Gametocyte Maturation,Exflagellation and Fertilization in Parahaemoproteus (=Haemoproteus) velans (Coatney & Roudabush) (Haemosporidia: Haemoproteidae): an Ultrastructural Study*

SYNOPSIS. The structural changes in macro and microgametocytes of Parahaemoproteus velans following removal of infected blood from the avian host were studied in the light and electron microscope. Gametocytes of both sexes round up and soon escape from their host cells. Shortly thereafter they assume a dumbbell shape. The microgametocyte undergoes exflagellation forming 8 slender microgametes. During fertilization the entire microgamete appears to enter the female. The most striking ultrastructural change in the formation of the macrogamete is the condensation and enclosure by a membrane of abundant amophorus dense material seen in the cytoplasm of the immature gametocyte. Maturation of the microgametocyte begins prior to its escape from the host cell. Axonemes are present in the cytoplasm and nuclear reorganization occurs while the parasite is intracellular. Bundles of microtubules associated with condensed chromatin are found in the peripheral cytoplasm of maturing forms and apparently participate in the formation of small compact microgamete nuclei. Each of these filiform structures consists of a dense, centrally located nucleus and a single axoneme lying in flocculent cytoplasm. The nucleus and axoneme of the microgamete are seen free in the cytoplasm of a fertilized macrogamete.     

Ultrastructural observations on fertilization and sporulation in Goussia iroquoina (Molnar and Fernando, 1974) in experimentally infected fathead minnows (Pimephales promelas, Cyprinidae)

The ultrastructural features of fertilization and sporogony of Eimeria iroquoina are described from the intestinal epithelium of experimentally infected fathead minnows (Pimephales promelas). Intact microgametes were observed in the cytoplasm of macrogametes. Within immature macrogametes the microgamete was segregated from the cytoplasm of the former by the plasma membrane of each cell plus additional membranes. Within mature macrogametes, only the plasma membranes separated the gametes. Fertilization by fusion of the limiting membrane of both gametes occurred after the entire microgamete lay within the cytoplasm of the macrogamete. The cytoplasm of the zygote cleaved into sporoblasts within cisternae of endoplasmic reticulum. The sporocystic wall was composed of an outer electron-lucent layer and an inner, thicker layer with periodic striations at right angles to the surface of the sporocyst. The sporocysts were bivalved and joined by a continuous suture. The sporozoites were morphologically similar to sporozoites and merozoites of other Coccidia. Due to the structure of the sporocyst, Eimeria iroquoina Molnar and Fernando, 1974 is amended to Goussia iroquoina (Molnar and Fernando, 1974).     

The Fine Structure of the Mature Gametes of Haemoproteus columbae Kruse*

SYNOPSIS. The filiform microgamete of Haemoproteus columbae consists of an elongate double-walled nucleus paralleled by 2 axonemes embedded in a homogeneous matrix. At one end of the gamete, the axonemes are sharply flexed back on themselves, but no conventional kinetosome has been recognized. No mitochondria have been seen. Single-walled vesicles occur in the matrix, and the entire gamete is surrounded by a single membrane. The large round macrogamete has a conspicuous central nucleus with its outer membrane drawn out into anastomosing evaginations which extend to the periphery of the cell. A moderately electron dense material fills the space between the 2 nuclear membranes and the lumina of the evaginations. Nucleolar material may occur in scattered masses within the nucleus. One or 2 axonemes appear to arise endogenously next to the nuclear membrane. The cytoplasm is filled with ribosomes and perhaps glycogen granules. Typical protozoan mitochondria and vesicles containing pigment retained from the erythrocytic stage are found in the peripheral cytoplasm. Accumulations of dense-walled vesicles occur in the cytoplasm in conjunction with evaginations of the nuclear membrane. Amid these vesicles triple-ringed discs resembling the cytostomes of merozoites are frequently seen. Several distinct layers of dense material surround the micro-gamete.     

