Use of micromanipulation and in vitro fertilization to investigate gamete interactions in the mouse.
Item
-
Title
-
Use of micromanipulation and in vitro fertilization to investigate gamete interactions in the mouse.
-
Identifier
-
AAI8821125
-
identifier
-
8821125
-
Creator
-
Talansky, Beth Ellen.
-
Contributor
-
Adviser: Jon W. Gordon
-
Date
-
1988
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, General
-
Abstract
-
In vitro fertilization and gamete micromanipulation can bypass one or more steps in mammalian fertilization and thereby allow for detailed study of the events involved in gamete fusion. In this thesis, these techniques were applied to a mouse model. Acrosome-reacted spermatozoa were inserted under the zona pellucida to determine if normal processes of sperm penetration are required for fertilization. Fusion did not occur after sperm were inserted in the perivitelline space, and the findings further suggested that mechanical manipulation of the flagellum resulted in membrane changes which rendered the sperm incapable of fertilisation. To determine if these changes were based on alterations of membrane potential, sperm were exposed to Na{dollar}\sp+{dollar}-K{dollar}\sp+{dollar} ATPase inhibitors which, at the concentrations used, would not interfere with motility, but would alter membrane potential. ATPase inhibitors blocked fertilization, results which indicate that a membrane potential is required for gamete fusion in the mouse. Because ATPase inhibitors are known to block the acrosome reaction, we induced the acrosome reaction prior to exposure. These experiments led to the novel observation that mouse sperm which have completed the acrosome reaction prior to zona binding cannot fertilize the egg.;Difficulties in fertilizing oocytes by sperm microinjection led us to devise a new approach for improving fertilization in vitro. A microneedle was used to dissolve a small hole in the zona. This "zona drilling" led to increased fertilization rates, even at reduced sperm counts. Polyspermy was rare, and transfer of embryos fertilized after drilling resulted in birth of normal live young. In a related study, oocytes were fertilized after drilling, cultured in vitro to blastocysts, and compared to controls with respect to sperm binding, time of fertilization, and patterns of cleavage development. Significant differences characteristic of the drilled embryos were observed at each stage.;In summary, the data in this dissertation identify difficulties with fertilization by sperm microinjection, elucidate the sequence of events required for sperm penetration in the mouse, indicate that the sperm membrane is important to gamete fusion, establish a new method for improving in vitro fertilization, and characterize fertilization and cleavage of eggs subjected to zona pellucida drilling.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
