Acrosomal Vesicle Research Articles

Structural and ultrastructural characterization of the spermatozoa in Pachycondyla striata and P. marginata

This study characterizes the male gametic cells in two ant species (Pachycondyla striata and Pachnoda marginata) by light and transmission electron microscopies. The sperm of both species is composed of head, flagellum, and transition regions. The head consists of the acrosome, tapered and bilayer, and a cylindrical nucleus. In P. marginata, the intranuclear material is homogeneously compacted, and in P. striata, there are translucent inclusions and density variations. In the transition region of both species, centriolar adjunct, nuclear base, and the modified basal body (structure of the spermatozoa equivalent to the spermatids’ centriole) coexist and are progressively replaced by two oval and symmetrical mitochondrial derivatives and an axoneme 9 + 9 + 2, respectively, in addition to two symmetrical triangular accessory bodies. Moreover, cytochemical analysis with Ethanolic Phosphotungstic Acid Method (E-PTA) revealed species-specific features not previously reported in the Formicidae, such as the presence of E-PTA negative acrosomal vesicle, E-PTA positive perforatorium, and E-PTA positive nucleus, all in P. marginata. Mitochondrial derivatives exhibited distinct behavior in its internal composition, with E-PTA negative mitochondrial cristae and E-PTA positive paracrystalline in both species. Sperm morphology in Pachycondyla has marked morphological similarities with other ant groups, and its specific features can be useful tools to understand phylogenetic relationships within the Formicidae and Hymenoptera.

Ultrastructural Localization of Intracellular Calcium During Spermatogenesis of Sterlet (Acipenser ruthenus).

Calcium regulates many intracellular events such as growth and differentiation during different stages of gamete development. The aim of this study was to localize and quantify the intracellular distribution of calcium during different developmental stages of spermatogenesis in sterlet, Acipenser ruthenus, using a combined oxalate-pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. In the spermatogonium and spermatocyte, calcium deposits were mainly localized in the nucleus and cytoplasm. The spermatid had calcium in the nucleus, developing acrosomal vesicle, and cytoplasm. Intracellular calcium transformed from scattered deposits in spermatogonia and spermatocyte stages into an unbound form in spermatid and the spermatozoon. The proportion of area covered by calcium increased significantly (p<0.05) from early to late stages of spermatogenesis. The largest proportion of area covered by calcium was observed in the nucleus of the spermatozoon. In conclusion, although most of the intracellular calcium is deposited in limited areas of the spermatogonium and spermatocyte, it is present an unbound form in the larger area of spermatids and spermatozoa which probably reflects changes in its physiological function and homeostasis during the process of male gamete production in spermatogenesis.

Ultrastructural differentiation of spermiogenesis in Scincus scincus (Scincidae, Reptilia).

BackgroundKnowledge of spermiogenesis in reptiles, especially in lizards, is very limited. Lizards found in Arabian deserts have not been considered for detailed studies due to many reasons and the paucity of these animals. Therefore, we designed a study on the differentiation and morphogenesis of spermiogenesis, at an ultrastructural level, in a rare lizard species, Scincus scincus.ResultsThe spermiogenesis process includes the development of an acrosomal vesicle, aggregation of acrosomal granules, formation of subacrosomal nuclear space, and nuclear elongation. A role for manchette microtubules was described in nuclear shaping and organelle movement. During head differentiation, the fine granular chromatin of the early spermatid is gradually replaced by highly condensed contents in a process called chromatin condensation. Furthermore, ultrastructural features of sperm tail differentiation in S. scincus were described in detail. The commencement was with caudal migration toward centrioles, insertion of the proximal centriole in the nuclear fossa, and extension of the distal centrioles to form the microtubular axoneme. Subsequently, tail differentiation consists of the enlargement of neck portion, middle piece, the main and end pieces.ConclusionsThis study aids in the understanding of different aspects of spermiogenesis in the lizard family and unfurls evolutionary links within and outside reptiles.

