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Mechanisms of fertilization elucidated by gene-manipulated animals.

Okabe M - Asian J. Androl. (2015 Jul-Aug)

Bottom Line: These phenomena were found more than 60 years ago.However, fundamental questions regarding the nature of capacitation and the timing of the acrosome reaction remain unsolved.Factors were postulated over time, but as their roles were not verified by gene-disruption experiments, widely accepted notions concerning the mechanism of fertilization are facing modifications.

View Article: PubMed Central - PubMed

Affiliation: Center for Genetic Analysis for Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565 0871, Japan.

ABSTRACT
Capacitation and the acrosome reaction are key phenomena in mammalian fertilization. These phenomena were found more than 60 years ago. However, fundamental questions regarding the nature of capacitation and the timing of the acrosome reaction remain unsolved. Factors were postulated over time, but as their roles were not verified by gene-disruption experiments, widely accepted notions concerning the mechanism of fertilization are facing modifications. Today, although in vitro fertilization systems remain our central research tool, the importance of in vivo observations must be revisited. Here, primarily focusing on our own research, I summarize how in vivo observations using gene-manipulated animals have elucidated new concepts in the mechanisms of fertilization.

No MeSH data available.


Factors involved in sperm-egg fusion. Izumo1, migrated outward from the outer acrosomal membrane to the sperm surface, tending to localize in the equatorial segment of spermatozoa. Various segments of Izumo1 were examined for their binding ability to eggs and residue 57–113 was indicated to contain an active binding site.49 Using the AVEXIS assay, JUNO was identified as an Izumo1 binding protein and its role in fusion was verified by gene-disruption experiments. JUNO is a 244-residue protein but is cleaved at 222 to form a GPI (glycosylphosphatidylinositol)-anchored protein. GPI-anchored proteins are initially formed on the cytosolic side and flipped over to the outer membrane side in the final maturation stage. The next helpful piece of information will be the elucidation of the active site of JUNO. Since Izumo1 (57–113) bound to Cd9-disrupted eggs normally, the elucidation of Cd9's role(s) will offer further clarification.
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Figure 2: Factors involved in sperm-egg fusion. Izumo1, migrated outward from the outer acrosomal membrane to the sperm surface, tending to localize in the equatorial segment of spermatozoa. Various segments of Izumo1 were examined for their binding ability to eggs and residue 57–113 was indicated to contain an active binding site.49 Using the AVEXIS assay, JUNO was identified as an Izumo1 binding protein and its role in fusion was verified by gene-disruption experiments. JUNO is a 244-residue protein but is cleaved at 222 to form a GPI (glycosylphosphatidylinositol)-anchored protein. GPI-anchored proteins are initially formed on the cytosolic side and flipped over to the outer membrane side in the final maturation stage. The next helpful piece of information will be the elucidation of the active site of JUNO. Since Izumo1 (57–113) bound to Cd9-disrupted eggs normally, the elucidation of Cd9's role(s) will offer further clarification.

Mentions: The fusing ability of Cd9-disrupted eggs was severely impaired, but it was not entirely lost, differing from the complete infertility seen in Izumo1 disruption. In addition, the binding of the putative functional fragment of Izumo1 in the N-terminus region (Izumo1: 57–113) to the egg surface was not altered by disruption of Cd9.49 Thus, Izumo1 binding to a protein other than Cd9 was expected on the egg surface. However, as the number of eggs that we can use for the experiment is quite limited, the purification of Izumo1 binding protein from eggs seemed difficult by conventional means. However, a method called the AVEXIS assay (avidity-based extracellular interaction screen) was invented.50 Using this method, a soluble, biochemically active, highly avid recombinant mouse Izumo1 ectodomain was prepared and the reactivity against HEK293 cells transfected with a normalized mouse oocyte cDNA expression library was analyzed and Bianchi et al. successfully identified the Izumo1 binding protein on the egg and named it JUNO after the goddess of marriage.51 The Juno-disrupted female mice were completely infertile. Now that interacting components Izumo1 and JUNO have been found, rapid progress in the elucidation of the sperm-egg fusion mechanism is expected to follow (Figure 2).


