169 MATURE OOCYTES TRIGGER THE RELEASE OF BOVINE SPERM FROM AN IMMOBILIZED OVIDUCT GLYCAN

2017 ◽  
Vol 29 (1) ◽  
pp. 193
Author(s):  
M. M. Elsokary ◽  
D. J. Miller
Keyword(s):  
Final Concentration ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Bovine Semen ◽  
Bovine Sperm ◽  
Sperm Release ◽  
Before And After ◽  
Treatment Data ◽  
Bovine Oocytes ◽  
Lewis A

After mating, sperm move from the site of semen deposition towards the oviduct, where sperm are stored before fertilization. Much of the literature indicates that oviduct glycans bind sperm to retain them in the oviduct reservoir. We have discovered that a specific oviduct glycan on the epithelium (sulfated Lewis A trisaccharide; suLeA) binds bovine sperm and maintains sperm viability. But the way in which sperm are released from oviduct epithelial glycans to fertilize oocytes is enigmatic. In this study, we tested the hypothesis that oocytes signal the release of sperm from an oviduct glycan (suLeA) on a bead and that the released sperm could fertilize the oocytes. Sepharose beads with avidin on their surface were used to immobilize biotinylated suLeA. After coupling, the beads were washed. Frozen bovine semen was thawed, and sperm were washed and allowed to bind to suLeA-coated beads for 1 h in capacitating medium at 39°C. Mature bovine oocytes were then prewashed with the same medium (without heparin) and added to droplets with sperm bound to beads and incubated for 18 h at 5% CO2 at 38.5°C. Bound sperm (mean = 4 per bead) were enumerated before and after oocyte addition. A control using dead (fixed) oocytes was also used. Three independent replicates were tested using 125 oocytes for each treatment. Data were analysed by ANOVA. The addition of live oocytes released 68.1% of the sperm bound to beads, whereas only 18.8% of sperm were released by addition of dead oocytes (control; P < 0.05). If heparin for sperm capacitation (3.125 g mL−1 final concentration) was added concurrently with the addition of live oocytes to sperm bound to beads, the majority of bound sperm were released (87.7%), whereas dead oocytes (control) released fewer sperm (67.3% sperm, P < 0.05). Thus, both heparin and oocytes released sperm from beads independently but, together, they released the greatest number of sperm. Altogether, our results indicate that, in addition to heparin, mature oocytes induce sperm release from immobilized oviduct glycans for fertilization. This is the first demonstration that oocytes can signal sperm release from oviduct glycans in any mammal.

scholarly journals Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rasoul Kowsar ◽  
Shahrzad Ronasi ◽  
Nima Sadeghi ◽  
Khaled Sadeghi ◽  
Akio Miyamoto
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dna Fragmentation ◽  
Acrosome Reaction ◽  
Developmental Competence ◽  
Ros Production ◽  
Sperm Capacitation ◽  
Bovine Sperm ◽  
Epidermal Growth ◽  
High Level

AbstractUpon insemination, sperm cells are exposed to components of the female reproductive tract (FRT) fluids, such as urea and epidermal growth factor (EGF). It has been shown that both urea and EGF use EGF receptor signaling and produce reactive oxygen species (ROS) that are required at certain levels for sperm capacitation and acrosome reaction. We therefore hypothesized that during bovine sperm capacitation, a high level of urea and EGF could interfere with sperm function through overproduction of ROS. High-level urea (40 mg/dl urea is equal to 18.8 mg/dl of blood urea nitrogen) significantly increased ROS production and TUNEL-positive sperm (sperm DNA fragmentation, sDF) percentage, but decreased HOS test score, progressive motility, acrosome reaction and capacitation. The EGF reversed the negative effects of urea on all sperm parameters, with the exception of ROS production and DNA fragmentation, which were higher in urea-EGF-incubated sperm than in control-sperm. The developmental competence of oocytes inseminated with urea-EGF-incubated sperm was significantly reduced compared to the control. A close association of ROS production or sDF with 0-pronuclear and sperm non-capacitation rates was found in the network analysis. In conclusion, EGF enhanced urea-reduced sperm motility; however, it failed to reduce urea-increased sperm ROS or sDF levels and to enhance subsequent oocyte competence. The data suggests that any study to improve sperm quality should be followed by a follow-up assessment of the fertilization outcome.

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Osamu Okitsu ◽  
Shuji Yamano ◽  
Toshihiro Aono
Keyword(s):  
Human Sperm ◽  
Human Spermatozoa ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Bovine Sperm ◽  
Female Pronucleus ◽  
Sperm Factor ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.

