Formation of sperm cells inGalium mollugo (Rubiaceae),Trichodiadema setuliferum (Aizoaceae), andAvena sativa (Poaceae)

The formation of sperm cells has been examined ultrastructurally in the tricellular pollen grains ofGalium mollugo L. (Rubiaceae).Trichodiadema setuliferum Schwantes (Aizoaceae), andAvena sativa L. (Poaceae). After detachement from the intine the generative cell of all three species lies free within the vegetative cytoplasm. The two sperm cells are built inTrichodiadema andAvena by a single separating wall, while inGalium mollugo two independent walls are formed. However, both mechanisms separate the two male gametes completely.     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

The spatial association of the sperm cells and vegetative nucleus in the pollen grain ofBrassica

Summary In mature viable pollen ofBrassica oleracea, the pair of sperm cells and the nucleus of the vegetative cell are linked to form a structured unit we term the male germ unit. The sperm cells are held within a common periplasm and have no cell walls. Each sperm cell has a central globular body containing the nucleus surrounded by several evaginations which provide the means for linkage between them. One sperm cell, usually that closest to the nucleus of the vegetative cell contains most of mitochondria profiles (plastids are absent). This sperm cell appears to be linked by its protoplasmic evaginations to the envelope of the vegetative nucleus. The role of this unit in interactions with the female gametic complex is considered.     

Antibodies to flowering-plant sperm

Summary The mechanisms by which sperm cells recognize and fuse with the egg and central cell during double fertilization in flowering plants are unknown. To identify membrane surface molecules that might function in fertilization, we immunized mice with isolated sperm ofBrassica campestris and screened the polyclonal sera and monoclonal hybridoma supernatants by immunocytochemistry for binding to isolated sperm cells. We identified three cell lines producing hybridoma supernatants which bind to sperm cell surfaces inB. campestris and further analyzed the properties of one of these, BRSP1. The molecular mass of the epitope to BRSP1 was 54 kDa, and was not glycosylated. Although the antibodies were immunoglobulin M, neither removal of carbohydrates nor competition with antibodies which recognize arabinogalactan decreased binding. BRSP1 recognized sperm ofPlumbago zeylanica, Nicotiana tabacum. Arabidopsis thaliana, andEruca vesicaria and generative cells ofLilium longiflorum and ofNarcissus tazetta but did not recognize sperm ofHelianthus annuus, Gerbera jamesonii, orZea mays. Antibodies to plant sperm allow us to probe sperm membranes for functional components.     

Fine structural observations of sperm resorption in the seminal vesicle of a marine snail,Littorina scutulata (Gould, 1849)

Summary Waste sperm and spermatozeugmata in the seminal vesicle of Littorina scutulata are phagocytised either by cell buds (large vesicles given off from the epithelial cells) or by the epithelial cells themselves. Cell buds containing sperm, are in turn engulfed by epithelial cells. In both cases, heterophagic vacuoles are formed inside the cell and subsequently the vacuoles are fused with primary lysosomes or lysosomal derivatives to become secondary lysosomes. Throughout this process the sperm are being digested. The second lysosome transforms further to telolysosome and finally to residual body when the sperm is completely digested.     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

Characterization of a sperm lysin of Volvox carteri

Summary A cell-wall degrading enzyme has been isolated from mature sperm packets of the green flagellate Volvox carteri (Poona strain). This sperm lysin (S-lysin) is a Ca²⁺-dependent protease of 34 kDa with an essential serine group in its active centre. Neither SH group-blocking reagents nor transition metal chelators inhibit its action. S-lysin degrades the hydroxyproline-rich glycoprotein structures of the cell walls of sheath cells and gonidia (eggs) of vegetative and sexual spheroids in a characteristic manner. In asexual spheroids the somatic envelope is totally disintegrated, whereas in sexual spheroids pores are formed by local lysis at sites of adjacent eggs. Although S-lysin is very similar to the G-lysin of the closely related Chlamydomonads, it is species specific and does not attack the mother or daughter cell walls of Chlamydomonas reinhardtii. S-lysin resembles the aerosin of animal sperm cells in some aspects of its action.Dedicated to Professor Richard C. Starr on the occasion of his 65th birthday. He called the piper and gave the tune     

Ultrastructure of sperm cells and the male germ unit in pollen tubes ofNicotiana tabacum