Mechanisms of fertilization elucidated by gene-manipulated animals.

Okabe M - Asian J. Androl. (2015 Jul-Aug)

Factors involved in sperm-egg fusion. Izumo1, migrated outward from the outer acrosomal membrane to the sperm surface, tending to localize in the equatorial segment of spermatozoa. Various segments of Izumo1 were examined for their binding ability to eggs and residue 57–113 was indicated to contain an active binding site.49 Using the AVEXIS assay, JUNO was identified as an Izumo1 binding protein and its role in fusion was verified by gene-disruption experiments. JUNO is a 244-residue protein but is cleaved at 222 to form a GPI (glycosylphosphatidylinositol)-anchored protein. GPI-anchored proteins are initially formed on the cytosolic side and flipped over to the outer membrane side in the final maturation stage. The next helpful piece of information will be the elucidation of the active site of JUNO. Since Izumo1 (57–113) bound to Cd9-disrupted eggs normally, the elucidation of Cd9's role(s) will offer further clarification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492058&req=5

Figure 2: Factors involved in sperm-egg fusion. Izumo1, migrated outward from the outer acrosomal membrane to the sperm surface, tending to localize in the equatorial segment of spermatozoa. Various segments of Izumo1 were examined for their binding ability to eggs and residue 57–113 was indicated to contain an active binding site.49 Using the AVEXIS assay, JUNO was identified as an Izumo1 binding protein and its role in fusion was verified by gene-disruption experiments. JUNO is a 244-residue protein but is cleaved at 222 to form a GPI (glycosylphosphatidylinositol)-anchored protein. GPI-anchored proteins are initially formed on the cytosolic side and flipped over to the outer membrane side in the final maturation stage. The next helpful piece of information will be the elucidation of the active site of JUNO. Since Izumo1 (57–113) bound to Cd9-disrupted eggs normally, the elucidation of Cd9's role(s) will offer further clarification.
Mentions: The fusing ability of Cd9-disrupted eggs was severely impaired, but it was not entirely lost, differing from the complete infertility seen in Izumo1 disruption. In addition, the binding of the putative functional fragment of Izumo1 in the N-terminus region (Izumo1: 57–113) to the egg surface was not altered by disruption of Cd9.49 Thus, Izumo1 binding to a protein other than Cd9 was expected on the egg surface. However, as the number of eggs that we can use for the experiment is quite limited, the purification of Izumo1 binding protein from eggs seemed difficult by conventional means. However, a method called the AVEXIS assay (avidity-based extracellular interaction screen) was invented.50 Using this method, a soluble, biochemically active, highly avid recombinant mouse Izumo1 ectodomain was prepared and the reactivity against HEK293 cells transfected with a normalized mouse oocyte cDNA expression library was analyzed and Bianchi et al. successfully identified the Izumo1 binding protein on the egg and named it JUNO after the goddess of marriage.51 The Juno-disrupted female mice were completely infertile. Now that interacting components Izumo1 and JUNO have been found, rapid progress in the elucidation of the sperm-egg fusion mechanism is expected to follow (Figure 2).

Bottom Line: These phenomena were found more than 60 years ago.However, fundamental questions regarding the nature of capacitation and the timing of the acrosome reaction remain unsolved.Factors were postulated over time, but as their roles were not verified by gene-disruption experiments, widely accepted notions concerning the mechanism of fertilization are facing modifications.

View Article: PubMed Central - PubMed

Affiliation: Center for Genetic Analysis for Biological Responses, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565 0871, Japan.

ABSTRACT
Capacitation and the acrosome reaction are key phenomena in mammalian fertilization. These phenomena were found more than 60 years ago. However, fundamental questions regarding the nature of capacitation and the timing of the acrosome reaction remain unsolved. Factors were postulated over time, but as their roles were not verified by gene-disruption experiments, widely accepted notions concerning the mechanism of fertilization are facing modifications. Today, although in vitro fertilization systems remain our central research tool, the importance of in vivo observations must be revisited. Here, primarily focusing on our own research, I summarize how in vivo observations using gene-manipulated animals have elucidated new concepts in the mechanisms of fertilization.

No MeSH data available.