93 CALCIUM REMOVAL INCREASES THE PROTECTIVE EFFECTS OF β-CYCLODEXTRIN PLUS CHOLESTEROL ON PORCINE SPERM DURING COLD SHOCK

2006 ◽  
Vol 18 (2) ◽  
pp. 155 ◽  
Author(s):  
H. Galantino-Homer ◽  
W. Zeng ◽  
S. Megee ◽  
M. Modelski ◽  
I. Dobrinski
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cold Shock ◽  
Cholesterol Content ◽  
Extracellular Calcium ◽  
Shock Treatment ◽  
Membrane Cholesterol ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Porcine sperm are extremely sensitive to the damaging effects of cold shock and cryopreservation. Cholesterol-binding molecules, such as 2-hydroxypropyl-�-cyclodextrin (HBCD), improve post-thaw and post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. Depending upon the environmental cholesterol content, HBCD can act either as a cholesterol shuttle or sink to increase or decrease, respectively, sperm plasma membrane cholesterol content. Increasing the sperm cholesterol to phospholipid ratio reduces cold shock sensitivity whereas decreasing the ratio initiates the process of sperm capacitation. An increase in protein tyrosine phosphorylation correlates with sperm capacitation and has been shown to be dependent upon the presence of extracellular calcium. Sperm intracellular calcium also increases during cold shock. The objective of this study was to determine the combined effects of extracellular calcium and membrane cholesterol manipulation on porcine sperm viability and protein tyrosine phosphorylation following cold shock (10�C for 10 min). Viability was assessed using CFDA/propidium iodide staining. Protein tyrosine phosphorylation, previously shown to correlate with porcine sperm capacitation, was evaluated via antiphosphotyrosine (clone 4G10) immunoblots. We report here that following cold shock, porcine sperm incubated in defined medium containing both 0.8 mM HBCD and 0.5 mM cholesterol 3-sulfate (ChS) incubated in the absence of added extracellular calcium and the presence of 6 mM EGTA have significantly improved viability (90.5 � 6.3%, n = 3) when compared with cold-shocked sperm incubated in either the same medium with calcium (46.1 � 3.8%), without HBCD or ChS (26.5 � 7.4% with calcium; 46.5 � 13.1% without calcium), or with HBCD alone (17.0 � 7.4% with calcium, 36.8 � 7.5% without calcium). As we have found previously, treatment with 0.8 mM HBCD plus 0.5 mM ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Although protein tyrosine phosphorylation correlates with porcine sperm capacitation, the ability of cold shock treatment to induce the same phosphorylation pattern indicates that other processes or pathways may contribute to its appearance. Removing extracellular calcium consistently decreased, but did not completely eliminate, the protein tyrosine phosphorylation induced by cold shock. These results indicate that cold shock-induced protein tyrosine phosphorylation is not dependent upon, but can be modulated by, extracellular calcium. The combined effects of calcium, HBCD and ChS on viability suggest that porcine sperm viability following cold shock is best maintained by removing extracellular calcium and increasing membrane cholesterol content via the cholesterol shuttle activity of HBCD. This work was supported by grants from PA Dept. Ag. (ME 443291) and the NIH (5-K08-HD041430).

92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 154
Author(s):  
J. Gadea ◽  
S. Martínez-Miró ◽  
G. Decuadro-Hansen ◽  
C. Matás
Keyword(s):  
Seminal Plasma ◽  
Acrosome Reaction ◽  
Sperm Viability ◽  
Lipid Packing ◽  
Lipid Disorder ◽  
Merocyanine 540 ◽  
Centrifuge Tube ◽  
Before And After ◽  
Centrifugation Technique

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

360 EVALUATION OF QUALITY OF IN VITRO-MATURED BOVINE OOCYTES BY SEDIMENTATION WITH PERCOLL

2007 ◽  
Vol 19 (1) ◽  
pp. 295
Author(s):  
K. Yotsushima ◽  
M. Shimizu ◽  
H. Kon ◽  
Y. Izaike
Keyword(s):  
Specific Gravity ◽  
Simple Method ◽  
Serum Free ◽  
Class A ◽  
Serum Supplementation ◽  
Before And After ◽  
Class C ◽  
Bovine Oocytes