Molecular characterization and sub-cellular distribution of JNK and JIP4 protein kinases in spermatogenesis and acrosome reaction of the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Brachyura: Varunidae)

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in controlling cell growth, differentiation, and apoptosis in mammalian testis and germ cells. As a scaffold protein of the JNK pathway, JNK-interacting protein 4 (JIP4) is found in some mammalian spermatozoa and participates in certain male reproductive processes. Few have reported on the roles of JNK and JIP4 in the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853. Based on transcriptome sequences from the testis of E. sinensis , we successfully cloned full-length cDNAs of JNK and JIP4 (designated Es-JNK and Es-JIP4 , respectively). Western blotting showed relatively high expression of Es -JNK and Es -JIP4 proteins in the testis. Expression levels of total Es -JNK, phosphorylated Es -JNK ( Es -p-JNK), and Es -JIP4 proteins decreased gradually during spermatogenesis. Es-JNK and Es-JIP4 mRNAs were mainly localized in the nucleus from the spermatogonium stage to the secondary spermatocyte stage and were widely distributed in the nuclear cup, apical cap, acrosomal tubule, and acrosomal vesicle of spermatids. Es -JNK, Es -p-JNK, and Es -JIP4 proteins were nevertheless mainly dispersed in the cytoplasm and intercellular substance of spermatogonium and primary spermatocytes, in the nucleus of secondary spermatocytes, and in the apical cap, acrosomal tubule, acrosomal vesicle, and nuclear cup of spermatids. During the acrosome reaction induced by the calcium ionophore A23187, Es -JNK, Es -p-JNK, and Es -JIP4 proteins were found mainly expressed in the apical cap and acrosomal vesicle by immunofluorescence microscopy. The results implicate functions for Es -JNK and Es -JIP4 in spermatogenesis and the acrosome reaction in E. sinensis .

Comparative sperm ultrastructure of twelve leptodactylid frog species with insights into their phylogenetic relationships

The spermatozoa of representatives of three Neotropical frog subfamilies, Leiuperinae, Leptodactylinae and Paratelmatobiinae, were observed using Transmission Electron Microscopy, with the aim of identifying ultrastructural traits that provide insights into the phylogenetic relationships among these anurans, which are currently unclear. In the leiuperines, spermatozoa of Physalaemus albifrons, P. cicada, P. deimaticus and P. feioi were characterized by an acrosomal vesicle covering the subacrosomal cone that was not observed in the spermatozoa of Physalaemus centralis and P. cuvieri. The tail of the spermatozoa of P. albifrons, P. centralis, P. cicada, P. cuvieri, P. deimaticus, and P. feioi presented a long undulating membrane, whereas Engystomops petersi and E. freibergi, which form a sister clade to Physalaemus, had an axial fiber, which were absent in Physalaemus. Other leiuperine, E. puyango had an abaxonemal bulb-like swelling distally to the paraxonemal rod, which were also absent in Physalaemus. These differences support the revalidation of Engystomops as a true taxon, distinct from Physalaemus. The tail of the spermatozoa of E. petersi and E. freibergi was similar to that of Paratelmatobius poecilogaster (Paratelmatobiinae). The spermatozoa of Leptodactylus natalenis (Leptodactylinae) had undulating membrane and axial fiber, in contrast with Adenomera marmorata, which lacked these structures. Morphological differences between A. marmorata and L. natalensis sperm cells appeared to validate the allocation of A. marmorata into a genus distinct from Leptodactylus. Overall, dissimilarities in the spermatozoa of the leptodactylids provided an important phylogenetic signal for the understanding of their taxonomic relationships.

