Acrosomal Vesicle Research Articles

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.

Spermatogenesis and gonadal cycle in male Tamoya haplonema and Chiropsalmus quadrumanus (Cnidaria, Cubozoa)

Spermatogenesis has been little studied in cubozoans. This work describes spermatogenesis of two cubozoan species from Brazil, Tamoya haplonema and Chiropsalmus quadrumanus, using light microscopy and histochemical techniques to highlight and contrast stages of the gonadal cycle. Specimens were collected by hand at the water surface or with shrimp trawls in three areas in the state of São Paulo (SE Brazil): Cananéia lagoon estuarine system (south), Santos and São Vicente Bay (central), and São Sebastião and Ubatuba (north). Male box jellyfish gonads are located between the mesoglea and gastrodermis in a thin and independent layer, organized in elongated follicles, macroscopically resembling a fingerprint pattern. After subsampling, serial sections were performed on male gonad tissue. The resulting slides were stained with hematoxylin and eosin and methods for staining proteins, acidic materials and neutral polysaccharides. Light microscopy revealed that the follicle wall of the male gonads of both cubozoan species is composed of spermatogonia. Spermatocytes are found peripherally near the follicle wall, while spermatids and spermatozoa are located towards the center. Sperm release occurs through rupture of the follicular wall into the gastric pockets. Four stages of gonad development were documented for T. haplonema and three for C. quadrumanus. The sperm of C. quadrumanus has a rounded head while in T. haplonema the sperm head is conical with an elongated apical projection. In both species, there was no positive histochemical reaction for putative acrosomal vesicles precursors.

Morphometry, frequency and ultrastructure of male germ cells in morphotypes of the freshwater prawn Macrobrachium amazonicum (Decapoda: Palaemonidae)

Males of the freshwater prawn species, Macrobrachium amazonicum, have been staged histologically into morphotypes, Translucent claw (TC), Cinnamon claw (CC) and Green claw (GC). However, information on reproductive system anatomy and spermatogenesis among morphotypes is scarce. Our aim was to describe the frequency of spermatogenic cells, morphometry of seminiferous tubules and spermatogenic cells, and spermiogenesis to establish any differences among morphotypes. Specimens were captured and sexed. Testes were dissected, processed, and analyzed using light, transmission, and scanning electron microscopy. Macroscopically, testes were symmetrical, elongated, and translucid, with long vasa deferentia ending in ampoules. Histologically, testes exhibited seminiferous tubules with the germinal epithelium containing male germ cells in different stages of development and supporting cells. The diameter of seminiferous tubules was largest (P < 0.05) in the GC morphotype. Germ cell types were evaluated based upon size, as well as their cytoplasm and nuclear chromatin organization. Spermatozoa were the only germ cells displaying no differences in size among morphotypes. According to shape and localization, we classified spermatids into three subtypes: St1 located in the germinal epithelium with scant cytoplasm, a concave nucleus, and condensed chromatin; St2 sickle-shaped, with a concave, thin nucleus; and, St3 cup-shaped, with a developing acrosomal vesicle in the convex portion of the cell. Notably, St2 and St3 were found in the lumen of the seminiferous tubules and vasa deferentia. Spermatids and spermatozoa were floating in a large amount of amorp's glycoprotein-rich material. Spermatozoa were cup-shaped and displayed an acrosome fashioning a long spike with regular and compact cross-striations. In summary, we established differences in size and frequency of male germ cells among morphotypes and described spermatid characteristics not yet observed in other prawn species. Altogether, this information contributes to expanding our knowledge on taxonomy, phylogenetic relationships, and reproduction among Palaemonidae species.

Ultrastructural investigations on spermatogenesis and spermatozoan morphology in the endangered Adriatic sturgeon, Acipenser naccarii (Chondrostei, Acipenseriformes).

