A Novel Membrane Protein-Specific Serine/Threonine Kinase: Tissue Distribution and Role in Sperm Maturation

Our recent studies have described for the first time the purification of an ectoprotein kinase to apparent homogeneity using caprine sperm as the model. Purified ectokinase (CIK) is a novel membrane protein-specific kinase that phosphorylates serine and threonine residues of ectophosphoproteins. This study, using ELISA based on ecto-CIK antibody demonstrates that ecto-CIK level is remarkably higher in the sperm membrane than in the cytosol. The epididymal sperm maturational event as well as sperm vertical velocity is associated with a significant increase in the ecto-CIK level. Ecto-CIK, the membrane protein-specific kinase, is also present in all the tissues tested and is predominantly localized in the cell membrane. Ubiquitous localization of the novel kinase on the mammalian cell membrane suggests that the kinase may play pivotal role in gamete as well as somatic cell regulation by modulating membrane biology through serine/threonine phosphorylation of specific membrane proteins located in the ectodomains

Sperm Motility Regulatory Proteins: A Tool to Enhance Sperm Quality

Sperm forward motility is an essential parameter in mammalian fertilization. Studies from our laboratory have identified and characterized a few unique sperm motility regulatory proteins/glycoproteins from the male reproductive fluids and mammalian blood serum. The purified sperm motility-initiating protein (MIP) from caprine epididymal plasma as well as the forward motility-stimulating factor (FMSF) and motility-stimulating protein (MSP) from buffalo and goat serum, respectively, have high efficacy to initiate or increase motility in nonmotile or less motile sperm. Antibody of sperm motility inhibitory factor (MIF-II) has the high potential to enhance sperm vertical velocity and forward motility by increasing intracellular cyclic adenosine monophosphate (cAMP) level. The appearance and disappearance of D-galactose–specific lectin and its receptor along the epididymis has been reported to be involved in motility regulation in spermatozoa. A novel synthetic cryopreservation method and role of lipid to protect membrane damage during cryopreservation have been demonstrated. Motility-promoting proteins may be extremely useful for improving cattle breeding and breeding of endangered species, thereby helping in enhanced production of animal products as well as in the conservation of animals. Isolated proteins and developed cryopreservation technology may also be beneficial in human infertility clinics to increase the chance of fertilization

Extracellular regulation of sperm transmembrane adenylyl cyclase by a forward motility stimulating protein.

Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation of progressive motility by a physiologic extracellular factor in a mammalian species

A clinicopathological study of mediastinal masses operated in a tertiary care hospital in Eastern India in 3 years with special reference to thymoma

Introduction: The mediastinum is the central portion of the thoracic cavity, limited by pleural cavities laterally, thoracic inlet superiorly, and the diaphragm inferiorly. Housing numerous organs, it is a veritable Pandora′s box, within which various lesions may develop. This study was conducted to assess the epidemiologic profile, clinicoradiological features, cytological, and histopathological findings in patients presenting with mediastinal masses in a tertiary care hospital over a period of 3 years. Materials and Methods: This is a retrospective study of cases presenting with mediastinal masses attending the Cardiothoracic Surgery Department of Medical College, Kolkata between May 2011 and April 2014. Detailed history, physical, and radiological findings were noted. Fine needle aspiration cytology (FNAC) was performed when feasible. Following surgery, histopathological, and immunohistochemical (IHC) examinations of the specimens were undertaken. Results: Of the 22 cases included in our study, ten were anterior, seven middle, and five posterior mediastinal masses. Fifteen cases were male and seven were female. Thymic pathology was detected in seven cases, lymphoma in five, extragonadal germ cell tumor (GCT) in three, schwannoma and pericardial cyst in two cases each and neurofibroma, ganglioneuroma, and retrosternal thyroid in one case each. The age group of the patients for each diagnostic category was found to be of significance. FNAC was done in 15 cases. IHC was required for classification of lymphoma cases (CD45, CD15, CD30, CD20, CD3, Tdt, CD34, and Ki-67). Conclusion: This study reflects the incidence of different mediastinal masses in West Bengal with their clinicopathologic correlation

A. Effect of PKA inhibitor in forward motility of the FMSF activated sperm cells.

<p>Duration of IP20 incubation was 30 min. B. Effect of tyrosine-kinase inhibitor on forward movement profile of FMSF stimulated spermatozoa. Duration of Genistein incubation was 30 min. Viable cell count throughout the different assay conditions remained ≥95%. <b>C. Phospho-kinase stimulating potential of FMSF.</b> Only absorbance values from ELISA results have been plotted, actual activity units have not been shown; anti-phospho-serine/threonine for Protein Kinase A and tyrosine antibodies for Tyrosine kinase were used in ELISA of the assayed treatments. All the data represent mean ± SEM for n = 3 samples. Hash mark (#) indicates statistically significant inhibition vs. only FMSF-treated (<i>p<0.01</i>), Asterisks (*, **) denote statistically significant difference vs. control (<i>p<0.05 and p<0.01</i>), respectively.</p

Elucidation of role of tmAC in quality of sperm forward movement.

