Characteristics of chimpanzee (Pan troglodytes) ejaculates collected by rectal probe electrostimulation and by artificial vagina

This study compares characteristics of ejaculates collected from 16 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Ejaculate weight, semen volume, and sperm number were significantly lower (P < 0.01) and percentage liquefaction was significantly higher (P < 0.01) in ejaculates collected by RPE. Percentages of motile sperm and of live sperm in semen did not differ significantly between the two collection methods. Total amounts of protein and of α-glucosidase activity were significantly lower (P < 0.01) in seminal fluid from RPE samples. For ejaculates collected by RPE, semen volume correlated positively with protein (r = 0.8640, P < 0.001), fructose (r = 0.6976, P < 0.001), and citrate (r = 0.6976, P < 0.001); sperm number correlated positively with α-glucosidase activity (r = 0.6547, P < 0.001); and protein correlated positively with fructose (r = 0.5906, P < 0.002), citrate (r = 0.5926, P < 0.002) and α-glucosidase activity (r = 0.6006, P < 0.001). For ejaculates collected by AV, semen volume correlated positively with percentage liquefaction (r = 0.6058, P < 0.001), protein (r = 0.8055, P < 0.001), fructose (r = 0.6606, P < 0.001), and citrate (r = 0.8272, P < 0.001); sperm number correlated positively with percentage of motile sperm (r = 0.4196, P 0.004); percentage of motile sperm correlated positively with percentage of live sperm (r = 0.4388, P < 0.002); and, protein correlated positively with fructose (r = 0.6947, P < 0.002) and with citrate (r = 0.5926, P < 0.002). These data show that there is a significant difference in semen parameters and in biochemical parameters of ejaculates obtained by RPE and by AV. © 1995 Wiley-Liss, Inc.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Evaluation of anesthetic protocol for the collection of semen from captive collared peccaries (Tayassu tajacu) by electroejaculation

The objective of this study is to verify and compare the effects of acepromazine–tiletamine–zolazepam and propofol used in anesthetic protocols for semen collection by electroejaculation from captive collared peccaries. Ten sexually mature animals were physically restrained and anesthetized by either intravenous administration of tiletamine–zolazepam (2 mg/kg) after acepromazine premedication, or a propofol dose of 5 mg/kg. The onset of anesthetic recovery was determined by the animals regaining consciousness and attempting to stand. Semen was collected by electroejaculation and evaluated for volume, pH, sperm concentration, progressive motility, morphology, percentage of live cells and functional membrane integrity. Six anesthetized animals with the acepromazine–tiletamine–zolazepam protocol showed erection, but semen could be collected in only four (40%) attempts. Of the animals anesthetized using propofol, nine showed erection, and the ejaculates were collected in eight (80%) attempts. Furthermore, propofol afforded rapid recovery of animals, and ejaculates with enhanced sperm motility and functional membrane integrity as compared with those collected by the other protocol (P < 0.05). In conclusion, use of propofol for anesthetic restraint of collared peccaries enhanced collection of semen by electroejaculation.     

Distribution of sperm in the storage organs of the Drosophila melanogaster female at various levels of insemination

Summary It has been shown in Drosophila melanogaster that when 50 or fewer sperm are stored in the female, some 58% are found in the spermathecae and 42% in the ventral receptacle. When between 51 and 100 are stored, some 34% are found in the spermathecae and 66% in the ventral receptacle. The more commonly encountered distribution of 10–20% in the spermathecae and 80–90% in the ventral receptacle hold for higher levels of insemination (greater than 100 sperm). The reason for the relatively high proportion of sperm stored in the spermathecae when the total number of sperm stored is small is unknown. It is pointed out that in experiments dealing with sperm: progeny ratios, careful counting of sperm in the spermathecae becomes of critical importance, especially at low levels of insemination.Supported by National Science Foundation research grant GB-3117.National Science Foundation Undergraduate Research Participant, Summer 1966.     

