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 Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction.

1979 ◽  
Vol 83 (3) ◽  
pp. 544-555 ◽  
Author(s):  
P M Saling ◽  
B T Storey
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Probe ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Sperm Head ◽  
Fertilization In Vitro ◽  
Ionophore A23187 ◽  
Mouse Sperm ◽  
Sperm Acrosome

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

Zygote ◽  
2000 ◽  
Vol 8 (4) ◽  
pp. 329-338 ◽  
Author(s):  
D.K. Saxena ◽  
I. Tanii ◽  
T. Oh-oka ◽  
K. Yoshinaga ◽  
K. Toshimori
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Formation Rate ◽  
Dependent Manner ◽  
Sperm Plasma Membrane ◽  
Dose Dependent

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm–zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm–egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 μg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm–oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.

scholarly journals Phospholipase Cδ4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

2003 ◽  
Vol 161 (1) ◽  
pp. 79-88 ◽  
Author(s):  
Kiyoko Fukami ◽  
Manabu Yoshida ◽  
Takafumi Inoue ◽  
Manabu Kurokawa ◽  
Rafael A. Fissore ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Calcium Imaging ◽  
Acrosome Reaction ◽  
Sperm Head ◽  
Wild Type ◽  
Imaging Studies ◽  
Mouse Sperm ◽  
Mammalian Fertilization ◽  
Important Enzyme

Zona pellucida (ZP)–induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCδ4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCδ4−/− sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCδ4−/− sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCδ4−/− sperm. These results indicate that PLCδ4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 15-24 ◽  
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Freeze Drying ◽  
Chelating Agents ◽  
Sperm Head ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Freeze Dried ◽  
Boar Sperm

SUMMARYSuccessful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

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.

scholarly journals EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ginto George ◽  
Satoshi Ninagawa ◽  
Hirokazu Yagi ◽  
Taiki Saito ◽  
Tokiro Ishikawa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Knockout ◽  
Covalent Bonding ◽  
Cxxc Motif ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Endoplasmic Reticulum Protein ◽  
Hct116 Cells

Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

Anchoring of FLT3 in the endoplasmic reticulum alters signaling quality

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3568-3576 ◽  
Author(s):  
Dirk Schmidt-Arras ◽  
Sylvia-Annette Böhmer ◽  
Sina Koch ◽  
Jörg P. Müller ◽  
Lutz Blei ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Transformation ◽  
Intracellular Location ◽  
Er Retention ◽  
Transforming Activity ◽  
Signaling Quality

Abstract The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

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