Impaired mitochondrial morphological plasticity and failure of mitophagy associated with the G11778A mutation of LHON.

Mitochondrial dynamics were examined in human dermal fibroblasts biopsied from a confirmed Leber's Hereditary Optic Neuropathy (LHON) patient with a homoplasmic G11778A mutation of the mitochondrial genome. Expression of the G11778A mutation did not impart any discernible difference in mitochondrial network morphology using widefield fluorescence microscopy. However, at the ultrastructural level, cells expressing this mutation exhibited an impairment of mitochondrial morphological plasticity when forced to utilize oxidative phosphorylation (OXPHOS) by transition to glucose-free, galactose-containing media. LHON fibroblasts also displayed a transient increase in mitophagy upon transition to galactose media. These results provide new insights into the consequences of the G11778A mutation of LHON and the pathological mechanisms underlying this disease.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in Rattus norvegicus†.

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).

Rorqual whale nasal plugs: protecting the respiratory tract against water entry and barotrauma.

The upper respiratory tract of rorquals, lunge-feeding baleen whales, must be protected against water incursion and 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, which demonstrate their importance and novelty during diving, where pressure becomes as important an issue as the danger of water entry.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

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 soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line 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 germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line 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.

Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.

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. Stem Cells 2017;35:2366-2378.

The caval sphincter in cetaceans and its predicted role in controlling venous flow during a dive.

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 that 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 that pinnipeds, which do not fluke, maintain low abdominal pressures. Simulations also showed that 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.

Discovery of a sensory organ that coordinates lunge feeding in rorqual whales.

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.

Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells.

The endoplasmic reticulum (ER) in Sertoli cells is a component of unique adhesion junctions (ectoplasmic specializations-ESs) and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. Using electron microscopy, we define two new ER-plasma membrane (PM) contact sites in apical Sertoli cell processes. One of these sites occurs at TBCs where flattened lamellar cisternae of ER envelope the swollen bulb regions of the complexes, and where the gap between adjacent membranes is 12 nm. The other is at the periphery of apical processes where the gap between membranes is 13-14 nm. Using immunolocalization at the light and electron microscopic levels, we demonstrate that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. Sarco/endoplasmic reticulum Ca2+-ATPase 2 (ATP2A2, SERCA2) is present at ESs; transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER-PM contacts associated with TBC bulbs; and stromal interacting molecule 1 (STIM1), Orai1 (ORAI1), and ATP2A2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling-a process necessary for sperm release.

Controlling thoracic pressures in cetaceans during a breath-hold dive: importance of the diaphragm.

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<0.001); and (3) pinnipeds, which do not locomote by vertical fluking, do not possess this subserosal collagen. These results strongly suggest that this collagen is associated with loads experienced during a dive, and they support the hypothesis that diving cetaceans experience periods during which abdominal pressures significantly exceed thoracic pressures. Our results are consistent with the generation of pressure differentials by fluking and by different compartmental equilibration rates. Pressure differentials during diving would affect venous and arterial perfusion and alter transmural pressures in abdominal arteries.

Novel clathrin/actin-based endocytic machinery associated with junction turnover in the seminiferous epithelium.

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.

Morphological analysis of Francisella novicida epithelial cell infections in the absence of functional FipA.

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.

An Alternative Model of Tubulobulbar Complex Internalization During Junction Remodeling in the Seminiferous Epithelium of the Rat Testis.

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.

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis.

The mouse protein phosphatase gene Ppp1cc is 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 the Ppp1cc mutant 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 the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout 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.

Videofluoroscopy of the aerodigestive tract in Phoca vitulina: reshaping perspectives on translational medicine.

Thousands of rescued harbor seals (Phoca vitulina) require rehabilitation worldwide. Many require resource intensive gavage feeding due to abandonment soon after birth. Little is known about seal swallowing, therefore, our primary objective was to determine the feasibility of conducting videofluoroscopic swallowing studies (VFS) on seal pups prior to their release. Secondarily, we propose swallowing phase descriptions. We adapted a VFS approach used in humans and our feasibility parameters included: bolus detection and consumption, and number of analyzable swallowing events. Unrestrained seals were imaged in a dry environment using a Siemens mobile c-arm fluoroscopy unit. Oral boluses were thawed herring injected with liquid barium suspension (105% w/v). Two independent raters described swallows using a standardized approach with results summarized descriptively. We successfully completed freely-behaving VFS with two infant seals (1 male: 8 wks, 3 d; 1 female: 5 wks, 3 d). Both consumed five boluses with six fully analyzable swallowing events. We describe four swallow phases: preparatory, prehension, oropharyngeal and esophageal. Airway protection likely occurs in two ways: (1) during the preparatory phase through modified corniculate cartilage contact with the glottis and (2) with soft palate contact to the base of tongue prior to swallow initiation. We have conducted a unique VFS approach on rehabilitated seals, prior to their release. We have described airway protection and suggest that swallowing is initiated earlier in the feeding process than described previously. This protocol success will afford: (1) collection of normative swallowing data, and (2) future knowledge translation from humans to seals.

Regulated expression of cyclic AMP-dependent protein kinase A reveals an influence on cell size and the secretion of virulence factors in Cryptococcus neoformans.

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.

A novel subcellular machine contributes to basal junction remodeling in the seminiferous 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.

Novel muscle and connective tissue design enables high extensibility and controls engulfment volume in lunge-feeding rorqual whales.

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 groove 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 behaviour of VGB muscles revealed a crimped microstructure in an unstrained, non-feeding state that is arranged in parallel with dense and straight elastin fibres. 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.

Prenatal ethanol exposure delays the onset of spermatogenesis in the rat.

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.