Distribution of a seminal plasma-associated protein kinase inhibitor in normal, oligozoospermic, and vasectomized men

Human sperm-free seminal plasma contains an inhibitor, which is protein in nature, of the histone kinase present in seminal plasma. Since protein kinase inhibitors have been observed to be present in spermatozoa, the objective of the present study was to determine whether this seminal plasma-associated enzyme inhibitor originates from the sperm, or whether it is a component of accessory secretion(s) comprising the seminal plasma. Sperm-free seminal plasma from normospermic (greater than 20 X 10(6) sperm/ml), oligozoospermic (less than or equal to 20 X 10(6) sperm/ml), and vasectomized donors was obtained, and inhibitor-enriched fractions were prepared by (NH4)2SO4 fractionation and gel filtration. Contamination of the sperm-free seminal plasma by spermatozoa or spermatozoan components was negligible as assessed by light microscopy, polyacrylamide gel electrophoresis, and measurement of the activity of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Specific (inhibitory units/mg protein) and total inhibitory activities were determined in each of the donors by constructing linear inhibition curves using various concentrations of inhibitor. The results were correlated with the initial sperm concentration. There was no apparent relationship between the amount of inhibitory activity present and the initial sperm concentration. The histone kinase inhibitor also did not appear to be associated with testicular or epididymal secretions since it was observed in the seminal plasma of vasectomized donors. It is concluded that this inhibitor of histone kinase originates from the accessory secretions comprising the human ejaculate.     

Production of diverse brGDGTs by Acidobacterium Solibacter usitatus in response to temperature,pH, and O2 provides a culturing perspective on brGDGT proxies and biosynthesis

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids that are frequently employed as paleoenvironmental proxies because of the strong empirical correlations between their relative abundances and environmental temperature and pH. Despite the ubiquity of brGDGTs in modern and paleoenvironments, the source organisms of these enigmatic compounds have remained elusive, requiring paleoenvironmental applications to rely solely on observed environmental correlations. Previous laboratory and environmental studies have suggested that the globally abundant bacterial phylum of the Acidobacteria may be an important brGDGT producer in nature. Here, we report on experiments with a cultured Acidobacterium, Solibacter usitatus, that makes a large portion of its cellular membrane (24 ± 9% across all experiments) out of a structurally diverse set of tetraethers including the common brGDGTs Ia, IIa, IIIa, Ib, and IIb. Solibacter usitatus was grown across a range of conditions including temperatures from 15 to 30°C, pH from 5.0 to 6.5, and O₂ from 1% to 21%, and demonstrated pronounced shifts in the degree of brGDGT methylation across these growth conditions. The temperature response in culture was in close agreement with trends observed in published environmental datasets, supporting a physiological basis for the empirical relationship between brGDGT methylation number and temperature. However, brGDGT methylation at lower temperatures (15 and 20°C) was modulated by culture pH with higher pH systematically increasing the degree of methylation. In contrast, pH had little effect on brGDGT cyclization, supporting the hypothesis that changes in bacterial community composition may underlie the link between cyclization number and pH observed in environmental samples. Oxygen concentration likewise affected brGDGT methylation highlighting the potential for this environmental parameter to impact paleotemperature reconstruction. Low O₂ culture conditions further resulted in the production of uncommon brGDGT isomers that could be indicators of O₂ limitation. Finally, the production of brGTGTs (trialkyl tetraethers) in addition to the previously discovered iso-C15-based mono- and diethers in S. usitatus suggests a potential biosynthetic pathway for brGDGTs that uses homologs of the archaeal tetraether synthase (Tes) enzyme for tetraether synthesis from diethers.     

Starfish and horseshoe crab egg factors cause elevations of cyclic nucleotide concentrations in spermatozoa from starfish and horseshoe crabs.

Factors collected from the eggs of the starfish (Pisaster giganteus) and the horsehoe crab (Limulus polyphemus) caused significant increases in the sperm cyclic nucleotide concentrations of the respective species. Sea urchin egg factors, at concentrations that resulted in maximal cyclic nucleotide elevations in sea urchin spermatozoa, had no effect on those of starfish or horseshoe crab, suggesting a species specificity with respect to egg factor-induced changes in sperm cyclic nucleotide metabolism.     

