Morphologic Features and Deep Learning-Based Analysis of Canine Spermatogenic Stages.

In nonclinical toxicity studies, stage-aware evaluation is often expected to assess drug-induced testicular toxicity. Although stage-aware evaluation does not require identification of specific stages, it is important to understand microscopic features of spermatogenic staging. Staging of the spermatogenic cycle in dogs is a challenging and time-consuming process. In this study, we first defined morphologic features for the eight spermatogenic stages in standard histology sections (H&E slides) of dog testes. For image analysis, we defined the key morphologic features of five stages/pooled stage groups (I-II, III-IV, V, VI-VII, and VIII). These criteria were used to develop a deep learning (DL) algorithm for staging of the spermatogenic cycle of control dog testes using whole slide images. In addition, a DL-based nucleus segmentation model was trained to detect and quantify the number of different germ cells, including spermatogonia, spermatocytes, and spermatids. Identification of spermatogenic stages and quantification of germ cell populations were successfully automated by the DL models. Combining these two algorithms provided color-coding visual spermatogenic staging and quantitative information on germ cell populations at specific stages that would facilitate the stage-aware evaluation and detection of changes in germ cell populations in nonclinical toxicity studies.

Ultrastructural research of spermiogenesis in two sponges, Crellomima imparidens and Hymedesmia irregularis (Demospongiae): New evidence of sperms with acrosome in sponges.

Details of spermatogenesis and sperm organization are often useful for reconstructing the phylogeny of closely related taxa of invertebrates. Here, the spermiogenesis and the ultrastructure of sperm were studied in two marine demosponges, Crellomima imparidens and Hymedesmia irregularis (order Poecilosclerida). In C. imparidens and H. irregularis, we found bundles of microtubules arranged along the nucleus during spermiogenesis. These bundles derived from the basal body of axoneme, reaching the apical pole of the cell. In C. imparidens, the microtubules surround the nucleus, forming the manchette. In H. irregularis, the microtubules pass along only one side of the cell periphery. During spermiogenesis, the nucleus stretches and elongates. In both species, the nucleus is twisted into a spiral structure. We suppose that the manchette of microtubules could be responsible for controlling the elongation and shaping of the sperm nucleus to a helical form and for the twisting and/or condensation of chromatin in these sponges. The spermatozoon of both species has an elongated shape. Its apical part has an acrosome, which is dome-shaped in C. imparidens and flattened and lenticular in H. irregularis. The cytoplasm of the spermatozoa contains some small mitochondria, and proximal and distal centrioles arranged at an angle to each other. There is a small volume of residual cytoplasm with dark glycogen-like granules. The axoneme of the spermatid and the flagellum of the sperm of both sponges is located in the deep tunnel-like cytoplasmic depression. The comparison of spermatozoa morphology of different species of the order Poecilosclerida demonstrates that the knowledge of variation within genera and families can give valuable insights into the significance of many characters proposed for phylogenetic studies of this order.

Spermiogenic chromatin condensation patterning in several hexapods may involve phase separation dynamics by spinodal decomposition or microemulsion inversion (nucleation).

We have examined published transmission electron microscopy (TEM). photomicrographs of chromatin condensation patterning in developing sperm nuclei from five species of entognathous hexapods within the Classes Protura, Collembola, Diplura and five species of ancestral wingless insects in the Orders Archaeognatha and Zygentoma as well as in fifteen species of the winged insects. Each species reproduces by internal fertilization. Spatially quantitative analysis indicates that spermiogenic chromatin condensation patterning in several of these species may be due to spinodal decomposition (SD) or to microemulsion inversion (chromatin-in-nucleoplasm → nucleoplasm-in-chromatin), also known as nucleation (Nc). These are two different dynamic mechanisms of liquid-liquid phase separation (LLPS). They might either occur independently or co-exist during the chromatin condensation associated with insect spermiogenesis. For example, the chromatin condensation pattern such as that observed in transverse sections of developing sperm nuclei from the wingless insect Anurida maritima (Collembola) is: granules → fibers → lamellae (SD) → nucleation (Nc) → condensed nuclei. Similar transitions are also observed in other more recently evolved species within the Class Insecta. From the limited but comprehensive sample of entognathus and ectognathus hexapods analyzed here, it appears that LLPS of sperm chromatin during spermiogenesis has occurred quite pervasively within the subphylum Hexapoda, including insects.

