP proliferation is significantly increased in SAT but not VAT in response to high-fat diet. Our results suggest that depot-specific differences in AP abundance and proliferation underlie whether a fat depot expands by hypertrophy or hyperplasia, and thus may have important implications on the development of metabolic disease. In addition, we provide the first evidence that dietary inputs can modulate the proliferation of adipogenic progenitors in adults.

The attaching and effacing (A/E) bacterial pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli and the related mouse pathogen Citrobacter rodentium colonize their hosts' intestines by infecting the apical surfaces of enterocytes, subverting their function, and they ultimately cause diarrhea. Surprisingly, little is known about the interactions of these organisms with goblet cells, which are specialized epithelial cells that secrete the protective molecules Muc2 and trefoil factor 3 (Tff3) into the intestinal lumen. C. rodentium infection leads to dramatic goblet cell depletion within the infected colon, yet it is not clear whether C. rodentium infects goblet cells or if this pathology is pathogen or host mediated. As determined by immunostaining and PCR, both the number of goblet cells and the expression of genes encoding Muc2 and Tff3 were significantly reduced by day 10 postinfection. While electron microscopy and immunostaining revealed that C. rodentium directly infected a fraction of colonic goblet cells, C. rodentium localization did not correlate with goblet cell depletion. To assess the role of the host immune system in these changes, Rag1 knockout (KO) (T- and B-cell-deficient) mice were infected with C. rodentium . Rag1 KO mice did not exhibit the reduction in the number of goblet cells or in mediator (Muc2 and Tff3) expression observed in infected immunocompetent mice. However, reconstitution of Rag1 KO mice with T and B lymphocytes from C57BL/6 mice restored the goblet cell depletion phenotype during C. rodentium infection. In conclusion, these studies demonstrated that while colonic goblet cells can be subject to direct infection and potential subversion by A/E pathogens in vivo, it is the host immune system that primarily modulates the function of these cells during infection.

Huntington disease (HD) is an adult onset, neurodegenerative disorder that results from CAG expansion in the HD gene. Recent work has demonstrated testicular degeneration in mouse models of HD and alterations in the hypothalamic-pituitary-gonadal (HPG) axis in HD patients. Here, we show that HD patients have specific testicular pathology with reduced numbers of germ cells and abnormal seminiferous tubule morphology. In the YAC128 mouse model, testicular degeneration develops prior to 12 months of age, but at 12 months, there is no evidence for decreased testosterone levels or loss of GnRH neurons in the hypothalamus. This suggests that testicular pathology results from a direct toxic effect of mutant huntingtin in the testis and is supported by the fact that huntingtin is highly expressed in the affected cell populations in the testis. Understanding the pathogenesis of HD in the testis may reveal common critical pathways which lead to degeneration in both the brain and testis.

The human attaching and effacing (A/E) intestinal pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and the murine A/E pathogen Citrobacter rodentium cause serious diarrhea in their hosts. These bacteria alter numerous host cell components, including organelles, the host cell cytoskeleton, and tight junctions during the infectious process. One of the proteins that contribute to the intermediate filament network in host cells, cytokeratin-18, is extensively altered during EPEC infections. Based on this, we tested the hypothesis that desmosomes, the only intercellular junctions that interact with intermediate filaments, are also influenced by A/E pathogen infections. We found that the desmosomal transmembrane proteins desmoglein and desmocollin, as well as the desmosome plaque protein desmoplakin, all remain unchanged during EPEC infection in vitro. This evidence is corroborated by the unaltered localization of desmoglein and desmoplakin in vivo in mice infected with C. rodentium for 7 days. Electron microscopic analysis of 7-day C. rodentium-infected murine colonocytes also show no observable differences in the desmosomes when compared to uninfected controls. Our data suggest that, unlike tight junctions, the desmosome protein levels and localization, as well as desmosome morphology, are unaltered during A/E pathogenesis.

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning, and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP, and EGF pathways are used to mediate germline-soma communication. Here we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg/Inx4 and Inx2 on the germline and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues.