Streptomycetes excreting dd-carboxypeptidases

Summary Excretion of exocellular dd-carboxypeptidases was tested using 128 strains of streptomycetes. Exocellular enzyme activity was shown in 13% of the trains investigated. Streptomyces strains showed low activity of excretion of dd-carboxypeptidases: 2.7–4.8 M of released C-terminal d-alanine (d-Ala) residue/1 culture supernatant per minute. Saccharopolyspora erythraea mutants produced considerably higher levels of exocellular enzymes, the dynamics of excretion depending upon the medium used. The highest activity of exocellular dd-carboxypeptidase production was 44 M d-Ala/1 culture supernatant per minute. The affinity of exocellular dd-carboxypeptidase of S. erythraea 64-575 for -lactam antibiotics was assessed by a statistical computer programme. The enzyme showed the lowest affinity for sodium cefotaxime, ID₅₀(M) = 7.5 × 10^–6, and the highest for potassium cephalosporin C, ID₅₀(M) = 5.0 × 10^–9, ID₅₀(M) representing the molar concentration of -lactan antibiotics which decreased by 50% the release of d-Ala. Offprint requests to: W. Kurzatkowski     

Comparative study of the enzymatic synthesis of l-valine by purified enzymes,crude extract,intact or permeabilized cells from Bacillus megaterium

Summary A detailed study on the reductive amination of -ketoisovalerate to l-valine by l-valine dehydrogenase using glucose dehydrogenase as an NADH regeneration enzyme was performed. The presence of both enzyme activities in Bacillus megaterium ATCC 39 118 permitted a direct and systematic comparison of the performances (initial l-valine production rate, productivity, molar conversion yield) of different types of conversion systems: purified enzymes or crude extract and whole cells, intact or permeabilized. A maximal l-valine productivity of 8 mmol·l^–1 · h^–1 was obtained using purified enzymes which constituted the most efficient system with a maximal rate of 0.87 mol · ml^–1 · min^–1 and a molar conversion yield of 0.91. Permeabilized cells were also an attractive system because of their easy preparation and of the good performances attained.Offprint requests to: F. Monot     

Comparative study of the thiourea carrier in erythrocytes

Summary A densimeter technique was used to measure the rate of exit of thiourea from erythrocytes of various species of mammals. The cells were first equilibrated with a 200mm thiourea solution in 1% NaCl. An aliquot of these cells was added to 1% NaCl containing 4.6–23.1mm thiourea. Facilitated diffusion was demonstrated in each case. Using exit times or initial rates, calculations of half-saturation constants () inmm and maximum transport rates (K) in isotones per min were made by three different methods. The following values were obtained: human –=60, 42, 35;K=1.2, 2.9, 0.9; rabbit –=46, 33, 32;K=0.8, 2.1, 0.8; mouse –=46,40, 30;K=3.4, 8.5, 3.2; rat –=65, 42, 23;K=6.1, 15.3, 3.7; ox –=107, 63, 88;K=0.6, 1.4, 0.4; sheep –=56, 38, 56;K=0.9, 2.2, 0.6; and pig –=110, 64, 49;K=1.6, 3.6, 1.1.     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Cell wall composition and synthesis via Golgi-directed scale formation in the marine eucaryote,Schizochytrium aggregatum,with a note on Thraustochytrium sp.

Summary Cell walls of Schizochytrium aggregatum and Thraustochytrium sp. were mechanically isolated and subjected to chemical analysis. On a dry weight basis the cell walls contain 21–36% carbohydrate and 30–43% protein. The principal sugar (>95%) of the Schizochytrium wall is l-galactose, while the Thraustochytrium cell wall contains l-galactose, d-galactose and xylose with l-galactose predominating. Ultrastructurally the cell walls of both organisms consist of a laminated structure which yields thin, flexible, nearly circular scales (0.5–1.1 in diameter) upon sonic disintegration. Structures presumed to be developing wall scales are found within cisternae of the Golgi apparatus in both organisms. The chemical composition and method of formation of the cell wall in these two protists is distinctly different from that found in the Saprolegniales (Oomycetes), the group with which these organisms have hitherto been aligned.     