Summary The structure of sperm cells and their association with the vegetative nucleus in pollen tubes ofNicotiana tabacum grown in styles were observed with the electron microscope, demonstrating the existence of a male germ unit. The two sperm cells are arranged in tandem and are closely associated with the vegetative nucleus, which always takes the lead. The leading sperm cell (SC 1) has a long and narrow cytoplasmic projection which lies within the enclaves of the much lobed vegetative nucleus, thus forming a physical association. The trailing sperm cell (SC 2) and the SC 1 are not only joined by a common transverse cell wall but also are surrounded by a periplasm bounded by the plasma membrane of the sperm cells and that of the vegetative cell, thus forming a structural connection. The sperm cells are elongated, with cytoplasmic projections at the anterior end of the SC 1 and at both ends of the SC 2. The cytoplasm of both sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, small vacuoles and axially oriented microtubules. No plastids were observed.Abbreviations DAPI 4,6-diamino-2-phenylindole - MGU male germ unit - MT microtubule - SC 1 the leading sperm cell physically associated with the vegetative nucleus - SC 2 the trailing sperm cell     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

Ultrastructural study of spermatogenesis in<Emphasis Type="Italic"> Phoronopsis harmeri</Emphasis> (Lophophorata,Phoronida)

The process of sperm development in Phoronopsis harmeri was studied by electron microscopy. Developing spermatogenical cells are aggregated around the capillaries of the haemal plexus. The spermatogonia, which are situated around the capillary walls of the caeca, are remarkable for the presence of germ-line vesicles and contain their centrioles near the cell membrane. The spermatocytes and spermatids are flagellated cells arranged in clusters. During spermiogenesis the basal body/flagellum complex migrates to the apical pole of the spermatid. The acrosome-like structure arises from material produced by the Golgi complex. It lacks a surrounding membrane and has a fibrillar content. The nucleus elongates and the condensation of chromatin is caused by an activation of 'initiation centres'. The late spermatid and the spermatozoon appear as two-armed 'V'-shaped cells in which one arm contains the nucleus and posteriorly located mitochondria, and the other one is the axoneme. Spermatogenesis of P. harmeri is an interesting example of gamete differentiation where advanced sperm structure is combined with a plesiomorphic pattern of sperm development characterized as 'flagellate spermatogenesis'. Communicated by H.-D. Franke     

Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.     

Isolation of two populations of sperm cells from the pollen tube of Torenia fournieri

The two sperm cells of Torenia fournieri are dimorphic. The dimorphic character suggests that they might be preferentially involved in fertilization during in vivo fusion with the egg cell and central cell. To probe the mechanism of preferential fertilization, it is necessary to use the most current molecular techniques. For this purpose, populations of >1000 individuals of the two dimorphic sperm cells, Sua (unassociated with the vegetative nucleus) and Svn (associated with the vegetative nucleus) were isolated from pollen tubes that had grown out of the cut ends of the styles. The two sperm cells released from pollen tubes remained attached to one another. When the two attached sperm cells were transferred into a solution containing 0.01% cellulose, 0.01% pectinase, and 5% mannitol, the connection between the two cells disappeared, and they were easily separated using a micromanipulator. The collection of these two individual populations containing over a thousand cells will permit research on gametic recognition at the molecular level.     

Mitosis and cytokinesis in androgonidia ofVolvox carteri f.weismannia

Summary Reproductive cells (androgonidia) ofVolvox carteri f.weismannia divide to form packets of 64 or 128 sperm cells. The androgonidium morphology, stages of mitosis, and cytokinesis were examined by electron microscopy. The biflagellate androgonidium loses its flagella before mitosis but the flagellar bases at the anterior end of the cell are retained. Two additional basal bodies are formed and the nucleus migrates from its central position to the area of the basal bodies before mitosis begins. A five-layered kinetochore is present on the chromosomes and remnant nucleolar material persists during mitosis. A furrow at the chloroplast end of the cell and the formation of phycoplast microtubules and vesicles signal the beginning of cytokinesis at early telophase. The cells maintain cytoplasmic connections until after the packet of sperm cells completes its development.     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

Microtubule organization in the sperm ofTradescantia virginiana

Summary Microtubules were visualized in the sperm ofTradescantia virginiana pollen tubes grownin vitro and processed for antitubulin immunocytochemistry. The sperm contain thick microtubule bundles from which emerge numerous branches of various dimensions disposed longitudinally and helically along the cell axis. Sperm are usually spindle or cigar-shaped, but cells of various sizes and shapes can be found. All contain microtubule arrays. No F-actin was detected in sperm using rhodamine-phalloidin staining. Sperm microtubules are discussed in terms of their potential roles in cell shaping and motility and their origin during generative cell division.Abbreviations DAPI 4,6-diamidino-2-phenylindole - IgG immunoglobulin G - M+W Mascarenhas and Walker medium - Mf microfilament - Mt microtubule - PBA phosphate-buffered saline     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

Successive observations on the fertilization of a centric diatomMelosira moniliformis var.octagona

The details of fertilization in the centric diatom,Melosira moniliformis var.octagona, were examined by light microscopy with a video system. The sperm penetrated into the egg cell through a temporary opening of the oogonium. The flagellum of the sperm was brought into the egg cell and it remained active for a few minutes. Three observations on the behavior of the sperm and egg cell during fertilization are shown.