The identification and differentiation of the mineral phases, hydroxyapatite (HA, Ca10(PO4)6(OH)2) and octacalcium phosphate (OCP, Ca8H2(PO4)6), remains challenging because of their similar composition and chemical structure. In this research, electron energy-loss spectroscopy (EELS) analyses revealed indicators to distinguish HA and OCP and these were applied to examine mineral development in enamel from mouse incisors. Reference EELS data for HA and OCP was established with commercial HA and synthesized OCP. An evaluation of electron damage and a mitigation strategy of multipass imaging was conducted, and the electron dose limitation of OCP was identified. New insights into the mechanism of electron beam damage on the apatite crystal were obtained. With the characterization of the energy-loss spectra and the EELS simulation, the oxygen K-edge was found to be one of the indicators for the differentiation of HA from OCP. The second indicator, the Ca/P ratio, was calculated with a calibrated experimental factor of K exp. Elemental mapping was done to establish the different Ca/P ratio of HA and OCP, and the boundary between these mineral forms. EELS analysis was performed on developing enamel in wild-type (WT) and Mmp20 knockout (KO) mice. This research establishes a protocol for EELS analysis on biological specimens and demonstrates the power and potential of EELS in biomaterial characterization.

Dentin sialophosprotein (DSPP) is cleaved into the N-terminal dentin sialoprotein (DSP) proteoglycan and the C-terminal dentin phosphoprotein (DPP; the most acidic proteins in humans). To define the functions of DSP and DPP, we generated a Dspp-DPP mouse model using CRISPR/Cas9 technology and compared tooth mineralization in Dspp-DPP and Dspp-/- mice in the C57BL/6 background. In both mice, the initiation of dentin mineralization was associated with matrix vesicles. Odontoblasts appeared normal with odontoblastic processes. In Dspp-/- mice, limited mineralized dentin was observed. In Dspp-DPP/-DPP mice, dentin globules (calcospherites) with varied mineral density and unmineralized interglobular dentin were observed throughout the circumpulpal dentin. The area of predentin was smaller compared to Dspp-/- mice, but larger than wild-type mice. In Dspp-/- and Dspp-DPP/-DPP mice, enamel formation was comparable to wild-type. In both mice, Dmp1 expression in differentiating and differentiated odontoblasts was altered. We propose a model for dentin mineralization in which DSP, enriched in the peritubular dentin, propagates mineralization within the hypomineralized calcospherites in the intertubular dentin while DPP is essential for the maturation of calcospherite mineralization in the circumpulpal dentin. We conclude that DSP and DPP are dispensable for the initiation of dentin and enamel mineralization, but critical for circumpulpal dentin mineralization.

The sodium–citrate cotransporter (NaCT) plays a crucial role in citrate transport during amelogenesis. Mutations in the SLC13A5 gene, which encodes the NaCT, cause early infantile epileptic encephalopathy 25 and amelogenesis imperfecta. We analyzed developing pig molars and determined that the citrate concentrations in secretory- and maturation-stage enamel are both 5.3 µmol/g, with about 95% of the citrate being bound to mineral. To better understand how citrate might enter developing enamel, we developed Slc13a5Flag reporter mice that express NaCT with a C-terminal Flag-tag (DYKDDDDK) that can be specifically and accurately recognized by commercially available anti-Flag antibodies. The 24-base Flag coding sequence was located immediately upstream of the natural translation termination codon (TAG) and was validated by Sanger sequencing. The general development, physical activities, and reproductive outcomes of this mouse strain were comparable to those of the C57BL/6 mice. No differences were detected between the Slc13a5Flag and wild-type mice. Tooth development was extensively characterized using dissection microscopy, bSEM, light microscopy, in situ hybridization, and immunohistochemistry. Tooth formation was not altered in any detectable way by the introduction of the Flag. The Slc13a5Flag citrate transporter was observed on all outer membranes of secretory ameloblasts (distal, lateral, and proximal), with the strongest signal on the Tomes process, and was detectable in all but the distal membrane of maturation-stage ameloblasts. The papillary layer also showed positive immunostaining for Flag. The outer membrane of odontoblasts stained stronger than ameloblasts, except for the odontoblastic processes, which did not immunostain. As NaCT is thought to only facilitate citrate entry into the cell, we performed in situ hybridization that showed Ank is not expressed by secretory- or maturation-stage ameloblasts, ruling out that ANK can transport citrate into enamel. In conclusion, we developed Slc13a5Flag reporter mice that provide specific and sensitive localization of a fully functional NaCT-Flag protein. The localization of the Slc13a5Flag citrate transporter throughout the ameloblast membrane suggests that either citrate enters enamel by a paracellular route or NaCT can transport citrate bidirectionally (into or out of ameloblasts) depending upon local conditions.

