In vitro initiated sperm forward motility in caput spermatozoa: weak and transient.

Testicular spermatozoa during journey through epididymis acquire forward motility, which is essential for fertility. To understand the biochemistry of sperm motility initiation, various initiation media have been developed that permitted high level of motility induction (55-60%) in the immature caput-spermatozoa in presence of activating principles: theophylline, bicarbonate and epididymal plasma (EP) when analysed microscopically. Here, we show for the first time using caprine model that stability and quality of in vitro-induced motility in the caput spermatozoa is insignificant in contrast to naturally induced motility in mature cauda spermatozoa. In vitro-induced motility of the immature spermatozoa was lost completely upon the removal of these activators by centrifugation. Selective withdrawal of either EP or HCO(3) by dilution retains 50-60% of the in vitro-induced motility. Spectrophotometric analysis revealed that in vitro-induced vertical motility in immature spermatozoa is too little when compared to mature spermatozoa. In in vitro-initiated caput spermatozoa, cyclic adenosine monophosphate level becomes doubled but lesser than cauda spermatozoa. This revelation concludes that scientific knowledge generated over the years on the basis of in vitro initiation method is insignificant and needs improvisation to delineate biochemical regulation of sperm motility which in turn has remarkable potential in wide biological fields, especially in infertility treatment.

Assessment of noise environment during construction of a major bridge and associated approach road.

In this paper a methodology to quantify the noise environment, during a major bridge construction and upgrading approach road connectivity, has been provided. Noise levels were monitored at eleven sites. These eleven sites have been classified into three categories - commercial, residential and silence zones. The study was carried out to measure the ambient noise levels in all the eleven sites falling in the above three categories during both day and night times considering both "working" and "non-working" days. It was found that the mean noise level during night time was more, compared to that during day time for commercial, residential as well as silence zones. The likely causes of more noise during night time have been explored. Appropriate remedial measures have been suggested to reduce the noise levels. In addition, the noise levels in the above three zones have been compared, wherever feasible statistically, with the respective zonal standards. Significance has been found in all the cases. The underlying causes and remedies have been provided.

Role of the major ecto-phosphoprotein in sperm flagellar motility using a cell electroporation method.

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.

Shedding off specific lipid constituents from sperm cell membrane during cryopreservation.

Membrane damage is one of the main reasons for reduced motility and fertility of sperm cells during cryopreservation. Using a model system of sperm cryopreservation developed in our laboratory, we have investigated the detailed changes due to cryopreservation in the plasma membrane lipid composition of the goat epididymal sperm cells. Total lipid and its components, i.e., neutral lipids, glycolipids and phospholipids decreased significantly after cryopreservation. Among neutral lipids sterols, steryl esters and 1-O-alkyl-2,3-diacyl glycerols decreased appreciably, while among phospholipids, major loss was observed for phosphatidyl choline and phosphatidyl ethanolamine. Unsaturated fatty acids bound to the phospholipids diminished while the percentage of saturated acids increased. The cholesterol:phospholipid ratio enhanced and the amount of hydrocarbon, which was unusually high, increased further on cryopreservation. The data indicates that profound increase of the hydrophobicity of the cell membrane is one of the major mechanisms by which spermatozoa acquire potential to resist or combat stress factors like cryodamage. The results are compatible with the view that for survival against cryodamage, sperm cells modulate the structure of their outer membrane by shedding off preferentially some hydrophilic lipid constituents of the cell membrane.

Observation of a near-threshold D(0)D[over](0)pi(0) enhancement in B-->D(0)D[over](0)pi(0)Kappa decay.

We report the first observation of a near-threshold enhancement in the D(0)D[over](0)pi(0) system from B-->D(0)D[over](0)pi(0)Kappa decays using a 414 fb(-1) data sample collected at the Upsilon(4S) resonance. The enhancement peaks at a mass M=3875.2+/-0.7(+0.3)/(-1.6) +/-0.8 MeV/c2 and the branching fraction for events in the peak is B(B-->D(0)D[over](0)pi(0)Kappa)=(1.22+/-0.31(+0.23)/(-0.30))x10(-4). The data were collected with the Belle detector at the KEKB energy-asymmetric e+ e- collider.

Evidence for B0 --> D+ D- and observation of B- --> D0D- and B- -->D0D*- decays.

We report evidence for B(0) --> D(0)D(-) and the first observation of the decay modes B(-) --> D(0)D(-) and B(-) --> D(0)D(*-) based on a sample of 152 x 10(6) BB events collected by the Belle detector at KEKB. The branching fractions for B(0) --> D(+) D(-), B--->D(0)D(-), and B--> D(0)D(*-) are found to be (1.91 +/- 0.51 +/- 0.30) x10(-4), (4.83 +/- 0.78 +/- 0.58) x 10(-4), and (4.57 +/- 0.71 +/- 0.56) x 10(-4), respectively. Charge asymmetries in the B---> D(0)D(-) and B(-) --> D(0)D(*-) channels are consistent with zero.

