Failure of acrosome assembly in a male sterile mouse mutant

Blind-sterile (bs) is a new autosomal recessive mutation of the mouse that causes sterility in males and bilenticular cataracts in both sexes. Sterile bs/bs males exhibited normal copulatory behavior, reduced testis weights, and few or no epididymal sperm. The effects of the bs mutation on spermatogenesis were examined by light and electron microscopy. All sperm present were morphologically abnormal with aberrant head shape. Adult bs/bs testes were characterized by germ cell depletion that resulted in profound alterations of the typical germ cell associations. Only 30% of the tubules contained relatively normal germ cell associations while 39% were extensively depleted, showing only Sertoli cells or Sertoli cells and spermatogonia. The most striking effect of the bs mutation on spermiogenesis was the failure of acrosome formation. Disorganized proacrosomic granules were detected up to step 3 of spermiogenesis by both periodic acid-Schiff staining and ultrastructural analysis. In over 3500 spermatids scored past steps 3-4 of spermiogenesis not a single acrosomal cap or fully developed acrosome was detected. Electron microscopy revealed a thickening of the nuclear envelope of elongating spermatids in the region where the acrosome should have been located; however, no acrosome was present. Chromatin condensation and nuclear elongation did occur in these acrosomeless spermatids, suggesting that caudal growth of the acrosome is not a mechanistic factor in these events.     

Chara tomentosa Antheridial Plasmodesmata at Various Stages of Spermatogenesis

Chara tomentosa antheridial plasmodesmata are described during proliferation and spermiogenesis. In antheridial filament cells which are cycling completely synchronously, unplugged plasmodesmata are filled with light cytoplasm. The same plasmodesmata are observed after cessation of mitotic division followed by the onset of synchronous spermiogenesis. Walls separating cells at different cell cycle stages and dividing antheridial filaments into asynchronous domains are plugged with a dense osmophilic substance. Similarly plugged plasmodesmata are present between antheridial cells of different types, e.g., capitular cells and antheridial filaments. In mid spermiogenesis when abundant endoplasmic reticulum (ER) appears temporarily it penetrates into plasmodesmata enabling cell-to-cell transport via ER cisternae. In late spermiogenesis there are no cisternae in plasmodesmata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Paired arrangement of nonhomologous centromeres during vertebrate spermiogenesis

Indirect immunofluorescence staining with human anti-kinetochore antibodies was used to study the position of centromeres during vertebrate spermiogenesis. Many species of Amphibia have a low chromosome number and very large spermatids and spermatozoa. The number of kinetochore dots correlates exactly with the haploid chromosome number. This implies that kinetochore duplication occurs in the interval between meiosis I and meiosis II. The nonhomologous centromeres are arranged in tandem during the entire course of spermiogenesis and in mature spermatozoa. A higher order centromere arrangement was found in spermiogenic cells of Anura and Urodela. In mammals, immunofluorescence analysis is complicated by the extreme condensation of chromatin during spermiogenesis and the high chromosome numbers. Nevertheless, centromere-centromere associations were observed in mammalian round spermatids and sporadically in testicular spermatozoa. This indicates that pair-wise association of centromeres is a universal principle of centromere arrangement at the postmeiotic stage.     

Kinetics of spermatogenesis in the wild squirrel Funambulus palmarum (Linnaeus).

The paper describes in detail the morphology and kinetics of germ cell associations, pattern of mitotic divisions, frequency distribution of different cellular associations (stages) and percent degeneration of various germ cells in the squirrel in which spermatogenesis in adults occurs all year round. Eighteen steps of spermiogenesis were identified based on the development of the acrosomal system using PAS-haematoxylin. These were appropriately divided into Golgi, acrosome, cap and maturation phases. Thirteen types of cellular associations or stages (I-XIII) were characterized along the length of the seminiferous tubule which repeated itself in space and time constituting the seminiferous epithelial cycle (CSE). Of the 18 steps of spermiogenesis, the first 13 were associated with stages I-XIII, respectively, and the rest with the first 9 stages. Spermiation occurred in stage IX. Seven types of spermatogonia [A0, A1, A2, A3, A4, intermediate (In) and B type] were identified based on their shape, size and nuclear morphology. A0 spermatogonia are pale in appearance with homogeneously distributed chromatin surrounded by a thin nuclear membrane. These are present in all stages. A1 are oval in shape and possess a thicker nuclear membrane. They are found in stages VI-X. The chromatin material undergoes progressive condensation from A1 to A4 making the last generation of spermatogonia appear darker. The In spermatogonia which are derived from A4 are morphologically similar to them but smaller in size. The B-type spermatogonia derived from the In types possess a typically round nucleus with uniformly condensed chromatin material underneath the nuclear membrane. The spermatogonia divide mitotically at fixed stages of the CSE giving rise to their next generations. Thus, A-type spermatogonia divide at stages X, XIII/I, IV and V, while In divide at stage VI. During each CSE of the squirrel, 5 peaks of mitosis occur. There is a single generation of B-type spermatogonia. These differentiate into primary spermatocytes and undergo meiosis or maturation divisions which enter to form spermatids. The A4 which divide differentially in stage VI give rise to In- and A1-type spermatogonia. Therefore, A4 spermatogonia form renewing stem cells. Based on the above pattern of spermatogonial mitosis a model for stem cell renewal in the squirrel is proposed. The percentage degeneration of germ cells varied with the cell type. During a single CSE of the squirrel, a total of 42.09% germ cells were found to degenerate. An attempt is made to compare and contrast the kinetics of spermatogenesis in the wild squirrel with that of the other rodents studied so far.     

