The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

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Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation

Development ◽

10.1242/dev.121.4.1129 ◽

1995 ◽

Vol 121 (4) ◽

pp. 1129-1137 ◽

Cited By ~ 6

Author(s):

P.E. Visconti ◽

J.L. Bailey ◽

G.D. Moore ◽

D. Pan ◽

P. Olds-Clarke ◽

...

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Tyrosine Phosphorylation ◽

Bovine Serum ◽

Female Reproductive Tract ◽

Dependent Manner ◽

Protein Tyrosine Phosphorylation ◽

Sperm Capacitation ◽

Protein Tyrosine

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

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224 SILDENAFIL ACCELERATES SPERM PENETRATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab224 ◽

2013 ◽

Vol 25 (1) ◽

pp. 260

Author(s):

H. Funahashi ◽

Q.-S. Wu

Keyword(s):

Adenosine Monophosphate ◽

Defined Medium ◽

Fluorescence Assay ◽

Chemically Defined Medium ◽

Sperm Capacitation ◽

Boar Sperm ◽

Sperm Penetration ◽

Type 3 ◽

Chlortetracycline Fluorescence Assay

Sperm capacitation, a cyclic-adenosine monophosphate-dependent phenomenon, is an important initiation step for penetration into oocytes. In porcine IVF, the use of caffeine, as a nonspecific phosphodiesterase (PDE) inhibitor, is common to accelerate sperm capacitation and penetration. The objective of this study was to examine the effects of PDE inhibitors (cilostamide, rolipram, and sildenafil as PDE type 3-, type 4-, and type 5-specific inhibitors, respectively) on the capacitation of boar sperm and the penetration into porcine oocytes in the absence of caffeine and other capacitation inducers in a chemically defined medium. After washing sperm samples collected from an ejaculated sperm-rich fraction of different individual Berkshires, the sperm were resuspended in capacitation inducer-free (theophylline- and adenosine-free) PGM-tac4 (mPGM-tac) at 5 × 105 cells mL–1. The suspension was cultured in mPGM-tac nonsupplemented or supplemented with 2.5 mM cilostamide, rolipram, or sildenafil for 90 min at 39°C in an atmosphere of 5% CO2 in air, and then the capacitation status was assessed by chlortetracycline fluorescence assay. Other sperm suspensions were used to co-culture with denuded in vitro-matured oocytes in the same medium for 8 h in an atmosphere of 5% CO2 in air and fixed, and sperm penetration was then examined. Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In our result from the chlortetracycline fluorescence assay, although the incidence of intact sperm was significantly reduced in the presence of rolipram (54.3%) and sildenafil (52.7%) as compared with controls (66.7%), there were no differences in capacitated sperm among experimental groups (24.3 to 34.3%). The incidence of acrosome-reacted sperm was higher in the presence of cilostamide (17.3%) than in the others (9.0 to 13.0%). High sperm penetration was observed only in the presence of sildenafil (76.6%) as compared with the control (0%) or the presence of rolipram (4.4%) or cilostamide (1.8%). These results demonstrate that inhibition of PDE type 5, but not PDE type 3 and type 4, significantly accelerates the penetration of boar sperm into the oocytes in a capacitation inducer-free chemically defined medium, whereas inhibition of PDE type 3 may induce an acrosome reaction.

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217 KINETICS OF SPERM PENETRATION IS CORRELATED WITH IN VITRO FERTILITY OF BUFFALO (BUBALUS BUBALIS) BULLS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab217 ◽

2009 ◽

Vol 21 (1) ◽

pp. 206 ◽

Cited By ~ 2

Author(s):

M. Rubessa ◽

M. Di Fenza ◽

E. Mariotti ◽

S. Di Francesco ◽

C. de Dilectis ◽

...

Keyword(s):

