Microscopic anatomy of the upper aerodigestive tract in harbor seals (Phoca vitulina): Functional adaptations to swallowing.

Abandoned harbor seal pups (Phoca vitulina) are frequently recovered by rehabilitation centers and often require intensive nursing, gavage feeding and swallowing rehabilitation prior to anticipated release. Seal upper aerodigestive tract (UAT) histology descriptions relevant to deglutition are limited, impacting advances in rehabilitation practice. Therefore, we examined the histological characteristics of the harbor seal UAT to understand species-specific functional anatomy and characterize adaptations. To this end, we conducted gross dissections, compiled measurements and reviewed histologic features of the UAT structures of 14 preweaned harbor seal pups that died due to natural causes or were humanely euthanized. Representative samples for histologic evaluation included the tongue, salivary glands, epiglottis, and varying levels of the trachea and esophagus. Histologically, there was a prominent muscularis in the tongue with fewer lingual papillae types compared to humans. Abundant submucosal glands were observed in lateral and pharyngeal parts of the tongue and rostral parts of the esophagus. When compared to other mammalian species, there was a disproportionate increase in the amount of striated muscle throughout the length of the esophageal muscularis externa. This may indicate a lesser degree of autonomic control over the esophageal phase of swallowing in harbor seals. Our study represents the first detailed UAT histological descriptions for neonatal harbor seals. Collectively, these findings support specific anatomic and biomechanical adaptations relevant to suckling, prehension, and deglutition. This work will inform rehabilitation practices and guide future studies on swallowing physiology in harbor seals with potential applications to other pinniped and otariid species in rehabilitation settings.

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).

ORP9 knockdown delays the maturation of junction-related endocytic structures in the testis and leads to impaired sperm release†.

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.

Laryngeal and soft palate valving in the harbour seal (Phoca vitulina).

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 (Phoca vitulina), and generate models for how valving mechanisms in the head and neck may function during breathing, phonating, diving and swallowing. Harbour seals have an elevated larynx and the epiglottis may rise above the level of the soft palate, particularly in pups when sucking. In addition, the corniculate and arytenoid cartilages with associated muscles form most of the lateral margins of the laryngeal inlet and vestibule, and move independently to facilitate airway closure. The corniculate cartilages flex over the laryngeal inlet beneath the epiglottis to completely close the laryngeal vestibule and inlet. The vocal folds are thick and muscular and the medial margin of the folds contains a small vocal ligament. The soft palate has well-defined levator veli palatini muscles that probably function to elevate the palate and close the pharyngeal isthmus during feeding. Our results support the conclusion that harbour seals have evolved UAT valving mechanisms as adaptations to a marine environment that are not seen in terrestrial carnivores.

Knockdown of IP3R1 disrupts tubulobulbar complex-ectoplasmic reticulum contact sites and the morphology of apical processes encapsulating late spermatids†.

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.

Internalization of Intact Intercellular Junctions in the Testis by Clathrin/Actin-Mediated Endocytic Structures: Tubulobulbar Complexes.

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.

The endoplasmic reticulum, calcium signaling and junction turnover in Sertoli cells.

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 are 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.

The Functional Anatomy of Nerves Innervating the Ventral Grooved Blubber of Fin Whales (Balaenoptera Physalus).

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.

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.

High Resolution Localization of Rab5, EEA1, and Nectin-3 to Tubulobulbar Complexes in the Rat Testis.

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.