The evolution of insect sperm − an unusual character system in a megadiverse group

Spermatozoa provide an unusual character system, with a limited number of components organized in a single cell. Similar spermatozoa occur in groups widely separated in the phylogenetic tree of Metazoa. Nevertheless, the character system contains phylogenetic information. Hexapoda have acquired spermatophores along with the switch from aquatic to terrestrial habitats, and related to this, a multitude of different sperm types. The aim of this study is a formal evaluation of the phylogenetic information content of spermatozoa. For the first time, sperm characters are coded for formal phylogenetic analyses. Different approaches are used and compared. Mainly due to a high level of homoplasy, the evaluation of sperm characters alone is insufficient for a reconstruction of the phylogeny of the group. Yet, a reliable reconstruction of the evolution of insect sperm is possible when character transformations are assessed using a phylogeny based on extensive molecular data. Important changes took place in the early evolution of Hexapoda. Sperm characters support some major clades (e.g. Hexapoda, Dicondylia, Polyneoptera, Psocodea), but important steps in the evolution are not reflected by transformations of spermatozoa, notably the rise of Pterygota or Holometabola. Important innovations are the formation of mitochondrial derivatives and the acquisition of accessory microtubules. Some features are conservative, whereas others evolved rapidly (e.g. presence or absence of the acrosome vesicle). Some groups are conservative in their sperm features (e.g. Odonata, Heteroptera), whereas the evolution of spermatozoa was distinctly accelerated in others (e.g. Ephemeroptera). The rate of evolution can change drastically in closely related groups. Profound changes in the morphologically uniform Zoraptera underline that sperm evolution can follow a pattern very different from the general somatic morphology. The mode of character reconstruction preferred here will be useful for the evaluation of specialized morphological character systems and strengthen the concept of evolutionary morphology.

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development.

This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI). Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS). No differences were observed in the zygote/cleavage/blastocyst rate between chemically activated and non-activated oocytes (50 vs. 45%, 11.6 vs. 10.1%; 1.8 vs. 1.1%, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45%, p < 0.01; 15.5 vs. 1.1%, p < 0.0001, respectively). Moreover, the percentage of PPS-derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2%). The ICSI blastocysts' development was also improved with PPS pre-treated with ionomycin (15.6%), but was completely impeded with PPS pre-treated with Triton X-100 (0%). Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.

Sperm morphology of Muscidifurax uniraptor (Hymenoptera: Chalcidoidea: Pteromalidae)

Sperm morphology of the parasitoid Muscidifurax uniraptor was investigated under light and transmission electron microscopy. M. uniraptor sperm are filiform, spiraled, approximately 150 μm in length, with a distinctive head, hooded by an extracellular sheath and a flagellum. This extracellular layer, from which many filaments radiate, measures approximately 90 nm in thickness and covers a small acrosome and the anterior nuclear region. The acrosome is composed of an acrosomal vesicle and a perforatorium with its base inserted in the nuclear tip. The nucleus is filled with homogeneously compacted chromatin. The centriolar adjunct extends towards the anterior portion in a spiral around the nucleus for 3.5 μm in length. The two mitochondrial derivatives begin exactly at the centriole adjunct base and, in cross-section, have a circular shape with equal areas that are smaller than the axoneme diameter. It is coiled, with 9 + 9 + 2 microtubules and begins from the centriole, just below the nuclear base. The axoneme is connected to the mitochondrial derivatives by two small irregularly shaped masses. Between the derivatives and the axoneme, the ‘center-flagellar material’ is observed. Overall, these characteristics are recognized in other Chalcidoidea, especially in the eurytomids, but together they form a set of species-specific data.

Three-dimensional reconstruction of black tiger prawn (Penaeus monodon) spermatozoa using serial block-face scanning electron microscopy.

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) was used in this study to examine the ultrastructural morphology of Penaeus monodon spermatozoa. SBF-SEM provided a large dataset of sequential electron-microscopic-level images that facilitated comprehensive ultrastructural observations and three-dimensional reconstructions of the sperm cell. Reconstruction divulged a nuclear region of the spermatophoral spermatozoon filled with decondensed chromatin but with two apparent levels of packaging density. In addition, the nuclear region contained, not only numerous filamentous chromatin elements with dense microregions, but also large centrally gathered granular masses. Analysis of the sperm cytoplasm revealed the presence of degenerated mitochondria and membrane-less dense granules. A large electron-lucent vesicle and "arch-like" structures were apparent in the subacrosomal area, and an acrosomal core was found in the acrosomal vesicle. The spermatozoal spike arose from the inner membrane of the acrosomal vesicle, which was slightly bulbous in the middle region of the acrosomal vesicle, but then extended distally into a broad dense plate and to a sharp point proximally. This study has demonstrated that SBF-SEM is a powerful technique for the 3D ultrastructural reconstruction of prawn spermatozoa, that will no doubt be informative for further studies of sperm assessment, reproductive pathology and the spermiocladistics of penaeid prawns, and other decapod crustaceans.

Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice.

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1-/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

Spermatogenesis in the rock oyster, Saccostrea forskali (Gmelin, 1791).

Morphology of the differentiating spermatogenic cells of the rock oyster Saccostrea forskali (Bivalve: Ostreidae) was investigated by light and transmission electron microscopy. The testis is formed by several branching acini containing developing spermatogenic cells, classified into 7 stages based on nuclear characteristics, patterns of chromatin condensation and cytoplasmic contents. The spermatogonium is characterized by a euchromatic nucleus with a prominent nucleolus. The cytoplasm contains several round granulo-fibrillar dense bodies surrounded by numerous mitochondria. The round nucleus of the primary spermatocyte contains patches of electron-dense heterochromatin, numerous proacrosomal vesicles, ribosomes and mitochondria. The secondary spermatocytes contain a reticulated chromatin pattern and reduced number of proacrosomal vesicles. The early spermatids contain a small amount of euchromatin among dense patches of heterochromatin. A large single acrosomal vesicle is located in the posterior part of the cell. The middle spermatid is characterized by the migration of an acrosomal vesicle to the anterior part of the nucleus. The late spermatids contain highly condensed heterochromatin blocks and the acrosomal vesicle becomes cup-shaped and invaginated at the basal part. The spermatozoon contains a barrel-shaped head covered with the cup-like acrosome. At this stage, the subacrosomal space contains an axial rod in subacrosomal materials. Three to four transverse bands appear at the anterior region of the acrosome. The middle piece consists of spherical mitochondria surrounding the proximal and distal centrioles. The flagellum consists of 9+2 axonemal microtubule doublets surrounded by the plasma membrane. Our electron microscopic study of spermatogenesis in the S. forskali provides important new information on the mechanism of development of spermatogenesis of this species.

Differences in sperm morphology in foam‐nesting leptodactyline frogs (Anura, Leptodactylidae)

AbstractSperm morphology is diverse among vertebrates and is influenced by the reproductive strategies adopted by species. In anurans, sperm morphology is associated with reproductive modes and mating systems. Here, we describe the sperm morphology of 11 frog species in the genus Leptodactylus and that of Lithodytes lineatus and discuss the relationship between sperm morphology and species' mating systems. We observed two distinct sperm morphotypes among the leptodactyline species, which differed mostly in head morphology. Type I sperm had triangular head, discrete acrosome vesicle with posterior margin not clearly visible; type II sperm had elongated head, clear acrosomal vesicle with posterior margin clearly visible. These sperm types do not seem to be associated with phylogeny; instead, type II sperm was observed in all polyandrous species analysed and in species with evidences of polyandry. Moreover, sperm of all species presented tail with undulating membrane connected to the axial fibre. We suggest that differences in sperm morphology might be associated with sperm competition to what polyandrous species are subjected. However, natural history observations on polyandrous mating in some species presenting type II sperm and phylogenetic comparative studies are need to elucidate the role of mating systems in the evolution of sperm morphology in leptodactylines.

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in Rat Spermatozoa.

The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-β-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization.

