Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B

The zona pellucida, a glycoprotein matrix surrounding the mammalian oocyte, hardens after intrusion of the first spermatozoon, thus protecting the embryo until implantation and preventing multiple fertilizations (polyspermy). Definitive zona hardening is mediated by the metalloprotease ovastacin, which is released from cortical granules of the oocyte upon sperm penetration. However, traces of ovastacin seep from unfertilized eggs to cause zona hardening even in the absence of sperm. These small amounts of protease are inactivated by the plasma protein fetuin-B, thus keeping eggs fertilizable. Once a sperm has penetrated the egg, ovastacin from cortical vesicles overrides fetuin-B and initiates zona hardening.

346 ULTRASTRUCTURE OF A HUMAN GIANT MII OOCYTE: CASE REPORT

The occurrence of giant oocytes has been reported with considerable frequency during IVF procedures in women of all ages and was associated with an increased response to gonadotrophin therapy. Previous studies suggested that giant oocytes, larger in diameter than normal oocytes and either at the GV, MI, or MII stage, might be a source of human digynic triploidy. However, to our knowledge there are no previous reports concerning the ultrastructure of human giant oocytes, and therefore the objective of this study was to characterize a human giant mature metaphase II oocyte from the ultrastuctural point of view. A giant oocyte with a visible polar body was used under informed consent, after controlled superovulation during a IVF treatment cycle. The oocyte was fixed with Karnovsky’s fixative, post-fixed in 2% OsO4 in 0.15 M cacodylate buffer, pH 7.2, dehydrated and embedded in Epon Semithin and ultrathin sections were cut with a diamond Diatome knife (Diatome, Hatfield, PA, USA) in a LKB ultramicrotome. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were collected on 200 mesh copper grids (Taab Laboratories Equipment Ltd, Aldermaston, UK) and stained with 3% aqueous uranyl acetate (20 min) and Reynolds lead citrate (10 min). Contrast ultrathin sections were observed in a JEOL 100XII transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 60 Kv. Cytogenetic analysis was not performed in this oocyte. At the time of collection, the giant oocyte was 1.4-fold larger than average size oocytes and contained a fragmented polar body. The zona pellucida had a normal loosely fibrillar structure and the perivitelline space was normal containing remnants of follicular cells. The cytoplasmic organelles were no uniformly dispersed with a large organelle-free zone (lake) observed in the periphery of the oocyte cortex and smaller focal lakes visible in the subcortex. Within the cortex the dense cortical vesicles were reduced in number and did not form one or two continuous rows beneath the oolemma. Smooth endoplasmic reticulum (SER) aggregates of tubules were also scarce but underdeveloped SER aggregates of tubules were present in the cortex. The distribution of the SER large and middle vesicles was abnormal: these organelles were nearly absent in the cortex but present in the inner cytoplasm. The content of some vesicles was denser than normal. We also observed the presence of several large secondary lysosomes filled with multiple small and medium lipid droplets (lipofuscin bodies), corresponding to retractile bodies located in the cortex. Large dense vesicles of different densities and volumes were found in the cortex and subcortex. The polar body showed very few cortical vesicles and did not contain mitochondria and SER large or small vesicles. The condensed chromosomes were aligned on the metaphase II plate, in the cortex of the oocyte. The ultrastructural alterations observed in this oocyte corroborated the conclusions of previous genetic studies and confirmed that giant oocytes are immature cytoplasmatically and would be associated with a higher frequency of abnormal development following fertilization. Thus, giant oocytes should not be used in IVF treatments. ASL was supported by FCT, Portugal.

Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

Twin malformation induced in chum salmon eggs by elevated water temperature, with a suggestion as to its mechanism

Inseminated eggs of chum salmon, Oncorhynchus keta, were incubated at 18 °C. In batches of eggs from different females, we regularly observed twinning in a proportion of the eggs (0.5–4%) continuously incubated at this temperature, although no twins were obtained at 8 °C. Twinning was, however, observed at 8 °C when inseminated eggs had been previously treated at 18 °C until the 2- to 4-cell stage. In contrast, eggs attaining telophase in the second meiosis at 8 °C did not show the twin malformation, even when they continued embryonic development at 18 °C. In sections of eggs developed as twins, we detected accumulations of PAS-positive vesicular bodies in the ooplasm between the two embryos. A small number of eggs showing impaired exocytosis of cortical vesicles (alveoli) during egg activation developed into twins. A similar malformation was also induced after part of the ooplasm was dislodged in activated eggs. We propose that vesicular bodies interfere with the convergent migration of the axial determinant during the early phase of embryonic development, which leads to the formation of multiple morphogenetic centers in the eggs incubated at 18 °C.

Calcium-regulated exocytosis is required for cell membrane resealing.

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.