Differential Expression and Localization of EHBP1L1 during the First Wave of Rat Spermatogenesis Suggest Its Involvement in Acrosome Biogenesis

The identification and characterization of new proteins involved in spermatogenesis is fundamental, considering that good-quality gametes are basic in ensuring proper reproduction. Here, we further analyzed the temporal and spatial localization during the first spermatogenic wave of rat testis of EHBP1L1, which is involved in vesicular trafficking due to the CH and bMERB domains, which bind to actin and Rab8/10, respectively. Western blot and immunofluorescence analyses showed that EHBP1L1 protein expression started at 21 days post-partum (dpp) concomitantly with the appearance of primary spermatocytes (I SPC). In subsequent stages, EHBP1L1 specifically localized together with actin in the perinuclear cytoplasm close to the acrosomal and Golgian regions of spermatids (SPT) during the different phases of acrosome biogenesis (AB). Moreover, it was completely absent in elongated SPT and in mature spermatozoa, suggesting that its role was completed in previous stages. The combined data, also supported by our previous report demonstrating that EHBP1L1 mRNA was expressed by primary (I) and secondary (II) SPC, lead us to hypothesize its specific role during AB. Although these results are suggestive, further studies are needed to better clarify the underlying molecular mechanisms of AB, with the aim to use EHBP1L1 as a potential new marker for spermatogenesis.

The Potential Function of KIF17 in Large Yellow Croaker (Larimichthys crocea) Spermatid Remodeling: Molecular Characterization and Expression Pattern During Spermiogenesis

KIF17, which belongs to the kinesin-2 protein family, plays an indispensable role in mammalian spermiogenesis. However, the role of KIF17 in fish spermatid remodeling during spermiogenesis remains poorly understood. Therefore, we aimed to study the role of KIF17 in spermatid remodeling during Larimichthys crocea (L. crocea) spermiogenesis. The kif17 cDNA sequence, 3247 bp in length, was cloned from L. crocea testis, which consisted of a 347 bp 5ʹ-untranslated region (UTR), 413 bp 3ʹ -UTR, and 2487 bp open reading frame. Bioinformatic analyses revealed that KIF17 obtained from L. crocea (Lc-KIF17) exhibited a high sequence identity compared with those from other teleosts and possessed the structural features of other kinesin-2 proteins. Based on structural similarity, we speculate that the role of Lc-KIF17 may be similar to that of KIF17 in other animals. Lc-kif17 mRNA was diffusely expressed in L. crocea tissues and was highly expressed in the testis, especially at stage IV testicular development. Immunofluorescence analysis revealed that Lc-KIF17 signals colocalized with β-tubulin signals and migrated from the perinuclear cytoplasm to the side of the nucleus where the tail forms during spermiogenesis. These findings revealed that KIF17 may be involved in L. crocea spermiogenesis. In particular, KIF17 may participate in spermatid remodeling by interacting with perinuclear microtubules during L. crocea spermiogenesis. Collectively, this study contributes to an improved understanding of the mechanism underlying L. crocea spermiogenesis and provides a basis for further research on L. crocea reproduction and development.

Characterization of Extracellular Vesicles from Cilia and Epithelial Cells of Ductuli Efferentes in a Turtle (Pelodiscus sinensis)

The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.

Fertilization in the cestode Echinococcus multilocularis (Cyclophyllidea, Taeniidae)

Fertilization in the taeniid cestode Echinococcus multilocularis with uniflagellate spermatozoa was examined by means of transmission electron microscopy (TEM). Fertilization in this species occurs in the oviduct lumen or in the fertilization canal proximal to the ootype, where the formation of the embryonic capsule precludes sperm contact with the oocytes. Cortical granules are not present in the cytoplasm of the oocytes of this species, however, several large bodies containing granular material where frequently observed. Spermatozoa coil spirally around the oocytes and syngamy occurs by lateral fusion of oocyte and sperm plasma membranes. In the ootype, one vitellocyte associates with fertilized oocyte, forming a membranous capsule which encloses both cell types. In this stage, the spirally coiled sperm body adheres partly to the external oocyte surface, and partially enters into the perinuclear cytoplasm. The electron-dense sperm nucleus becomes progressively electron-lucent within the oocyte cytoplasm after penetration. Simultaneously with chromatin decondensation, the elongated sperm pronucleus changes shape, forming a spherical male pronucleus, which attains the size of the female pronucleus. Cleavage begins immediately after pronuclear fusion.