Bubalus Bubalis ◽

Blastocyst Stage ◽

Optimal Time ◽

High Fertility ◽

Chi Square ◽

Sperm Penetration ◽

Blastocyst Rate ◽

Kinetics Of ◽

Time Point

It was previously demonstrated that the kinetics of early cleavage could be used to discriminate between bovine bulls with high and low field fertility (Ward F et al. 2001 Mol. Reprod. Dev. 60, 47–55). Marked differences exist in the kinetics of sperm penetration between bulls, and this may be a useful predictor of field fertility in cattle (Ward F et al. 2002 Theriogenology 57, 2105–2117). It is well known that the ability to fertilize oocytes in vitro and to sustain embryo development varies significantly among buffalo bulls. Therefore, the aim of this work was to evaluate whether the speed of oocyte penetration after IVF was correlated with the blastocyst rates obtainable with different bulls in buffalo species. In Experiment 1, in vitro-matured buffalo oocytes were co-incubated with MitoTracker-labeled spermatozoa (Ward F et al. 2002 Theriogenology 57, 2105–2117) from 6 different bulls, over 2 replicates. Oocytes were subsequently fixed every 3 h (up to 18 h) postinsemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight, and stained with 4′,6-diamidino-2-phenylindole for nuclei examination under a fluorescence microscope. In Experiment 2, in vitro-matured oocytes were fertilized with sperm from the same 6 bulls and were cultured to the blastocyst stage, over 4 replicates. Bulls were tested, collectively, on each batch of ovaries in both experiments. Differences in the percentages of monospermic penetration among bulls were analyzed by chi-square test. Correlation and multiple regression analyses were also carried out between the speed of penetration and blastocyst yields. Marked differences in the kinetics of sperm penetration were found among buffalo bulls, as shown in Table 1. Interestingly, a correlation was found between the blastocyst rate and the percentage of oocytes penetrated at 6 h (r = 0.71; P < 0.01), at 9 h (r = 0.65; P < 0.05), at 12 h (r = 0.77; P < 0.01), and at 18 h pi (r = 0.59; P < 0.05). Regression analysis showed that the optimal time of penetration for predicting the blastocyst rate was 12 h pi (R2 = 0.6). In conclusion, the kinetics of sperm penetration may be a useful marker to predict the in vitro-fertilizing ability of buffalo bulls. The great variability in the speed of oocyte penetration suggests inserting this assessment in the preliminary screening of bulls before their utilization in IVF programs. This may be helpful in selecting high-fertility bulls and identifying the optimal gamete co-incubation times for each bull used. Table 1.Percentage of oocytes penetrated at each time point (hpi, h postinsemination) by different bulls1

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The influence of sperm concentration, length of the gamete co-culture and the evolution of different sperm parameters on the in vitro fertilization of prepubertal goat oocytes

Zygote ◽

10.1017/s0967199410000055 ◽

2010 ◽

Vol 18 (4) ◽

pp. 345-355 ◽

Cited By ~ 3

Author(s):

M.J. Palomo ◽

T. Mogas ◽

D. Izquierdo ◽

M.T. Paramio

Keyword(s):

Acrosome Reaction ◽

Penetration Rate ◽

Sperm Concentration ◽

Semen Parameters ◽

Oxygen Species ◽

Vitro Fertilization ◽

Rate Of Penetration ◽

Sperm Penetration ◽

Kinetics Of

SummaryThe aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 106 to 4 × 106 spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus–oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 106 sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8–12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12–14 h, with a concentration of 4 × 106 sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

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Effects of activin A and follistatin on developmental kinetics of bovine embryos: cinematographic analysis in a chemically defined medium

Reproduction ◽

10.1530/reprod/118.1.119 ◽

2000 ◽

pp. 119-125 ◽

Cited By ~ 7

Author(s):

K Yoshioka ◽

C Suzuki ◽

S Iwamura

Keyword(s):

Defined Medium ◽

Activin A ◽

Lag Phase ◽

Chemically Defined Medium ◽

Bovine Embryos ◽

Cell Stage ◽

Oviduct Fluid ◽

Kinetics Of ◽

Number Of Cells

The effects of recombinant human activin A and follistatin on the developmental kinetics of bovine presumptive zygotes matured and fertilized in vitro using time-lapse cinematography were investigated. The presumptive zygotes were cultured for 9 days in a chemically defined medium (modified synthetic oviduct fluid, control) and modified synthetic oviduct fluid supplemented with activin A or follistatin. Development under cine-recording conditions was similar to that in an incubator. Addition of activin A to modified synthetic oviduct fluid increased, while addition of follistatin decreased, the percentage of zygotes that developed to morulae and blastocysts. Follistatin significantly prolonged the timing of development to the 9-16-cell stage compared with the control and activin A media. Activin A significantly shortened the duration of the third cell cycle compared with the control, but follistatin significantly prolonged the fourth cell cycle compared with the control and activin A. Developmental arrest ('lag-phase') during the 4-8-cell stage was observed in 95% of embryos developed to more than the 9-16-cell stage in all treatments. The greater the number of cells at the onset of the lag-phase, the earlier the onset of the phase and the shorter the duration of the phase, the further embryos were able to develop by day 9 in all treatments. The number of cells at the onset of the lag-phase in the medium containing activin A was significantly higher than it was in control or follistatin-containing media. Moreover, activin A significantly shortened the duration of the lag-phase compared with follistatin. The present results indicate that activin A may enhance in vitro development of bovine embryos by improving developmental kinetics, especially by increasing the number of cells at the onset of the lag-phase and shortening the duration of this phase.

