Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

The manchette in Stage 14 rat spermatids: A possible structural relationship with the redundant nuclear envelope

Summary The manchette or caudal tube has been examined in Stage 14 rat spermatids. The microtubules of the caudal tube have been found to be partially sheathed by smooth endoplasmic reticulum which appears to be continuous with the outer nuclear membrane of the redundant nuclear envelope. The microtubules in caudal regions of the manchette have been noted to be interconnected by links of unusual size and morphology. It is suggested that the caudal tube consists at this stage of development of two structures, membrane and microtubules and that the links between the microtubules appear to play a role in the structural order noted in the position of the tubules of the manchette. The possible significance of these links in relation to motility is discussed.Supported by a grant to E. A. MacKinnon by the Medical Research Council of Canada.     

Presence and localization of the 60 KD calicin in human spermatozoa presenting postacrosomal sheath defects: preliminary results

Using an indirect immunofluorescence technique, we studied the anticalicin antibody distribution in four teratozoospermic human sperm samples with a high rate of spermatozoa with incomplete postacrosomal sheath structure (PAS) detected at the ultrastructural level. The calicin distribution obtained in these samples is compared with the distribution obtained in two normal human sperm samples with a high proportion of normal PAS and in 3 human sperm samples without PAS ("round-headed syndrome"). In contrast to the homogeneous calicin distribution found in the majority of the normal group, similar to that reported by Longo et al. (J Cell Biol 105:1105-1120, 1987) and Paranko et al. (Differentiation 38:21-27, 1988), and the absence of calicin in the three non-PAS samples, we observed new patterns of calicin distribution in the four sperm samples studied. In the light of these preliminary results, the use of anticalicin antibody as a tool for human postacrosomal region evaluation is discussed.     

Unusual structures in the spermatozoa of the ascidians Lissoclinum perforatum and Diplosoma listerianum (Didemnidae)

Summary Sperm and spermiogenesis of L. perforatum and D. listerianum have been investigated by light- and electron microscopy. The study revealed basic ascidian organization of sperm cells with the nucleus flanked by a unique mitochondrion, but with specializations not previously reported: a dense groove on the head surface, extended nucleus-mitochondrion association, and well developed endoplasmic derivatives.The groove is a long furrow of the plasmalemma stretching the nucleus towards the cell periphery. At least two parallel, 20 nm high, ridges emerge from the bottom of the groove. At the opposite side of the groove the long mitochondrion embraces the nucleus by extending its outer membrane over the nuclear envelope; a restricted cytoplasmic space is always found between the two organelles. Filamentous inclusions organized in two strands are present in the mitochondrial matrix. Endoplasmic derivatives appear as a single, extended cistern in L. perforatum, and series of long tubules in D. listerianum. They appear to originate in the spermatid from the Golgi complex and endoplasmic reticulum. The spermatozoon lacks an evident acrosomal complex, but the possible homology of small apical densities with an acrosomal complex is considered. It is suggested that the functional role of these specialized structures is related to the special mode of internal fertilization of these species.     

A highly conserved microsatellite in the dystrophin gene of diverse mammalian species

The presence of a CA repeat within the 3'-untranslated region (UTR) of the dystrophin gene has been reported previously in several species. Because microsatellites showing high cross-species homology can be conveniently used as markers in those species for which detailed linkage maps have not yet been developed, we evaluated whether the CA repeat could be amplified from a wide variety of mammalian species. Using a single pair of canine-specific oligonucleotide primers, we successfully amplified the 3'-UTR from 18 different carnivore and six additional species (human, chimpanzee, goat, cow, rabbit and mouse) and show conservation of the CA repeat in the dystrophin gene from a wide range of evolutionarily diverse mammalian species.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white-tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37 °C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t(0)) and after 4 hr (t(4)) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t(0) and t(4) were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1-to-protamine 2 ratios.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday     

Carrier design: Synthesis and conformational studies of poly (L-lysine) based branched polypeptides with hydroxyl groups in the side chains

In the present study the development of a new series of branched polypeptides that contain hydroxyl groups in side chains is reported. Serine or threonine were attached by 1-hydroxy-benzotriazole catalyzed active ester method to the N-terminals of oligo (DL -alanine) chains grafted to a polylysine backbone resulted in poly[Lys-(Ser₁-DL -Ala_m)] (SAK) and poly-[Lys-(Thr_i-DL -Ala_m)] (TAK). Ser was coupled also directly to the η-amino groups of polylysine followed by polymerization of N-carboxy-DL -alanine anhydride resulting oligo (DL -Ala) chain terminals. In this way a reverse sequence was built up in the side chain corresponding to the poly[Lys-(DL -Ala_m-Ser_i)] (ASK). The number of hydroxyl groups in the polymer was increased by the synthesis of a branched polypeptide with oligo (DL -serine) branches instead of oligo (DL -alanine) ones—poly[Lys-(DL -Ser_m)] (SK). Classification of solution conformations of branched polypeptides was carried out by CD spectroscopy performed in water solution of various pH values and ionic strengths. Incorporation of single Ser residues in poly[Lys-(X_i)]-type polypeptides markedly promotes the formation of ordered structure without resulting precipitation even in high salt concentration. The presence of branches with multiple DL -Ser residues resulted in a slightly decreased ability of the polypeptide backbone to adopt an ordered conformation. Comparison of the CD properties of the SAK-ASK pair demonstrates that these compounds are similar, showing an increased tendency to form an ordered spatial arrangement in solution at elevated pH or ionic strength; however, differences in their CD spectra suggest that SAK has higher capability to form regular conformation under comparable conditions. The replacement of Ser by the Thr residue in poly[Lys-(X_i-DL -Ala_m)] induced a conformational transition and TAK exhibited a more helical structure. These results might indicate that not only hydrophobic or ionic attraction, but also H-bond interaction, can play a role in the formation and/or stabilization of ordered conformation of branched polypeptides. Findings with the hydroxyl group containing polymers reported in this paper can also explain their prolonged shelf stability and high water solubility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 719–730, 1997     