<p><b>A.</b> Effect of ddAdo (dideoxyadenosine) and db-cAMP (dibutyryl cAMP) in relation with FMSF-induced forward motility. <b>B.</b> Effect of ddAdo and KH7 in relation with FSK induced forward motility. Incubation periods with ddAdo, KH7, FSK and db-cAMP were of 30 min each. All the data represent mean ± SEM for n = 6 samples. Double asterisks (**) denote statistically significant difference vs. control (<i>p<0.01</i>), hash mark (#) indicates statistically significant inhibition vs. FMSF-treated (<i>p<0.01</i>), double hash mark (##) indicates statistically significant inhibition vs. control (<i>p<0.01</i>), asterisk (*) denotes statistically significant difference vs. (ddAdo+FMSF) (<i>p<0.05</i>), <i>nsd</i> denotes not significant difference vs. control. Viable cell count throughout the different assay conditions remained ≥95%. <b>C.</b> Effect of different doses of ddAdo on modulation of intracellular cAMP level in presence of FMSF (0.5 µM). Duration of ddAdo and FMSF incubation were 30 and 2 min, respectively. All the data represent mean ± SEM for n = 4 samples. Hash mark (##) indicates statistically significant inhibition vs. ddAdo-untreated (<i>p<0.01</i>).</p

Flow-cytometric analysis of the phosphorylation status of spermatozoa.

<p><b>Panel I–IV:</b> Ser/Thr phosphorylation status; <b>I</b>- Control, <b>II-</b> db-cAMP (0.5 mM), <b>III</b>- FMSF (0.5 µM), <b>IV</b>- Genistein (250 µM). <b>Panel V–VI</b>: Tyr phosphorylation status; <b>V</b>- Control, <b>VI</b>- FMSF (0.5 µM). The figure is representative and the data represent mean ± SEM for n = 3 samples. The R2 region signifies fluorescence of stained cells (R1, not indicated denotes the entire region); percentage in the figure is of gated cells in the R2 region and corresponding Mean Fluorescence Intensity (MFI) are given beside.</p

Role of transmembrane adenylyl cyclase in mediation of FMSF activity.

<p><b>A.</b> Dose-response curve of ddAdo on effect on forward motility of caudal spermatozoa; <b>B.</b> Results of Spectrophotometric vertical motility assay of caprine mature spermatozoa. Viable cell count throughout the different assay conditions remained ≥95%. <b>C.</b> Dose-dependency of tmAC activity in presence of FMSF (light bars). Involvement of G_α-subunit in manifestation of FMSF activity represented by dense bars where GTP_γS was used at a concentration of 10 µM. Pre-treatment periods for both ddAdo and GTP_γS were of 30 min. Data for figure A and B represent mean ± SEM for n = 5 samples, while that of figure C is of n = 3 samples. Asterisks (***) and hash marks (#, ##, ###) denote statistically significant difference vs. control (<i>p<0.001</i>) and vs. only FMSF-treated (<i>p<0.05, p<0.01 and p<0.001</i>), respectively.</p

Study of kinetic properties of adenylyl cyclase activity of purified sperm membrane fraction.

<p>‘Complete’ assay medium for membrane protein AC-activity contains the following: 50 mM Tris-Cl, pH 8.5, 2 mM MgCl_2, 0.5 mM NaF, 0.5 mM phosphocreatinine, 1.5 mM ATP-Na_2. Results represent mean ± SEM for n = 5 experiments.</p><p>Study of kinetic properties of adenylyl cyclase activity of purified sperm membrane fraction.</p

MALDI-TOF spectrum of trypsin digested peptides of FMSF.

<p>Three concentrations of the same sample were spotted on the CHCA matrix for analysis, which produced qualitatively identical results. All the peaks on the spectrum were checked for homology in the online protein database; only those with a score >65 were considered significant for discussion in the result section.</p