Ultrastructure of Gametogenesis and Sporogony of Haemogregarina (sensu lato) myoxocephali (Apicomplexa: Adeleina) in the Marine Leech Malmiana scorpii

ABSTRACT The sexual and sporogonic development of Haemogregarina (sensu lato) myoxocephali , an apicomplexan blood parasite of longhorn sculpin, Myoxocephalus octodecemspinosus , was studied by transmission electron microscopy. All stages of development were observed epicellularly within intestinal epithelial cells of the leech Malmiana scorpii . During microgametogenesis nuclear division was characterized by a transnuclear cytoplasmic channel containing the spindle microtubules. Four aflagellate microgametes were formed. During fertilization, a single microgamete nucleus was associated with the endoplasmic reticulum in the macrogamete, followed by fusion of the nuclear envelopes of the gametes. Sporogony involved peripheral budding of sporozoite anlagen and subsequent development to form approximately 32 sporozoites in mature oocysts.     

Ultrastructure of gamonts and gametes and fertilization of Sarcocystis sp. from the roe deer (capreolus capreolus) in dogs

Gamonts of Sarcocystis sp. from the roe deer were examined in the intestine of dogs 10 h after inoculation. Early macrogamonts were limited by a three-membranous pellicle, and situated in a parasitophorous vacuole. Female sexual stages during fertilization, the macrogametes, were limited by five membranes, and microgametes were observed in the parasitophorous vacuole. The outer membranes of the microgamete and macrogamete fuse, and the nucleoplasm of the microgamte enters the cytoplasm of the macrogamete. No wall-forming bodies were observed in macrogamonts and macrogametes.     

Gametogenesis, fertilization and ookinete differentiation of Leucocytozoon smithi

Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1-2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the zygote elongated. Development of sausage-shaped ookinetes was completed within 6-8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double-layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.     

DNA synthesis,reduction, and elimination during life cycles of the eimeriine coccidian, Eimeria tenella and the haemogregarine, Hepatozoon domerguei

The occurrence of zygotic nuclear reduction and the haploid state of postzygotic stages were demonstrated in Eimeria tenella (Coccidia, Eimeriina) and Hepatozoon domerguei (Coccidia, Adeleina) by direct measurement of DNA by microdensitometry. Nuclear divisions, which were probably mitotic, were demonstrated in the macrogametocytes of a species of Hepatocystis (Coccidia, Haemosporina). The macrogametocytes of E. tenella were Feulgen negative but gametocytes of both sexes of H. domerguei were Feulgen positive and had DNA values estimated at about twice the haploid amount. This latter finding was interpreted as DNA synthesis preceding a maturation process which involved mitotic divisions and led to micro- and macrogamete formation. In macrogametogenesis this may be an atavistic trait, recapitulating the evolution of sexuality or a method for increased RNA synthesis.     

The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Interactions between endoplasmic reticulum and nuclear membranes of different types of cells during repair of damaged Amoeba nuclei

Repair of amoeba nuclear envelopes that have been damaged microsurgically involves the association of pieces of endoplasmic reticulum with the damaged nuclear membranes. The capacity of endoplasmic reticulum of one type of cell to interact with the nuclear membranes of a different type was tested by placing the damaged nucleus of one kind of amoeba into the cytoplasm of another. Damaged nuclei from Amoeba proteus underwent repair in the cytoplasm of A. discoides or A. indica, as was the case in the reciprocal combinations of these nuclei and cytoplasms. In samples prepared 30 min after operation, heterologous endoplasmic reticulum was associated with holes in the nuclear membranes and appeared to fuse with the nuclear membranes at the margins of the holes. By 5 h after operation, almost all of the cells survived, and the nuclear membranes were largely intact, indicating that repair had occurred. In contrast, when an Amoeba dubia nucleus was damaged and placed in A. proteus cytoplasm there was no evidence of repair and many cells died within a few hours. The results indicate that endoplasmic reticulum and nuclear membranes from different types of cells can interact during repair of damaged nuclear membranes. There appears to be a specificity to this interaction, however, since in a combination of relatively dissimilar cells no association of endoplasmic reticulum with damaged nuclear envelopes was observed and repair did not occur.     

