Fresh sperm (extract and release?)

2016 ◽  
pp. 72-96
Keyword(s):  
Sperm Extract

scholarly journals Activation of Xenopus Eggs by Cynops Sperm Extract is Dependent upon Both Extra-and Intra-Cellular Ca Activities

1995 ◽  
Vol 12 (5) ◽  
pp. 573-581 ◽  
Author(s):  
Yasuhiro Iwao ◽  
Michiko Kobayashi ◽  
Akiko Miki ◽  
Hiroshi Y. Kubota ◽  
Yasuaki Yoshimoto
Keyword(s):  
Sperm Extract ◽  
Cellular Ca

scholarly journals Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 71-79 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
M Maeda ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Acrosome Reaction ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Immunoblot Analysis ◽  
Calpain Inhibitor ◽  
Cytoplasmic Matrix ◽  
Sperm Extract

At the avian body temperature of 40 °C, intact fowl spermatozoa require Ca2+for the initiation of motility and a combination of both Ca2+and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1–100 μmol/l, neither PD 150606 (a Ca2+-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca2+-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca2+and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca2+, as well as motility initiated by calyculin A – a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 °C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Analysis of Mn2+/Ca2+influx and release during Ca2+oscillations in mouse eggs injected with sperm extract

Cell Calcium ◽  
2001 ◽  
Vol 29 (5) ◽  
pp. 311-325 ◽  
Author(s):  
T. Mohri ◽  
H. Shirakawa ◽  
S. Oda ◽  
M.S. Sato ◽  
K. Mikoshiba ◽  
...  
Keyword(s):  
Sperm Extract ◽  
Mouse Eggs

Role of membrane phosphotyrosine proteins in human spermatozoal function

1991 ◽  
Vol 99 (1) ◽  
pp. 157-165
Author(s):  
R.K. Naz ◽  
K. Ahmad ◽  
R. Kumar
Keyword(s):  
Physiological State ◽  
Human Sperm ◽  
Kinase Assay ◽  
Relative Molecular Mass ◽  
Sperm Cells ◽  
Western Blots ◽  
Tyrosine Residues ◽  
Sperm Extract ◽  
32P Incorporation

The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/− 3000 and 46,000 +/− 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/− 3000, 46,000 +/− 3000, 25,000 +/− 7000 and 12,000 +/− 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/− 3000 Mr proteins and 17% less incorporation into 12,000 +/− 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/− 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins and Heparin Binding Proteins of Spermatozoa and Seminal Plasma in Beetal Bucks: Purification, Characterization and Role in In vivo Fertility

2020 ◽  
Author(s):  
Ranjna S. Cheema ◽  
Navjot S. Dhillon ◽  
Sumit Singhal
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Proteome Analysis ◽  
Fertility Rate ◽  
Heparin Binding ◽  
Sds Page ◽  
Sperm Extract ◽  
Sperm Membrane ◽  
Special Relevance

Background: The proteome analysis of seminal plasma and spermatozoa is of special relevance in livestock. Heparin binding proteins (HBPs) found in the seminal plasma of several mammals are shown to bind to sperm membrane and affect a series of events that contribute to normal fertility, such as sperm capacitation, formation of the oviduct reservoir and binding to the oocyte. Profiles of HBPs from seminal plasma and sperm membranes have been associated with sperm fertility. Although, HBPs present in the SP are described in several species, but little is known about HBPs in buck. Methods: Seminal plasma (SP) and sperm extracts (SE) of 13 bucks were subjected to heparin-sepharose affinity chromatography. Sperm extract, seminal plasma and purified HBPs and Non-HBPs were fractionated by SDS-PAGE. Total 78 females (6 per buck) were mated with 13 bucks. Bucks were divided into two groups, G-I (high fertile, 83.3-100% FR) and G-II (low fertile, 50-66.7% FR). Relationship between HBPs and fertility rate was observed. Result: SDS – PAGE of SP and SE resulted in resolution of 22 (10-240 kDa) and 21 (10-270 kDa) bands, respectively. Based on fertility rate 15 and 13 kDa proteins were absent in SP of higher number of GI-compared to G-II bucks. Fourteen bands ranging from 10 – 180 kDa and 10 – 150 kDa were separated from SP-NHBP and SP-HBP. SP-HBPs of 75, 35, 30, 28, 25 and 13 kDa were present in higher (28.6%, 42.5%, 26.2%, 40.5%, 14.3% and 36.2%) number of high fertile than low fertile bucks. NHBP and HBP purified from SE resolved into 11 bands ranging from 10 – 135 kDa and 10 – 120 kDa, respectively. SE-HBP of 53 kDa, 50/45 kDa and 25 kDa were present in higher percentage of high fertile than low fertile bucks.