Cytological evaluation of spermatogenesis within the germinal epithelium of the male European wall lizard, Podarcis muralis

The annual cytological changes to the male germinal epithelium were investigated in an introduced population of European wall lizards (Podarcis muralis). Testicular tissues were collected, embedded, sectioned by an ultramicrotome, and stained with the PAS procedure followed by a toluidine counterstain. Spermatogenesis in the lizard is divided into the proliferative, meiotic, and maturational phases. Wall lizards have a prenuptial pattern of spermatogenesis, where sperm development begins immediately prior to and continues through the months of breeding (April-June). The testis then involutes, undergoes a short period of quiescence, and recrudescence commences in mid-July. Germ cells undergo proliferation, meiosis, and the early stages of spermiogenesis (maturation) from late July through December. However, the late stages of spermiogenesis are retarded from December through February. Spermiogenesis continues at an accelerated pace from March through May, leading to a single massive spermiation event through the month of June. Although spatial relationships are seen between germ cells within the seminiferous epithelium, accumulation of spermatids during winter and acceleration of elongation in spring prevents determination of consistent cellular associations between early and late developing germ cells within the wall lizard testis. This temporal germ cell development is different from the consistent spatial development seen within seasonally breeding birds and mammals and may represent an evolutionary intermediate in terms of amniotic germ cell development.     

Ordered progress of spermiogenesis to the fertilizable sperm of the medaka fish, Oryzias latipes, in cell culture

Spermiogenesis is significant for producing sperm with equipment for achieving fertilization. Although multiple events occur in a particular order during spermiogenesis, it is unclear how the timing of those events is controlled. In the present study, we found that primary spermatocytes obtained from the spermatogenic testes of Oryzias latipes synchronously differentiated into sperm without contact with somatic cells in culture. Because those sperm can fertilize with mature eggs (Saiki et al., 1997), any events of spermiogenesis that are essential for achieving fertilization are completed in the in vitro spermiogenesis. In the in vitro spermiogenesis, the protamine gene expression was observed in the early period and mitochondrion localization was established in the same period. Those results suggest that both nuclear remodeling and organelle replacement begin in the early period of spermiogenesis. The cytoplasmic lobe was formed after the mitochondrion localization had been established. In most spermatids differentiated in cell culture, a flagellum began to elongate during the early period and continued to elongate up to 3 days. These results revealed the timings of the spermiogenetic events under the intrinsic control of the cultured spermatids toward the formation of fertilizable sperm in O. latipes.     

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Defect of sperm assembly in a neurological mutant of the mouse, wobbler (WR)

In the wobbler (WR) mouse, a neuromuscular mutant characterized by a motoneuron degeneration and male infertility, the cellular basis of the defect in spermiogenesis was studied by light and electron microscopy as well as by lectin binding. Spermatozoa of the wobbler mutant had rounded heads, and their motility was reduced. In histological sections of WR testes, spermatogenesis appeared normal up to the stage of round spermatids, but the elongation and flattening of the nucleus during late spermiogenesis did not occur. Numbers of spermatid nuclei in WR testes were reduced to 70%-80% of controls. The acrosomal marker glycoprotein, peanut agglutinin receptor, was synthesized, but the acrosomal membrane did not attach to the nucleus. The disturbance in spermiogenesis of the wobbler mouse is not due to impaired descent of the testis, nor to a lack of testosterone, and is distinct from that observed in other mouse mutants (quaking, QK; Purkinje cell degeneration, PCD) with combined neurological and spermiogenesis defects.     