The temperature coefficient of the duration of life in Tubularia Crocea

Zusammenfassung Der Temperaturkoeffizient für die Lebensdauer vonTubularia crocea wurde für das Temperaturintervall 36°–25° C. bestimmt. Es wurde gefunden, daß derselbe etwa 2 für einen Temperaturunterschied von 1° beträgt. Das ist derselbe Temperaturkoeffizient, denLoeb für die Lebensdauer der Seeigeleier für das Temperaturintervall 32°–20° C. gefunden hat.

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I wish to thank ProfessorLoeb for his many helpful suggestions and criticisms.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Differential sensitivity of pneumolysin-induced channels to gating by divalent cations

Summary The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (<50 pS to >1 nS at 0.1 m KCl). Channels can be categorized on the basis of their K⁺:C^– selectivity: the smallest channels are strongly cation selective, with t₊ (the cation transference number) approaching 1.0; the largest channels are unselective (t₊ 0.5). Channels tend to remain open at all voltages (–150 to 150 mV); only the smallest channels exhibit any rectification.In the presence of divalent cations (1–5 mm Zn²⁺; 10–20 mm Ca²⁺), small (<50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones.It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation: anion selectivity and by their sensitivity to closure by divalent cations.We are grateful to Dr. G. J. Boulnois and T. J. Mitchell forfruitful discussion and supplies of pneumolysin, and to G. M. Alder for technical assistance. YEK is grateful to Dr. A. A. Lev for leave of absence and to the USSR Academy of Sciences and the Global Network for Molecular and Cell Biology (UNESCO) for support of travel and accommodation, respectively. The work was supported by the Cell Surface Research Fund.     

Effects of catecholamines on electrolyte transport in cortical collecting tubule

Summary We examined the direct effects of isoproterenol (ISO) andl-norepinephrine (NE) on electrolyte transport in isolated rabbit cortical collecting tubules (CCT) perfusedin vitro. The addition of either ISO (10^–6 m) or NE (10^–6 m) to the bath decreased transepithelial potential difference (PD), on average by 51 and 25%, respectively. These effects of ISO and NE were abolished by prior addition of the -adrenergic blocker,l-propranolol. ISO (10^–5 m) had no effect from lumen. Also, osmotic water permeability was not influenced by ISO. Ouabain and ISO had additive effects on PD. Elimination of chloride from both perfusate and bath, or addition of acetazolamide, abolished the effect of ISO on PD. Although isotopic sodium flux from lumen to bath was not influenced by ISO, chemical net chloride absorption increased from 1.1±0.4 to 2.7±0.6 peq·cm^–1·sec^–1 (n=8,p<0.005). In conclusion, both ISO and NE are capable of decreasing PD in rabbit CCT perfusedin vitro. This effect is mediated by -adrenergic receptors and is accompanied by the increase in net chloride absorption. Although the mechanism responsible for this decrease in PD with ISO is unclear, active chloride absorption, active hydrogen secretion, or membrane chloride permeability changes may account for the effects of ISO.     

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters

Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-l-capacity mechanically-stirred bioreactors containing 61 medium (LP, von Arnold and Eriksson) with 30 mm sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with fresh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mm to 60 mm resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 gl^–1 and 3076 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 10–12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 gl^–1 and 2278 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 6–8 days culture. For all sucrose concentrations, fresh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.Correspondence to: D. I. Dunstan     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8%) and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%), in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml) and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹) compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.     

Outwardly rectifying chloride channels in lymphocytes

Summary Outwardly rectifying Cl^– channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1–6 min at +80 mV, 20°C). The single-channel current at +80 mV was 4.5 ± 0.3 pA and at –80 mV, it was 1.0 ± 0.4 pA. After activation, the probability of being open (P ₀)for the lymphocyte channel was voltage independent. Activation of the Cl^– channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20°C exhibited Cl^– channel activity. In contrast, 49% of recordings made at 30°C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30°C than at 20°C. There was no marked change in the single-channel conductance at 30°C. Open-channel conductance was blocked by 200 m indanyloxyacetic acid (IAA) or 1 mm SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl^– channels thought to be involved in fluid secretion