Fine Structure of Microgametocytes and Macrogametes of Eimeria nieschulzi

SYNOPSIS. An electron microscope study of microgametocytes and macrogametes of Eimeria nieschulzi Dieben, 1924 revealed that they lie within vacuoles bounded by a host unit membrane. The vacuole surrounding the microgametocyte contains granular material. The vacuole around the macrogamete is narrower and contains vesicles and membranes. Micropores were seen on the surface of the plasma membrane of microgametocytes and macrogametes. Microtubules were seen in macrogametes. Young microgametocytes and macrogametes have a similar cytoplasmic matrix, mitochondria and nuclei. Glycogen granules apparently develop around vacuoles in both microgametocytes and macrogametes. Glycogen granules were also seen along the margins of parallel bundles of fibers in microgametocytes. As nuclei of the microgametocyte divide, they move to the periphery of the parasite. Three basal bodies, each with 9 fibers in triplet form, develop in association with each nucleus. Microgametes have 2 free flagella and a central short, attached flagellum. Basal granules lie along the outer fibers of the central flagellum. Each microgamete has an elongate mitochondrion in close contact with the nucleus. In macrogametes wall-forming bodies develop in lacunae in the cytoplasm. Smaller dark bodies with areas of low density were also seen. Wall-forming bodies and dark bodies move to the periphery of mature macrogametes.     

Ultrastructure of the gametes ofCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary As part of an investigation on the developmental biology ofCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), the ultrastructure of the male and female gametes was studied. The nucleus is central and conical in shape except for a basal spur that curves back towards the large plate-like mitochondrion. A nuclear cap of ribosomes sits on the flat anterior end of the nucleus. Approximately seven lipid globules are partially embedded in the mitochondrion and are interconnected by membrane cisternae. The lipid globules are covered by a single fenestrated microbody and a backing membrane lies between the microbody and the gamete plasma membrane. The kinetosome is at the base of the nucleus and is connected to a single, posterior, whiplash flagellum. A nonkinetosomal centriole is absent. In the peripheral cytoplasm of both mating types there is a paracrystalline body of unknown composition and function. No significant ultrastructural differences were found between the male and female gametes.     

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : II. Zygote Formation by Gamete Membrane Fusion

An earlier paper showed that in Saccoglossus the acrosomal tubule makes contact with the egg plasma membrane. The present paper includes evidence that the sperm and egg plasma membranes fuse to establish the single continuous zygote membrane which, consequently, is a mosaic. Contrary to the general hypothesis of Tyler, pinocytosis or phagocytosis plays no role in zygote formation. Contact between the gametes is actually between two newly exposed surfaces: in the spermatozoon, the surface was formerly the interior of the acrosomal vesicle; in the egg, it was membrane previously covered by the egg envelopes. The concept that all the events of fertilization are mediated by a fertilizin-antifertilizin reaction seems an oversimplification of events actually observed: rather, the evidence indicates that a series of specific biochemical interactions probably would be involved. Gamete membrane fusion permits sperm periacrosomal material to meet the egg cytoplasm; if an activating substance exists in the spermatozoon it probably is periacrosomal rather than acrosomal in origin. The contents of the acrosome are expended in the process of delivering the sperm plasma membrane to the egg plasma membrane. After these membranes coalesce, the sperm nucleus and other internal sperm structures move into the egg cytoplasm.     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

FERTILIZATION IN OEDOGONIUM. III. KARYOGAMY

Karyogamy is described in Oedogonium cardiacum from ultrastructural studies. Close proximity of the two gamete nuclei in the fusion cell is established by plasmogamy, whereas karyogamy appears to be initiated by multiple contacts formed between the outer membranes of the adjoining nuclear envelopes. Blebs of endoplasmic reticulum (ER) originate from the outer membrane of each nuclear envelope; these ER blebs presumably contact and fuse with the outer membrane of the nuclear envelope of the opposing nucleus. This is followed by the fusion of the inner membranes of the opposing nuclear envelopes, thereby resulting in a series of small connective bridges between the two gamete nuclei. It is estimated that in this manner 30–50 bridges are formed, perhaps many more. Several of these bridges enlarge relative to the others; one presumably becomes the major connection between the fusing nuclei. As it continues to enlarge, any organelles positioned between the fusing nuclei are pushed aside. There is also evidence, particularly in later stages of karyogamy, that the smaller connective bridges fuse to form larger ones. Temporary cytoplasmic channels often result at the juncture of fusion. In other instances, isolated inclusions of cytoplasm may be delimited by remnants of nuclear envelope deep within the developing zygote nucleus; these inclusions disappear with subsequent development. Throughout karyogamy the contribution of the male gamete nucleus is readily recognized by the characteristic appearance of its highly condensed chromatin. Ultimately, however, this distinction is lost and the content of the mature zygote nucleus assumes a more uniform appearance very similar to that of an egg nucleus. The complete process of fertilization in Oedogonium may occur within 15 min of mixing the spermatozoids with eggs.     