Spermatogenesis was investigated in the Adriatic sturgeon, Acipenser naccarii, by light and electron microscopy. The testis of the unrestricted type had a germinal compartment composed of lobules containing germ cells and Sertoli cells, and separated by a basal lamina from the interstitial compartment, in which Leydig and myoid cells were detected for the first time in Acipenseridae. Spermatogenesis occurred in spermatocysts produced when Sertoli cells became associated with type A spermatogonia of subsequent generations, which produced a clone of synchronized aligned spermatogonia. In primary spermatocytes at zygo-pachytene stage, the large spherical nucleus contained synaptonemal complexes. The smaller secondary spermatocytes were ovoid with a central round nucleus and scarce cytoplasm. Spermatids were interconnected by cytoplasmic bridges until early spermiogenesis. Chromatin initially condensed as long, twisted, and nonhomogeneous fibers and finally as a compact structure made of thick filaments. Early spermatids showed the flagellum, the primordia of centriole complex and of "implantation fossa," followed by the acrosomal vesicle formed by Golgi complexes and a fibrous body associated to centriole complex. The spermatozoan head had 10 postero-lateral projections and a trapezoidal nucleus, a cylindrical midpiece with six to eight mitochondria, the centriole complex, and a "9 + 2" flagellum with a pair of lateral fins. Three helical endonuclear canals crossed the nucleus from the acrosome base to the implantation fossa; their spiralization and that of chromatin fibers suggest a spiral twisting of the nucleus during spermiogenesis. The Sertoli cells performed phagocytosis of degenerating spermatids and spermatozoa. Significant interindividual differences were detected in most morphological parameters of spermatozoa. Data on spermatogenesis in A. naccarii and morphometric measurements on mature spermatozoa provide information about the reproductive biology of the species useful not only for phylogenetic studies but also for evaluation of sperm quality for artificial reproduction projects and restocking of this and other critically endangered sturgeon species.

Ultrastructure of spermatozoa of members of Calappidae, Aethridae and Menippidae and discussion of their phylogenetic placement

AbstractThe families Aethridae and Calappidae were originally considered as part of the same family; however, their morphology and molecular biology separate them into two families. In this context, we describe the ultrastructure of spermatozoa of species of the Calappidae, Aethridae and Menippidae to elucidate the relationships among taxa. The vasa deferentia were submitted to routine protocols for transmission electron microscopy. Our results indicate that the morphology of the spermatozoa of Hepatus pudibundussupports its exclusion from the Superfamily Calappoidea due to the presence of the apical striated layer. The spermatozoa of Menippe nodifrons is very similar to H. pudibundus and corroborates the recent phylogenetic analysis using sequence data of nuclear genes. Moreover, our results evidence two morphological patterns of spermatozoa within Calappidae. Calappa ocellata and C. cinerea show spermatozoa with a wide acrosome vesicle, a thick operculum shaped as a shallow “W” and a large thickened ring. Calappa gallusand C. hepatica show spermatozoa with a longer acrosome vesicle, a pointed operculum and a slender thickened ring. Our ultrastructure results conform with previous molecular proposal and show that spermatozoa ultrastructure can be an effective tool to adjust phylogenetic relationship when used in association with molecular data.

Fine structure of the gametes and spermiogenesis in Spiophanes uschakowi (Annelida: Spionidae) from the Sea of Japan, with comments on fertilization biology in broadcast-spawning spionids

Spiophanes uschakowi is a common polychaete living in tubes in sandy sediments in shallow waters of the Sea of Japan. Females and males release their gametes into the water where fertilization and holopelagic, planktotrophic larval development occur. In females, oogenesis is intraovarian: vitellogenesis occurs when the oocytes grow in paired ovaries attached to genital blood vessels in fertile segments. The developed oocytes are accumulated in the coelomic cavity prior to spawning. The newly released oocytes are lentiform, 185–200 μm in diameter, with honey-combed envelopes 5–7 μm thick. Each oocyte has 41–49 cortical alveoli regularly arranged in a peripheral circle, a nucleus 80–83 μm in diameter, and a single nucleolus about 30 μm in diameter. In males, spermatogonia proliferate in testes and the rest of spermatogenesis occurs in the coelomic cavity. During spermiogenesis, the acrosomal vesicle migrates from the posterior to the anterior part of the spermatid. The spermatozoa are ect-aquasperm with a plate-like acrosome 0.58 ± 0.06 μm thick and 2.14 ± 0.13 μm in diameter, barrel-shaped nucleus 2.23 ± 0.13 μm long and 3.18 ± 0.13 μm in diameter, short midpiece 0.93 ± 0.09 μm long with five spherical mitochondria, two centrioles and one small lipid droplet, and a flagellum 62–63 μm long with 9 × 2 + 2 organization of microtubules. The acrosome is a complex heterogeneous structure with 4–6 subspherical apical bodies, and numerous small branched basal cisternae. The anterior end of the nucleus is truncate, while its posterior end has wide shallow depressions accommodating the mitochondria. The centrioles are situated in the center of the midpiece between mitochondria and oriented obliquely to each other. The structure of the gametes of broadcast-spawning spionids is reviewed and the roles of surface granules in species-specific attraction of sperm toward eggs by releasing chemical signals (sperm chemotaxis), and cortical alveoli as a place of penetration of spermatozoa into oocytes (micropyle) are suggested. The lentiform oocytes of Spiophanes spp. are unique among Spionidae by their shape, while spermatozoa are unique by their plate-like acrosomes.