The human laminin family is composed of five α, four β, and three γ chains. Laminins are heterotrimers of α, β, and γ chains. Laminins play critical roles during organogenesis, mostly as basement membrane components. The expression of all and the localization of most laminin chains were characterized in mouse developing teeth. Primary laminin isoforms in basement membranes along the inner enamel epithelium before the secretory stage and outside of the outer enamel epithelium were laminins 111 (α1β1γ1) and 511. The mouse laminin α3 chain has two variants, α3A and α3B. Although a basement membrane structure is absent, laminin 3A32 was localized along the secretory surface of the secretory stage ameloblast Tomes’ processes. Laminin 3A32 was localized along the atypical basement membrane of maturation stage ameloblasts and the specialized basement membrane of junctional epithelium facing the enamel surface. The endothelial basement membrane in the dental papilla and outside of the enamel organ contained laminins 411 and 511. Laminin 332 was detected in the extracellular matrix but not the basement membrane of the apical loop. Laminin 111 was localized in the extracellular matrix of the apical dental papilla without forming a visible basement membrane. These findings suggest the multifaceted functions of laminins in tooth development and set the foundation for functional investigations.

Dentin sialophosphoprotein ( DSPP ) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5’ mutations affecting targeting and trafficking, and 3’ − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of Dspp P19L and Dspp −1fs mice that replicate the two categories of human DSPP mutations. In Dspp P19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp −1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of Dspp P19L and Dspp −1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp −1fs mice may be valuable for the study of autophagy and ER-phagy.

Journal Article The Characterization of Hydroxyapatite and Octa-calcium Phosphate with Electron Energy Loss Spectroscopy Get access Ya-Hsiang Hsu, Ya-Hsiang Hsu Department of Materials Science and Engineering, The Ohio State University, Columbus, OH, USA Search for other works by this author on: Oxford Academic Google Scholar Asra Hassan, Asra Hassan Center for Electron Microscopy and Analysis, The Ohio State University, Columbus, OH, USAMaterials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH, USA Search for other works by this author on: Oxford Academic Google Scholar Amanda Trout, Amanda Trout Center for Electron Microscopy and Analysis, The Ohio State University, Columbus, OH, USA Search for other works by this author on: Oxford Academic Google Scholar John D Bartlett, John D Bartlett Division of Biosciences, Ohio State University College of Dentistry, Columbus, OH, USA Search for other works by this author on: Oxford Academic Google Scholar Charles E Smith, Charles E Smith Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, USADepartment of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada Search for other works by this author on: Oxford Academic Google Scholar James P Simmer, James P Simmer Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, USA Search for other works by this author on: Oxford Academic Google Scholar David W McComb David W McComb Department of Materials Science and Engineering, The Ohio State University, Columbus, OH, USACenter for Electron Microscopy and Analysis, The Ohio State University, Columbus, OH, USA Corresponding author: mccomb.29@osu.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1223–1225, https://doi.org/10.1093/micmic/ozad067.629 Published: 22 July 2023

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Human ACP4 (OMIM*606362) encodes a transmembrane protein that belongs to histidine acid phosphatase (ACP) family. Recessive mutations in ACP4 cause non-syndromic hypoplastic amelogenesis imperfecta (AI1J, OMIM#617297). While ACP activity has long been detected in developing teeth, its functions during tooth development and the pathogenesis of ACP4 -associated AI remain largely unknown. Here, we characterized 2 AI1J families and identified a novel ACP4 disease-causing mutation: c.774_775del, p.Gly260Aspfs*29. To investigate the role of ACP4 during amelogenesis, we generated and characterized Acp4 R110C mice that carry the p.(Arg110Cys) loss-of-function mutation. Mouse Acp4 expression was the strongest at secretory stage ameloblasts, and the protein localized primarily at Tomes’ processes. While Acp4 heterozygous ( Acp4 +/R110C ) mice showed no phenotypes, incisors and molars of homozygous ( Acp4 R110C/R110C ) mice exhibited a thin layer of aplastic enamel with numerous ectopic mineralized nodules. Acp4 R110C/R110C ameloblasts appeared normal initially but underwent pathology at mid-way of secretory stage. Ultrastructurally, sporadic enamel ribbons grew on mineralized dentin but failed to elongate, and aberrant needle-like crystals formed instead. Globs of organic matrix accumulated by the distal membranes of defective Tomes’ processes. These results demonstrated a critical role for ACP4 in appositional growth of dental enamel probably by processing and regulating enamel matrix proteins around mineralization front apparatus.