The Fine Structure of Microgametogenesis in Haemoproteus columbae Kruse*

SYNOPSIS. The structural changes leading to the formation of motile microgametes from a single immobile intracellular gametocyte have been examiued in the electron microscope. After pigeon blood infected with Haemoproteus columbae was exposed to the air at room temperature for a few minutes axonemes appeared in the parasite's cytoplasm and the cytoplasm itself appeared less dense. The axonemes were connected with bundles of intranuclear microtubules that were perhaps spindle fibers. No conventional kinetosomes or centrioles have been observed. After the microgametocyte left the erythrocyte, it assumed the shape of a polarized slug or a dumb-bell. Half of the organism was surrounded by a single membrane and filled by part of the nucleus. The other half was surrounded by the remains of the multiple membranes of the gametocyte and contained pigment granules, mitochondria, axonemes and nuclear extensions. The axonemes and nuclear extensions were segregated at the periphery of the cell, exterior to the gametocyte's inner membrane, and were assembled in situ into microgametes. The mature microgamete appeared to peel off from the gametocyte, leaving a residual body.     

Fine structural observations on nuclear maturation during spermiogenesis in Hydra littoralis

Cytodifferentiation during spermiogenesis in Hydra littoralis was studied at the fine structural level. Concentration of nuclear material as well as specific orientation of granular and filamentous nuclear elements are apparent in two regions of the early spermatid: where the nuclear envelope is in contact with mitochondrial membranes at one pole of the cell and at an opposite region where the nucleus is closely apposed to the plasma membrane. Ultimately the mass of condensed nuclear material becomes concentrated at the mitochondrial pole of the cell. Additional electron-dense material is extruded from the nucleus into a large vacuole which is in continuity with the nuclear membrane as well as associated with Golgi lamellae and vesicles. Eventually all residual cytoplasm is sloughed, leaving the nucleus, mitochondria, and flagellum. These observations are suggestive of nucleocytoplasmic interactions during development, especially influences of mitochondria and plasma membranes on chromatin condensation.     

Fine Structure of Gametogony and Oocyst Formation in Sarcocystis sp. in Cell Culture

Fine structure of gametocytes and oocyst formation of Sarcocystis sp. from Quiscalus quiscula Linnaeus grown in cultured embryonic bovine kidney cells was studied. Microgametocytes measured up to ～5 μm diameter. During nuclear division of the microgametocyte, dense plaques were found adjacent to the nucleus just beneath the pellicle; occasionally microtubules were present within these plaques. These microtubules subsequently formed 2 basal bodies with a bundle of 4 microtubules between them. Microgametocytes also contained numerous mitochondria, micropores, granules, vacuoles, and free ribosomes. Each microgamete was covered by a single membrane and consisted of 2 basal bodies, 2 flagella, a bundle of 4 microtubules, a perforatorium, a mitochondrion, and a long dense nucleus which extended anteriorly and posteriorly beyond the mitochondrion. The bundle of 4 microtubules is thought to be the rudiment of a 3rd flagellum. Macrogametes were covered by a double membrane pellicle, and contained a large nucleus (～2.5 μm), vacuoles, and a dilated nuclear envelope connected with the rough endoplasmic reticulum (ER). In young macrogametes (～4 μm), the ER was arranged in concentric rows in the cortical region, and several sizes of dense granules were found in the cytoplasm. However, in later stages (～8 μm) the ER was irregularly arranged and was dilated with numerous cisternae; only large dark granules remained and a few scattered polysaccharide granules were found. No Golgi apparatus or micropores were observed. After the disappearance of dark granules 5 concentric membranes appeared. Four of these fused to form an oocyst wall composed of a dense outer layer (～66 nm thick) and a thin inner layer (～7 nm). The 5th or innermost membrane surrounded the cytoplasmic mass which was covered by a 2-layered pellicle and contained a nucleus, small amounts of ER, large vacuoles, and mitochondria. The sexual stages described greatly resemble those of Eimeria and Toxoplasma.     

Differentiation of Male Gametes in Gracilaria verrucosa (Rhodophyta, Gracilariales)

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.     

FERTILIZATION IN PLUMBAGO ZEYLANICA: GAMETIC FUSION AND FATE OF THE MALE CYTOPLASM

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.