AU040320 deficiency leads to disruption of acrosome biogenesis and infertility in homozygous mutant mice

Study of knockout (KO) mice has helped understand the link between many genes/proteins and human diseases. Identification of infertile KO mice provides valuable tools to characterize the molecular mechanisms underlying gamete formation. The KIAA0319L gene has been described to have a putative association with dyslexia; surprisingly, we observed that homozygous KO males for AU040320, KIAA0319L ortholog, are infertile and present a globozoospermia-like phenotype. Mutant spermatozoa are mostly immotile and display a malformed roundish head with no acrosome. In round spermatids, proacrosomal vesicles accumulate close to the acroplaxome but fail to coalesce into a single acrosomal vesicle. In wild-type mice AU040320 localises to the trans-Golgi-Network of germ cells but cannot be detected in mature acrosomes. Our results suggest AU040320 may be necessary for the normal formation of proacrosomal vesicles or the recruitment of cargo proteins required for downstream events leading to acrosomal fusion. Mutations in KIAA0319L could lead to human infertility; we screened for KIAA0319L mutations in a selected cohort of globozoospermia patients in which no genetic abnormalities have been previously identified, but detected no pathogenic changes in this particular cohort.

Ultrastructure of spermatophoral sperm in the freshwater gastropod Thiara amarula (Linnaeus, 1758) (Cerithioidea: Thiaridae): potential taxonomic features including eusperm nuclear content differentiation

The ultrastructure of spermatophoral sperm is investigated for the first time in a cerithioidean gastropod. Thiara amarula (Linnaeus, 1758), a widespread Indo-West Pacific species of the Thiaridae, produces uniaxonemal euspermatozoa and multiaxonemal paraspermatozoa. Euspermatozoa possess a laterally-flattened acrosomal vesicle (with eccentrically positioned invagination and subacrosomal material), a tubular, laterally-compressed nucleus, an elongate midpiece (with four, straight, equal-sized mitochondria each with parallel cristae), an elongate glycogen piece and an end piece. The eusperm nucleus exhibits or is associated with a number of unusual features including: (1) nuclear contents differentiated into two components (fibrous, highly electron-dense material, enclosing a pair of less-dense, finely granular tracts); (2) nuclear invagination with two elongate grooves aligned at 90 degrees to the axonemal central microtubules and parallel with the 1 and 5 axonemal doublets; (3) a periodically-banded rootiet associated with the centriolar complex near the nuclear apex; (4) an electron-dense structure attaching the nucleus to the axoneme at the nucleus-midpiece junction. Feature (1) has been reported elsewhere in Melanoides tuberculata (Thiaridae), but in no other examined gastropods (or other molluscs) and its purpose is unknown. Paraspermatozoa of T. amarula possess an elongate head region (up to 14 axonemes and large, irregular mitochondria enclosed by sheath of spherical, dense vesicles), a short 'glycogen' region and a posterior tuft of glycogen tails. The two sperm types do not form spermatozeugmata and observations suggest that neither type undergoes any noticeable structural change while inside the spermatophore. Sperm features suggest a close relationship between T. amurula and M. tuberculata and more broadly with other 'Group T cerithioidean families exhibiting four equal-sized eusperm mitochondria, including Potamididae, Modulidae, Scaliolidae, Melanopsidae, Semisulcospiridae and Pleuroceridae.

