Surgery – Reproduction and fertilization techniques
Reexamination Certificate
1998-10-23
2003-11-04
Paras, Peter (Department: 1632)
Surgery
Reproduction and fertilization techniques
C424S561000, C435S002000, C435S348000, C435S349000, C435S350000, C435S351000, C435S352000, C435S353000, C435S354000, C435S363000, C435S366000, C435S374000
Reexamination Certificate
active
06641526
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the freeze-drying of spermatozoa, the fertilization of oocytes with reconstituted freeze-dried spermatozoa, and the development of live offspring therefrom.
The successful cryopreservation of sperm by the use of cryoprotectants and the ability to store the frozen sperm for long periods of time has produced dramatic improvements in animal husbandry as well as human reproductive medicine. It has been found that cryoprotected frozen-thawed spermatozoa often regain their motility and fertilize almost as efficiently as fresh spermatozoa.
Long term storage of spermatozoa in liquid nitrogen (−196° C.) has been routinely used to cryopreserve cattle and human spermatozoa. However, conventional sperm preservation is very expensive in the long term because of the need for a constant supply of liquid nitrogen. Moreover, in certain areas of the world it may be inconvenient and/or expensive to store spermatozoa in liquid nitrogen, such as in developing countries where liquid nitrogen (or even dry ice) may not be readily accessible. Transporting of conventionally frozen spermatozoa is also problematic in that it may require the transport of large liquid nitrogen tanks or the use of special shipping containers containing liquid nitrogen or dry ice. Thus, many attempts have been made to preserve spermatozoa without requiring storage in liquid nitrogen. For example, if fertilization-competent spermatozoa could be stored in a freeze-dried state at ambient temperatures or in ordinary refrigerators, the reduction in the maintenance and shipping costs could be enormous.
It is believed that the first recorded attempt to freeze-dry spermatozoa was in 1949, when one milliliter (ml) of fowl semen was mixed with an equal volume of Ringer's solution containing 20-30% glycerol, spread as a thin layer in a distillation flask, and “freeze-dried” by removing 90% of the water. When the preparations were rehydrated within 2 hours after being brought back to room temperature, it was reported that up to 50% of spermatozoa regained motility. The fertility of the rehydrated spermatozoa, however, was never determined.
Subsequent attempts to produce live offspring using freeze-dried spermatozoa have not been successful. It has been reported, for example, that a live calf was born following artificial insemination of a cow with bull spermatozoa that were reconstituted immediately after freeze-drying and showed 50% sperm motility. It was also reported that twelve litters of normal rabbits were obtained by insemination using freeze-dried spermatozoa. However, neither of these results could be repeated by the originators or duplicated and confirmed by others working in the field.
From studies of the properties and fertility of normal and frozen-thawed spermatozoa, it is known that spermatozoa are not required to be “alive” in the conventional sense (i.e., having intact plasma membranes) in order for them to support normal embryonic development. For example, in an intracytoplasmic sperm injection (ICSI) technique, motile human spermatozoa are selected. They are immobilized (“killed”) immediately before injection into an oocyte by aggressive abrasion of their tails resulting in damage to the plasma membrane. It has been reported that sperm immobilization significantly increases the rate of successful fertilization by ICSI. It has also been reported that when mouse spermatozoa were suspended in media without any cryoprotectant, then immediately frozen in liquid nitrogen, 100% of the spermatozoa were “dead” as judged by live/dead cell staining, and yet normal embryonic development occurred after microinjection of the heads of the thawed spermatozoa into oocytes. There has also been a report of the birth of two normal calves after microinjection into oocytes of spermatozoa killed by freeze-thawing without any cryoprotectants.
Although it is known that freeze-dried hamster and human sperm heads injected into hamster oocytes can form normal looking pronuclei, it has never been determined whether such sperm heads can support normal embryonic development. Moreover, it has been demonstrated that loss of motility, acrosome damage and enzyme release occur when spermatozoa are frozen and dried to a moisture content of less than 30%, 7% and 0.5%, respectively. Evidence has also been obtained that cellular proteins in spermatozoa are altered by dehydration below 6% moisture.
In view of the foregoing, there is a need for a reliable and reproducible method for freeze-drying spermatozoa, by which the freeze-dried sperm retain their fertilization competency during long periods of storage at ambient, or ordinary refrigerator temperatures or below. There is a further need for a method for sperm injection that employs rehydrated freeze-dried sperm to fertilize recipient oocytes, resulting in normal live offspring.
SUMMARY OF THE INVENTION
The invention provides a method for freeze-drying spermatozoa to obtain at least one reconstituted spermatozoon whose head (nucleus) is capable of fertilizing an oocyte to produce a live offspring. The method of the invention comprises the steps of: (a) collecting live spermatozoa; (b) suspending the spermatozoa in a suspension medium; (c) freezing the suspension of spermatozoa; and (d) drying the suspension of spermatozoa to a moisture level that is less than 1%, preferably less than 0.01%, more preferably less than 0.001%, and especially less than 0.00001%. The method may further comprise the step of rehydrating the freeze-dried suspension of spermatozoa, wherein at least one rehydrated spermatozoon head retains its genetic integrity and is capable of fertilizing an oocyte to produce a live offspring. As discussed below, the method may further comprise the step of storing the freeze-dried spermatozoa prior to rehydrating.
To obtain a live offspring, at least the head (nucleus) of a rehydrated spermatozoon is inserted into an isolated oocyte to form a fertilized oocyte. The sperm head is inserted into the oocyte by microinjection, preferably by piezo electrically-actuated microinjection. Preferably, the insertion of the nucleus takes place within one hour of rehydration. The fertilized oocyte is then allowed to develop into an embryo and implanted into the uterus of a foster mother where it develops into a live offspring.
In some species (e.g., most eutherian mammals, including humans), normal embryonic development of the fertilized oocyte also requires the insertion of the paternal centrosome associated with the sperm nucleus. The centrosome is normally attached either to the posterior end of the sperm head or to the anterior end of the sperm tail when the head and tail are separated. Thus, in embodiments of the invention, a sperm-associated centrosome from another spermatozoon may be inserted simultaneously with the sperm head, or may be inserted by means of simultaneous or consecutive insertion of the sperm tail. Alternatively, insertion of the sperm nucleus and centrosome may be accomplished by insertion of an entire rehydrated spermatozoon into the oocyte.
It has been discovered that spermatozoa that are freeze-dried by the method of the invention may be stored for at least three months or, more preferably, for at least a year, at temperatures ranging from ambient (e.g., room temperature) to refrigerator temperatures (e.g., about 4° C.) without losing their genetic integrity or fertilization capability. Thus, the freeze-dried spermatozoa prepared by the method of the invention, can retain their competency during shipping to virtually anywhere in the world at ambient or refrigerator temperatures, and may also undergo short term storage at ambient or refrigerator temperatures in locations where liquid nitrogen or dry ice are not readily available. Preferably, long term (e.g., indefinite) storage of the freeze-dried sperm takes place at temperatures of less than 4° C. (e.g., −20° C. or below).
The method of the invention may be used to freeze-dry spermatozoa of both invertebrate and vertebrate species including, but not limited to, in
Wakayama Teruhiko
Yanagimachi Ryuzo
Day Jones
Paras Peter
University of Hawai'i
LandOfFree
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