Variation in seminal plasma alters the ability of ram spermatozoa to survive cryopreservation.

Variation in the effect of seminal plasma on sperm function and fertility has been hypothesised to be due to differences between males and their seminal plasma composition. The freezing resilience of individual rams (n=17) was investigated to characterise inter-male variation. This was determined by measuring the degree of change in motility induced by cryopreservation (Experiment 1). Experiment 2 examined the effect of pooled seminal plasma from rams identified as having high or low resilience to freezing on the cryosurvival of washed spermatozoa from either high (n=3) or low (n=3) sperm freezing resilience rams. Immediately after thawing and throughout the incubation period (0-4h), spermatozoa from high-resilience rams frozen with high-resilience seminal plasma demonstrated superior motility to spermatozoa from high-resilience rams frozen with low-resilience seminal plasma (P<0.001). Similarly, spermatozoa from low-resilience rams frozen with high-resilience seminal plasma exhibited higher motility than spermatozoa from low-resilience rams frozen with low-resilience seminal plasma immediately after thawing (0h; P<0.001). The present study shows that variation in freezing resilience of ram spermatozoa is related to the source and composition of the seminal plasma.

The identification of proteomic markers of sperm freezing resilience in ram seminal plasma.

The source and composition of seminal plasma has previously been shown to alter the ability of spermatozoa to survive cryopreservation. In the present study, the ionic and proteomic composition of seminal plasma from rams with high (HSP; n = 3) or low (LSP; n = 3) freezing resilient spermatozoa was assessed. 75 proteins were identified to be more abundant in HSP and 48 proteins were identified to be more abundant in LSP. Individual seminal plasma proteomes were established for each of the six rams examined. For each ram, correlations were conducted between previously recorded freezing resilience [1] and individual spectral counts in order to identify markers of freezing resilience. 26S proteasome complex, acylamino acid releasing enzyme, alpha mannosidase class 2C, heat shock protein 90, tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase and transitional endoplasmic reticulum ATPase were found to be positively correlated (r(2) > 0.7) with freezing resilience. Cystatin, zinc-2-alpha glycoprotein, angiogenin-2-like protein, cartilage acidic protein-1, cathepsin B and ribonuclease 4 isoform 1 were found to be negatively correlated (r(2) > 0.7) with freezing resilience. Several negative markers were found to originate from the accessory sex glands, whereas many positive markers originated from spermatozoa and were part of or associated with the 26S proteasome or CCT complex.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

CD4 is expressed by epidermal Langerhans' cells predominantly as covalent dimers.

Langerhans' cells (LC) of skin are CD4 expressing, dendritic, antigen-presenting cells, that are essential for activation of primary immune responses and are productively infected by HIV. We have shown previously that lymphocytes and monocytes express CD4 both as monomers and covalently linked homodimers. In those cells the 55-kDa monomer structure predominates. LC in un-fractionated human epidermal cell (EC) suspension also expresses both forms of CD4, but in EC the dimer form is predominant. Because isolation of LC into single cell suspension by trypsin, as is routinely used for LC isolation, degrades CD4, a systematic study for an alternate procedure for LC isolation was performed. Thus it was found that collagenase blend F treatment can efficiently release LC into suspension, under conditions of only minimal degradation of control soluble recombinant CD4 or CEM-T4 or THP-1 cell CD4, or importantly of LC surface CD4. SDS-PAGE immunoblotting of purified LC extracted from EC by collagenase confirmed CD4 structure as predominantly 110-kDa dimers, with only minimal 55-kDa monomers. The suitability of LC prepared thus for functional studies was demonstrated with binding of functional ligand HIV gp120. It remains to be determined, however, why tissue embedded LC express mainly CD4 dimers, but single-celled blood lymphocytes and monocytes mainly monomers.

Direct evidence for native CD4 oligomers in lymphoid and monocytoid cells.

CD4 is expressed by T lymphocytes and monocytes and is generally considered a monomer even though its structure was originally modelled on the REI Bence-Jones homodimer. However, native CD4 was demonstrated as both monomer and dimers of 55 and 110 kDa in lymphoid and monocytoid cells by immunoprecipitation and immunoblotting after solubilization with alkylating (iodoacetamide) or reducing (dithiothreitol, 2-mercaptoethanol) reagents. Full reduction yielded only the 55-kDa monomeric form. Purified CD4 oligomers from CEM-T4 cells were also resolved as homodimers by MALDI-Tof mass fingerprinting after tryptic digestion. Cell treatment with the membrane impermeable, free-thiol reactive, 5,5'-dithiobis-2-nitrobenzoic acid enhanced cell surface CD4 dimers and tetramers. The interaction sites producing dimerization were probably in the D4 domain as OKT4 inhibited self association of recombinant CD4 (rCD4). Oligomerization of rCD4 by glutathione and thioredoxin indicates that thiol exchange interactions were responsible. Enhanced CD4 dimer expression was also observed after PMA (20 ng/ml) activation of THP-1 cells. These findings demonstrate that different quaternary forms of CD4 such as monomers, homodimers and tetramers are expressed by T lymphocytes and monocytes/macrophages.

