scholarly journals Progress of sperm IZUMO1 relocation during spontaneous acrosome reaction

Reproduction ◽  
2014 ◽  
Vol 147 (2) ◽  
pp. 231-240 ◽  
Author(s):  
Natasa Sebkova ◽  
Lukas Ded ◽  
Katerina Vesela ◽  
Katerina Dvorakova-Hortova
Keyword(s):  
Fusion Protein ◽  
Acrosome Reaction ◽  
Mating Behaviour ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Sperm Head ◽  
High Rate ◽  
Field Mice ◽  
Species Specific

It has been recently shown in mice that sperm undergo acrosome reaction (AR) by passing through cumulus cells; furthermore, the acrosome-reacted sperm can bind to zona pellucida and consequently fertilise the egg. During AR, the relocation of the primary fusion protein IZUMO1 into the equatorial segment is crucial for sperm–egg fusion. There is a high rate of spontaneous AR in rodents, with up to 60% in promiscuous species. The aim of this study was to clarify whether the IZUMO1 relocation in sperm after spontaneous and induced AR is the same, and whether there is a correlation between the speed of IZUMO1 relocation and species-specific mating behaviour in field mice. Immunofluorescent detection of IZUMO1 dynamics during the in vitro capacitation, spontaneous, calcium ionophore and progesterone-induced AR was monitored. Our results show that during spontaneous AR, there is a clear IZUMO1 relocation from the acrosomal cap to the equatorial segment, and further over the whole sperm head. In addition, there is positive tail tyrosine phosphorylation (TyrP) associated with hyperactive motility. Moreover, the beginning and the progress of IZUMO1 relocation and tail TyrP positively correlate with the level of promiscuity and the acrosome instability in promiscuous species. The findings that crucial molecular changes essential for sperm–egg fusion represented by dynamic movements of IZUMO1 also happen during spontaneous AR are vital for understanding fertilisation in mice.

Sperm from beta 1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4121-4131 ◽  
Author(s):  
Q. Lu ◽  
B.D. Shur
Keyword(s):  
Acrosome Reaction ◽  
Direct Evidence ◽  
Calcium Ionophore ◽  
Wild Type ◽  
Sperm Surface ◽  
Gamete Recognition ◽  
Dependent Signal ◽  
Relative Inability ◽  
Egg Coat

A variety of sperm surface components have been suggested to mediate gamete recognition by binding to glycoside ligands on the egg coat glycoprotein ZP3. The function of each of these candidate receptors is based upon varying degrees of circumstantial and direct evidence; however, the effects on fertilization of targeted mutations in any of these candidate receptors have not yet been reported. In this paper, we describe the effects of targeted mutations in beta1,4-galactosyltransferase, the best studied of the candidate receptors for ZP3. Surprisingly, galactosyltransferase-null (gt[−/−]) males are fertile; however, sperm from gt(−/−) males bind less radiolabeled ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either ZP3 or anti-galactosyltransferase antibodies, as do wild-type sperm. In contrast, gt(−/−) sperm undergo the acrosome reaction normally in response to calcium ionophore, which bypasses the requirement for ZP3 binding. The inability of gt(−/−) sperm to undergo a ZP3-induced acrosome reaction renders them physiologically inferior to wild-type sperm, as assayed by their relative inability to penetrate the egg coat and fertilize the oocyte in vitro. Thus, although ZP3 binding and subsequent induction of the acrosome reaction are dispensable for fertilization, they impart a physiological advantage to the fertilizing sperm. A second strain of mice was created that is characterized by a loss of of the long galactosyltransferase isoform responsible for ZP3-dependent signal transduction, but which maintains normal levels of Golgi galactosylation. Sperm from these mice show that the defective sperm-egg interactions in gt(−/−) mice are due directly to a loss of the long galactosyltransferase isoform from the sperm surface and are independent of the state of intracellular galactosylation during spermatogenesis.