Molecular characterization, dynamic transcription, and potential function of KIF3A/KIF3B during spermiogenesis in Opsariichthys bidens.

Spermiogenesis is the final phase of spermatogenesis, wherein the spermatids differentiate into mature spermatozoa via complex morphological transformation. In this process, kinesin plays an important role. Here, we observed the morphological transformation of spermatids and analyzed the characterization, dynamic transcription, and potential function of kinesin KIF3A/KIF3B during spermiogenesis in Chinese hook snout carp (Opsariichthys bidens). We found that the full-length cDNAs of O. bidens kif3a and kif3b were 2544 and 2806 bp in length comprising 119 bp and 259 bp 5' untranslated region (UTR), 313 bp and 222 bp 3' UTR, and 2112 bp and 2325 bp open reading frame encoding 703 and 774 amino acids, respectively. Ob-KIF3A/KIF3B proteins have three domains, namely N-terminal head, coiled-coil stalk, and C-terminal tail, and exhibit high similarity with homologous proteins in vertebrates and invertebrates. Ob-kif3a/kif3b mRNAs were ubiquitously expressed in all tissues examined, with the highest expression in the brain and stage-IV testis. Immunofluorescence results showed that Ob-KIF3A was co-localized with tubulin and the mitochondria. Particularly, in early spermatids, Ob-KIF3A, tubulin, and the mitochondrial signals were evenly distributed in the cytoplasm, whereas in middle spermatids, they were distributed around the nucleus. In the late stage, the signals were concentrated on one side of the nucleus, where the tail is formed, whereas in mature sperms, they were detected in the midpiece and flagellum. These results indicate that Ob-KIF3A/KIF3B may participate in nuclear reshaping, flagellum formation, and mitochondrial aggregation in the midpiece during spermiogenesis.

A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores.

We examined whether a tool for determining Johnsen scores automatically using artificial intelligence (AI) could be used in place of traditional Johnsen scoring to support pathologists' evaluations. Average precision, precision, and recall were assessed by the Google Cloud AutoML Vision platform. We obtained testicular tissues for 275 patients and were able to use haematoxylin and eosin (H&E)-stained glass microscope slides from 264 patients. In addition, we cut out of parts of the histopathology images (5.0 × 5.0 cm) for expansion of Johnsen's characteristic areas with seminiferous tubules. We defined four labels: Johnsen score 1-3, 4-5, 6-7, and 8-10 to distinguish Johnsen scores in clinical practice. All images were uploaded to the Google Cloud AutoML Vision platform. We obtained a dataset of 7155 images at magnification 400× and a dataset of 9822 expansion images for the 5.0 × 5.0 cm cutouts. For the 400× magnification image dataset, the average precision (positive predictive value) of the algorithm was 82.6%, precision was 80.31%, and recall was 60.96%. For the expansion image dataset (5.0 × 5.0 cm), the average precision was 99.5%, precision was 96.29%, and recall was 96.23%. This is the first report of an AI-based algorithm for predicting Johnsen scores.

Process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei: Polarized development of the spermatid and discarding of the residual body.