scholarly journals Cell cycle-dependent repetitive Ca(2+)waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization

2000 ◽  
Vol 113 (19) ◽  
pp. 3453-3462 ◽  
Author(s):  
A. McDougall ◽  
M. Levasseur ◽  
A.J. O'Sullivan ◽  
K.T. Jones
Keyword(s):  
Spatial Pattern ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Egg Activation ◽  
Second Phase ◽  
Protein Factor ◽  
Sperm Extract ◽  
Two Phases ◽  
Cycle Dependent ◽  
Cytosolic Factor

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+)waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

27 EFFECT OF ACTIVATION METHODS AND BIOPSY SAMPLING ON NUCLEAR TRANSFER BLASTOCYST PRODUCTION AND CLONING EFFICIENCY IN A HORSE

2008 ◽  
Vol 20 (1) ◽  
pp. 94
Author(s):  
Y. H. Choi ◽  
D. L. Hartman ◽  
R. A. Fissore ◽  
S. J. Bedford ◽  
K. Hinrichs
Keyword(s):  
Skin Biopsy ◽  
Nuclear Transfer ◽  
Control Method ◽  
Tissue Sample ◽  
Injection Pressure ◽  
Biopsy Sample ◽  
Control Treatment ◽  
Cloning Efficiency ◽  
Blastocyst Development ◽  
Sperm Extract

In the horse, rates of blastocyst production after nuclear transfer are low. This study was conducted to examine the effect of activation via injection of different volumes of sperm extract or via injection of murine mRNA for PLC-ζ, a sperm-specific protein which induces Ca2+ oscillations in all species thus far studied, on blastocyst development after nuclear transfer. Two biopsy samples from the same horse were also examined for cloning efficiency. Donor fibroblasts cultured from a skin biopsy taken from a 19-year-old mare were treated with 15 µm R-roscovitine for 18 to 24 h before direct injection into enucleated ooplasts. Ionomycin treatment, 5 µm for 4 min, was used in all activation treatments, as the combination of ionomycin with sperm extract provided the highest blastocyst development and foaling rates in our previous report (Hinrichs et al. 2007 Reproduction 134, 319–325). All treatments were followed by incubation in 2 mm 6-dimethylaminopurine for 4 h. Differences in blastocyst development between treatments were analyzed using Fisher's exact test. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 s at a fixed pipette diameter and injection pressure, and then treated with ionomycin. The blastocyst rate (9.8%) for 0.1 s was significantly higher than that for 0.2 s (0%) or 0.8 s (1.4%). In Experiment 2, murine PLC-ζ mRNA (0.25 µg µL–1) was injected into reconstructed oocytes 20 to 30 min before or after ionomycin treatment and compared with a control treatment (injection of 2 to 4 pL sperm extract 20 to 50 min after ionomycin exposure). There were no differences in blastocyst development among treatments (0, 4.5, and 5.6%, respectively). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in 5 pregnancies: however, all were lost before 70 days of gestation. In Experiment 3, a second skin biopsy was obtained from the same mare and cells from this tissue sample were used for nuclear transfer concurrently with cells from the first sample, using the control method above. Blastocyst production was higher using cells from the second biopsy sample (4/23 v. 0/23; P = 0.05). Transfer of these four blastocysts yielded four pregnancies, two of which continued to term and produced viable foals. These results indicate that blastocyst development after injection of sperm extract is dependent upon the volume injected, that injection of murine PLC-ζ mRNA does not improve blastocyst formation under the given conditions, and that the efficiency of cloning may vary with biopsy sample even from the same animal.

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