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. Secreted proteins are critical for this process. Enam −/− and Ambn −/− mice fail to form enamel. We characterize enamel ribbon formation in wild-type (WT), Amelx −/− and Mmp20 −/− mouse mandibular incisors using focused ion beam scanning electron microscopy (FIB-SEM) in inverted backscatter mode. In Amelx −/− mice, initial enamel mineral ribbons extending from dentin are similar in form to those of WT mice. As early enamel development progresses, the Amelx −/− mineral ribbons develop multiple branches, resembling the staves of a Japanese fan. These striking fan-shaped structures cease growing after attaining ~ 20 µm of enamel thickness (WT is ~ 120 µm). The initial enamel mineral ribbons in Mmp20 −/− mice, like those of the Amelx −/− and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. Remarkably, Mmp20 −/− mineral ribbons also form fan-like structures that extend to ~ 20 µm from the dentin surface. However, these fans are subsequently capped with a hard, disorganized outer mineral layer. Amelogenin cleavage products are the only matrix components absent in both Amelx −/− and Mmp20 −/− mice. We conclude that MMP20 and amelogenin are not critical for enamel mineral ribbon initiation, orientation, or initial shape. The pathological fan-like plates in these mice may form from the lack of amelogenin cleavage products, which appear necessary to form ordered hydroxyapatite.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein ( DSPP ): 5′ mutations affecting an N-terminal targeting sequence and 3′ mutations that shift translation into the − 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp −1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a Dspp P19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. Dspp P19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. Dspp P19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp −1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp +/+ and Dspp P19L dentin. The Dspp −1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5′ and 3′ Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

Purpose: To evaluate surgical complications and oncological outcomes of patients submitted to primary radical inguinal surgical debulking (PRISD) and myocutaneous pediculate flap reconstruction (MPFR) for locally advanced penile cancer (PC). Materials and Methods: Forty-two patients with ulcerated and/or fixed bulky inguinal masses underwent unilateral or bilateral PRISD with MPFR. Tensor fascia lata flap (TFL) was the standard of care for all patients. Additional use of the gracilis flap (GF) was carried out when necessary. Contra-lateral radical inguinal lymphadenectomy (RIL) was conduced when PRISD was performed unilaterally. Surgical complications were analyzed and stratified into minor and major according to the Bevan-Thomas classification. Adjunctive treatments were assessed and oncological outcomes analyzed. Results: Of the 42 patients evaluated, 10 (23.8%) underwent bilateral PRISD and 32 (76.2%) unilateral PRISD with contra-lateral RIL, totaling 84 lymphadenectomies. A total of 62 MPFRs were performed, 52 with TFL and 10 with GF. A total of 53 complications were identified, 49 related to PRISD with MPFR and 4 to RIL. Adjuvant chemotherapy was carried out in 16 patients. Median follow-up was 10.8 months with a median overall survival (OS) of 14.0 months against 6.0 months (p=0.006) for patients submitted to PRISD with adjuvant chemotherapy in relation to surgery alone. Conclusions: PRISD alone for advanced loco-regional PC is unlikely to promote long-term survival, although it can lead to temporary local control of the disease. Despite the feasibility of the procedure, it is related to high incidence of complications. Surgical treatment with adjuvant chemotherapy is associated with improved OS.

Enamel was one of the first biological tissues to be examined in the light microscope and has since fascinated and mystified researchers with its complexity. The compartmentalization of the ameloblast life cycle into stages implies that enamel development occurs in precisely defined steps and that extracellular matrix proteins are produced and released exclusively at one point in time – the secretory stage. The bulk of the enamel matrix is produced during the secretory stage. The literature on enamel proteolytic enzymes, like the enamel matrix proteins, has been controversial, with little agreement between various investigators as to the exact class(es) of enzymes present in developing enamel. Besides the concepts of dual categories of enamelins and amelogenins (EP), and of extracellular proteinases, another idea which has had significant impact on enamel research has been the hypothesis that ameloblasts resorb intact or partially degraded EPs directly from maturing enamel and dispose of them within their lysosomes.

Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2-45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to "unbend" the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 +/- 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 +/- 5.6 vs. 5.9 +/- 4.0 rods per row, p < 0.01). There was no relationship between the length of a focal stack and its location within the inner enamel. All results were consistent with the notion that focal stacks travel from the DEJ to the outer enamel the same as the longer and decussating companion rows to which they are paired. The spatial distribution of focal stacks within the inner enamel was not spatially random but best fit a null model based on a heterogenous Poisson point process dependent on regional location within the transverse plane of the enamel layer.