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Stimulation of rat ovarian ornithine decarboxylase in vitro by hCG, amino acids and bovine serum albumin

Acta Endocrinologica ◽

10.1530/acta.0.0940571 ◽

1980 ◽

Vol 94 (4) ◽

pp. 571-576 ◽

Cited By ~ 3

Author(s):

Kirsti Käpyaho

Keyword(s):

Amino Acids ◽

Bovine Serum Albumin ◽

Cyclic Amp ◽

Serum Albumin ◽

Ornithine Decarboxylase ◽

Bovine Serum ◽

Defined Medium ◽

Decarboxylase Activity ◽

Ornithine Decarboxylase Activity

Abstract. Rat ovarian ornithine decarboxylase activity could be stimulated in vitro by a variety of factors, which apparently have different modes of action. Ovarian cells prepared from pre-pubertal rats by collagenase dispersion exhibited a low but detectable ornithine decarboxylase activity after a 6-h incubation in a defined medium. The enzyme activity was markedly enhanced in vitro by hCG, which also produced increased accumulation of cyclic AMP and stimulated the secretion of progesterone. In addition to the gonadotrophin, ovarian ornithine decarboxylase activity was strikingly stimulated by some non-essential amino acids, and especially by bovine serum albumin. While markedly enhancing ornithine decarboxylase activity, none of the latter additions increased the accumulation of cyclic AMP or enhanced the secretion of progesterone. Bovine serum albumin enhanced powerfully ornithine decarboxylase activity in vitro at very small concentrations (from 0.75 μm). The half-life of the enzyme remained unchanged (26—28 min) upon stimulation indicating that the stimulation mechanism did not involve any stabilization of the enzyme.

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Effect of serum on the in vitro maturation of canine oocytes

Reproduction Fertility and Development ◽

10.1071/rd00012 ◽

1999 ◽

Vol 11 (8) ◽

pp. 387 ◽

Cited By ~ 35

Author(s):

Takeshige Otoi ◽

Maya Fujii ◽

Aya Ooka ◽

Masaki Tanaka ◽

Tatsuyuki Suzuki

Keyword(s):

Bovine Serum Albumin ◽

Bovine Serum ◽

In Vitro Maturation ◽

Culture Medium ◽

The Other ◽

Serum Supplement ◽

Meiotic Competence ◽

Positive Effect ◽

Serum Type

The meiotic competence of canine oocytes cultured for 72 h in medium supplemented with three different concentrations (5, 10 and 20%) of anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine serum albumin (BSA), was examined. The oestrous serum supplement had a positive effect on the resumption of meiosis, compared with the other supplements (P<0.05). The number of oocytes that reached metaphase I (MI) and metaphase II (MII) was significantly higher (P<0.05) with the oestrous serum supplement than with the anoestrous serum supplement. There were no significant differences among the three different concentrations in each serum type with respect to the proportion of oocytes that completed meiosis (MI to MII). The number of oocytes that resumed meiosis in the 10% oestrous serum supplement was significantly higher (P<0.05) than those of each concentration of the anoestrous and metoestrous serum supplements, and of the 0.3% BSA supplement. Moreover, a higher number of oocytes reached MII in the presence of the 10% oestrous serum supplement than with the 10% anoestrous serum supplement. These results suggest that supplementation of the culture medium with 10% oestrous serum is the optimal treatment for in vitro maturation of canine oocytes.

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Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro

Reproduction ◽

10.1530/rep.1.00374 ◽

2005 ◽

Vol 129 (1) ◽

pp. 51-60 ◽

Cited By ~ 50

Author(s):

Roberto Gualtieri ◽

Raffaele Boni ◽

Elisabetta Tosti ◽

Maria Zagami ◽

Riccardo Talevi

Keyword(s):

Tyrosine Phosphorylation ◽

Ionophore A23187 ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Phosphorylated Proteins ◽

Bovine Sperm ◽

Surface Remodeling ◽

Bovine Species

In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]i than adhering sperm; 2. heparin induces a [Ca2+]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.

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