Identification of goat sperm ecto-cyclic AMP independent protein kinase substrate localized on sperm outer surface.

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.

Isolation and identification of a novel motility-inhibiting factor from goat cauda sperm plasma membrane.

A motility inhibiting factor (MIF) in sperm plasma membrane of mammalian spermatozoa (goat) has been demonstrated. This factor has been purified to apparent homogeneity by Sepharose-6B affinity chromatography and DEAE-cellulose ion-exchange chromatography. The molecular weight of the isolated factor has been estimated as 98 kDa by molecular sieving and analytical HPLC. SDS-polyacrylamide gel electrophoresis of MIF gave a single band of 100 kDa, indicating that the factor is a monomer. MIF is a thermo-stable factor and it inhibited the spermatozoa motility in a dose dependent manner. It is a glycoprotein as it binds with high affinity to Sepharose-6B and the affinity matrix-bound factor can be eluted with D-galactose. Data show that the motility inhibiting activity is lost completely when treated with beta-galactosidase indicating that its sugar side chain is essential for its activity. Addition of MIF antibody caused significant enhancement of forward motility of the caput and cauda-spermatoza. This antibody may thus be useful for solving some of the problems of human infertility due to low sperm motility. The motility inhibiting protein may also be useful as a vaginal contraceptive.

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium.

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

First measurement of gamma(D*(+)).

We present the first measurement of the D*(+) width using 9/fb of e(+)e(-) data collected near the Upsilon(4S) resonance by the CLEO II.V detector. Our method uses advanced tracking techniques and a reconstruction method that takes advantage of the small vertical size of the Cornell Electron-positron Storage Ring beam spot to measure the energy release distribution from the D*(+)-->D(0)pi(+) decay. We find gamma(D*(+)) = 96+/-4 (stat)+/-22 (syst) keV. We also measure the energy release in the decay and compute Delta m identical with m(D*(+))-m(D(0)) = 145.412+/-0.002 (stat)+/-0.012 (syst) MeV/c(2).

Branching fraction and photon energy spectrum for b --> s gamma.

We have measured the branching fraction and photon energy spectrum for the radiative penguin process b-->s gamma. We find Beta(b-->s gamma) = (3.21+/-0.43+/-0.27(+0.18)(-0.10))x10(-4), where the errors are statistical, systematic, and from theory corrections. We obtain first and second moments of the photon energy spectrum above 2.0 GeV, = 2.346+/-0.032+/-0.011 GeV, and -(2) = 0.0226+/-0.0066+/-0.0020 GeV(2), where the errors are statistical and systematic. From the first moment, we obtain (in the modified minimal subtraction renormalization scheme, to order 1/M(3)(B) and beta(0)alpha(2)(s)) the heavy quark effective theory parameter Lambda = 0.35+/-0.08+/-0.10 GeV.

Hadronic mass moments in inclusive semileptonic B meson decays.

We have measured the first and second moments of the hadronic mass-squared distribution in B-->X(c)l nu, for P(lepton)>1.5 GeV/c. We find = 0.251+/-0.066 GeV(2), <(M(2)(X)-)(2)> = 0.576+/-0.170 GeV(4), where M macro(D) is the spin-averaged D meson mass. From that first moment and the first moment of the photon energy spectrum in b-->s gamma, we find the heavy quark effective theory parameter lambda(1) (in the modified minimal subtraction renormalization scheme, to order 1/M(3)(B) and beta(0)alpha(2)(s)) to be -0.24+/-0.11 GeV(2). Using these first moments and the B semileptonic width, and assuming parton-hadron duality, we obtain absolute value of V(cb) = 0.0404+/-0.0013.

Search for the decay upsilon(1S) --> gammaeta(').

We report on a search for the radiative decay Upsilon(1S)-->gammaeta(') in 61.3 pb(-1) of data taken with the CLEO II detector at the Cornell Electron Storage Ring. Three decay chains were investigated, all involving eta(')-->pi(+)pi(-)eta, followed by eta-->gammagamma, eta-->pi(0)pi(0)pi(0), or eta-->pi(+)pi(-)pi(0). We find no candidate events in any of the three cases and set a combined upper limit of 1.6x10(-5) at 90% C.L., significantly smaller than the previous limit. We compare our result to other radiative Upsilon decays, to radiative J/psi decays, and to theoretical predictions.

Study of tau decays to six pions and a neutrino.