The 2D arrangement of rows of enamel rods with alternating (decussating) tilt angles across the thickness of the inner layer in rat and mouse incisor enamel is well known and assumed to occur in a uniform and repetitive pattern. Some irregularities in the arrangement of rows have been reported, but no detailed investigation of row structure across the entire inner enamel layer currently exists. This investigation was undertaken to determine if the global row pattern in mouse mandibular incisor enamel is predominately regular in nature with only occasional anomalies or if rows of enamel rods have more spatial complexity than previously suspected. The data from this investigation indicate that rows of enamel rods are highly variable in length and have complex transverse arrangements across the width and thickness of the inner enamel layer. The majority of rows are short or medium in length, with 87% having < 100 rods per row. The remaining 13% are long rows (with 100-233 rods per row) that contain 46% of all enamel rods seen in transverse sections. Variable numbers of rows were associated with the lateral, central and mesial regions of the enamel layer. Each region contained different ratios of short, medium and long rows. A variety of relationships was found along the transverse length of rows in each region, including uniform associations of alternating rod tilts between neighboring rows, and instances where two rows having the same rod tilt were paired for variable distances then moved apart to accommodate rows of opposite tilt. Sometimes a row appeared to branch into two rows with the same tilt, or conversely where two rows merged into one row depending upon the mesial-to-lateral direction in which the row was viewed. Some rows showed both pairing and branching/merging along their length. These tended to be among the longest rows identified, and they often crossed the central region with extensions into the lateral and mesial regions. The most frequent row arrangement was a row of petite length nestled at the side of another row having the same rod tilt (30% of all rows). These were termed 'focal stacks' and may relate to the evolution of uniserial rat and mouse incisor enamel from a multilayered ancestor. The mesial and lateral endpoints of rows also showed complex arrangements with the dentinoenamel junction (DEJ), the inner enamel layer itself, and the boundary area to the outer enamel layer. It was concluded that the diversity in row lengths and various spatial arrangements both within and between rows across the transverse plane provides a method to interlock the enamel layer across each region and keep the enamel layer compact relative to the curving DEJ surface. The uniserial pattern for rows in mouse mandibular incisors is not uniform, but diverse and very complex.

AbstractBackgroundAmeloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel‐specific with respect to its essential functions. Biallelic AMBN defects cause non‐syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin.MethodsWe characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS‐lacZ (AmbnlacZ/lacZ) knockin mice.ResultsNo AMBN protein was detected using immunohistochemistry in null mice. ß‐galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. AmbnlacZ/lacZ 7‐week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the AmbnlacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the AmbnlacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS‐lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X‐gal.ConclusionsAmeloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

BackgroundTruncation FAM83H mutations cause human autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), an inherited disorder characterized by severe hardness defects in dental enamel. No enamel defects were observed in Fam83h null mice suggesting that Fam83h truncation mice would better replicate human mutations. MethodsWe generated and characterized a mouse model (Fam83h(Tr/Tr)) expressing a truncated FAM83H protein (amino acids 1-296), which recapitulated the ADHCAI-causing human FAM83H p.Tyr297* mutation. ResultsDay 14 and 7-week Fam83h(Tr/Tr) molars exhibited rough enamel surfaces and slender cusps resulting from hypoplastic enamel defects. The lateral third of the Fam83h(Tr/Tr) incisor enamel layer was thinner, with surface roughness and altered enamel rod orientation, suggesting disturbed enamel matrix secretion. Regular electron density in mandibular incisor enamel indicated normal enamel maturation. Only mildly increased posteruption attrition of Fam83h(Tr/Tr) molar enamel was observed at 7-weeks. Histologically, the Fam83h(Tr/Tr) enamel organ, including ameloblasts, and enamel matrices at sequential stages of amelogenesis exhibited comparable morphology without overt abnormalities, except irregular and less evident ameloblast Tomes' processes in specific areas. ConclusionsConsidering Fam83h(-/-) mice showed no enamel phenotype, while Fam83h(Tr/Tr) (p.Tyr297*) mice displayed obvious enamel malformations, we conclude that FAM83H truncation mutations causing ADHCAI in humans disturb amelogenesis through a neomorphic mechanism, rather than haploinsufficiency.