Giant spermatozoa and a huge spermatheca: A case of coevolution of male and female reproductive organs in the ground louse Zorotypus impolitus (Insecta,Zoraptera)

The male and female genital apparatus of the recently discovered ground louse Zorotypus impolitus were examined using light and electron microscopy. The rounded testes and a large seminal vesicle are connected with a complex of four accessory glands by a long tapering ejaculatory duct. Two accessory glands have the same whitish coloration, whereas the third one is pale blue, and the elongated and cylindrical fourth one translucent. The sperm are the largest known in Hexapoda, 3 mm long and 3 μm wide, with a volume of ca. 21,000 μm³; the ratio between the diameter of the axoneme and the width of the main body of the sperm ranges between 1:10 and 1:13. The exceptional width of the spermatozoa is due to an extreme enlargement of the mitochondrial derivatives and accessory bodies. A single sperm is contained in a small globular spermatophore (100 μm). The highly unusual external transfer correlates with an atypical mating behavior. The male produces several to many spermatophores during the mating process. As in other zorapterans the ovaries are panoistic and the eggs bear two micropyles. An exceptionally large apical spermathecal receptacle is present; it is connected with the vagina by a long spermathecal duct, which varies structurally along its course. A correlation between the sperm size and the size of the spermatheca is likely. Ultrastructural features of different species support two strikingly different models of male and female reproductive apparatus in the small order Zoraptera. This is in stark contrast to the extreme uniformity of their external morphology. It is likely that sexual selection played a decisive role in the evolution of the reproductive system.     

Self-assembly of three novel mixed-ligand coordination polymers: Formation of 2-D grids, 1-D chains and ladders and crystal band structure

Hydrothermal reaction of the carboxylate-based ligands with metal salts (or oxide) and 4,4′-bipyridine as a second linker, afforded three new coordination polymers, namely, [Co(PCPA)₂(4,4′-bpy)]_n (1) with 2-D rectangle grids, Cu(PCPA)₂(4,4′-bpy)]_n (2) with a linear chain, [Ag(PCPA)(PCPAH)(4,4′-bpy) · H₂O]_n (3) with 1-D molecular ladder (4,4′-bpy = 4,4′-bipyridine; PCPA = p-chlorophenoxyacetate; PCPAH = p-chlorophenoxyacetic acid). It is noticeable that compound 3 is also a supramolecular framework built by coordination bonds, weak interactions between Ag ions, π-π stacking interactions and hydrogen-bonded interactions. The three compounds with different structure motifs have been characterized by elemental analyses, IR spectra, ultraviolet-visible diffuse reflection integral spectra, fluorescent spectra and single crystal X-ray diffraction analysis. Furthermore, the bonding properties of compound 3 were investigated in terms of the absorption spectrum, as well as the calculated band structures and density of states.     

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples

MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.     

Glycosylation of IgG B cell receptor (IgG BCR) in multiple myeloma: relationship between sialylation and the signal activity of IgG BCR

Little is known about the glycosylation of the isotype switched B cell receptor (BCR) in multiple myeloma, and the way it might affect receptor function. In this work IgG BCRs isolated from the individual lysates of peripheral blood lymphocytes (PBL) of 32 patients with IgG multiple myeloma and healthy controls were investigated for the expression of sialic acid (SA), galactose (Gal) and N-acetylglucosamine (GlcNAc), the sugars known to specify the glycoforms of human serum IgG. The degree of glycosylation and signaling status of all 32 isolated myeloma IgG BCRs were correlated and compared with the glycosylation of the IgG paraproteins isolated from sera of the same patients. It was shown that BCR IgG in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of IgG BCR is associated with higher levels of tyrosine phosphorylation (signaling activity) of the IgG BCR supramolecular complex and that BCR IgG and serum IgG paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. The results suggest that the development of the malignant clone in MM from post-switch B cells expressing IgG BCR at their surfaces to plasma cells secreting IgG paraprotein may be followed by permanent glycosylation changes in the IgG molecules.     

Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication events in the DUF606 family

X-ray crystallography has revealed that many integral membrane proteins consist of two domains with a similar fold but opposite (antiparallel) orientation in the membrane. The proteins are believed to have evolved by gene duplication and gene fusion events from a dual topology ancestral membrane protein, that adapted both orientations in the membrane and formed antiparallel homodimers. Here, we present a detailed analysis of the DUF606 family of bacterial membrane proteins that contains the entire collection of intermediate states of such an evolutionary pathway: single genes that would code for dual topology homodimeric proteins, paired genes coding for homologous proteins with a fixed but opposite orientation in the membrane that would form heterodimers, and fused genes that encode antiparallel two-domain fusion proteins. Two types of paired genes can be discriminated corresponding to the order in which the genes coding for the two oppositely oriented proteins occur in the operon. On the protein level, the heterodimers resulting from the two types of gene pairs are indistinguishable. In contrast, two types of fused genes corresponding to the two possible orders in which the oppositely oriented domains are present in the encoded proteins, do result in discernible types of proteins. The large number of genetic and protein states in the DUF606 family allowed for a detailed phylogenic analysis that revealed a total of nine independent duplication events in the DUF606 family, five of which resulted in paired genes, and four resulted in fused genes. Noticeably, there was no evidence for a sequential mechanism in which fusions evolve from a pair of genes. Rather, an evolutionary mechanism is proposed by which antiparallel two-domain proteins are the direct result of a gene duplication event. Combining the phylogeny of proteins and hosting microorganisms allowed for a reconstruction of the evolutionary pathway.     

Evidence for the ultraviolet-B (280–320 nm) radiation induced structural reorganization and damage of photosystem II polypeptides in isolated chloroplasts

Direct evidence for the possible loss of photosystem II (PS II) activity in chloroplasts of Vigna sinensis L. cv. Walp after ultraviolet-B (UV-B, 280–320 nm) radiation treatment was provided by polyacrylamide gel electrophoretic analysis of PS II polypeptides. A 30 min UV-B treatment of chloroplasts caused a 50% loss of PS II activity. The artificial electron donor. Mn²⁺ failed to restore UV-B radiation induced loss of PS II activity, while diphenyl carbazide (DPC) and NH₂OH only partially restored activity. Such a loss in PS II activity was found to be primarily due to a loss of 23 and 33 kDa extrinsic polypeptides. UV-B treatment induced the synthesis of a few polypeptides and a 29 kDa light-harvesting chlorophyll protein.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

Influence of amino acid specificities on the molecular and supramolecular organization of glycine-rich elastin-like polypeptides in water

Elastin-like polypeptides adopt complex supramolecular structures, showing either a hydrophobic or a hydrophilic surface, depending on their surrounding environment and the supporting substrate. The preferred organization is important in many situations ranging from biocompatibility to bio-function. Here we compare the n-repeat pentamer LeuGlyGlyValGly (n = 7) with the analogue ValGlyGlyValGly (n = 5), as water suspensions and as deposits on silicon substrates. These sequences contain the repeat XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) motif belonging to the hydrophobic glycine-rich domain of elastin and represent a simplified model from which to obtain information on molecular interactions functional to elastin itself. The compounds studied differ only by the presence of the -CH(2)- spacer in the Leu moiety and thus the work was aimed at revealing the influence of this spacer element on self assembly. Both polypeptides were studied under identical conditions, using combined techniques, to identify differences in their conformational states both at molecular (CD, FTIR) and supramolecular (XPS, AFM) levels. By these means, together with a Congo Red spectroscopic assay of β-sheet formation in water, a clear correlation between amino acid sequences (sequence specificity) and their kinetics and ordering of aggregation has emerged. The novel outcomes of this work are from the supplementary measurements, made to augment the AFM and XPS studies, showing that the significant step in the self assembly of both polypeptides takes place in the liquid phase and from the finding that the substitution of Val by Leu in the first position of the pentapeptide effectively inhibits the formation of amyloidal fibers.     

The effects of climate change on a mega‐diverse country: predicted shifts in mammalian species richness and turnover in continental Ecuador

Ecuador has some of the greatest biodiversity in the world, sheltering global biodiversity hotspots in lowland and mountain regions. Climate change will likely have a major effect on these regions, but the consequences for faunal diversity and conservation remain unclear. To address this issue, we used an ensemble of eight species distribution models to predict future shifts and identify areas of high changes in species richness and species turnover for 201 mammals. We projected the distributions using two different climate change scenarios at the 2050 horizon and contrasted two extreme dispersal scenarios (no dispersal vs. full dispersal). Our results showed extended distributional shifts all over the country. For most groups, our results predicted that the current diversity of mammals in Ecuador would decrease significantly under all climate change scenarios and dispersal assumptions. The Northern Andes and the Amazonian region would remain diversity hotspots but with a significant decrease in the number of species. All predictions, including the most conservative scenarios in terms of dispersal and climate change, predicted major changes in the distribution of mammalian species diversity in Ecuador. Primates might be the most severely affected because they would have fewer suitable areas, compared with other mammals. Our work emphasizes the need for sound conservation strategies in Ecuador to mitigate the effects of climate change     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.