The elimination of the spermatid cytoplasm during spermiogenesis enables the sperm to acquire a streamlined architecture, which allows for unhindered swimming. While this process has been well described in vertebrates, it has rarely been reported in invertebrates. In this study, we observed the process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei (Mollusca, Cephalopoda) using light microscopy, transmission electron microscopy, and immunofluorescence. In the early spermatid, the cell is circular, and the nucleus is centrally located. With spermatid development, the cell becomes polarized. The nucleus gradually elongates and moves toward the end of the cell where the tail is forming. As a result, the cytoplasm moves past the nucleus at the anterior region of the future sperm head (the foreside of the acrosome). Following this, during the late stage of spermiogenesis, the cytoplasm condenses and collects on the foreside of the acrosome until finally the residual body is discarded from the top of the sperm head. This represents a distinct directionality for the development of cytoplasmic polarity and discarding of residual body compared with that reported for vertebrates (in which the cytoplasm of the elongating spermatids is polarized toward the caudal region). The fact that the cytoplasm also becomes concentrated suggests that water pumps may be involved in the elimination of water from the cytoplasm before the residual body is discarded. Furthermore, we found that microtubules, forming a manchette-like structure, are involved not only in reshaping of the nucleus but also in the transport of mitochondria and vesicles to the foreside of the acrosome, subsequently allowing them to be discarded with the residual body. This study broadens our understanding of the development of polarization and elimination of cytoplasm from spermatids in animals.

Spermiogenesis in the cattle egret (Bubulcus ibis).

Avian species comprise more than half of all vertebrates yet there is a dearth of information regarding spermatid development in this class of animals. This report of spermiogenesis in the cattle egret, Bubulcus ibis, is the first in the order Pelecaniformes. Five sexually mature and reproductively active male cattle egrets were captured in the wild, humanely euthanized, the reproductive organs dissected out, and tissues from the testes routinely prepared for transmission electron microscopy. Twelve steps of spermatid development, using the step-wise system, were determined. Acrosomogenesis in the egret results in a relatively short, solid, bullet-shaped acrosome that ends bluntly anteriorly and flat posteriorly or basally. The nucleus displays remarkable morphological changes, with the anterior end of the mature spermatid becoming flat, lacking a rostrum and an endonuclear canal. A perforatorium does not develop. It is noteworthy that a longitudinal, but not a circular, manchette develops during spermiogenesis in this bird. The proximal centriole is attached to the nucleus, at the implantation fossa, by means of well-formed, electron dense struts of material. An amorphous fibrous sheath develops in the principal piece. The interesting development and peculiar features of the acrosome and nucleus, as well as the absence of the perforatorium and circular manchette in the spermatozoon of the cattle egret, may be of phylogenetic significance.

Ultrastructural aspects of spermatogenesis in Calyptogena pacifica Dall 1891 (Vesicomyidae; Bivalvia).

The process of spermatogenesis and spermatozoon morphology was characterized from a deep-sea bivalve, Calyptogena pacifica (Vesicomyidae, Pliocardiinae), a member of the superfamily Glossoidea, using light and electron microscopy. Spermatogenesis in C. pacifica is generally similar to that in shallow-water bivalves but, the development of spermatogenic cells in this species has also some distinguishing features. First proacrosomal vesicles are observed in early spermatocytes I. Although, early appearance of proacrosomal vesicles is well known for bivalves, in C. pacifica, these vesicles are associated with electron-dense material, which is located outside the limiting membrane of the proacrosomal vesicles and disappears in late spermatids. Another feature of spermatogenesis in C. pacifica is the localization of the axoneme and flagellum development. Early spermatogenic cells lack typical flagellum, while in spermatogonia, spermatocytes, and early spermatids, the axoneme is observed in the cytoplasm. In late spermatids, the axoneme is located along the nucleus, and the flagellum is oriented anteriorly. During sperm maturation, the bent flagellum is transformed into the typical posteriorly oriented tail. Spermatozoa of C. pacifica are of ect-aqua sperm type with a bullet-like head of about 5.8 µm in length and 1.8 µm in width, consisting of a well-developed dome-shaped acrosomal complex, an elongated barrel-shaped nucleus filled with granular chromatin, and a midpiece with mainly four rounded mitochondria. A comparative analysis has shown a number of common traits in C. pacifica and Neotrapezium sublaevigatum.

