Spermatogenesis in the Watase's shrew, Crocidura watasei--a light electron microscopic study.

Spermatogenesis in the Watase's shrew, Crocidura watasei, was investigated by light and transmission electron microscopy. The cycle of the seminiferous epithelium was divided into 12 stages using the development of spermatids as a main criterion. The steps of spermatids were characterized by morphological changes of the nucleus and acrosomal structure. The relative frequencies of the stages 1 to x 11 were 11.0, 10.3, 6.8, 10.6, 24.0, 6.4, 4.4, 7.9, 6.4, 4.9, 3.7 and 3.6%, respectively. Four types of spermatogonia (A1, A2, In and B) could be discerned by the observation of whole mount samples. The development of spermatids was divided into four phases (Golgi, cap, acrosome and maturation phases), as in other mammals. In Golgi phase of the spermatid, several acrosomal granules were encountered. In cap phase, the acrosome gradually spread over the nuclear surface. In early acrosome phase, the acrosome began to elongate and reached the maximal length in step 8 spermatids. The acrosome of step 8 spermatids was twice as long as that of spermatozoa. In late acrosome phase, the acrosome was on the way of shrinkage. Finally, the fan-shaped acrosome was formed in maturation phase. These findings suggested that the process of acrosomal formation was quite characteristic in the Watase's shrew in that the spermatid acrosome elongated most prominently in the mammals hitherto examined.     

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in the process of fertilization. The molecular mechanism underlying the biogenesis of this lysosome-related organelle (LRO) is still largely unknown. Here, we show that germ cell-specific Atg7-knockout mice were infertile due to a defect in acrosome biogenesis and displayed a phenotype similar to human globozoospermia; this reproductive defect was successfully rescued by intracytoplasmic sperm injections. Furthermore, the depletion of Atg7 in germ cells did not affect the early stages of development of germ cells, but at later stages of spermatogenesis, the proacrosomal vesicles failed to fuse into a single acrosomal vesicle during the Golgi phase, which finally resulted in irregular or nearly round-headed spermatozoa. Autophagic flux was disrupted in Atg7-depleted germ cells, finally leading to the failure of LC3 conjugation to Golgi apparatus-derived vesicles. In addition, Atg7 partially regulated another globozoospermia-related protein, Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), during acrosome biogenesis. Finally, the injection of either autophagy or lysosome inhibitors into testis resulted in a similar phenotype to that of germ cell-specific Atg7-knockout mice. Altogether, our results uncover a new role for Atg7 in the biogenesis of the acrosome, and we provide evidence to support the autolysosome origination hypothesis for the acrosome.     

Evolution of octopod sperm II: comparison of acrosomal morphogenesis in Eledone and Octopus

The first stages of acrosome development during Eledone cirrhosa spermiogenesis are similar to that in Octopus vulgaris, and comprise the initial elongation of both organelles. However, the acrosome in E. cirrhosa does not continue its elongation as it does in O. vulgaris. Instead, its length remains fixed and it undergoes a process of helicoidization that includes the entire organelle. In each spermatid, helicoidization of the E. cirrhosa acrosome occurs simultaneously with helicoidization of the nucleus. The acrosome is associated with special structures that probably are involved in the helical torsion of the organelle. We propose a hypothesis to explain the evolutionary relationship between the acrosomes of O. vulgaris and E. cirrhosa, particularly as it is influenced by nucleomorphogenesis and microtubular contraction.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998     

Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.     

The acrosomal zonule

A structure associated with the developing acrosome during spermiogenesis in the rat and the Chinese hamster is described. The acrosomal zonule first appears in the cap phase of spermiogenesis and can be detected at all stages of development until after spermiation. The role of the acrosomal zonule is discussed.     

Changes in acrosome morphology during cooling and freezing of dog semen

Sixteen semen samples, collected from purebred beagles over a period of 12 months, were diluted, frozen and thawed. In an attempt to monitor any changes occurring to the acrosome during processing, smears made at various stages of the handling procedure were stained with Spermac stain and examined under 1000X magnification. Most acrosomal damage appeared to occur during the freeze/thaw processes. Sperm motility did not correlate well with acrosomal integrity.     

SPERMIOGENESIS IN BARNACLES WITH SPECIAL REFERENCE TO ORGANIZATION OF THE ACCESSORY BODY*

Ultrastructural changes during spermiogenesis in the barnacles, Balanus amphitrite albicostatus, Balanus tintinnabulum rosa, Balanus trigonus and Tetraclita squamosa japonica, and organization of the sperm with special reference to the accessory body were studied. The Golgi complex organizes both the acrosome and the accessory body at different stages during spermiogenesis; the former is formed at the mid-spermatid stage and the latter is formed at the late spermatid stage. The arrangement of the components in the mature filiform sperm is quite unique, with the acrosome, the basal body just behind the acrosome, the axial filament parallel to a long nucleus, and a slender long mitochondrion behind the nucleus. The sperm in the anterior and posterior half of the ejaculatory duct differ from each other in form in that the sperm in the anterior duct are not equipped with the accessory body and the sperm in the posterior duct are. The accessory body can be artificially broken down by some treatments (1 M urea, alkaline sea water: pH 9.0-9.7, low ionic concentration of sea water). The loss of the accessory body from the sperm is assumed to be related to the ferti-lizability of the sperm.     

