Electron microscopic examination of sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe

A homothallic haploid strain of the fission yeast Schizosaccharomyces pombe initiates sexual reproduction (mating, meiosis and sporulation) in nitrogen-free sporulation medium. Cellular fine structures of eleven sporulation-deficient mutants (spo2, spo3, spo4, spo5, spo6, spo13, spo14, spo15, spo18, spo19 and spo20) of S. pombe in sporulation medium were examined by serial section-electron microscopy. The striking features of these spo mutants were: 1) the disappearance of the spindle pole bodies (SPBs) after the second meiotic division, and 2) the accumulation of unorganized structures. Based on histochemical staining, these structures were presumably unorganized spore wall precursors. In some mutants (spo3, spo5, spo6, spo19 and spo20), diploid zygotes contained four spore-like bodies which had walls similar to complete spore walls but failed to enclose any nuclei. After completion of the second meiotic division the nuclei were abnormally distributed in zygotic diploid cells. In the spo5, spo13, spo14, spo15 and spo19 mutants, the nuclei remained attached to each other. In spo5 and spo19, the inner membrane of the nuclear envelope was separated, but its outer membrane was shared by two sister nuclei. These observations suggest that the spo⁺ gene products play important roles in spatial and temporal organization of cellular structures during ascospore development.Abbreviations SPB spindle pole body - PTA-Cr phosphotungstic acid and chromic acid - PATAg periodic acid, thiocarbohydrazide and silver proteinate     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.     

Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

Nud1p links astral microtubule organization and the control of exit from mitosis

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the gamma-tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell-cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.     

烤后不同霉变程度烟叶际真菌群落组成与多样性分析

【目的】为了解烤后不同霉变程度烟叶真菌群落组成与多样性。【方法】基于IlluminaMiSeq高通量测序平台对烤后烟叶叶柄和叶片叶际样品的真菌群落组成进行了分析。【结果】霉变重烟叶叶片(BQ)、霉变重烟叶叶柄(BZ)、霉变轻烟叶叶片(JQ)、霉变轻烟叶叶柄(JZ) 4类样品真菌分布于子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、接合菌门(Zygomycota)等7个菌门、27个纲、58个目、104个科、171个属的360个真菌分类单元(OTUs)。不同霉变程度烟叶叶际真菌群落组成、分类单元的相对丰度及优势分类单元皆存在不同程度差异。在属的水平上,霉变重烟叶叶片(BQ)的优势菌属为Aspergillus (89.64%)、Myrothecium (2.54%)、Rhodotorula (2.48%)、Gibberella (1.49%);霉变重烟叶叶柄(BZ)的优势菌属为Aspergillus(96.93%)和Alternaria(1.92%);霉变轻烟叶叶片(JQ)的优势菌属为Aspergillus(40.13%)、Rhodotorula(31.81%)、Alternaria(16.75%);霉变轻烟叶叶柄(JZ)的优势菌属为Aspergillus (62.77%)、Alternaria (9.74%)、Rhodotorula (5.20%)。【结论】霉变轻烟叶叶片样品真菌群落的多样性最高,霉变轻烟叶叶柄的真菌群落丰富度最高。霉变重烟叶样品的叶片部位的真菌群落丰富度和多样性均高于叶柄。烟叶霉烂为烤房常见病害,其病原真菌种类多样,且广泛分布于烟叶和环境中。该研究结果为烤房烟叶霉烂病的防治可根据不同发病程度的不同部位的真菌群落构成特征,制定相应的防治方案提供了参考依据。     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Identification and expression pattern analysis of Piwi genes during the spermiogenesis of Portunus trituberculatus

The Piwi genes have an important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwi complete cDNA sequences from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species.     

The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38)

A consequence of the acrosome reaction is to expose the inner acrosomal membrane (IAM), which is a requirement for the sperm's ability to secondarily bind to and then penetrate the zona pellucida (ZP) of the mammalian oocyte. However, the proteins on the IAM responsible for binding and presumably penetrating the zona have not been identified. This issue can be resolved if direct information is made available on the composition of the IAM. For this purpose, we devised a methodology in order to obtain a sperm head fraction consisting solely of the IAM bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, we defined an electron dense protein layer that we termed the IAM extracellular coat (IAMC), which was visible on sonicated and acrosome-reacted sperm of several mammalian species. High salt extraction removed the IAMC coincident with the removal of a prominent 38 kDa polypeptide, which we termed IAM38. Antibodies raised against this polypeptide confirmed its presence in the IAMC of intact, sonicated and acrosome-reacted sperm. By immunoscreening of a bovine testicular cDNA library and sequencing the resulting clones, we identified IAM38 as the equivalent of porcine Sp38 [Mori, E., Kashiwabara, S., Baba, T., Inagaki, Y., Mori, T., 1995. Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida. Dev. Biol., 168, 575-583], an intra-acrosomal protein with ZP-binding ability, whose precise localization in sperm was unknown. The blockage of IVF at the level of the zona with anti-IAM38 antibodies and the retention of IAM38 after sperm passage through the zona support its involvement in secondary sperm-zona binding. This study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM for identifying other candidates for sperm-zona interactions.     

Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication

Spindle pole bodies (SPBs), like nuclear pore complexes, are embedded in the nuclear envelope (NE) at sites of fusion of the inner and outer nuclear membranes. A network of interacting proteins is required to insert a cytoplasmic SPB precursor into the NE. A central player of this network is Nbp1 that interacts with the conserved integral membrane protein Ndc1. Here, we establish that Nbp1 is a monotopic membrane protein that is essential for SPB insertion at the inner face of the NE. In vitro and in vivo studies identified an N-terminal amphipathic α-helix of Nbp1 as a membrane-binding element, with crucial functions in SPB duplication. The karyopherin Kap123 binds to a nuclear localization sequence next to this amphipathic α-helix and prevents unspecific tethering of Nbp1 to membranes. After transport into the nucleus, Nbp1 binds to the inner nuclear membrane. These data define the targeting pathway of a SPB component and suggest that the amphipathic α-helix of Nbp1 is important for SPB insertion into the NE from within the nucleus.