The Golgi apparatus of germ cells of the testis of adult rats was isolated and subjected to a proteomics analysis with 1318 proteins characterized and 20 localized in situ by light microscope immunocytochemistry. The data revealed unexpected temporal distributions of the 20 germ cell Golgi localized proteins from chaperones to protein-folding enzymes involved in protein maturation. Some proteins were selective for the Golgi apparatus of germ cells spanning the greater part of germ cell differentiation (spermatogenesis). Others were selective for spermatocytes and/or spermatids during acrosome formation. A segregation of two classes of Golgi proteins was also noted during acrosome formation, with some localizing to both the Golgi and acrosome, while others were restricted solely to the Golgi apparatus. Several Golgi markers defined the postacrosome Golgi migration. In addition, some proteins were expressed selectively during the last step of germ cell differentiation (i.e., step 19 spermatids) at a time when the Hermes body (cytoplasmic droplet) was being formed. In the case of the isolated Hermes body of epididymal sperm, 30 non-Golgi proteins were chosen from 1511 characterized by proteomics. These proteins were mapped according to the 14 stages of the cycle of the seminiferous epithelium, with a correlation being noted with specific germ cell organelles that may lead to an understanding of the functional significance of these proteins. In addition, the expression profile of the 30 non-Golgi proteins was compared to that of the 20 Golgi localized proteins according to the 14 stages of the cycle. In this way, similarities in waves of expression of these two different classes of proteins could be compared to reveal functional implications.

The Hermes body (HB), previously called the cytoplasmic droplet, is a focal dilated component of the sperm flagellum with unique structural features that have been in search of an identity since first discovered in 1909. The HB is an evolutionarily conserved structure unique to epididymal spermatozoa where it has been observed in over 30 vertebrate species including humans. As sperm traverse the epididymis, they acquire their maturational features, motility and fertility, an event coincident with the presence of the HB on sperm suggesting a cause-effect relationship. Recent studies have enabled a postulation as to the significance of this structure in the acquisition of sperm motility due its abundant concentration of the glucose transporter 3 (GLUT3), in addition to all glycolytic enzymes. The internal membranes have characteristics not only of Golgi apparatus, but also endoplasmic reticulum and endosomes, in addition to COPI and COPII coats, clathrin, SNAREs, annexins, AAA + proteins and GTPases for vesicular trafficking and membrane fusion. Ribosomal, cytoskeletal and stress proteins along with proteins serving a protective function were also noted. Thus the Hermes body contains a panoply of proteins that may be involved in sperm maturation. Step 19 spermatids of the testis just prior to their release as sperm into the seminiferous tubule lumen were also uncovered as a key step in germ cell differentiation, where many proteins, and some for the first time, were expressed in the forming Hermes body, where they were concentrated in sperm as they traversed the epididymal duct on route to maturation. Taken together the data indicate that far from being a cell devoid of a translational machinery, epididymal sperm may be reassessed as in a dynamic state of protein turnover.

Considerable descriptive information about the overall organization of mouse mandibular incisor enamel is available but almost nothing is known about the quantitative characteristics of enamel rod arrangement and distribution in these teeth. This has important implications concerning cell movement during the secretory stage because each ameloblast makes one enamel rod. Knowing how many enamel rods are cut open in a cross-section of the enamel layer could provide insights into understanding the dynamics of how groups of ameloblasts form the enamel layer. In this study, cross-sections of fully mineralized enamel were cut on 24 mandibular mouse incisors, polished and etched, and imaged by scanning electron microscopy in backscatter mode. Montaged maps of the entire enamel layer were made at high magnification and the enamel rod profiles in each map were color-coded based upon rod category. Quantitative analyses of each color layer in the maps were then performed using standard routines available in imagej. The data indicated that that there were on average 7233 ± 575 enamel rod profiles per cross-section in mandibular incisors of 7-week-old mice, with 70% located in the inner enamel layer, 27% located in the outer enamel layer, and 3% positioned near the mesial and lateral cementoenamel junctions. All enamel rod profiles showed progressive increases in tilt angles, some very large in magnitude, from the lateral to mesial sides of the enamel layer, whereas only minor variations in tilt angle were found relative to enamel thickness at given locations across the enamel layer. The decussation angle between alternating rows of rod profiles within the inner enamel layer was fairly constant from the lateral to central labial sides of the enamel layer, but it increased dramatically in the mesial region of the enamel layer. The packing density of all rod profiles decreased from lateral to central labial regions of the enamel layer and then in progressing mesially, decreased slightly (inner enamel, mesial tilt), increased slightly (outer enamel layer) or almost doubled in magnitude (inner enamel, lateral tilt). It was concluded that these variations in rod tilt angle and packing densities are adaptations that allow the tooth to maintain a sharp incisal edge and shovel-shape as renewing segments formed by around 7200 ameloblasts are brought onto the occluding surface of the tooth by continuous renewal.