Scanning electron microscopy of post-ejaculatory spermiogenesis in the tick Ornithodoros moubata

The final stages of spermiogenesis in ticks occur in the female genital tract. Scanning electron microscopy was used to follow the morphologic changes that occur in the sperm during this post-ejaculatory spermiogenesis in the African soft tick, Ornithodoros moubata, and to determine a time sequence for its occurrence in vivo. Characteristic features of the maturing and mature cell described include (1) differentiation and detachment of the operculum, (2) changes in cell shape corresponding to different developmental stages, (3) passive migration of the nucleus and acrosome from an anterior to a posterior position, and (4) eversion of that portion of the acrosomal canal containing the nucleus and acrosome. A possible fate for the remainder of the acrosomal canal is suggested by extrusion and detachment of spherical structures, the ‘posterior bubbles’, from the posterior end of the mature supermatozoon. A mechanism for cellular elongation resulting from contractions of the outer sheath is proposed.     

Immunocytological characterization of the outer acrosomal membrane (OAM) during acrosome reaction in boar

In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phase-contrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60-120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Immunocytological characteriziation of the outer acrosomal membrane (OAM) during acrosome reaction in boar

Summary In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phasecontrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60–120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Vesicular traffic and golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture

Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes beta-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both beta-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

TELEOGRYLUS EMMA精子体外顶体反应的超微结构比较研究

采用相同种类卵水诱导的方法对直翅目,蟋蟀科,黄脸油葫芦的受精囊精子的顶体反应过程进行系统观察.发现黄脸油葫芦精子顶体反应可划分为3个阶段,第1阶段,精子质膜膨胀、断裂或丢失;第2阶段,顶体复合体的顶体外层与顶体本体外膜发生融合,囊泡化;第3阶段,顶体复合体大部分脱落,只留有短锥状的顶体位于核前端.据观察,蟋蟀精子质膜不参与囊泡形成,此结果与家蝇及哺乳类的猪、牛、绵羊、猕猴精子的顶体反应结果很相似.经过比较发现卵水对受精囊内精子的诱导率明显高于精巢内,据分析,可能与精子的生理成熟有关,即便受精囊内精子比精巢内精子更趋于成熟.与其他学者的实验结果相比,蟋蟀精子顶体反应率与家蝇的相似,但明显低于其他动物.这可能与动物的授精方式有关.     

Maturation of mammalian spermatozoa: modifications of the acrosome and plasma membrane leading to fertilization

The mammalian spermatozoon is a terminally differentiated cell. Its surface is covered by a continuous plasma membrane that is divided into distinct domains in which functional molecules are distributed. The acrosome is an internal organelle located at the anterior head and contains hydrolytic enzymes. The anterior acrosome participates in the acrosome reaction, which is an indispensable event during fertilization. These surface domains and the acrosome are formed during spermiogenesis, during which associated molecules are transported and organized. Many of the molecules thus arranged are functionally immature but gradually become mature during epididymal maturation. Some of them are further altered and redistributed during the fertilization process and play various roles. Here, the sequential changes in the acrosome and plasma membrane during spermatozoan maturation are reviewed, particular attention being paid to molecules derived from the testis.     

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.     

Electron microscopic localization of a fucose-binding protein in acrosome reacted boar spermatozoa by the fucosyl-peroxidase-gold method

In this study we have examined the behaviour and the localization of the fucose-binding protein (FBP) in boar spermatozoa during ionophore induced acrosome reaction (AR) by means of normal TEM and specimen preparation in toto. During early stages of AR the FBP is first localized at the border between equatorial segment and anterior acrosome. With the propagation of the AR the FBP is dramatically expressed and visible over the entire surface of the acrosome and equatorial segment. TEM pictures of this stages show that the FBP is associated with the OAM. At later stages of AR, when acrosomal ghost formation occur, the FBP is associated with the acrosomal ghost, and equatorial segment and to a very low degree also with the IAM. It is concluded from this data that the FBP is responsible for the specific binding of the ghost-sperm unit to the zona pellucida.     

Electron microscopic localization of a fucose-binding protein in acrosome reacted boar spermatozoa by the fucosyl-peroxydase-gold method

Summary In this study we have examined the behaviour and the localization of the fucose-binding protein (FBP) in boar spermatozoa during ionophore induced acrosome reaction (AR) by means of normal TEM and specimen preparation in toto. During early stages of AR the FBP is first localized at the border between equatorial segment and anterior acrosome. With the propagation of the AR the FBP is dramatically expressed and visible over the entire surface of the acrosome and equatorial segment. TEM pictures of this stages show that the FBP is associated with the OAM. At later stages of AR, when acrosomal ghost formation occur, the FBP is associated with the acrosomal ghost, and equatorial segment and to a very low degree also with the IAM. It is concluded from this data that the FBP is responsible for the specific binding of the ghost-sperm unit to the zone pellucida.