THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

Molecular Distributions in the Inner Regions of Protostellar Disks

The distributions of molecules in the inner regions of a protostellar disk are presented. These were calculated using an uncoupled chemical/dynamical model, with a numerical integration of the vertical disk structure. A comparison between models with and without the effects of X-ray ionisation is made, and molecules are identified which are good tracers of the ionisation level in this part of the disk, notably CN and C_2H. In the region considered in this paper (r

The Masculine Habitus as 'Distributed Cognition': A Case Study of 5- to 6-Year-Old Boys in an English Inner-City, Multi-Ethnic Primary School

This article provides a case study demonstrating the active role that 5- to 6-year-old boys in an English inner-city, multi-ethnic primary school play in the appropriation and reproduction of their masculine identities. It is argued that the emphasis on physicality, violence and racism found among the boys cannot be understood without reference to the immediate contexts of the local community and the school within which they are located. In making this argument the article draws upon and applies the concept of the habitus and develops this with the notion of 'distributed cognition' as proposed in sociocultural theory. Some of the implications of this analysis for working with boys in early years settings are discussed in the conclusion.

Dramatic structure in the photodetachment of inner shells of negative ions: Li-

Deep inner-shell photodetachment of negative ions is shown to differ qualitatively and quantitatively from the corresponding process in neutral atoms and positive ions. As an example, calculations of the photodetachment of Li- out of the 1s shell, using an augmented R-matrix methodology, are presented and show numerous structures over a broad energy region including a new phenomenon: Auger decay of a shape resonance. Li- was selected because it is the simplest multishell negative ion and is amenable to experimental scrutiny.

Inner-shell photoexcitation of Fe XV and Fe XVI

The configuration-interaction method as implemented in the computer code CIV3 is used to determine energy levels, electric dipole radiative transition wavelengths, oscillator strengths and transition probabilities for inner-shell excitation of transitions in Fe XV and Fe XVI. Specifically, transitions are considered of the type 1s(2) 2s(2) 2p(6) 3s(2) -1s(2) 2s(2) 2p(5) 3l3l' 3l" (l, l' and l" = s,p or d) in FeXV and 1s(2) 2s(2) 2p(6) 3s- 1s(2) 2s(2) 2p(5) 3l3l' (l and l' = s,p or d) in FeXVI, using the relativistic Breit-Pauli approach. An assessment of the accuracy of the derived atomic data is performed.

A chemical analysis of five hot stars towards the Galactic centre

High resolution echelle spectroscopy is presented for thirteen stars lying in the direction of the Galactic centre which, on the basis of photographic photometry and low dispersion spectroscopy, have been classified as early-B-type. Eight of these stars have large rotational velocities which preclude a detailed analysis. The five stars with moderate to low projected rotational velocities have been analysed using model atmosphere techniques to determine atmospheric parameters and chemical compositions. Two of these stars appear to be evolved blue horizontal branch objects on the basis of their chemical compositions and small projected rotational velocity. The evolutionary status of a third is ambiguous but it is probably a post-asymptotic-giant branch star. The remaining two objects are probably young massive stars and show enhanced abundances of N, C, Mg and Si, consistent with their formation in the inner part of the Galactic disk. However their O abundances are normal, confirming results found previously for other early- type stars, which would imply a flat abundance gradient for this element in the inner region of our Galaxy.

Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.

The VLT-FLAMES survey of massive stars: evolution of surface N abundances and effective temperature scales in the Galaxy and Magellanic Clouds

We present an analysis of high resolution VLT-FLAMES spectra of 61 B-type stars with relatively narrow-lined spectra located in 4 fields centered on the Milky Way clusters; NGC 3293 and NGC 4755 and the Large and Small Magellanic cloud clusters; NGC 2004 and NGC 330. For each object a quantitative analysis was carried out using the non-LTE model atmosphere code TLUSTY; resulting in the determination of their atmospheric parameters and photospheric abundances of the dominant metal species (C, N, O, Mg, Si, Fe). The results are discussed in relation to our earlier work on 3 younger clusters in these galaxies; NGC 6611, N11 and NGC 346 paying particular attention to the nitrogen abundances which are an important probe of the role of rotation in the evolution of stars. This work along with that of the younger clusters provides a consistent dataset of abundances and atmospheric parameters for over 100 B-type stars in the three galaxies. We provide effective temperature scales for B-type dwarfs in all three galaxies and for giants and supergiants in the SMC and LMC. In each galaxy a dependence on luminosity is found between the three classes with the unevolved dwarf objects having significantly higher effective temperatures. A metallicity dependence is present between the SMC and Galactic dwarf objects, and whilst the LMC stars are only slightly cooler than the SMC stars, they are significantly hotter than their Galactic counterparts.