Sperm ultrastructure in honeycomb (foam) oysters (Mollusca, Bivalvia, Gryphaeidae, Pycnodontinae): comparison with other Ostreoidea and taxonomic implications

AbstractSperm ultrastructural features of the honeycomb (foam) oysters Hyotissa hyotis, H. sinensis, and H. mcgintyi (Gryphaeidae) are described and compared with other Ostreoidea and more generally with other pteriomorphian Bivalvia. Spermatozoa of H. sinensis and H. mcgintyi (the type species of Parahyotissa Harry 1985) exhibit (1) a broad, low‐conical acrosomal vesicle; (2) subacrosomal material (very electron‐dense granular material and an almost electron‐lucent axial rod); (3) a spheroidal nucleus with a wide anterior invagination (filled with subacrosomal components); (4) a midpiece composed of four spherical mitochondria surrounding a pair of centrioles (rootlet associated with proximal centriole); and (5) a flagellum. Sperm of Hyotissa hyotis (type species of Hyotissa Stenzel 1971) differ markedly from those of H. sinensis and H. mcgintyi, in having (1) a conical acrosomal vesicle showing coarse granular texture anteriorly; (2) a very electron‐dense axial rod; (3) a barrel‐shaped nucleus with a long, narrow anterior invagination (filled with both subacrosomal components) and a basal invagination partly housing the proximal centriole; and (4) five midpiece mitochondria and no proximal centriolar rootlet. Results indicate that H. sinensis should be relocated to another genus, possibly a revised genus Parahyotissa, and also show that the sperm of H. sinensis and H. mcgintyi show many similarities to those of the Ostreidae, with the exception that the ‘axial rod’ component of the subacrosomal material is less electron‐dense than the surrounding substance (more dense in Ostreidae, as in H. hyotis). No family defining sperm features of the Gryphaeidae can be identified.

Ultrastructural observations on spermiogenesis in the peanut worm, Phascolosoma esculenta (Sipuncula: Phascolosomatidea)

Ultrastructural characteristics of spermiogenesis in the peanut worm, Phascolosoma esculenta (Phascolosomatidea), were observed by transmission electron microscopy (TEM). The spermiogenesis principally occurs in the coelom and can be divided into three stages (the early, middle and late stages) based on the chromatin morphology, acrosome development, spermatozoon midpiece and flagellum formation. Spermatids within a given spermatid mass develop synchronously. With the spermiogenesis proceeding, the spermatid masses become loosely structured, and later, adjacent spermatids are interconnected at one extremity of the cells. Gradually, condensation of the chromatin accelerates and is almost completed in late spermiogenesis, leaving the late spermatids with highly condensed homogeneous chromatin. In the spermatid head, the conical acrosome is generally composed of an acrosomal vesicle which is formed by the coalescence of small proacrosomal vesicles within the cytoplasm, a subacrosomal space that situates between the acrosomal vesicle and nucleus, and an acrosomal rod which develops from a bunch of filamentous material within the subacrosomal space. Certain mitochondria move posteriorly towards the nucleus, thus constituting the spermatozoon midpiece. The flagellum, originated from the distal centriole, appears in the early spermiogenesis. Ultimately, mature spermatidium dissociates into numerous spermatozoa, which are subsequently released as a single cell from the coelom into the nephridia. The spermatozoon has a prominent head, containing an acrosome and nucleus, a short midpiece and a slender tail. When compared with other sipunculans or invertebrates with external fertilization, the spermiogenesis of P. esculenta, presumably, is closely associated with its biological adaptations for the reproductive strategy.

Spermatozoon ultrastructure and semen parameters of Brycon vermelha (Characiformes, Characidae)

This study analyzed semen parameters and the ultrastructure of spermatozoa of Brycon vermelha. The semen was white and viscous, with a mean volume of 5.0±2.6mL/kg body weight and mean spermatozoon concentration of 4.3±0.8×1010 spermatozoa/mL. The estimated motility rate was 90%, with 50% of spermatozoa motile at 35.0±0.1s and 100% immotile by 46.5±0.1s. The spermatozoon of B. vermelha had a distinct head, midpiece, and flagellum. The ovoid head measured 1.9±0.2μm by 1.3±0.1μm, with its volume almost completely occupied by the nucleus, and was enveloped by an irregular nuclear membrane, with no acrosome vesicle. The nuclear fossa held the centriole complex and the initial segment of flagellum. The midpiece was symmetrical and measured 1.3±0.3μm. Mitochondria were scarce and restricted to the anterior region, while vesicles were absent. The posterior region of the midpiece was characterized by the absence of mitochondria and the presence of the cytoplasmic sheath. The flagellum, enclosed by the flagellar membrane, measured 29.6±3.4μm, and possessed an axial filament containing a 9+2 microtubule pattern. The spermatozoa of B. vermelha appeared similar in structure to those of fish that breed through external fertilization, thus classifying them as uniflagellate anacrosomal aquasperm, or Type 1 aquasperm.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Ultrastructure of the Spermatogenic Process in the PenshellAtrina maura(Bivalvia: Pinnidae)

