Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Identification and validation of QTLs conferring resistance to sorghum downy mildew (Peronosclerospora sorghi) and Rajasthan downy mildew (P. heteropogoni) in maize

We have mapped the quantitative trait loci (QTLs) conferring resistance to sorghum downy mildew (Peronosclerospora sorghi; SDM) and Rajasthan downy mildew (P. heteropogoni; RDM), two species of DM prevalent throughout India. QTL mapping was carried out on a backcross population of 151 individuals derived from a cross between CM139 (susceptible parent) and NAI116 (highly resistant to both SDM and RDM). Heritability estimates were 0.74 for SDM and 0.67 for RDM. Composite interval mapping combined with a linkage map constructed with 80 simple sequence repeat (SSR) markers resulted in the identification of three QTLs (one each on chromosomes 2, 3 and 6) for SDM resistance and two QTLs (one each on chromosomes 3 and 6) for RDM resistance, all of which were contributed by NAI116. The significance of the major QTL on chromosome 6 (bin 6.05) that confers resistance to diverse DMs in tropical Asia, including SDM and RDM in India, was also verified. The results confirmed that some common QTLs contribute to both SDM and RDM resistance, while additional loci might specifically govern resistance to SDM. The QTL information generated in this study provide information that will aid in undertaking an integrated breeding strategy for the transfer of resistance to SDM and RDM in maize lines using marker-assisted selection.     

Dynamics of amino acids in the conditioning film developed on glass panels immersed in the surface seawaters of Dona Paula Bay

The conditioning film developed on glass panels immersed in surface seawater over a period of 24 h was analysed for total organic carbon (OC), total organic nitrogen (ON), and total hydrolyzable amino acid (THAA) concentrations and composition. The concentrations of C and N and THAA increased, whereas the C/N ratio decreased over the period of immersion. The amino acid-C and N accounted for 3.7-6.7% and 10.3-65.3% of OC and ON, respectively. The relative contribution of glycine plus threonine and serine to the total amino acids decreased while that of valine, phenylalanine, isoleucine and leucine increased over the period of immersion. Principal component analysis (PCA) based on mole% amino acid composition showed that the degradation indices (DI) for the conditioning film organic matter increased over the period of immersion. A high C/N ratio, a low %THAA-C, % THAA-N and DI values and the abundance of glycine plus threonine and serine in the conditioning film organic matter during the first few hours following immersion imply that the adsorbed organic matter was mostly derived from degraded organic matter.     

Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy

Mutations in the gene encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) have been reported in patients with congenital generalized lipodystrophy (CGL). AGPAT2, a 278 amino acid protein, belongs to the acyltransferase enzyme family, and has two conserved motifs, NHX(4)D and EGTR, involved in the enzymatic activity. The AGPATs catalyze acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) during the biosynthesis of glycerophospholipids and triglycerides from glycerol-3-phosphate. The present studies were designed to determine the enzymatic activity of AGPAT2 mutants found in CGL patients to provide a molecular explanation for the phenotype and to obtain additional information about the structure-function relationship of AGPAT2 protein. The enzymatic activities of the wild type AGPAT2 and mutants were determined in cell lysates of overexpressing Chinese hamster ovary cells by measuring the conversion of [(3)H]LPA to [(3)H]PA in the presence of oleoyl-coenzyme A. Whereas, the R68X, 221delGT, 252delMRT, D180fsX251, and V167fsX183 mutants had markedly reduced enzymatic activity (median <15% of the wild type), the mutants, 140delF, G136R, and L228P, retained median activity ranging from 15% to 40% of the wild type enzyme. However, the missense mutant, A239V, had 90% of the wild type activity. We suggest that reduction in AGPAT2 enzymatic activity underlies the loss of adipose tissue in CGL. Our observations reveal an important role of various carboxy-terminal residues in determining the enzymatic activity of AGPAT2.     

Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.     

Evaluation of tocolytic efficacy of selective beta2 adrenoceptor agonists on buffalo uterus

Present study was conducted on prostaglandin F2alpha (PGF2alpha), oxytocin, (OT), potassium chloride (KCI) and barium chloride (BaCl2) pre-contracted perimetrial uterine strips of dioestrus and pregnant buffaloes to evaluate the tocolytic efficacy of selective beta2 adrenoceptor agonists-albuterol (salbutamol) and terbutaline. Cumulative concentration-response curves of both the beta2 adrenoceptor agonists were constructed and the mean effective concentration (EC50) values determined and compared statistically. Based on the comparative EC50 values in relaxing the pre-contracted uterine strips with different spasmogens, the rank order potency of albuterol was found to be--PGF2alpha > BaCl2 > OT > KCl on uterine strips from dioestrus animals, while OT> BaCl2> PGF2alpha >KCl on the uterine strips of pregnant buffaloes. The rank order potency of terbutaline on uterine strips from dioestrus stage animals was- BaCl2 > OT > KCl > PGF2alpha, while BaCl2 > PGF2alpha > KCl > OT on uterine tissues of pregnant animals. Thus, irrespective of the state of uterus, whether gravid or non-gravid, KCl-depolarized uterine tissues required comparatively higher concentrations of albuterol or terbutaline to produce tocolytic effect. High concentrations of K+ in biophase may have interfered with the beta2 adrenoceptor agonists-induced outward K+ current and hyperpolarization. From the results of present study, it was evident that selective beta2 adrenergic agonists had good tocolytic efficacy on the uterus of buffaloes. Further, indirectly the possibility of existence and activation of K(Ca) channels by selective beta2 adrenoceptor agonists in mediating tocolysis of buffalo myometrium can not be ruled out, however, detailed studies using specific K(Ca) channel blockers are required for characterizing the nature of such channels in buffalo uterus.