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Biomedical subjects

F J Longo

Publications and source records attributed to F J Longo.

At least 19 recordsLinked to original sources
PubMed1995

Ultrastructural localization of acrosome reaction-inducing substance (ARIS) on sperm of the starfish Asterias amurensis.

Using colloidal gold tagged ligands we have identified the ultrastructural site of ARIS binding to intact and acrosome-reacted starfish sperm. In intact sperm, colloidal gold conjugated ARIS was specifically localized to a single domain (0.1-0.3 micron in diameter) on the plasma membrane. This site was located on the anterior-lateral aspect of the sperm head, that is, just peripheral to the region occupied by the acrosomal vesicle and periacrosomal components. When sperm were labeled with colloidal gold conjugated ARIS, washed to remove unbound label, and then induced to undergo the acrosome reaction, the labeled patch remained associated with the plasma membrane and was positioned just lateral to the acrosomal process. However, when sperm were suspended in labeled ARIS and induced to undergo the acrosome reaction, label was observed along the entire anterior aspect of the sperm head with the exception of the acrosomal process. Labeling along the entire anterior aspect of the sperm head in this case was deemed to be nonspecific and due to binding of colloidal gold tagged molecules to components formerly located within the acrosomal vesicle, as the same pattern was obtained using colloidal gold tagged bovine serum albumin. Quantitative and qualitative aspects of ARIS binding observed here by electron microscopy are in agreement with measured binding characteristics previously reported (Ushiyama et al., 1993a: Zygote 1:121-127; Ushiyama et al., 1993b: J Reprod Dev 39:53-54), and indicate that the site of labeled ARIS binding represents a specific plasma membrane domain occupied by ARIS receptors.

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PubMed1995

A periodic network of G protein beta gamma subunit coexisting with cytokeratin filament in starfish oocytes.

Heterotrimeric G proteins are membrane-bound and carry signals from activated receptors on plasma membranes to cytoplasmic effector enzymes and channels. In starfish oocytes, the beta gamma subunit of G protein mediates 1-methyladenine stimulation of oocyte maturation. In order to investigate the localization of beta gamma subunits in starfish oocytes during oocyte maturation, we raised a monoclonal antibody against the beta subunit. By immunofluorescence microscopy using the antibody, immature oocytes show a network of fibers in the cytoplasm. The staining of fibers is beaded with a periodicity of 0.7 microns. The same staining pattern is obtained by anti-gamma subunit antibody. In addition, the fibers are stained by anti-cytokeratin antibody. These results indicate that the G protein beta gamma subunit coexists with cytokeratin filaments in starfish oocytes. Stimulation of oocyte maturation by 1-methyladenine causes the beta gamma subunit to be disassembled.

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PubMed1992

Preparation of individual electrically and video-recorded eggs for integrated temporal and electron microscopic analyses.

A method for correlative studies of early fertilization events that integrates techniques of intracellular electrophysiological recording, video-imaging, and electron microscopy is described. A key feature of the method is its ability to identify the fertilizing sperm and to record the moment of egg excitation. Since the site of gamete interaction is recognizable throughout all stages of preparation, difficulties associated with locating the site of fertilization and determining specimen orientation for microtomy and electron microscopic examination are eliminated. Virtually all samples yield useful information. An example of interacting gametes fixed 4 sec after initiation of the fertilization potential and serial sectioned is described. The method is applicable to systems other than fertilizing eggs when functional, temporal, and spatial relationships of individual cells need to be correlated with changes in ultrastructure.

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PubMed1992

Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids.

DNase I-hypersensitivity of rat spermatogenic cells was analyzed 1) to establish overall patterns of hypersensitivity in individual cell types, 2) to correlate these patterns with known changes in chromatin organization and function, and 3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from "checkerboard" in steps 7-8 round spermatid nuclei to "lamellar" in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids. DHR were localized throughout the nuclei or in a graded manner--increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicators the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.

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PubMed1992

Cortical localization of a calcium release channel in sea urchin eggs.

We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.

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PubMed1991

Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs.

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.

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PubMed1991

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides.

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.

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PubMed1991

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs.

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.

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PubMed1991

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin.

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.

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PubMed1990

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.

Acrosome↗
PubMed1989

Incorporation and fate of specific sperm plasma membrane components following insemination as revealed by ultrastructural immunocytochemistry.

Studies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctulata) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold-tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalized.

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PubMed1989

Membrane specializations associated with the acrosomal complex of sea urchin sperm as revealed by immunocytochemistry and freeze fracture replication.

Observations, employing freeze fracture replication and electron microscopic immunochemistry, have been carried out to determine structural correlations of the plasma membrane domain occupied by a 210 kDa protein involved in the acrosomal reaction of sea urchin sperm and recognized by the monoclonal antibody, J10/14 (Trimmer et al.: Cell 40:697-703, 1985; Proceedings of the National Academy of Sciences of the United States of America 83: 9055-9059, 1986). Immunogold-J10/14 staining of acrosome-intact sperm was intense along the flagellum and a narrow collar just posterior to the sperm apex that surrounded the acrosomal complex (acrosomal vesicle and subjacent anterior nuclear fossa containing g-actin). Counts of gold particles revealed a density (average number of particles/micron2 of surface area) eightfold greater along the plasma membrane associated with the acrosomal complex than membrane delimiting the remainder of the sperm head. The collar of J10/14 staining was isomorphic with a dense aggregation of intramembranous particles in the P-face of the plasma membrane and a thin cytoplasmic region that surrounded the acrosomal complex. In acrosome-reacted sperm, intense J10/14 staining was distributed along the flagellum and sperm head; prominent anterior staining was not apparent in all specimens. The density of gold particles associated with plasma membrane delimiting components of the former acrosomal complex, nucleus and mitochondrion, as well as the total average number of particles along the entire sperm surface, were increased in sperm acrosome-reacted with A-23187. Concomitant with this change in staining was the disappearance/reduction of the collars of intramembranous particles and cytoplasm. These observations indicate that plasma membrane components (210 kDa protein and intramembranous particles) and the collar of cytoplasm which are associated with the acrosomal complex are functionally, as well as structurally related. Analyses of particle density distributions along acrosome- and non-acrosome-reacted sperm suggest that the different staining patterns observed may be brought about by the recognition of cryptic sites at the time of the acrosomal reaction.