Roles of three Es-Caspases during spermatogenesis and Cadmium-induced apoptosis in Eriocheir sinensis.

Functions of Caspases remain obscure in Crustacea. We studied the existence and participations of apoptosis-related factors in Eriocheir sinensis testis. Three Es-Caspases (Es-Caspase 3/ 7/ 8) in E. sinensis were cloned and characterized. We observed that three es-caspases mRNA had specific expression patterns during spermiogenesis, with weak signal around the nucleus and invaginated acrosomal vesicle in early-stage spermatids, became stronger in middle-stage, finally focused on the acrosomal tube and nucleus in mature sperm. We then investigated the immunostaining intensity and positional alterations of Es-Caspase 3, Es-Caspase 8 and p53 during spermatogenesis, which were correlated with the differential tendencies of cells to undergo apoptosis and specific organelles shaping processes. After apoptotic induction by Cadmium, Es-Caspase 8 increased gradually, while Es-Caspase 3 increased firstly and then decreased, Es-p53 initially decreased and then increased. These results implies that Es-Caspase 3/ Es-Caspase 8/ p53 may play roles in Cadmium-induced apoptosis during spermatogenesis, and Caspase 8-Caspase 3-p53 pathway may interact with extrinsic or intrinsic pathways to regulate the destiny of sperm cells. Our study revealed the indispensable roles of Caspases during spermatogenesis and the possible molecular interactions in response to the Cadmium-induced apoptosis in E. sinensis, which filled the gap of apoptotic mechanisms of crustacean.

Acrosomal alkalization triggers Ca2+ release and acrosome reaction in mammalian spermatozoa.

The sperm acrosome reaction (AR), an essential event for mammalian fertilization, involves Ca2+ permeability changes leading to exocytosis of the acrosomal vesicle. The acrosome, an intracellular Ca2+ store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pHacr ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca2+ concentration ([Ca2+ ]i ) through Ca2+ release from internal stores, mainly the acrosome. Catsper, a sperm specific Ca2+ channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca2+ ]i by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pHacr , trigger Ca2+ release from the acrosome, and induce the AR in both mouse and human sperm. To our surprise, μM concentrations of NNC55-0396 induced AR even in nominally Ca2+ free media. Our findings suggest that alkalization of the acrosome is critical step for Ca2+ release from the acrosome that leads to the acrosome reaction.

Electron microscopy observations on testis and spermatozoa of Leptodactylus chaquensis (Anura, Leptodactylidae)

Scanning and transmission electron microscopy were used to investigate the fine structure of the testis and spermatozoa of toad Leptodactylus chaquensis. Our observations indicate that germinal compartment contains Sertoli (SC) and germ cells. The tight junctions and desmosomes among SC indicate the existence of an hematotesticular barrier in charge of maintaining the differences in the composition of the germinal and interstitial compartments. During spermatogenesis, SC acts as a structural support for germ cells. Secondary spermatogonias, spermatocytes and spermatids are joined by cytoplasmic bridges that allow communication between cells in the same cyst. Spermatids at the subcellular level two well-defined morphological stages can be observed: primary spermatids are rounded cells with an acrosomal vesicle attached to the nucleus which has a diameter of about 4.39±0.36μm. Secondary spermatids are elongated with a nucleus of about 19.50±0.92μm in diameter and the acrosome and the axoneme are located in opposite poles of the cells. At the apical end of the spermatozoon we can observe a large arrowhead-shaped acrosome (6.26±0.28μm in length) that takes up about one third of the gamete head with 17.33±0.29μm in length. The proximal centriole is located in the nuclear fossa while the distal centriole gives rise to the flagellar axoneme. The flagellum exhibits a typical pattern of “9+2” and adjacent to it is the axial fiber, and an undulating membrane stretches between both structures. Transmission electron microscopy observations allowed us to produce a diagram of the structure of the spermatozoon of L. chaquensis. Leydig cells, located in the interstitial compartment, show scarce cytoplasm, mitochondria and large-sized lipid droplets which would provide the raw matter for the synthesis of steroid hormones.

