Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.
Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first t...
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| Format: | Article |
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4388664?pdf=render |
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| author | Xiaojing Yang Yuewen Zhao Xiaolong Yang Frederick W K Kan |
| author_facet | Xiaojing Yang Yuewen Zhao Xiaolong Yang Frederick W K Kan |
| author_sort | Xiaojing Yang |
| collection | DOAJ |
| description | Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function. |
| first_indexed | 2024-12-16T11:52:53Z |
| format | Article |
| id | doaj.art-585f46a3294e411aa41ba00d7b7d7d50 |
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| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T11:52:53Z |
| publishDate | 2015-01-01 |
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| spelling | doaj.art-585f46a3294e411aa41ba00d7b7d7d502022-12-21T22:32:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01104e012300310.1371/journal.pone.0123003Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.Xiaojing YangYuewen ZhaoXiaolong YangFrederick W K KanStudies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.http://europepmc.org/articles/PMC4388664?pdf=render |
| spellingShingle | Xiaojing Yang Yuewen Zhao Xiaolong Yang Frederick W K Kan Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. PLoS ONE |
| title | Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. |
| title_full | Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. |
| title_fullStr | Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. |
| title_full_unstemmed | Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. |
| title_short | Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding. |
| title_sort | recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm oocyte binding |
| url | http://europepmc.org/articles/PMC4388664?pdf=render |
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