BIOLOGY OF REPRODUCTION 59, 100–104 (1998)
The First Polar Body Can Be Used for the Production of Normal Offspring in Mice1
Teruhiko Wakayama3,4 and R. Yanagimachi2,3
Department of Anatomy and Reproductive Biology,3 University of Hawaii Medical School, Honolulu, Hawaii 96822
Department of Veterinary Anatomy,4 Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT
imals used in this study were maintained in accordance with
the guidelines of the Laboratory Animal Service at the University of Hawaii and with those prepared by the Committee on Care and Use of Laboratory Animals of the Institute
of Laboratory Resources National Research Council
(DHEW publication no. [NIH] 80–23, revised in 1985).
The protocol of our animal handling and treatments was
reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
The purpose of this study was to determine whether chromosomes in the first polar body can participate in normal embryonic development. In the mouse the majority of first polar
bodies degenerate soon after ovulation, but a few remain viable
for 10 h or more. When the contents of a live polar body were
injected into an enucleated mature oocyte and examined 2 h
later, the polar body chromosomes were arranged on a metaphase plate as seen prior to the secondary meiotic division. Such
oocytes were fertilized normally by sperm injection. When 2cell embryos were transferred to foster females, 30–57% developed into fertile offspring. This outcome supports a longstanding belief that chromosomes ejected into the first polar
body have the same genetic potential as those remaining in the
oocyte after the first meiotic division. As the chromosomes in
the second polar body are known to have full potential to participate in normal embryonic development, it is theoretically
possible to reproduce four offspring by using chromosomes in
one oocyte.
Media
Oocytes and fertilized eggs were cultured in a bicarbonate-buffered CZB medium [4] at 37.58C under 5% CO2 in
air. All oocyte manipulations were carried out in Hepes-buffered CZB (Hepes-CZB) [5] at room temperature (23–258C)
in air. The pH of both media was approximately 7.4.
Micromanipulation
Oocyte-holding and injecting pipettes were prepared according to Hogan et al. [6] except that the tip of the injection pipette was left flush after breaking. The inner diameter
of this pipette at its tip was approximately 10 mm for oocyte
enucleation and was 7–8 mm for suction and injection of
the first polar body. The injection pipette was attached to
a piezo electric pipette-driving unit (Prima Meat Packers,
Tsuchiura, Japan). Drilling of the zona pellucida and the
injection of polar body (or a spermatozoon) into oocytes
were performed as described previously [5, 7].
INTRODUCTION
The mammalian primary oocyte emits the first polar
body before ovulation. The second polar body is extruded
from the oocyte as a consequence of oocyte activation triggered by the fertilizing spermatozoon. Normally, the chromosomes within both the first and the second polar bodies
degenerate without contributing to embryonic development.
Speculation that polar body chromosomes have the same
genetic potential as their sister chromosomes remaining in
the oocyte [1] was proven experimentally by Wakayama et
al. [2] and Feng and Hall [3], who obtained live mouse
offspring by fusing the second polar bodies with fertilized
eggs from which female pronuclei had been removed. We
report here that chromosomes in the first polar body are
also able to participate in normal embryonic development
if they are allowed to complete the second meiotic division
within an enucleated oocyte, then allowed to mingle with
chromosomes of an injected spermatozoon.
Preparation of Recipient Oocytes
B6D2F1 females were superovulated by consecutive injections of eCG (5 IU) and hCG (5 IU) 48 h apart. About
14 h after hCG injection, oocyte-cumulus complexes were
released from oviducts into Hepes-CZB. Cumulus cells
were dispersed by 5-min treatment with 0.1% bovine testicular hyaluronidase (300 USP units/mg; ICN Pharmaceuticals, Costa Mesa, CA) in Hepes-CZB. Cumulus-free oocytes were kept in CZB at 37.58C under 5% CO2 in air for
less than 1 h before further treatments.