Spermatogenesis in free living mite, Pergamasus viator Hala?. (Parasitidae, Mesostigmata). II. Spermiogenesis.

The paper presents the results of ultrastructural studies on the spermiogenesis in the mite, Pergamasus viator Hala?kova. The cysts containing 16 spermatids per cyst, are localized in the anterior part of the saccular gonad. The process of spermatid maturation has been divided into three stages: of the early spermatid, middle spermatid and late spermatid. The modifications of spermatid occuring during the spermiogenesis include: a change of cell shape, modifications of its organelles and formation of new structures like the superficial layer of cellular processes, striated bodies, granular bodies, flattened cisternae and canaliculi, central canaliculi, and stiff bands. Within the nucleus the chromatin condenses to threads or lamellae, to form subsequently several electron-dense granules. The remaining nucleoplasm is filled with an electron-dense material, which appears in the middle spermatid and gradually accumulates. The above modifications occuring in the course of spermiogenesis and their relation to the data available from the literature concerning the spermatogenesis of allied groups of animals are discussed in length.     

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.     

The sperm of Matsucoccus feytaudi (Insecta,Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure – a short cylinder provided with a central hub surrounded by several microtubules with a dense wall – is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.     

Spermatological characters of monozoic tapeworms (Cestoda: Caryophyllidea), including first data on a species from the Indomalayan catfish

The ultrastructure of spermiogenesis and mature spermatozoon in Lytocestus indicus (Cestoda: Lytocestidae) is described; this is the first representative of this group of monozoic, presumably most basal, tapeworms (Eucestoda) from the Indomalayan region to be documented in this manner. Similarly, as in other caryophyllideans, its spermiogenesis involves the formation of a conical differentiation zone with 2 centrioles associated with striated roots and an intercentriolar body. In the course of the process, 1 of the centrioles develops a free flagellum, which fuses with a cytoplasmic protrusion, whereas the other remains oriented in a cytoplasmic bud. Spermiogenesis is also characterized by the presence of electron-dense material in the early stages of spermiogenesis and a slight rotation of the flagellar bud. The mature spermatozoon of L. indicus is a filiform cell tapered at both extremities that lacks mitochondria; its nucleus has parallel disposition to the axoneme and does not reach up to the posterior extremity of the spermatozoon, which is typical for spermatozoa of the type III pattern. The new data confirm that caryophyllideans share the same type of spermiogenesis that is considered to be plesiomorphic in the Eucestoda. The existing information on spermatological ultrastructure of 8 members for 3 of 4 caryophyllidean families from different host groups (cyprinids and catostomids, both Cypriniformes, and mochokids and clariids, both Siluriformes) from 4 zoogeographical regions (Palearctic, Neotropic, Ethiopian, and Indomalayan regions) demonstrates great uniformity in spermiogenesis and sperm ultrastructure, which does not reflect different taxonomic position of the species studied.     

Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis

Spermiogenesis in Caenorhabditis elegans involves the conversion of spherical, sessile spermatids into bipolar, crawling spermatozoa. In males, spermiogenesis is induced by mating, while in hermaphrodites, spermiogenesis occurs before the first oocytes are fertilized. Alternatively, spermiogenesis can be induced in vitro by treatment with monensin triethanolamine, or pronase. Treatment with the calmodulin inhibitors, trifluoperazine, chlorpromazine, or W7, also induces spermiogenesis in vitro with a half maximal effect at 20 microM. Upon initial activation, spermatids extend long, thin spikes and undergo extensive cellular movements. Eventually, a single motile pseudopod forms through the restructuring of one or more of these spikes. These transient spikes can be prolonged in vitro by removing triethanolamine as soon as the spermatids first form spikes. Spermatids from spe-8 and spe-12 spermatogenesis-defective (spe) mutants activate in vivo with male but not hermaphrodite sperm activator. In vitro, the mutant spermatids arrest spermiogenesis at the spike stage when activated with pronase, but form normal spermatozoa if subsequently or initially treated with monensin or triethanolamine. We present a model of spermiogenesis in which the mutant defects and the action of the pharmacological agents are ordered relative to one another.     