Role of G proteins in mouse egg activation: stimulatory effects of acetylcholine on the ZP2 to ZP2f conversion and pronuclear formation in eggs expressing a functional m1 muscarinic receptor.

Sperm-mediated egg activation may be analogous to ligand-mediated signal transduction through G protein-coupled receptors. We investigated this possibility in the mouse egg by microinjecting mouse oocytes with an m1 muscarinic receptor mRNA. Following oocyte maturation in vitro, the metaphase II-arrested eggs were treated with acetylcholine and its effect was examined on zona pellucida modifications and pronuclear formation, which are end points of early and late egg activation, respectively. Treatment of these eggs with acetylcholine reveals that both the ZP2 to ZP2f conversion and pronuclear formation occur. Atropine and microinjected GDP beta S block the acetylcholine-induced ZP2 conversion, suggesting that the acetylcholine effects are mediated via a functional G protein-coupled m1 receptor. The acetylcholine-induced ZP2 conversion, however, is not inhibited by pertussis toxin under conditions in which greater than 90% of the endogenous Gi is inactivated by ADP ribosylation. The presence of a pertussis toxin-insensitive G protein, Gq, is detected by immunoblotting; this G protein could be a candidate to mediate the pertussis toxin-insensitive effects of acetylcholine. Results of these experiments are consistent with the hypothesis that receptor-mediated G protein activation may play a role in egg activation.     

Effects of phorbol esters and a diacylglycerol on mouse eggs: inhibition of fertilization and modification of the zona pellucida

Sperm penetration of the zona pellucida and fertilization are inhibited in mouse eggs treated with phorbol esters and the diacylglycerol, sn-1,2-dioctanoyl glycerol. The effect appears mediated by the zona pellucida, since zona-free eggs treated with these compounds are fertilized to the same extent as untreated eggs. Moreover, the binding of sperm to isolated zonae incubated in the absence or presence of biologically active phorbol esters is similar. Last, sperm treated with phorbol esters or sn-1,2-dioctanoyl glycerol bind to eggs and undergo the acrosome reaction to the same extent as untreated sperm. The inhibitory effect on fertilization is correlated with an egg-induced modification of at least ZP2, as manifested by a change in its electrophoretic mobility in polyacrylamide gels. In addition, changes in the biological properties of the treated zonae occur, such that sperm binding is not altered, but that the final stage(s) of the zona-induced acrosome reaction is inhibited. Zonae obtained from phorbol ester- or diacylglycerol-treated eggs should provide a system to study both the structural modifications of the zona proteins that are involved in induction of the acrosome reaction, as well as, delineating the sequence of events that comprise the acrosome reaction.     

Cutting edge: lymphoproliferative disease in the absence of CTLA-4 is not T cell autonomous.

Mice deficient for the expression of CTLA-4 develop a lethal lymphoproliferative syndrome and multiorgan inflammation leading to death at about 4 wk of age. Here we show that RAG2-deficient mice reconstituted with CTLA-4-deficient bone marrow do not develop a lymphoproliferative syndrome despite lymphocyte infiltration mainly into pericardium and liver. Moreover, RAG2-deficient mice reconstituted with a mixture of normal and CTLA-4-deficient bone marrow remain healthy and do not develop any disease. Thus, the lethal disease observed in CTLA-4-deficient mice is not T cell autonomous and can be prevented by factors produced by normal T cells.     

Determination of preexcision surgical margins of melanomas from fixed-tissue specimens

The shrinkage of cutaneous surgical specimens of 199 malignant melanomas was analyzed. A formula was derived that makes it possible to calculate the in vivo (preexcision) specimen diameter from the in vitro (fixed-tissue) specimen diameter. The age of the patient was found to significantly influence specimen shrinkage and was incorporated into this shrinkage formula. The calculated in vivo specimen diameter was then used to determine the width of the in vivo surgical margins with reasonable accuracy. Thus this method permits calculation of the width of surgical margins from fixed-tissue specimens.