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Sperm Extracts (sperm + extract)

Distribution by Scientific Domains

Life Sciences	58%

Selected Abstracts

Isolation and preliminary characteristics of ,- N -acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)

JOURNAL OF APPLIED ICHTHYOLOGY, Issue 4 2008
B. Sarosiek
Summary The aim of this study was to characterize the enzyme ,- N -acetyglucosaminidase (,-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed ,-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing ,-NAGase activity were found in rainbow trout milt plasma. The molecular mass of ,-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the ,-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4,4.8 for milt plasma and sperm extract from rainbow trout. Km value of the ,-NAGases was 0.212, 0.563, 0.779 mm for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The ,-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma. [source]

Novel human testis-specific cDNA: Molecular cloning, expression and immunobiological effects of the recombinant protein

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001
Ramasamy Santhanam
Abstract A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-,gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122,124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S -transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the ,17 kDa recombinant TSA-1, and a ,24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility. Mol. Reprod. Dev. 60: 1,12, 2001. © 2001 Wiley-Liss, Inc. [source]

Isolation and preliminary characteristics of ,- N -acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae

MOLECULAR ECOLOGY, Issue 7 2001
F. Tripet
Abstract Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M ×S hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow. [source]

Inhibition of a novel sperm gelatinase in prawn sperm by the male reproduction-related kazal-type peptidase inhibitor

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2008
Ye Li
Abstract Previously, we have identified and characterized a male reproduction-related kazal-type peptidase inhibitor (MRPINK) gene from the prawn, Macrobrachium rosenbergii. In the present study, MRPINK was discovered to have an inhibitory effect on the gelatinolytic activity of M. rosenbergii sperm and immunofluorescence analysis revealed it bound specifically onto the base of sperm. The proteolytic activity of sperm extracts to vitelline coat components was also detected to be interfered by MRPINK. Furthermore, a novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M. rosenbergii sperm gelatinase (MSG). MSG was then isolated and purified by reversed-phase high performance liquid chromatography combining with gelatinolytic assay. By amino-terminal amino acid sequence analysis and molecular cloning, the primary structure of MSG was determined. The data presented in this study provided evidence that MRPINK has an inhibitory effect on the gelatinolytic activity as well as proteolytic activity of prawn sperm and specifically blocks the activity of MSG. Mol. Reprod. Dev. 75: 1327,1337, 2008. © 2008 Wiley-Liss, Inc. [source]

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006
Antonio Medrano
Abstract Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L -lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7,±,0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3,±,0.1 mIU/mg protein to 0.60,±,1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15,0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface. Mol. Reprod. Dev. 1179,1194, 2006. © 2006 Wiley-Liss, Inc. [source]

Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoa

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006
Antonio Medrano
Abstract Incubation of boar spermatozoa in Krebs,Ringer,Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO2 to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source]

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2005
Consuelo Pasten
Abstract In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head. Mol. Reprod. Dev. 71: 209,219, 2005. © 2005 Wiley-Liss, Inc. [source]

ORIGINAL ARTICLE: In situ Reconstruction of Humoral Immune Response Against Sperm: Comparison of SCID and NOD/SCID Mouse Models

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2009
Beata Grygielska
Problem, Comparison of two types of immunocompromised mouse strains (SCID and NOD/SCID) for production of human antisperm antibodies (AsA). Method of study, Human peripheral blood lymphocytes (PBL) were grafted to mouse peritoneal cavity and sensitized with natively glycosylated and N-deglycosylated sperm extracts. Results and conclusion, NOD/SCID mice inoculated with hu-PBLs exhibited higher AsA titres with a tendency for greater sperm agglutination than human AsA raised in SCID mouse model. A comparison between ,native' and deglycosylated sperm extracts revealed higher agglutination titres by sera induced with the latter ones. Inhibitory effect of human polyclonal AsA in sperm penetration assay, however, produced opposite results to that for agglutination. Western immunoblotting was used to evaluate reactive sperm antigens prior to and after in situ sensitization showing multiple bands different from positive reactions brought by original sera of in vivo primed AsA-positive males. It seems that in situ generated AsA recognized sperm entities different from those revealed by blood donor's sera samples. [source]

Embryo development of porcine oocytes after injection with miniature pig sperm and their extracts

ANIMAL SCIENCE JOURNAL, Issue 6 2009
Daizou MATSUURA
ABSTRACT This study examined embryo development of porcine oocytes after microinjection of sperm extracts (SE) in porcine intracytoplasmic sperm injection (ICSI). SE was prepared from miniature pig sperm by a nonionic surfactant, and various concentrations (0.02, 0.04 and 0.08 mg/mL) of SE were injected into the matured oocytes with a first polar body. In the pronuclear stage, the rate of oocytes with two pronuclei and a second polar body (21.4%) in the sperm and SE (0.04 mg/mL) injection group was significantly higher (P < 0.05) compared to other groups. The rate of 2,4-cell stage in sperm and SE (0.04 mg/mL) injection group was 38.1%, and it was significantly higher than that in the sperm injection group (22.9%). The rate of blastocyst stage in sperm and SE (0.04 mg/mL) injection group was 21.4%, the value was significantly higher than those in SE (0.08 mg/mL) injection group (0%), sperm injection group (5.7%), and sperm and SE (0.08 mg/mL) injection group (2.6%). These results suggest that SE induces activation of porcine oocytes and their further embryonic development, and that SE is effective for porcine ICSI. [source]