Figure 1.
The MII Oocyte Is Preferentially Fertilized in the pb Half Sphere Independent of Intrinsic Polarity
(A) Distribution of the site of fertilization with respect to the 2pb in vivo and in vitro after cumulus cell removal. Zones I–VI (depicted in yellow), defined as 30° wide each, categorize the site of fertilization with respect to the 2pb. The proportion of the surface area for each zone is depicted in red.
(B) Distribution of the position of fertilization in vitro after zona pellucida removal.
(C) Distribution of sperm binding to defined regions of the ZP with respect to the meiotic chromosomes at MII stage.
(D) Distribution of the point of ZP penetration by the sperm with respect to the 2pb.
Results are all presented in the order of Zone I to VI. Scale bars represent 20 μm.
Figure 2.
Sperm Fertilizes at a Point Far Distant from Its Site of ZP Penetration
(A) Zygote with an oil drop marking the position of the fertilization cone.
(B) The embryo after hypotonic treatment. The cytoplasmic protrusion marks the site of ZP penetration by the sperm. Arc distance (θ) between the point of ZP penetration and fertilization is measured.
(C) Distribution of the arc distance between the sites of ZP penetration and fertilization.
Scale bar in (A) represents 20 μm.
Figure 3.
Sperm Swims Around within the PVS Prior to Fertilization
(A) Typical morphology of a MII oocyte with the meiotic chromosomes located in one “shoulder.” (B and C) Sequential DIC (differential interference contrast) images of embryos during in vitro fertilization. (1) Sperm penetrates the ZP. (2 and 3) Sperm swims around the PVS. (4) Sperm hits the 1pb. (5) Sperm binds the oocyte membrane. (6) 2pb extrudes and the fertilization cone forms. White arrowheads indicate the sperm, red arrowheads the 1pb, and yellow arrowheads the collapsing (B) or rotating (C) 1pb. White and red asterisks mark the fertilization cone and the 2pb, respectively. Scale bars in (A) and (B1) represent 20 μm.
Figure 4.
Experimental Enlargement of the PVS in the Non-pb Half Increases the Probability of Fertilization in This Area
(A–C) Removal of cytoplasm at MII stage and fertilization in non-pb half of a manipulated embryo. (A) MII oocyte. (B) Manipulated oocyte (left) following removal of half of its original volume (right). (C) Fertilized zygote. (D–G) 2pb transplantation and fertilization in non-pb half of manipulated embryo. (D) 2pb removal from donor zygote. (E) 2pb transfer to another oocyte. (F) MII oocyte with additional 2pb and PVS. (G) Fertilized zygote. (H) Classification of embryos according to their site of fertilization with respect to the 2pb under control and manipulated conditions. Zones I–VI are defined as in Figure 1.
Red arrowheads indicate the 1pb, red asterisks indicate the 2pb, white asterisks indicate the fertilization cone, green arrowheads indicate MII chromosomes, green arrows indicate condensed female chromosomes, and yellow arrows indicate the transplanted 2pb. Scale bars in (A) and (F) represent 20 μm.