Prohibitin-mediated mitochondrial ubiquitination during spermiogenesis in Chinese mitten crab Eriocheir sinensis.

The sperm of Eriocheir sinensis has a cup-shaped nucleus that contains several mitochondria embedded at the opening of the cup. The acrosome vesicle also contains derivants of mitochondria. The mitochondria distribution pattern involves a decrease in the number and changes in the structure and transportation of these organelles. The decreased number of sperm mitochondria is achieved through autophagy or the ubiquitination pathway. Prohibitin (PHB), the mitochondria inner membrane protein, is an evolutionarily highly conserved protein, is closely associated with spermatogenesis and sperm quality control and is also a potential substrate of ubiquitination. However, whether PHB protein mediates the ubiquitination pathway of sperm mitochondria in crustacean animals remains poorly understood. In the present study, we revealed that PHB, a substrate of ubiquitin, participates in the ubiquitination and degradation of mitochondria during spermiogenesis in E. sinensis. To confirm this finding, we used shRNA interference to reduce PHB expression and an overexpression technique to increase PHB expression in vitro. The interference experiment showed that the reduced PHB expression directly affected the polyubiquitination level and mitochondria status, whereas PHB overexpression markedly increased the polyubiquitination level. In vitro experiments also showed that PHB and its ubiquitination decide the fate of mitochondria.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Sperm Ultrastructure of the Protobranchia: Comparison with Other Bivalve Mollusks and Potential Taxonomic and Phylogenetic Significance

Sperm ultrastructure of nine species of protobranch bivalves, representing three of four extant orders (Solemyida, Nuculida, Nuculanida), is discussed. Greatest diversity occurs in Solemyida (acrosomal vesicle low-conical, tall-conical, or very elongate, with radial plates; nucleus rod-shaped, teardrop-shaped, or very elongate; four, five, or six mitochondria) and the least in Nuculida (acrosomal vesicle low- to tall-conical; lacking radial plates; nucleus rod-shaped, five or six mitochondria) followed by Nuculanida (short, conical acrosomal vesicle with radial plates; spheroidal nucleus; four or five mitochondria). The wide variety of shapes in Solemyidae suggests taxonomic potential, especially in resolution and/or recognition of supraspecific taxa, but no diagnostic family characters were identified. Taxonomic potential exists for Nuculida (acrosomal shape) and Nuculanida (mitochondrial number). Protobranch sperm is highly diverse, and no defining character of the whole group was found. Support was found for the Nuculida and Nuculanida as natural groups but not for their close relationship. Nuculanida and Solemyida exhibit radial plates in the acrosomal vesicle but otherwise share no derived characters. The striking similarity of most sperm features of Nuculanida with certain pteriomorphians, especially Pectinoidea, a relationship also suggested by some mitochondrial DNA sequence data, poses interesting questions concerning their relationships and/or shared functional constraints.

Sugars Require Rigid Multivalent Displays for Activation of Mouse Sperm Acrosomal Exocytosis.

As a prerequisite to mammalian fertilization, the sperm acrosomal vesicle fuses with the plasma membrane and the acrosome contents are exocytosed. Induction occurs through engagement of the sperm receptors by multiple sugar residues. Multivalent polymers displaying mannose, fucose, or GlcNAc are effective synthetic inducers of mouse sperm acrosomal exocytosis (AE). Each carbohydrate is proposed to have a distinct binding site on the sperm cell surface. To determine the role of the scaffold structure in the efficiency of AE induction, different polymer backbones were employed to display the different activating sugar residues. These glycopolymers were prepared by ruthenium-catalyzed ring-opening metathesis of 5-substituted norbornene or cyclooctene. The conformations of the glycopolymers were characterized by small-angle X-ray scattering. Polynorbornene displaying mannose, fucose, or GlcNAc forms flexible cylinders in aqueous solution. However, polycyclooctenes displaying any of these same sugars are much more flexible and form random coils. The flexible polycyclooctenes displaying fucose or GlcNAc were less effective inducers of AE than their norbornene counterparts. In contrast, polycyclooctene displaying mannose was the most effective AE inducer and had a more collapsed spherelike structure. Our results suggest that the AE efficacy of fucose, GlcNAc, and mannose polymers relies on a relatively rigid polymer that can stabilize receptor signaling complexes.