scholarly journals Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 725-735
Author(s):  
Julieta Gabriela Hamze ◽  
María Jiménez-Movilla ◽  
Raquel Romar
Keyword(s):  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
In Vitro Fertilisation ◽  
Sperm Binding ◽  
Fertilisation Rate ◽  
Gamete Recognition ◽  
Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

Evidence for calcium inactivation during hormone release in the rat neurohypophysis

1976 ◽  
Vol 65 (3) ◽  
pp. 669-683
Author(s):  
J. J. Nordmann
Keyword(s):  
Hormone Secretion ◽  
Calcium Ionophore ◽  
High Rate ◽  
Hormone Release ◽  
45Ca Uptake ◽  
High K ◽  
K Concentration ◽  
The Relationship ◽  
Internal Calcium

1. A study has been made of the relationship between 45Ca uptake into and hormone release from isolated rat neurohypophyses incubated in vitro. 2. Hormone secretion is triggered by high-K (56 mM) but long exposure to the stimulus does not generate a maintained release of hormone. 3. When hormone release began to wane, addition of Ba of La increased hormone output which suggests that the decline in output did not result from depletion of the neurosecretory granules at the nerve terminals. 4. 45Ca uptake is enhanced in the presence of high-K concentration, but the initial high rate declines during long exposure to the potassium stimulus with a time constant similar to that of the decline in hormone release. 5. After a period of incubation in a K-rich, calcium-free medium, addition of calcium to the medium induced hormone release. The magnitude of this release was dependent on the time of exposure to excess potassium. 6. After inactivation of secretion, mobilization of internal calcium by means of a calcium ionophore increased hormone release.

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

2003 ◽  
Vol 51 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Ö. Uçar ◽  
T. J. Parkinson
Keyword(s):  
Phase Contrast ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Contrast Microscopy ◽  
The Relationship ◽  
In Vitro Induction ◽  
Artificial Vagina

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0µM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3± 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P<0.001) increased after incubation with A23187. After incubation with 0.1µM/l A23187 for 45 and 60min there were 22.4±3.0% and 31.7±4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0µM/l A23187 for 45 and 60min there were 46.2±6.5% and 53.8±5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0±2.7% and 22.3±4.2%. There was also a significant (P<0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.

33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor

2018 ◽  
Vol 30 (1) ◽  
pp. 156
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
B. C. Lee
Keyword(s):  
Calcium Ionophore ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Serum Progesterone ◽  
Significance Level ◽  
Chi Squared ◽  
Pregnancy And Delivery ◽  
Developmental Rates

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos. This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 152
Author(s):  
M. A. Lagares ◽  
H. S. Martins ◽  
M. R. Souza ◽  
C. F. A. M. Penna ◽  
F. O. P. Leme ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Propidium Iodide ◽  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Peanut Agglutinin ◽  
Binding Assays ◽  
Frozen Semen ◽  
Fertilizing Ability

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P < 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P > 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P < 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P < 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

scholarly journals 106EVALUATION OF ADDITION OF REDUCED GLUTATHIONE TO COOLING MEDIUM ON IN VITRO FERTILITY AND ACROSOME REACTION IN BOAR SPERMATOZOA

2004 ◽  
Vol 16 (2) ◽  
pp. 175
Author(s):  
C. Matás ◽  
J. Gadea ◽  
F. García-Vázquez ◽  
J.C. Gardón ◽  
S. Cánovas
Keyword(s):  
In Vitro Fertilization ◽  
Acrosome Reaction ◽  
Reduced Glutathione ◽  
Membrane Integrity ◽  
Calcium Ionophore ◽  
Egg Yolk ◽  
Ros Generation ◽  
Vitro Fertilization ◽  
Cooling Medium

