One‐step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large‐scale preparation

Yamatoya, Kenji; Ito, Chizuru; Araki, Motoyuki; Furuse, Ryoji; Toshimori, Kiyotaka

doi:10.1007/s12522-011-0075-8

Cited by 14 publications

(14 citation statements)

References 32 publications

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“…3A. The ‘zona-intact’ oocytes isolated from ovulated oocytes were subjected to treatment with collagenase (Yamatoya et al, 2011) to remove extracellular components such as ZP and PVS (hereafter referred to as ‘ZP + PVS’). The resulting two fractions, namely ‘ZP + PVS’ and ‘denuded oocytes’, were next subjected to immunoblotting (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To count the number of sperm fused per oocyte, zona-free oocytes were prepared according to the procedure described by Yamatoya et al (Yamatoya et al, 2011). They were then preincubated with 4′,6-diamidino-2-phenylindole (DAPI; Wako Pure Chemical Industries, Ltd., Osaka, Japan) at the final concentration of 10 µg/ml in TYH medium for 20 min at 37°C, and washed 3 times in separate drops of TYH medium before insemination.…”

Section: Methodsmentioning

confidence: 99%

“…After zona-intact oocytes were treated with collagenase (Wako Pure Chemical Industries, Ltd.) at the final concentration of 1 mg/ml in a 30 µl drop of medium (Yamatoya et al, 2011) for 20 min at 37°C, denuded oocytes were removed from the drop with a microcapillary attached to a mouth piece and the remaining solution (<30 µl) was collected as extracellular components of oocytes. The collected components were incubated with anti-CD9 (0.5 µg/ml) or anti-CD81 (0.5 µg/ml) for 2 hours at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

et al. 2012

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SummaryWhen a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

et al. 2012

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“…Thus, the idea that MMP2, a type IV collagenase, could play a role in lytic digestion of the ZP by the sperm is not surprising considering its wide range of cleavage site motifs and substrates (Dean and Overall 2007 ) and that normally, like all MMPs, it is secreted at the cell surface where it is activated to aid in invasion and regulation of the extracellular matrix (Hulboy et al 1997 ; Nagase and Woessner 1999 ). In fact, an alternative to the trypsin or acidified Tyrode’s digest methods of preparing ZP-free oocytes is by pre-incubation of oocytes in type I collagenase (Yamatoya et al 2011 ).…”

Section: Discussionmentioning

confidence: 99%

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

et al. 2012

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Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-012-1429-1) contains supplementary material, which is available to authorized users.

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“…Sixteen hours after hCG injection, oocytes were collected from the oviduct and placed in 50 μl of TYH medium. The zona pellucida was removed from the oocytes via treatment with 1.0 mg ml −1 of collagenase (FUJIFILM Wako Pure Chemical Corporation) (Yamatoya et al, 2011). Zona-free oocytes were incubated with 0.5 µg ml −1 MZ3-FITC and 0.25 µg ml −1 TH6-Alexa647 with 1 μg ml −1 of Hoechst 33342 (Thermo Fisher Scientific) for 1 h at 37°C in TYH medium.…”

Section: Fluorescence Imaging For Fixed Eggsmentioning

confidence: 99%

Unveiling a novel function of CD9 in surface compartmentalization of oocytes

2020

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Gamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1–oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

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One‐step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large‐scale preparation

Abstract: The one-step collagenase method is an easy preparation method for large numbers of high-quality ZP-free eggs.

Cited by 14 publications

References 32 publications

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

Unveiling a novel function of CD9 in surface compartmentalization of oocytes

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