Tubulobulbar complexes (TBCs) are elongate subcellular machines responsible for internalizing intercellular junctions during sperm release. Each complex consists of a double-membrane tubular core terminating in a clathrin-coated pit. The core is surrounded by a network of actin filaments, and a distinct swelling or bulb, which lacks an association with actin, develops in the distal third of the structure. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. The relationship of the actin cuff to the formation and budding of the bulb is not known. To gain insight into this relationship, we perturbed the actin networks of TBCs with cytochalasin D. When isolated testes were perfused with a physiological buffer containing cytochalasin D, apical TBCs at stage VII of spermatogenesis were associated with lower levels of actin compared to controls. At the ultrastructural level, the actin networks in cytochalasin D-treated testes appeared patchy, and ectopic bulbs and swollen tubular regions occurred. When normal untreated samples at early stage VII were analyzed, large elongate bulbs and short tubular sections were observed. Together, these results suggest a new model for TBC vesiculation in which the actin network begins to disassemble and the tubular region begins to swell into a bulb. As actin disassembly continues, the coated pit and most of the tubular region are incorporated into the enlarging bulb. The remaining short neck of the bulb near the base of the complex undergoes scission, and the bulb is internalized.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli are non-invasive attaching/effacing (A/E) bacterial pathogens that infect their host's intestinal epithelium, causing severe diarrhoeal disease. These bacteria utilize a type III secretion apparatus to deliver effector molecules into host cells, subverting cellular function. Mitochondrial associated protein (Map) is a multifunctional effector protein that targets host cell mitochondria and contributes to infection-induced epithelial barrier dysfunction in vitro. Unfortunately, the relevance of these actions to the pathogenesis of EPEC-induced disease is uncertain. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that Map colocalized with host cell mitochondria, and that in vivo infection led to a disruption of mitochondrial morphology in infected colonocytes as assessed by electron microscopy. Histochemical staining for the mitochondrial enzyme succinate dehydrogenase also revealed a significant loss of mitochondrial respiratory function in the infected intestinal epithelium; however, both pathologies were attenuated in mice infected with a Deltamap strain. C. rodentium Map was also implicated in the disruption of epithelial barrier function both in vitro and in vivo. These studies thus advance our understanding of how A/E pathogens subvert host cell functions and cause disease, demonstrating that Map contributes to the functional disruption of the intestinal epithelium during enteric infection by C. rodentium.

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined

During spermiogenesis, significant morphological changes occur as round spermatids are remodeled into the fusiform shape of mature spermatozoa. These changes are correlated with a reorganization of microfilaments and microtubules in the head and tail regions of elongating spermatids. There is also altered expression of specialized actin- and tubulin-associated proteins. We report the characterization of a novel, spermatid-specific murine paralog of the actin-bundling protein fascin (FSCN1); this paralog is designated testis fascin or FSCN3. Testis fascin is distantly related to fascins but retains its primary sequence organization. cDNA clones of mouse testis fascin predict a 498 amino acid protein of molecular mass 56 kD that shares 29% identity with mouse fascin. Mapping of murine and human FSCN3 genes shows localization to the 7q31.3 chromosome. Northern analysis indicates that FSCN3 expression is highly specific to testis and that in situ hybridization further restricts expression to elongating spermatids. Antibodies raised against recombinant FSCN3 protein identify a band at 56 kD in testis, epididymis, and epididymal spermatozoa, suggesting that testis fascin persists in mature spermatozoa. In accord with the in situ hybridization results, immunofluorescent microscopy localizes testis fascin protein to areas of the anterior spermatid head that match known distributions of F-actin in the dorsal and ventral subacrosomal spaces. It is possible that testis fascin may function in the terminal elongation of the spermatid head and in microfilament rearrangements that accompany fertilization.

There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 μmol/kg/day or bisphenol A at 400 or 800 μmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.

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.

From puberty and throughout adult spermatogenesis, retinoid signalling is essential for germ cell differentiation and male fertility. The initiation of spermatogonial differentiation and germ cell meiosis occurs under the direction of local retinoid signalling in the testis, and corresponds with the final phase of somatic Sertoli cell differentiation at puberty. Here, we consider the cellular and molecular basis of retinoid actions upon Sertoli cell differentiation. Primary rat Sertoli cells were isolated during the pubertal proliferative and quiescent phases at postnatal days 10- and 20- respectively, and cultured with all-trans-retinoic acid. We show that retinoid signalling can potently suppress activin-induced proliferation by antagonising G1 phase progression and entry into the cell cycle. Retinoid signalling was also found to initiate tight junction formation in primary Sertoli cells, consistent with a pro-differentiative role. This study implicates retinoid signalling in the differentiation of both somatic and germ cells in the testis at puberty.

