P. Talbot

Cell Adhesion and Fertilization: Steps in Oocyte Transport, Sperm-Zona Pellucida Interactions, and Sperm-Egg Fusion

Abstract Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte “pick-up,” in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.

Cigarette smoke inhibits oocyte cumulus complex pick-up by the oviduct in vitro independent of ciliary beat frequency

The purpose of this study was to quantify the effects of acute exposure to mainstream (MS) and sidestream (SS) smoke solutions on oocyte cumulus complex pick-up rate in explants of hamster oviducts using a newly developed in vitro assay. Experiments were performed in handmade perfusion chambers using infundibula from hamster oviducts and oocyte cumulus complexes harvested from mature ovarian follicles. Oocyte cumulus complex pick-up rate was measured by placing a stained oocyte cumulus complex at the base of the infundibulum and recording the length of time needed for the complex to traverse a defined path to the ostium. Addition of either whole MS or SS smoke solutions to the perfusion chamber caused a dose dependent decrease in oocyte cumulus complex pick-up rate. Unexpectedly, upon washout of smoke solutions with control medium, oocyte cumulus complex pick-up rate continued to decline. The gas phase of MS smoke is more inhibitory than the particulate phase, while SS gas and particulate phases inhibit oocyte cumulus complex pick-up rate at equivalent doses. Ciliary beat frequency and oocyte cumulus complex pick-up rate were measured using the same infundibular explants to determine if smoke solutions decrease oocyte cumulus complex pick-up rate by inhibiting ciliary beat frequency. Ciliary beat frequency decreased in MS smoke solutions and recovered either partially or completely after washout of the smoke solutions. SS smoke solutions either produced no change in ciliary beat frequency or stimulated ciliary beat frequency. Oocyte cumulus complex pick-up rate decreased in both MS and SS smoke solutions and further declined during washout when ciliary beat frequency was equivalent to or higher than controls. These data show that oocyte cumulus complex pick-up rate and ciliary beat frequency can be uncoupled and that smoke solutions inhibit oocyte cumulus complex pick-up rate by affecting factors in addition to ciliary beat frequency. Possible reasons for the smoke induced decrease in oocyte cumulus complex pick-up rate are discussed. These results may explain the increased incidence of tubal infertility and ectopic pregnancy observed in women who smoke.

The structure of sperm from Panulirus, the spiny lobster, with special regard to the acrosome.

The fine structure of mature sperm from the spiny lobster (Panulirus) has been examined. Each sperm comprises a nucleus, lamellar region, and acrosome. The nucleus (Feulgen-positive region) contains uncondensed chromatin and is limited by a nuclear envelope. A variable number of spikes extend from the nucleus. The spikes are lined by evaginated nuclear envelope and are packed with microtubules, which span the nucleus proper. The spikes are stationary, and the sperm are immotile. The lamellar body, which lies at one side of the base of the acrosome and external to the nuclear envelope, contains numerous stacks of membrane and small mitochondria-like bodies. The acrosomal vesicle (PAS-positive region) is structurally complex and can be divided into four discrete zones, which are homogeneous, scrolled, crystalline, and flocculent in appearance. The vesicle is surrounded by periacrosomal material which is flocculent near the base of the acrosome and filamentous at the apex. The structural features of Panulirus sperm are compared to those of other reptantians (lobsters, crayfish, crabs) and natantians (prawns, shrimp), and it is concluded that at least two basic structural plans for decapod sperm exist.

Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase.

Ovulated opossum oocytes are surrounded by a zona pellucida, but not by cumulus cells. Opossum sperm carry at least four acrosomal hydrolases (hyaluronidase, acrosin, N-acetylhexosaminidase, and arylsulfatase); the functions of these enzymes in opossum fertilization are uncertain. To identify possible substrates for these hydrolases, the ultrastructure of opossum oocytes was examined after fixation in the presence of ruthenium red which stabilizes extracellular matrices. This oocyte is unusual in having a wide perivitelline space containing a highly structured extracellular matrix (ECM). The ECM is comprised of granules and filaments, and it resembles matrices known to contain hyaluronic acid in other systems. Hydrolases, known to be present in opossum acrosomes, were tested for their effect on the ultrastructure of the zona pellucida and matrix of the perivitelline space. Trypsin dissolved the zona pellucida and decreased the size of the granules in the perivitelline space. Streptomyces hyaluronidase, which specifically attacks hyaluronic acid, removed only matrix filaments. Arylsulfatase, N-acetylhexosaminidase, and beta-glucuronidase did not affect the zona pellucida or ECM in our assay. These observations are consistent with the ideas that (1) opossum sperm must penetrate two oocyte investments, the zona pellucida and ECM of the perivitelline space; (2) the ECM contains hyaluronic acid (filaments) and protein (granules); (3) opossum sperm acrosin may function in penetration of the zona pellucida and ECM; and (4) opossum sperm hyaluronidase may function in penetration of the ECM by degrading hyaluronic acid (filaments). Dissolution of the granules and filaments from oocyte microvilli is probably necessary to permit close apposition and fusion of the sperm and oocyte membranes. The evolutionary significance of these results is discussed.