Spermiogenesis in the African sideneck turtle (Pelusios castaneus): Acrosomal vesicle formation and nuclear morphogenesis

Testicular samples were collected from African sideneck turtles (Pelusios castaneus) at the peak of spermiogenesis in order to describe spermatid acrosomal vesicle formation and nuclear morphogenesis. Acrosomal vesicle formation commences with a Golgi transport vesicle attaching to a round spermatid, followed by the emergence of an acrosome granule. This is followed by the development of the sub-acrosomal space, which becomes enlarged as nuclear elongation and condensation continue. The round spermatid elongates and the emerging elongating spermatid successively becomes surrounded by circular, longitudinal and slanting microtubules of the manchette. The acrosomal vesicle becomes visible with an acrosome granule resting on the base of the electron dense material. Acrosomal vesicle morphogenesis in the African sideneck turtle results in a highly compartmentalized acrosome divisible into the acrosomal cortex and medulla. The future position of the flagellum starts to develop, being encircled by mitochondria while the distal centriole becomes obvious and the emerging flagellum grossly divisible into the connecting piece, midpiece, principal piece and endpiece. Although acrosomal vesicle formation and nuclear morphogenesis during spermiogenesis in the turtle are consistent with other reptilian species, a few differences were observed. The major difference observed was the formation of a single acrosome granule, which manifests prior to the attachment of the acrosomal vesicle to spermatid nucleus. The other differences observed were the emergence of two endonuclear canals in the elongating spermatid and the presence of slanting microtubules of the manchette. The observed developmental variations are expected to be valuable in future phylogenetic studies and potentially serve to test certain hypotheses concerning the reproductive status of turtle species. Findings from this study add to the growing database of spermatid morphology in turtles, thereby providing insights into variations in mature sperm morphology in the species.

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.

Globozoospermia and lack of acrosome formation in GM130-deficient mice

Globozoospermia is a common reproductive disorder that causes male infertility in humans, and the malformation or loss of acrosomes is the prominent feature of this disease. Although the acrosome is thought to be derived from the Golgi apparatus, the detailed molecular mechanisms remain unclear. GM130 is a cis-side localized Golgi matrix protein,whereas the physiological functions of this protein remain elusive. Here we showed that inactivation of GM130-caused male infertility in mouse model. The primary defects were the absence of acrosomes, round sperm heads, and aberrant assembly of the mitochondrial sheath, which comprise the characteristic features of human globozoospermia. Further investigation indicated that loss of GM130 did not affect the secretion of pro-acrosomic vesicles, whereas the vesicles failed to fuse into a single large acrosome vesicle. Co-localization of the adaptor protein complex AP1 and trans-Golgi network (TGN) protein TGN46 was disrupted, suggesting that the malformation of acrosomes is most likely due to the defect in the sorting and coating of Golgi-derived pro-acrosomic vesicles. Thus, the GM130-deficient mouse provides a valuable model for investigating the etiology of human globozoospermia.

Morphology of the Male Reproductive System and Spermiogenesis of Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae).

Studying the reproductive attributes of pests is central to understanding their life cycle history and in crafting management strategies to regulate, if not bring down, their population below threshold levels. In this article, the morphology of the male reproductive tract, topology of the spermatozoa, and salient features of spermiogenesis in the Chinese white pine beetle, Dendroctonus armandi Tsai and Li was studied to provide baseline information for further pest management studies. Results showed that male reproductive tract of this species differs from those documented in other Coleopterans by having 20 testicular tubules in each testis and the presence of two types of accessory glands. The spermatozoon is seen having peculiar characteristics such as an “h”-shaped acrosomal vesicle with a “puff”-like expansion, one centriole, one large spongy body, and two accessory bodies. Despite with some morphological differences of the male reproductive organ, spermatogenesis in this organism is similar to other Coleopterans. Overall, detailed studies regarding the components of the primary male reproductive organ of this beetle species would expand the knowledge on the less-understood biology of Coleopteran pests and would help in designing regulatory measures to conserve endemic and indigenous pine trees in China.

Interactome of the inhibitory isoform of the nuclear transporter Importin 13

Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in diverse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.