The tau decays to six-pion final states have been studied with the CLEO detector at the Cornell Electron Storage Ring. The measured branching fractions are B(tau(-)-->2pi(-)pi(+)3pi(0)nu(tau)) = (2.2+/-0.3+/-0.4)x10(-4) and B(tau(-)-->3pi(-)2pi(+)pi(0)nu(tau)) = (1.7+/-0.2+/-0.2)x10(-4). A search for substructure in these decays shows that they are saturated by intermediate states with eta or omega mesons. We present the first observation of the decay tau(-)-->2pi(-)pi(+)omega(nu)tau and the branching fraction is measured to be (1.2+/-0.2+/-0.1)x10(-4). The measured branching fractions are in good agreement with the isospin expectations but somewhat below the conserved-vector-current predictions.

Observation of new states decaying into Lambda+(c)pi(-)pi(+).

Using 13.7 fb(-1) of data recorded by the CLEO detector at the Cornell Electron Storage Ring, we investigate the spectrum of charmed baryons which decay into Lambda+(c)pi(-)pi(+) and are more massive than the Lambda+(c)(2625) baryon. We find evidence for two new states: one is broad and has an invariant mass roughly 480 MeV above that of the Lambda+(c) baryon; the other is narrow with an invariant mass of 596+/-1+/-2 MeV above the Lambda+(c) mass.

Bounds on the CP asymmetry in b --> sgamma decays.

We have measured the CP asymmetry A(CP) identical with[gamma(b-->sgamma)-gammab-->sgamma)]/[gamma(b-->sgamma)+gamma(b-->sgamma)] to be A(CP) = (-0.079+/-0.108+/-0.022) (1.0+/-0.030), implying that, at 90% confidence level, A(CP) lies between -0.27 and +0.10. These limits rule out some extreme non-standard-model predictions, but are consistent with most, as well as with the standard model.

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system.

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.

Observation of the Omega(0)(c) Charmed Baryon at CLEO.

The CLEO experiment at the CESR collider has used 13.7 fb(-1) of data to search for the production of the Omega(0)(c) (css ground state) in e(+)e(-) collisions at square root of (s) approximately 10.6 GeV. The modes used to study the Omega(0)(c) are Omega(-)pi(+), Omega(-)pi(+)pi(0), Xi-K-pi(+)pi(+), Xi0K-pi(+), and Omega(-)pi(+)pi(+)pi(-). We observe a signal of 40.4+/-9.0(stat) events at a mass of 2694.6+/-2.6(stat)+/-1.9(syst) MeV/c(2), for all modes combined.

Alteration of goat sperm ecto-phosphoprotein phosphatase activity and its distribution on the sperm surface during epididymal maturation.

Phosphoprotein phosphatase (ecto-PPase) of goat epididymal sperm outer surface showed a significant increase in its activity at the initial stage of epididymal sperm maturation (up to the proximal corpus region) followed by a sharp fall towards the terminal phase of the maturation event. PPase activity showed nearly the same profile when estimated in intact cells as well as in isolated sperm plasma membrane. The ecto-PPase was purified to apparent homogeneity by using various biochemical fractionation procedures, such as solubilization with Triton X-100, sephadex gel filtration chromatography, concanavalin A-sepharose affinity chromatography and diethylaminoethyl-cellulose ion-exchange chromatography. The isolated PPase has a molecular mass of approximately 36 kDa and an isoelectric point of 5.95. Sperm surface topography of the enzyme was investigated using fluorescein isothiocyanate-conjugated antibody of the purified PPase. The immunofluorescent studies have demonstrated that the isolated PPase is localized on the external surface of viable sperm. Immunocytochemical studies also revealed a marked topographical alteration of ecto-PPase during epididymal transit of the male gametes. Immunoreactivity was observed all over the surface of caput sperm, but was restricted primarily to the anterior tip of the head in the corpus sperm and to the posterior part of the head in cauda sperm cells. The maturation-dependent decrease in PPase activity was also confirmed by immunofluorescent studies. This remarkable maturation-dependent modification of ecto-PPase activity, as well as its distribution on sperm surface, suggest that the ecto enzyme may play an important role in sperm function by regulating the phosphorylation states of the membrane-associated and reproductive fluid phosphoprotein substrates.

Observation of B --> K(+/-) pi(0) and B --> (K)0 pi(0), and evidence for B --> pi(+)pi(-).

We have studied charmless hadronic decays of B mesons into two-body final states with kaons and pions and observe three new processes with the following branching fractions: beta(B-->pi(+)pi(-)) = (4.3(+1. 6)(-1.4)+/-0.5)x10(-6), beta(B-->K(0)pi(0)) = (14.6(+5.9+2.4)(-5.1-3. 3))x10(-6), and beta(B-->K(+)/-pi(0)) = (11.6(+3.0+1.4)(-2.7-1.3))x10(-6). We also update our previous measurements for the decays B-->K(+)/-pi(-/+) and B+/--->K(0)pi(+/-).