Anatomical, Histochemical and Ultrastructural Adaptations of the Alimentary Canal of the Uromastyx aegyptius and the Spalerosophis diadema to their Food Habits

This study deals with anatomical, histochemical and ultrastructural adaptations of the alimentary canal of the Uromastyx aegyptius and the Spalerosophis diadema to their food habits. Proteins and nucleic acids are highly pronounced in the alimentary tract mucosal cells of the studied two species. A variable distribution of proteins and nucleic acids was observed in the different regions of the alimentary tract mucosa of the studied two species. The activity of alkaline phosphatase showed obvious variations not only among different organs, but also between the two species. At the ultrastructural level, the oesophageal mucosal cells contained oval shaped euchromatic nucleus with condensed chromatin and the perinuclear cytoplasm contained some electron-light vesicles. The gastric mucosal cells contained oval shaped euchromatic nucleus with condensed chromatin and the cytoplasm contained many rough endoplasmic reticulum, also many tonofilaments formed thick bundles which converged at the adherence junction in the lateral membranes. The small and large mucosal intestinal cells contained oval euchromatic nuclei and their cytoplasm contained few electron-light vesicles, also their lateral membranes showed many interdigitations. In spite of their difference in taxonomy, habitat, mode of feeding and their vital activities, they show more or less a similarity in the histochemical and ultrastructural patterns of their alimentary tract mucosa. This study can be applied to distinguish between different species of reptiles and for establishment of natural reserves.

NeuN As a Neuronal Nuclear Antigen and Neuron Differentiation Marker

The NeuN protein is localized in nuclei and perinuclear cytoplasm of most of the neurons in the central nervous system of mammals. Monoclonal antibodies to the NeuN protein have been actively used in the immunohistochemical research of neuronal differentiation to assess the functional state of neurons in norm and pathology for more than 20 years. Recently, NeuN antibodies have begun to be applied in the differential morphological diagnosis of cancer. However, the structure of the protein, which can be revealed by antibodies to NeuN, remained unknown until recently, and the functions of the protein are still not fully clear. In the present mini-review, data on NeuN accumulated so far are summarized and analyzed. Data on the structure and properties of the protein, its isoforms, intracellular localization, and hypothesized functions are reported. The application field of immunocytochemical detection of NeuN in scientific and clinical studies, as well as the difficulties in the interpretation of the obtained experimental data and their possible causes, is described in details.

Reinvestigation of vitellogenesis in Caryophyllaeus laticeps (Pallas, 1781) (Cestoda, Caryophyllidea, Caryophyllaeidae), monozoic tapeworm of Abramis brama (Pisces, Teleostei)

Reinvestigation of vitellogenesis in the caryophyllidean cestode Caryophyllaeus laticeps (Pallas, 1781) has been performed using light microscope (LM) and transmission electron microscopy (TEM), and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. Vitellogenesis is generally similar to that reported in the past, however, some new observations were made. The present study reveals the first evidence of: (i) interstitial tissue in the vitelline follicles, (ii) lipid droplets in maturing and mature vitellocytes from vitelline follicles, and (iii) lamellar bodies in vitellocytes from the vitelloduct in C. laticeps. Projections of interstitial tissue surround each vitellocyte and the follicle periphery. The perinuclear cytoplasm of the interstitial tissue contains granular endoplasmic reticulum and vesicles of various size and density. Cytoplasmic osmiophobic lipid droplets and lamellar bodies, previously believed to be absent in most caryophyllid cestodes, are readily apparent in vitellocytes of C. laticeps. The origin and presumed function of these inclusions are discussed. On the other hand, the formation and storage of massive amounts of glycogen in the nucleus and large amounts in the cytoplasm of mature vitelline cells are similar to the condition found in other caryophyllids. Results are compared and contrasted with previous studies on vitellogenesis in other monopleuroid cestodes (Amphilinidea and Gyrocotylidea) as well as polypleuroid cestodes (Spathebothriidea) and the remaining strobilated Eucestoda.

The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells

Phosphorylation of serine 256 (S256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-PK1 cells that express point mutations of AQP2 S261 and S256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 (S261A) and AQP2 (S261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, S261A, S261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of S256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of S261. Finally, blocking AQP2 endocytosis by methyl-β-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of S256 and S261, although as previously reported, the S256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of S261. Together, our data indicate that the phosphorylation state of AQP2 at S261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of S261 phosphorylation/dephosphorylation in vasopressin action remains to be determined.