The process of cooling to 5°C prior to freezing produces physical and chemical stress on the sperm membrane associated with oxidative stress and reactive oxygen species (ROS) generation that reduces sperm viability and fertilizing ability. The addition of antioxidants to cooling medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of addition of reduced glutathione (GSH) to cooling extenders on (1) plasma membrane integrity, (2) acrosome reaction induction by ionophore A 23187 or progesterone, and (3) in vitro fertilization. Ejaculate-rich fractions from three mature pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15°C over 2h (group C). Thereafter, sperm were centrifuged and diluted in lactose/egg-yolk extender with 0mM (group 0), 1mM (group 1) or 5mM (group 5) of GSH, cooled to 5°C over 2h. The acrosome reaction was then induced by 1μM calcium ionophore or 10μM progesterone in TALP medium and incubated in 5% CO2, 38.5°C for 30 or 45min, respectively. Membrane integrity was evaluated by propidium iodide, and acrosomal status was monitored by means of FITC-labeled peanut agglutinin. Finally, in vitro fertilization was performed with these four spermatozoa groups as described previously (Matás et al. 2003 Reproduction 125, 133–141). ANOVA analysis revealed that the addition of GSH had no effect on the membrane integrity (ranged 58.8 to 66.9) or acrosome reaction induction (ranged 24.3 to 28.2, and 55.7 to 41.4 for progesterone and calcium ionophore, respectively). However, the results of the penetration assay revealed that the cooling affected the penetration rate and the number of sperm per oocyte (Table 1), and this assay is better than the others to predict changes in the spermatozoa functionality (Gadea J and Matás C 2000 Theriogenology 54, 1343–1357). In conclusion, the cooling process affects the in vitro fertilization, but the addition of GSH to the medium did not influence the parameters studied. Supported by AGL2000-0485-CO2-01. Table 1 Homologous in vitro penetration

scholarly journals ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0251973
Author(s):  
Yoku Kato ◽  
Satheesh Kumar ◽  
Christian Lessard ◽  
Janice L. Bailey
Keyword(s):  
Endoplasmic Reticulum ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Porcine Model ◽  
Sperm Head ◽  
Mouse Sperm ◽  
Phosphorylated Proteins ◽  
Boar Sperm

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

scholarly journals 28 CLONED EMBRYOS CAN BE PRODUCED USING DONOR CELLS OBTAINED FROM A 72-HOUR COOLED CARCASS

2005 ◽  
Vol 17 (2) ◽  
pp. 164 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
H. Odaman Mercan ◽  
A. Dinnyes
Keyword(s):  
Calf Serum ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Penicillin G ◽  
Developmental Potential ◽  
Viable Cells ◽  
Donor Cells ◽  
A Cell ◽  
Vitro Development

There are few reports on the use of cells from a dead mammal for nuclear transfer (NT). So far, most calves have been cloned from live adult cows or fresh fetal samples. The ability to produce cloned animals using postmortem tissue can provide an additional application to the field of NT. This study was conducted to investigate whether viable cells could be obtained from tissues chilled for 72 h and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 50 μg/mL sodium pyruvate, 1% v:v penicillin-streptomycin (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin), 10 ng/mL EGF, 0.5 μg/mL FSH, and 5 μg/mL LH. A cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 72 h. Tissues from muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 medium supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18 h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 μm for 25 μs. After fusion, NT units were activated using a combination of calcium ionophore (5 μM), cytochalasin D (2.5 μg/mL) and cycloheximide (10 μg/mL) and cultured for 7 days in BARC or G1.3-G2.3 medium. Differences (developmental potential and cell numbers) among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from muscle of a cow carcass stored at cold temperature for 72 h and that these cells have ability to generate NT blastocysts at rates similar to those obtained with fresh GCs. In addition, G1.3 and G2.3 culture medium supported embryo development better than BARC medium. Table 1. In vitro development of NT embryos This study was supported by a grant from TUBITAK, Turkey (VHAG-1908 and Turkey-Hungary bilateral project VHAG-2022).

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