Morphological features of the acrosome reaction of lobster (Homarus) sperm and the role of the reaction in generating forward sperm movement

Morphological features of the acrosome reaction of sperm from Homarus americanus have been examined using light and electron microscopy. The reaction, which was induced with ionophore A23187, occurs in 2 stages: (1) eversion of the acrosomal vesicle, and (2) ejection of the subacrosomal and nuclear material into the cavity created by acrosomal eversion. There are five consequences of the reaction: (1) the everted acrosomal contents form a fuzzy coat on the outside of the sperm; (2) the apical cap lies at the base of the sperm and is constricted; (3) the subacrosomal material forms a prominent filament at the leading edge of the sperm; (4) most of the nucleus and part of the spikes are pulled into the cavity surrounded by the everted acrosome; and (5) the sperm undergoes a net forward movement of about 18 μm. Ultrastructural changes which accompany this reaction are described in detail, and the position of the plasma, acrosomal, and nuclear membranes before and after reaction is discussed. Data show that one function of the acrosome reaction of lobster sperm is to generate forward movement of this otherwise immotile cell. The importance of this forward movement in sperm penetration of the chorion is discussed.

Assay and Importance of Adhesive Interaction Between Hamster (Mesocricetus auratus) Oocyte-Cumulus Complexes and the Oviductal Epithelium

Abstract Adhesion between the oocyte-cumulus complex and infundibulum plays an important, but poorly understood, role in oocyte pick-up. The purposes of this study were to determine which components of the oocyte-cumulus complex and oviductal epithelium function in adhesion, to measure adhesion under physiological conditions, and to examine the effect of modulation of adhesion on oocyte-cumulus complex pick-up rate. Oocyte-cumulus complexes containing an expanded matrix were readily transported into the oviduct, while unexpanded complexes lacking an extracellular matrix were not picked up, indicating that the matrix is necessary for pick-up. Transmission electron microscopy revealed that during pick-up, adhesion occurred specifically between the ciliary crowns of the oviduct and the granules and filaments of the cumulus matrix. An assay was developed using vacuum from a low-flow peristaltic precision pump, modified for bi-directional flow, to measure the strength of adhesion between the oocyte-cumulus complex and the oviductal epithelium, and adhesion was measured during physiological conditions. The lectin wheat germ agglutinin and the polycation poly-l-lysine were then used to modulate adhesion, and the effects of increasing or decreasing adhesion on oocyte pick-up rate and ciliary beat frequency were examined. The data show that 1) the matrix of the oocyte-cumulus complex and the ciliary crowns of the oviduct function in adhesion during pick-up and that adhesion is necessary for pick-up, 2) adhesion can be assayed quantitatively and is very uniform among control infundibula, and 3) decreasing or increasing adhesion decreases oocyte pick-up rate and in some cases prevents pick-up without affecting ciliary beat frequency.

Light and electron microscopic studies on osmoregulatory tissue in the developing brown shrimp, Penaeus Aztecus

Abstract In larval and early postlarval brown shrimp, Penaeus aztecus , portions of the branchial chamber are lined by a tissue which appears ultrastructurally to be modified for osmoregulation. The distribution of this tissue, the larval stages in which it occurs, and its appearance with the light and electron microscope are presented. The significance of the distribution and ultrastructure of this modified tissue is discussed.

The structure of sperm from the lobster, Homarus americanus.

The structure of sperm from the lobster, Homarus americanus, was examined using light and electron microscopy. These sperm consist of an acrosome, subacrosomal region, collar, nucleus, and three spikes which are extensions of the nucleus. The acrosome is a cylinder projecting from the sperms antierior pole. It is comprised of inner acrosomal material, outer acrosomal material, and an apical cap, and is limited by a single continuous membrane. The subacrosomal material is finely granular with most of it lying in a fossa in the base of the acrosome. The collar, a region subjacent to the subacrosomal material, contains small mitochrondria and a pair of centrioles. The subacrosomal material and collar are in direct continuity with the chromatin of the nucleus. The nucleus itself is limited by a highly folded, thick membrane which probably represents a composite of the plasma membrane and nuclear envelope. The chromatin is granular or fibrillar and is uncondensed. The spikes are compartments of the nucleus and contain microtubules ensheathed by sheets of membrane. Ionophore A23187 which is fluorescent inserts into membranes of cells and was used to study the distribution of spike membranes in sperm with the aid of a fluorescent microscope. The microtubule/membrane complexes of the spikes converge in the region of the collar and interconnect to form a three-sided vault the apex of which terminates immediately beneath the base of the acrosome. These lobster sperm are similar in structure to other reptantian sperm.

An impurity of malathion alters the morphology of rat lung bronchiolar epithelium

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity in technical malathion, caused morphological changes in the bronchiolar epithelium of rat lungs. OOS-treated rat lungs had fewer but larger Clara (non-ciliated) cells than lungs from control rats given either corn oil or purified malathion. Moreover, lactate dehydrogenase (LDH) activity in bronchopulmonary lavage fluid was significantly higher in OOS than in control rats. We interpret these data to mean that OOS, and/or its metabolite(s) causes a lesion in the lung. Because of the widespread agricultural use of technical malathion, future work should address the significance of our findings and the possible toxic effect of OOS on lung tissue.

In vitro fertilization with non-motile spermatozoa of the brown shrimp Penaeus aztecus

The sperm of the brown shrimp Penaeus aztecus are nonflagellated and non-motile. In vitro fertilization can be obtained when mature eggs removed from female shrimp are added to dense sperm suspensions in seawater and agitated in a manner simulating natural spawning.