A simple method to evaluate the quality of in vitro-matured bovine oocytes is available for development of an in vitro embryo production system. Oocyte quality relates closely to oocyte fatty acid composition and mitochondrial distribution. The purpose of this study was to examine the influence of the quality of cumulus–oocyte complexes (COCs) and serum supplementation in IVM medium on the distribution of bovine oocyte specific gravities by sedimentation with Percoll before and after IVM. COCs were aspirated from abattoir-derived ovaries and were classified as classes A to D by the morphology of their cumulus cell layers as follows: class A, compact and more than 3 layers thick; class B, compact but &lt;3 layers; class C, partially naked and &lt;3 layers; and class D, naked or expanded. The classified COCs were cultured in TCM-199 supplemented with 0.1% BSA, 5 µg mL−1 insulin, 10 µg mL−1 transferrin, and 10 ng mL−1 transforming growth factor-α (M199-BITT) for 22–24 h. To evaluate the influence of serum supplementation, oocytes from classes A and B were also incubated in M199-BITT as serum-free culture or TCM-199 supplemented with 10% fetal calf serum as serum-supplemented culture. Percoll solutions were prepared by diluting Percoll with PBS supplemented with 0.3% BSA, 1 mg mL−1 glucose, and 0.2 mM sodium pyrvate to 20, 17.5, 15, 12.5, 10, 7.5, and 5% solutions. After removal of cumulus cells, denuded oocytes were put on the surface of Percoll solution for 3 min, and the precipitated oocytes were transferred to stepwise high density solution. The percent of Percoll solution just before buoyancy was considered as the oocyte specific gravity value. Statistical analysis was performed by one-way ANOVA. Oocytes from class A had the highest specific gravities before and after IVM in all classes (Table 1). After IVM, oocyte specific gravities from classes A and C were higher than those of oocytes before IVM (class A: P &lt; 0.05, class C: P &lt; 0.001). The specific gravities of in vitro-matured oocytes cultured in serum-free medium were higher than those cultured in serum-supplemented medium (15.3 ± 0.3%, n = 71, and 14.0 ± 0.3%, n = 58; P &lt; 0.01). These results show that the specific gravity was affected by the morphological quality of COC, and the culture conditions for IVM may profile the metabolic activity of oocytes during IVM. Table 1.Specific gravities of the bovine oocytes classified by morphology of COC before and after IVM

Fractal Reconstruction of Breast Perfusion Before and After Hyperthermia Treatments

2002 ◽  
Author(s):  
Oana I. Craciunescu ◽  
Shiva K. Das ◽  
Terrence Z. Wong ◽  
Thaddeus V. Samulski
Keyword(s):  
Dynamic Mri ◽  
Relevant Information ◽  
Fractal Interpolation ◽  
Hyperthermia Treatment ◽  
Data Set ◽  
Fractal Interpolation Functions ◽  
Before And After ◽  
Treatment Data ◽  
Mri Sequences ◽  
The Difference

Thermal modeling for hyperthermia breast patients can provide relevant information to better understand the temperatures achieved during treatment. However, human breast is much perfused, making knowledge of the perfusion crucial to the accuracy of the temperature computations. It has been shown that the perfusion of blood in tumor tissue can be approximated using the relative perfusion index (RPI) determined from dynamic contrast-enhanced magnetic resonance imaging (DE-MRI). It was also concluded that the 3D reconstruction of tumor perfusion can be performed using fractal interpolation functions (FIF). The technique used was called piecewise hidden variable fractal interpolation (PHVFI). Changes in the protocol parameters for the dynamic MRI sequences in breast patients allowed us to be able to acquire more spatial slices, hence the possibility to actually verify the accuracy of the fractal interpolation. The interpolated slices were compared to the imaged slices in the original set. The accuracy of the interpolation was tested on post-hyperthermia treatment data set. The difference between the reconstruction and the original slice varied from 2 to 5%. Significantly, the fractal dimension of the interpolated slices is within 2–3% from the original images, thus preserving the fractality of the perfusion maps. The use of such a method becomes crucial when tumor size and imaging restrictions limits the number of spatial slices, requiring interpolation to fill the data between the slices.

PKA Mediates PKCα Down Regulation and PI3Kα Activation During Bovine Sperm Capacitation.

2008 ◽  
Vol 78 (Suppl_1) ◽  
pp. 187-187
Author(s):  
Nir Etkovitz ◽  
Tali Rotman ◽  
Sara Rubinstein ◽  
Haim Breitbart
Keyword(s):  
Sperm Capacitation ◽  
Down Regulation ◽  
Bovine Sperm

Assessment of binder of sperm protein 1 (BSP1) and heparin effects on in vitro capacitation and fertilization of bovine ejaculated and epididymal sperm

Zygote ◽  
2020 ◽  
Vol 28 (6) ◽  
pp. 489-494
Author(s):  
Paula Rodríguez-Villamil ◽  
Daiane Mentz ◽  
Felipe Ledur Ongaratto ◽  
Luis Henrique Aguiar ◽  
Jose Luiz Rodrigues ◽  
...  
Keyword(s):  
Control Group ◽  
Sperm Capacitation ◽  
Epididymal Sperm ◽  
Sperm Protein ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
Development Rates ◽  
Developmental Rates

SummaryThe present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen–thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

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