Differential tropism and chemokine receptor expression of human immunodeficiency virus type 1 in neonatal monocytes, monocyte-derived macrophages, and placental macrophages.

Laboratory-adapted (LA) macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1) isolates (e.g., HIV-1(Ba-L)) and low-passage primary (PR) isolates differed markedly in tropism for syngeneic neonatal monocytes, monocyte-derived macrophages (MDMs), and placental macrophages (PMs). Newly adherent neonatal monocytes and cultured PMs were highly refractory to infection with PR HIV-1 isolates yet were permissive for LA M-tropic isolates. Day 4 MDMs were also permissive for LA M-tropic isolates and additionally, were permissive for over half the PR isolates tested. Qualitative differences in PR HIV-1 infection of monocytes/MDMs could not be correlated with CD4 levels alone, and in all three cell types the block to PR HIV-1 strain replication preceded reverse transcription. Neonatal monocyte susceptibility to PR HIV-1 strains correlated with increasing CCR-5 expression during maturation. CCR-5 could not be detected on newly adherent (day 1) neonatal monocytes, in contrast to adult monocytes (H. Naif et al., J. Virol. 72:830-836, 1998), but was readily detectable after 4 to 7 days of culture. However, moderate CCR-5 mRNA levels were present in day 1 neonatal monocytes and remained constant during monocyte maturation. CCR-5 was not detectable on the surface of PMs, yet the receptor was present within permeabilized cells. Notably, two brain-derived PR HIV-1 isolates from a single patient, differing in their V3 loops, were discordant in their abilities to infect neonatal monocytes/MDMs and PMs, yet both isolates could infect newly adherent adult monocytes. Together these data strongly suggest that LA HIV-1 isolates are able to infect neonatal monocytes at earlier stages of maturation and lower-level expression of CCR-5 than PR isolates. The differences between neonatal and adult monocytes in susceptibility to PR isolates may also be related to the level of CCR-5 expression.

Analysis of recombinant and native CD4 by one- and two-dimensional gel electrophoresis.

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.

Creation of state-level Medicare database for healthcare evaluation applications.

The Health Care Quality Improvement Initiative (HCQII) of the Health Care Financing Administration (HCFA) calls for Professional Review Organizations (PROs) to undertake pattern analysis of large administrative datasets for the purposes of quality of care assessment. The limitations of such administrative databases (primarily the MEDPAR file and derivatives thereof) include impoverished information regarding clinical attributes of Medicare enrollees and the process and outcome of their healthcare. This paper describes preliminary efforts to address this problem by the creation of a database, the PRO Concatenated Database (PCD), from the pooled implicit judgment review data of four Peer Review Organizations (PROs). The data elements comprising the PCD were carefully selected to provide important information regarding quality and appropriateness of care. Preliminary inter-state comparative studies employing the PCD are discussed. A method is also described by which the analytical power of state-level databases may be enhanced by linkage to state-level Modeled MEDPAR data which are issued by HCFA and contain patient-level risk-adjusted mortality data. This approach to the acquisition of data whose clinical content is enriched may prove to be particularly useful to the PRO community during the pattern analysis phase of the HCQII. Such analyses will evolve into more detailed studies involving primary data collection followed by dissemination of the results to local healthcare providers. In this manner, the PCD may facilitate rapid feedback regarding the effectiveness of healthcare delivery to the local community.

Thrombospondin expression in traumatized skeletal muscle. Correlation of appearance with post-trauma regeneration.

Biochemical and immuno-microscopic techniques were used to study temporal involvement of thrombospondin in relation to fibrinogen in muscle regeneration using a rat skeletal muscle-wound model. In undamaged control muscle, no fibrinogen and minimal thrombospondin antigen was found. Following crushing injury, fibrin networks appear immediately, followed by a gradual ordered accumulation of thrombospondin (within a few hours) in the vicinity of the vascular bed and adjacent endomysial connective tissue. Later, thrombospondin becomes associated with connective tissue and basal laminae around muscle fibers throughout the damaged muscle, maximal labelling occurring 3-6 days post-injury. Thrombospondin immunoreactivity decreased thereafter to near normal levels after 7 days post-injury, coincident with the appearance of regenerating muscle fibers. In contrast, little fibrin material remained by five days after injury. Quantitative radioimmunoassay of soluble thrombospondin antigen and radioimmune labelling of thick frozen sections reinforced the qualitative immuno-microscopic observations, with levels peaking at 3-4 days post-trauma, 10-fold over control levels. SDS-PAGE immunoblotting of non-reduced muscle extracts three days after a crush assault shows that the bulk of the thrombospondin incorporated into the injury site exists in a polymerized state (less than or equal to 1000 kD). These results demonstrate that the temporal appearance and disappearance of thrombospondin in the healing of a crushing lesion in muscle is related more closely to the regeneration phase of muscle than to the coagulation phase.