Acrosome↗
PubMed1989

Incorporation and dispersal of sperm surface antigens in plasma membranes of inseminated sea urchin (Arbacia punctulata) eggs and oocytes.

Monoclonal antibodies have been generated to determine the fate of sperm-specific surface components of the sea urchin, Arbacia punctulata, subsequent to gamete fusion. Monoclonal antibody-7 (MAB-7) reacted with two polypeptide bands having apparent molecular masses of 33 and 35 kDa derived from the sperm plasma membrane; similar reactivity was not detected in egg preparations. Eggs and oocytes were prepared for electron microscopy at periodic intervals following insemination and reacted with MAB-7 followed by antimouse antibodies conjugated to colloidal gold. In samples prepared 30 to 60 sec postinsemination (PI) colloidal gold was confined to the surface of fused sperm with only a few gold particles associated with the egg plasma membrane. In later samples (2-10 min PI) label was present on increasingly greater areas of the egg/oocyte surface away from the site of gamete fusion so that by 20 min PI particles were located along one hemisphere of inseminated eggs and oocytes. These results demonstrate the incorporation of sperm plasma membrane polypeptides and their intermixing with egg plasmalemmal components subsequent to gamete membrane fusion. From measurements of labeled oocytes at different times PI, the diffusion coefficient for sperm surface polypeptides detected by MAB-7 was estimated to be 0.7 to 2.4 x 10(-9) cm2/sec.

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PubMed1988

Sperm nuclear enlargement in fertilized hamster eggs is related to meiotic maturation of the maternal chromatin.

Experiments were conducted to test the hypothesis that sperm nuclear expansion in fertilized hamster eggs is correlated with meiotic processing of the maternal chromatin. In vitro fertilized hamster eggs were fixed at regular intervals following insemination, stained with the DNA specific fluorochrome, Hoechst 33342, and the extent of sperm nuclear expansion measured. Sperm nuclei enlarged in multiple, distinct phases (A-E) that were temporally correlated with meiotic processing of the maternal chromosomes: phase A, metaphase II; phase B, early anaphase II; phase C, late anaphase II, and phases D and E, female pronuclear development and expansion. During phase A, sperm nuclei were unchanged but enlarged at different rates during phases B (272 microns2/min), D (106 microns2/min) and E (29 microns2/min), and condensed during phase C (rate = -102 microns2/min). Area increases of both sperm nuclei and female pronuclei during phase D were significantly less in polyspermic and polygynic zygotes. If sperm nuclear enlargement and the status/activity of the maternal chromatin were correlated, it would be anticipated that alterations in the normal progression of meiotic maturation would be manifested in sperm nuclear expansion. The following agents affected both meiotic maturation and sperm nuclear enlargement: colchicine, antimycin A, and puromycin. These observations suggest that processes attending sperm nuclear transformations, the completion of maternal meiotic maturation, and development of the female pronucleus are coupled and may be linked by common regulatory agents.

Animals↗
PubMed1988

Sperm nuclear transformations consist of enlargement and condensation coordinate with stages of meiotic maturation in fertilized Spisula solidissima oocytes.

Rates of sperm nuclear expansion were measured and correlated with processing of the maternal chromatin in synchronous populations of fertilized surf clam (Spisula solidissima) oocytes fixed at regular intervals following insemination and stained with the DNA fluorochrome Hoechst 33342. Sperm nuclei expanded in four distinct phases each temporally coordinate with events of meiotic maturation: germinal vesicle stage (phase A), germinal vesicle breakdown (phase B), polar body formation (phase C), and female pronuclear development (phase D). Sperm nuclei were essentially unchanged during phase A (rate = 0.1 micron2/min, enlarged during phases B (rate = 8.2 microns2/min) and D (rate = 6.2 microns2/min), and condensed during phase C (rate = -1.9 micron2/min). Sperm nuclear enlargement during phase D was significantly less in polyspermic and polygynic zygotes. The effects of various treatments (temperature, microtubule disruption, pH alterations, and metabolic and protein synthesis inhibitions) which perturbed sperm nuclear enlargement and meiotic processing of the maternal chromatin indicated that the two processes are coupled and may be linked by common regulatory agents.

Animals↗
PubMed1987

Rates of male pronuclear enlargement in sea urchin zygotes.

Rates of male pronuclear enlargement were determined using synchronous populations of fertilized sea urchin eggs (Arbacia punctulata and Lytechinus variegatus), fixed at regular intervals following insemination and stained with Hoechst 33342. The rate of male pronuclear expansion in both Arbacia and Lytechinus zygotes was linear and significantly affected by polyspermy, temperature, metabolic inhibitors (dinitrophenol, potassium cyanide, sodium azide, antimycin A, and oligomycin), and blockage of cytoplasmic alkalinization. Inhibitors of protein synthesis (emetine and puromycin) and disrupters of cytoskeletal elements (colchicine, nocodazole and cytochalasin B) had no effect on the rate of male pronuclear enlargement.

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