The ultrastructural features of the spermatogenic development and mature spermatozoa morphology of Atrina maura were investigated through transmission electron microscopy. The testis displays a diffuse tubular structure in which gametes develop within a concentric arrangement according to the degree of development. Within the tubules also appear Sertoli cells, which contain a large amount of lipid droplets and glycogen granules in the cytoplasm, suggesting a nutritive role in the first spermatogenesis stage (spermatogonia). Four gamete maturation stages co-occurred in the same individual: spermatogonia (4–8 μm), spermatocytes (3–4 μm) (including primary and secondary), spermatids (2.5 μm) and spermatozoa (∼ 2 μm head length). The proacrosomal vesicle and flagellum occur only until the first stages of spermiogenesis, contrasting with the early formation of both structures in spermatogonia and spermatocytes in other bivalves. In A. maura, the material in the acrosomal vesicle of mature spermatozoa shows a characteristic electron density pattern, which seems to be an exclusive taxonomic feature of the family Pinnidae. However, A. maura displayed only four mitochondria in the midpiece, contrasting with five in all other species in this family.

Aberrant expression of TAR DNA binding protein-43 is associated with spermatogenic disorders in men.

Loss of function of TAR DNA-binding protein (TDP-43) has been implicated in neurodegenerative disorders in both humans and animal models. TDP-43 has also been shown to be cis-acting transcriptional repressor of the acrosome vesicle (Acrv) gene in mice. In the present study, we investigated the expression of the TDP-43 transcript (TARDBP) and protein in germ cells from 11 fertile and 98 subfertile men to verify its potential association with poor seminograms. The expression profile of TDP-43 was characterised in immature germ cells and spermatozoa from semen from fertile and subfertile men using reverse transcription-polymerase chain reaction, western blotting and immunofluorescence. Although germ cells from subfertile men tested negative for TARDBP, the full-length message of the same was detected in fertile men. TDP-43 was detected in spermatozoa from fertile men using western blot analysis and immunofluorescence. The expression of this protein was negligible in spermatozoa from men with primary spermatogenic dysfunction. We conclude that a deficiency in the TDP-43 expression is associated with defective spermatogenesis and male infertility. We propose that TDP-43 could be used as a marker of male factor infertility.

Ultrastructure developments during spermiogenesis in Polydora ciliata (Annelida: Spionidae), a parasite of mollusca

Spionid worms of Polydora ciliata inhabit the shells of many commercially important bivalves and cause disease in molluscan aquaculture. Their sperm structure is closely related to their fertilization method. To give an insight into the sperm structure and spermatogenesis, ultrastructure details of the subcellular components of germ cells during spermiogenesis of Polydora ciliata are detected by transmission electron microscopy (TEM). In P. ciliata, during spermiogenesis, chromatin is regularly arranged as dense fibrils and becomes more condensed when the nucleus elongates. Microtubules do not surround the nucleus during its elongation. The Golgi phase is characterized by the formation of proacrosomal granules within the Golgi apparatus. The proacrosomal granules fuse to form a single, spherical acrosomal vesicle that migrates to the anterior pole of the cell. At the time of nuclear condensation, mitochondria become reduced in number but increased in size, causing deep indentation at the base of the nucleus. The mid-piece has a few mitochondria. The cap phase includes the spreading of the acrosomal granule over the surface of the nucleus of the differentiating spermatid. The acrosomal phase of spermiogenesis is typically associated with changes in the shape of the nucleus, acrosome and tail. The relationship of sperm ultrastructure to spermiogenesis in spionidae species was discussed.