B protein of factor XIII: differentiation between free B and complexed B.

Plasma factor XIII is a complex of A and B proteins noncovalently linked in a tetramer, A2B2, Enzyme-linked immunosorbent assays (ELISA) were developed to measure the separate factor XIII proteins and the complex. All of the A protein in plasma is in the zymogen complex. The B assay measures the total amount of B protein in plasma (both free B and complexed B). This was confirmed by nondenaturing gel electrophoresis and immunoblotting, which showed two bands for B in plasma with this antibody. Two assays were developed to measure A2B2 complex specifically. One assay used a monoclonal antibody to B to bind antigen and measured B protein in the zymogen complex only and hence the concentration of the complex. The specificity of this antibody was also shown by immunoblotting. In the second assay, the capture antibody was to B and the tag antibody was to A. These two assays gave identical results for the concentration of A2B2 (0.07 mumol/L, 21.6 micrograms/mL in normal plasma). Thus, for the first time, differentiation and quantitation of free B and complexed B in plasma was possible. The assays were used to measure factor XIII proteins in plasma from normal controls, homozygous-deficient factor XIII patients, and their heterozygous relatives. The normal concentration of A in plasma is 0.13 to 0.16 mumol/L (approximately 11 micrograms/mL), all of which is in A2B2. The total B concentration is 0.26 to 0.28 mumol/L (approximately 21 micrograms/mL), half of which is complexed. The free B concentration is 0.13 mumol/L (approximately 10 micrograms/mL). Homozygous-deficient patients have essentially no A protein, but their free B concentration is 0.11 mumol/L. Heterozygotes have decreased A2B2, but their free B is 0.11 mumol/L. These results indicate that the concentration of free B is remarkably constant and does not depend on the concentration of A2 or A2B2.

Thrombin-independent activation of platelet factor XIII by endogenous platelet acid protease.

Platelets contain factor XIII, an A subunit zymogen form of transglutaminase (TGase), that is activated by thrombin. In addition a thrombin-independent TGase (A#) was observed. A# was formed in platelet preparations lysed at acid pH, and its generation inhibited by protease inhibitors and alkaline pH. When maximal A# activity was generated in acidified lysates no further TGase activity could be induced by subsequent treatment with thrombin. Both FXIII zymogen and A# copurified as for FXIII, from either alkaline or from acidified platelet lysates respectively, by the conventional procedure. The pH optima, Km's for NN dimethyl casein, molecular weights, heat lability of active forms, requirements for calcium and reducing agents, and immunological characteristics of both TGases were the same. Studies with inhibitor substrates suggested that a thrombin-like cathepsin C or carboxypeptidase was responsible for A# formation. Purified FXIII zymogen could be activated directly by cathepsin C. Thus, the predominant, and probably only, TGase of platelets is factor XIII, which may be activated either by thrombin or by endogenous platelet acid protease(s).

Factor XIII A is synthesized and expressed on the surface of U937 cells and alveolar macrophages.

Factor XIII A subunit was detected in U937 cells and human alveolar macrophages by immunohistology and Western blotting. U937 cells synthesize factor XIII A subunit de novo under serum-free, platelet-free conditions, as indicated by 35S-methionine labeling and immunoprecipitation. Thrombin-dependent activity was demonstrated to account for 98% of the total transglutaminase activity in U937 cells (1.5 micrograms per 0.5 X 10(6) cells/mL). Intact U937 cells and alveolar macrophages and homogenates from these cells cross-linked fibrin to form gamma-gamma and alpha-polymers. Factor XIII A was detected on the surface of intact U937 cells and macrophages by flow cytometry and 125I-labeling and immunoprecipitation. Cell surface expression of factor XIII A was augmented in the presence of several soluble macrophage activators; however, no concurrent increase in its biosynthesis was observed. The presence and cell surface expression of factor XIII A subunit within macrophages suggest new pathways by which these cells may function in clotting and in the remodeling of the extracellular matrix during inflammation and wound healing.

Characterization of thrombospondin as a substrate for factor XIII transglutaminase.

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.