Cytological features of spermatogenesis in Opsariichthys bidens (Teleostei, Cyprinidae).

Spermatogenesis is important for male fertility, but has not been well-studied in Opsariichthys bidens, an economically important freshwater fish in China. In this study, there was investigation of the cytological features of spermatogenesis in O. bidens using light microscopy, transmission electron microscopy, and immunofluorescence detection of microtubules. O. bidens has tubular testis. Spermatogenesis in O. bidens is of the cystic type, in which the spermatogenic cells develop into spermatozoa in cysts. There was asynchronous development of primary spermatocytes within a single cyst. Spermiogenesis was classified as Type I, which develops into a Type I aquasperm with an oval nucleus, a small and simple midpiece, a flagellum and no acrosome. There was a nuage in spermatogonia, spermatocytes, and spermatids in different developmental stages of spermatids which may have important functions in fish spermatogenesis. Furthermore, microtubule dynamics may be involved in spermatid reshaping, material transport, and polar distribution of organelles during spermiogenesis.

Spermatogenesis in the inseminating African butterflyfish Pantodon buchholzi (Teleostei: Osteoglossiformes: Pantodontidae) with the revision of residual bodies formation.

The aim of this study was to analyse spermatogenesis in the African butterflyfish, Pantodon buchholzi, using transmission electron microscopy and scanning electron microscopy. P. buchholzi is the most basal teleost that exhibits insemination and produces a highly complex introsperm with the most elongate midpiece known in teleost fishes. Their early stages (spermatogonia and spermatocytes) do not differ greatly from those of other fishes, with the exception of Golgi apparatus degradation appearing as spindle-shaped bodies (SSBs). In round, early spermatids, the development of the flagellum begins after the migration of the centriolar complex towards the nucleus. Later, the elongation of the midpiece coincides with the displacement of the mitochondria and their fusion to produce nine mitochondrial derivatives (MDs). In these spermatids, the nucleus is situated laterally to the midpiece, with condensing chromatin in the centre of the nucleus. Within the midpiece, the flagellum is located within a cytoplasmic canal and is surrounded by a cytoplasmic sleeve containing fibres, MDs and a great amount of cytoplasm located on one side. During the next phase, nuclear rotation, the highly condensed chromatin is displaced to a position above the centriolar apparatus, whereas chromatin-free nucleoplasm is transferred to the cytoplasm. Later, this nucleoplasm, still surrounded by the nuclear membrane, is eliminated into the cyst lumen as the nucleoplasmic packet. Within the highly elongate spermatids, other excess organelles (SSBs, endoplasmic reticulum and mitochondria) are eliminated as residual bodies (RBs). Fully developed spermatozoa, which contain conical-shaped nuclei, eventually coalesce to form unencapsulated sperm packets (spermatozeugmata) that are surrounded by RBs at the level of the extremely elongate midpieces. Later, RBs are removed at the periphery of the cyst by means of phagocytosis by Sertoli cells.

Blocking the Bromodomains Function Contributes to Disturbances in Alga Chara vulgaris Spermatids Differentiation.

Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 µM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.

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.

Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

Individual sperm cells are resolved from a syncytium during late step of spermiogenesis known as individualization, which is accomplished by an Individualization Complex (IC) composed of 64 investment cones. mulet encodes Tubulin-binding cofactor E-like (TBCEL), suggesting a role for microtubule dynamics in individualization. Indeed, a population of ∼100 cytoplasmic microtubules fails to disappear in mulet mutant testes during spermatogenesis. This persistence, detected using epi-fluorescence and electron microscopy, suggests that removal of these microtubules by TBCEL is a prerequisite for individualization. Immunofluorescence reveals TBCEL expression in elongated spermatid cysts. In addition, testes from mulet mutant males were rescued to wild type using tubulin-Gal4 to drive TBCEL expression, indicating that the mutant phenotype is caused by the lack of TBCEL. Finally, RNAi driven by bam-GAL4 successfully phenocopied mulet, confirming that mulet is required in the germline for individualization. We propose a model in which the cytoplasmic microtubules serve as alternate tracks for investment cones in mulet mutant testes.This article has an associated First Person interview with the first author of the paper.

Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota.

Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.

Spermatid differentiation, with particular reference to acrosomogenesis, in the passeridan bird, Carib grackle (Quiscalus lugubris).

Only a few studies on the development of the passerine spermatozoon are available, yet species variations in the conformation as well as structure of the generally helical acrosome have been reported. This study of spermiogenesis in the Carib grackle (Quiscalus lugubris) intended to provide a deeper understanding of the development of the sperm, and in particular to investigate the bi-partite nature and development of the acrosome as well as its relationship with the nucleus, in the absence of a perforatorium that is found in most non-passerine birds. The acrosomal vesicle already displays a bi-partite nature in the acrosomal granule within the Golgi complex, and the attachment of the dense granule (future acrosomal core) within the crest part (future acrosomal crest) establishes polarity as it approaches and attaches to the nucleus. Thereafter, they develop variably. The acrosomal crest leads the elongation and spiraling of the acrosome, and the core portion contributes significantly to the formation of the keel of the crest part. The rounded, core-bearing part of the base of the acrosome progressively indents and fits into the concavity, thus formed, at the anterior part of the nucleus. The possible homology of the acrosomal complex (including the perforatorium) and the nucleus between non-passerine and passerine birds was discussed. The centriolar complex comprises both the proximal and distal centrioles in all spermatids and spermatozoa. The mitochondria undergo a number of morphological changes, including size and electron-density, from the round spermatid through to the mature spermatid; changes that are probably influenced by their functional states in the different evolving phases of the spermatids.

Distribution of actin filaments in the seminiferous epithelium of the Habu, Trimeresurus flavoviridis.

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.

Telomere Dynamics Throughout Spermatogenesis.

Telomeres are repeat regions of DNA that cap either end of each chromosome, thereby providing stability and protection from the degradation of gene-rich regions. Each cell replication causes the loss of telomeric repeats due to incomplete DNA replication, though it is well-established that progressive telomere shortening is evaded in male germ cells by the maintenance of active telomerase. However, germ cell telomeres are still susceptible to disruption or insult by oxidative stress, toxicant exposure, and aging. Our aim was to examine the relative telomere length (rTL) in an outbred Sprague Dawley (SD) and an inbred Brown Norway (BN) rat model for paternal aging. No significant differences were found when comparing pachytene spermatocytes (PS), round spermatids (RS), and sperm obtained from the caput and cauda of the epididymis of young and aged SD rats; this is likely due to the high variance observed among individuals. A significant age-dependent decrease in rTL was observed from 115.6 (±6.5) to 93.3 (±6.3) in caput sperm and from 142.4 (±14.6) to 105.3 (±2.5) in cauda sperm from BN rats. Additionally, an increase in rTL during epididymal maturation was observed in both strains, most strikingly from 115.6 (±6.5) to 142 (±14.6) in young BN rats. These results confirm the decrease in rTL in rodents, but only when an inbred strain is used, and represent the first demonstration that rTL changes as sperm transit through the epididymis.

Ultrastructure of spermiogenesis and the distribution of spermatozoal nuclear histones in the Japanese mantis shrimp, Oratosquilla oratoria (Crustacea: Stomatopoda).