The morphological study of the Odontocete organ of Corti, together with possible alterations associated with damage from sound exposure, represents a key conservation approach to assess the effects of acoustic pollution on marine ecosystems. By collaborating with stranding networks from several European countries, 150 ears from 13 species of Odontocetes were collected and analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Based on our analyses, we first describe and compare Odontocete cochlear structures and then propose a diagnostic method to identify inner ear alterations in stranded individuals. The two species analyzed by TEM (Phocoena phocoena and Stenella coeruleoalba) showed morphological characteristics in the lower basal turn of high-frequency hearing species. Among other striking features, outer hair cell bodies were extremely small and were strongly attached to Deiters cells. Such morphological characteristics, shared with horseshoe bats, suggest that there has been convergent evolution of sound reception mechanisms among echolocating species. Despite possible autolytic artifacts due to technical and experimental constraints, the SEM analysis allowed us to detect the presence of scarring processes resulting from the disappearance of outer hair cells from the epithelium. In addition, in contrast to the rapid decomposition process of the sensory epithelium after death (especially of the inner hair cells), the tectorial membrane appeared to be more resistant to postmortem autolysis effects. Analysis of the stereocilia imprint pattern at the undersurface of the tectorial membrane may provide a way to detect possible ultrastructural alterations of the hair cell stereocilia by mirroring them on the tectorial membrane.

Tubulobulbar complexes are actin-filament-related structures that form at intercellular junctions in the seminiferous epithelium of mammalian testis. The structures occur both at adhesion junctions between Sertoli cells and the maturing spermatids in apical regions of the epithelium, and at junction complexes between neighboring Sertoli cells near the base of the epithelium. Here, we review the general morphology and molecular composition of tubulobulbar complexes, and also include a description of tubulobulbar complex structure in the human seminiferous epithelium. Although tubulobulbar complexes are unique to the seminiferous epithelium, they have the molecular signature of clathrin-based endocytosis machinery present generally in cells. We review the evidence that tubulobulbar complexes internalize intact intercellular junctions and are significant components of the sperm-release mechanism and the process by which spermatocytes translocate from basal to adluminal compartments of the epithelium.

Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids, the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here we explore the possibility that tubulobulbar complexes that form at the blood-testis barrier are subcellular machines that internalize basal junction complexes. Using electron microscopy, we confirmed that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and nectin-2 (PVRL2) react with linear structures at the light level that we interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. We also demonstrated, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells and that like their in vivo counterparts, the structures contain junction proteins. We use this culture system together with transfection techniques to show that junction proteins from one transfected cell occur in structures that project into adjacent nontransfected cells as predicted by the junction internalization hypothesis. On the basis of our findings, we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.

Summary Muscle serves a wide variety of mechanical functions during animal feeding and locomotion, but the performance of this tissue is limited by how far it can be extended. In rorqual whales, feeding and locomotion are integrated in a dynamic process called lunge-feeding where an enormous volume of prey-laden water is engulfed into a capacious ventral oropharyngeal cavity that is bounded superficially by skeletal muscle and ventral grooved blubber (VGB). The great expansion of the cavity wall presents a mechanical challenge for the physiological limits of skeletal muscle, yet its role is considered fundamental in controlling the flux of water into the mouth. Our analyses of the functional properties and mechanical behavior of VGB muscles revealed a crimped microstructure in an unstrained, non-feeding state that is arranged in parallel with dense and straight elastin fibers. This allows the muscles to accommodate large tissue deformations of the VGB yet still operate within the known strain limits of vertebrate skeletal muscle. VGB transverse strains in routine-feeding rorquals were substantially less than those observed in dead ones, where decomposition gas stretched the VGB to its elastic limit, evidence supporting the idea that eccentric muscle contraction modulates the rate of expansion and ultimate size of the ventral cavity during engulfment.

Cyclic AMP-dependent protein kinase A (PKA) regulates elaboration of the virulence factors melanin and polysaccharide capsule in Cryptococcus neoformans. A mutation in PKA1 encoding the catalytic subunit is known to reduce virulence in mice while a defect in PKR1 encoding the regulatory subunit enhances disease. Here, we constructed strains with galactose-inducible and glucose-repressible versions of PKA1 and PKR1 by inserting the GAL7 promoter upstream of the genes. As expected, no capsule was found in dextrose-containing media for the P(GAL7):PKA1 strain, whereas a large capsule was formed on cells grown in galactose. Along with capsule thickness, high PKA activity also influenced cell size, ploidy and vacuole enlargement, as observed in previous reports of giant/titan cell formation. We employed the regulated strains to test the hypothesis that PKA influences secretion and found that elevated PKA expression positively regulates extracellular protease activity and negatively regulates urease secretion. Furthermore, proper PKA regulation and activity were required for wild-type levels of melanization and laccase activity, as well as correct localization of the enzyme. The latter phenotype is consistent with the discovery that PKA regulates the organization of intracellular membrane compartments. Overall, these results indicate that PKA influences secretion pathways directly related to virulence factor elaboration.