Spermiogenesis in the rainbow trout (Salmo gairdneri)

In an ultrastructural study on the spermiogenesis of the rainbow trout (Salmo gairdneri R.) four spermatogenetic stages were identified. In young round spermatids, the nuclear chromatin was first heterogeneous (euchromatin and heterochromatin). Subsequently, it became more homogeneous and started to condense in the form of coarse granules and fibers and then into fibrils associated in ribbon-like elements which eventually partly fused together. During early spermiogenesis, a juxtanuclear vacuole appeared in the area where the nuclear envelope was specialized due to condensation of material between the two envelopes and a slight accumulation of nuclear material. This area was finally located in the anterior part of spermatids and spermatozoa; it probably plays a role during fertilization. A flagellar rootlet appeared early in spermiogenesis; it may play a role in the attachment of the flagellum to the nucleus since it persisted until the centriolar complex was definitively fixed in the implantation fossa. The flagellum did not display a plasma membrane and was first located in the cytoplasm, but when it was later extruded from the cell, it acquired a membrane. The cytoplasm was rich in ribosomes (free or in small groups) but poor in membranous organelles. The few mitochondria polarized around the centriolar complex were finally organized into an annular mid-piece. The spermatids remained connected by intercellular bridges until the end of spermiogenesis. The complexity of trout spermiogenesis is intermediate between that in poecilids and that in carp and pike, which have very simple spermatozoa. The role of the material from the nucleus and the cytoplasm reaching the Sertoli cell in the control of spermatogenesis has been discussed.     

A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.     

Cell surface topography duringMarsilea spermiogenesis: Flagellar reorientation and membrane particle arrays

Summary Changes in the cell surface during spermiogenesis in the fern,Marsilea, have been investigated by freeze-fracture. Early in development 150 or more flagella appear on the surface of the spermatid cell. As they grow in length, they change orientation in relation to the spermatid cell surface and to each other. While the flagella are growing, a band of membrane particles surrounds each flagellum at the transition zone. These particles disappear near the end of development and are not seen in mature sperm. Other particles are associated with the plasma membrane during development. One set of particles is found early in spermiogenesis in hexagonal arrays. At the end of spermiogenesis, these are no longer observed, but clusters of particles, with no particular order, appear around the flagellar bases, following the line of the flagellar band.     

Fine structure of spermatozoa and spermatogenesis ofSchizomus palaciosi,Reddell and Cokendolpher, 1986 (Arachnida: Uropygi,Schizomida)

Summary Spermatogenesis ofSchizomus palaciosi occurs in cysts in paired tubular testes located ventrally in the opisthosoma. Only few germ cells comprise one cyst. In early spermiogenesis an acrosomal complex composed of a spherical vacuole and a short acrosomal filament is established opposite of which a 9×2+3 flagellum emerges from a flagellar tunnel. The latter, however, is only a short-lasting structure. A manchette of microtubules surrounds nucleus and part of the acrosomal vacuole. The alterations in the arrangement of the microtubules during spermiogenesis are described. The spermatid finally is an elongate cell with a slender acrosomal vacuole on top of the helical nucleus. A deep implantation fossa filled with dense material is encountered. The acrosomal vacuole is accompanied by an intricate paracrosomal lattice structure not known at present of otherArachnida. This structure disappears during final spermiogenesis. The acrosomal filament (perforatorium) reveals filamentous subunits arranged in a regular pattern. Large ovoid mitochondria do not establish a distinct middle piece. Finally the elongate spermatid is coiled to form the mature spherical spermatozoon.The results are discussed under functional and taxonomical aspects.     

Effects on spermiogenesis in the mouse of a male sterile neurological mutation,Purkinje cell degeneration

Purkinje cell degeneration (pcd) is a neurological mutation in the mouse that causes male sterility, but not female sterility. In order to assess the effects of this mutation on spermiogenesis, the structure of the testis and of epididymal spermatozoa was examined by transmission and scanning electron microscopy. In the mutant males, the sperm count was reduced, sperm were nonmotile, and 93% of the sperm were characterized by structural abnormalities of the head, the tail, or both. In the testes of mutant mice, Sertoli cell structure was normal, as were also the early stages of spermiogenesis. However, the elongating and maturing spermatids were characterized by abnormally shaped heads and tails with extraneous and ectopic outer dense fibers. These defects were common in the testes of the mutant mice and rare in the testes of the littermate control mice. It was concluded that the structural abnormalities of the pcd sperm occurred during spermiogenesis and were not due to degeneration of the sperm in the epididymis. These structural abnormalities are similar to those found in all other reported male sterile mutants of the mouse; therefore, although they are caused by the expression of the pcd gene, they are not unique to the expression of this gene.