The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of China, the Philippines, the Malay Peninsula, and the Hawaiian Islands. Early studies described certain characteristics of spermatogenesis and the sperm ultrastructure in Stomatopoda, but the composition of sperm basic nuclear proteins (SBNPs) remains completely unknown. We studied the sperm ultrastructure of O. oratoria using transmission electron microscopy and the histone composition using immunofluorescence and immunoelectron microscopy. We found that the spherical nucleus is adjacent to the electron translucent external coat, which occurs in early spermatids. The acrosomal structure begins to form at the junction of the nucleus and the external coat. At the mid-spermatid stage, part of the chromatin appears to be more electron-dense than the external coat side. The aflagellate sperm of O. oratoria, are rounded or slightly ovoid in shape and have a consistent granular nucleus, an acrosome structure of pushpin shape and a spherical vesicular body in which faintly granular material is scattered. The acrosome consists of an acrosomal vesicle, perforatorium, and subacrosomal material. The sperm contains histones H2A, H2B, H3, H4, H3.3, H2AX, and H2AZ as well as some histone modifications, that is, H3K9me3, H3K4me2, H3S10ph, H4Kac, and H2A + H4S1ph. Histones are localized not only in the nucleus of the sperm but also in other structures outside the nucleus. The results may provide new perspectives for systematic studies of crustaceans and their sperm chromatin components. These findings extend the study of the sperm structure of Stomatopoda and provide basic data to elucidate the epigenetic mechanism of fertilization.

First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae).

Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using transmission electron microscopy and cytochemical staining for glycogen. General pattern of spermiogenesis is essentially like that of other caryophyllideans, i.e., apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of free flagellum and a flagellar bud, and proximo-distal fusion. Interestingly, rotation of a free flagellum and flagellar bud to the median cytoplasmic process (MCP) has been observed unconventionally at > 90° only in progenetic stages. Typical striated roots associated with the centrioles occur rarely in A. sieboldi, and only in form of faint structures in advanced stages of spermiogenesis. In contrast to most caryophyllideans studied to date, penetration of the nucleus into the spermatid body has started before the fusion of the free flagellum with the MCP. This feature has been reported rarely but exclusively in the family Caryophyllaeidae. The unipartite mature spermatozoon of A. sieboldi is composed of one axoneme of the 9 + '1' trepaxonematan pattern with its centriole, parallel nucleus, and parallel cortical microtubules which are situated in a moderately electron-dense cytoplasm with glycogen particles. An unusual arrangement of cortical microtubules in the two parallel rows in region I of the spermatozoon is described here for the first time in the Caryophyllidea. Ultrastructural data on spermiogenesis and the spermatozoon in A. sieboldi from tubuficids and carp are compared and discussed with those in other caryophyllideans and/or Neodermata.

Increase of germ cell nuclear factor expression in globozoospermic Gopc-/- knockout mice.

BACKGROUND: Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC) is a Golgi protein that plays a role in vesicular transport and intracellular protein trafficking and degradation. Mice deficient in GOPC protein have globozoospermia and are infertile. The germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily which is expressed in male germ cells, from spermatocytes and spermatids, both in the nucleus and the acrosomal region. It is not known if its expression could be altered in Gopc-/- mice with defective acrosomes. OBJECTIVES: The aim of the present work was to analyze the distribution of GCNF protein in spermatids of Gopc-/- knockout mice. MATERIALS AND METHODS: We have analyzed the expression and distribution during spermatogenesis of GCNF and its deregulation in Gopc-/- mutant mice by RT-qPCR, Western blot, immunohistochemistry and immunogold. RESULTS: Germ cell nuclear factor was localized in the nucleus of all the cell types in the seminiferous tubules. Despite being a nuclear protein, it was also located in the acrosome and in the manchette of elongating spermatids. We have found that in the absence of GOPC, the expression of GCNF was increased in the nucleus of spermatocytes, mainly in leptotene, and in the nucleus and the manchette during the spermatid elongation. DISCUSSION AND CONCLUSION: Gopc-/- mice have defective acrosome and manchette. The manchette is involved in the transport of proteins through the cytoplasm and the nucleus. Considering that the GCNF protein is normally transported to the acrosome and the nucleus, it can be thought that transport deficiencies in Gopc-/- mice are responsible for the increased expression of this protein.