MATERIALS AND METHODS
Animals
Enucleation of Recipient Oocytes
B6D2F1 female mice (black), 8–10 wk old, were used
as the donors of oocytes and polar bodies. C3H females
(agouti; 10 wk old) and CD1 females (albino; 10–15 wk
old) were also used as polar body donors. Spermatozoa
were collected from caudae epididymides of B6D2F1
males, 10 wk old. Foster mothers were CD1 females. An-
Enucleation of mature oocytes was performed in HepesCZB containing 5 mg/ml cytochalasin B [8]. The oocytes
were kept in this medium for about 10 min (258C) before
enucleation. An oocyte, held by a holding pipette, was rotated until detection of a small, translucent ooplasmic
spot—the location of metaphase II chromosomes. After the
zona pellucida was drilled with the enucleation pipette
(about 10-mm inner diameter) by application of a few piezo
pulses [5], its tip was advanced until it reached the translucent spot in the ooplasm. The translucent ooplasm (with
metaphase II chromosomes) was sucked into the pipette
without breaking the plasma membrane and was gently
pulled away from the oocyte until a stretched cytoplasmic
Accepted February 25, 1998.
Received January 16, 1998.
1
Supported by NIH grants (HD-03402 and HD-34362). T.W. was the
recipient of a postdoctoral fellowship from the Japanese Association of
Promotion of Science.
2
Correspondence: R. Yanagimachi, Department of Anatomy and Reproductive Biology, University of Hawaii Medical School, Honolulu, HI
96822. FAX: (808) 956-5474; e-mail: [email protected]
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FIRST POLAR BODY
101
FIG. 1. Live (A) and dead (B) polar bodies (arrows) seen with interference-contrast optics. A9, B9) Same as above, but seen with phase-contrast optics.
Live polar bodies with intact plasma membranes have relatively clear cytoplasm, whereas dead polar bodies, with broken or missing plasma membranes,
have granular cytoplasm.
bridge was pinched off. As assessed by fixing and staining
of the oocytes [9] or Hoechst 33342 staining, the efficiency
of enucleation was 100%.
Identification of ‘‘Live’’ and ‘‘Dead’’ First Polar Bodies
Oviductal oocytes were collected from B6D2F1, CD1,
and C3H females between 13 and 27 h after hCG injection.
The viability of polar bodies was assessed using a commercially available cell viability test kit (Live/dead
FertiLight; Molecular Probes, Inc., Eugene, OR) that differentiates between plasma membrane-intact (‘‘live’’) and
damaged (‘‘dead’’) cells according to the fluorescence
staining pattern under a UV microscope. The chromosomes
in live polar bodies with intact plasma membranes fluoresced green whereas those in dead polar bodies fluoresced
bright orange-red. The results of our primary observations
revealed that all live polar bodies had a sharply defined,
smooth membrane and clear cytoplasm (Fig. 1A). Their
chromosomes were scattered, stretched, or adherent to each
other. Some dead polar bodies had a smooth plasma membrane, but in most the membrane was rough or missing.
The most easily recognizable feature of the dead polar body
was a very granulated cytoplasm, regardless of the state of
the plasma membranes and chromosomes (Fig. 1B).
Transfer of First Polar Body Chromosomes into
Enucleated Oocytes and Subsequent Sperm Injection
The technique for transfer and injection was similar to
that used for injection of spermatid nuclei into oocytes [7].
An oocyte with a live first polar body was selected, and its
zona pellucida was drilled with a piezo-driven injection pi-
pette. The plasma membrane of the polar body was broken
by sucking it into the pipette. The entire contents of the
broken polar body were immediately injected into an enucleated oocyte. In a separate series of experiments, the entire contents of a dead polar body were injected. Polar
body-injected oocytes were incubated in CZB for 2 h at
37.58C under 5% CO2 in air before a second injection of a
spermatozoon. Immediately before sperm injection, individual spermatozoa were decapitated by applying a few piezo pulses to the neck region. A single sperm head was
injected into each oocyte as described by Kuretake et al.
[10] except that the operation was performed at room temperature (23–258C) rather than at 17–188C.
Oocyte Examination and Embryo Transfer
Some oocytes were examined between 10 min and 2 h
after injection to determine how polar body chromosomes
behaved within the oocyte’s cytoplasm. Other oocytes were
examined 5–6 h later for incidence of normal fertilization.
Those with one second polar body and two pronuclei were
considered normally fertilized and were cultured in CZB
overnight. Regular 2-cell stage embryos were then transferred into oviducts of recipient females that had been mated with vasectomized males during the previous night.
In a series of experiments, C3H mice were used as polar
body donors. All the recipient oocytes and spermatozoa
were from B6D2F1 mice. C3H mice are homologous in
four hair color genes, A (agouti), B (brown), C (albino),
and D (dilute), i.e., A/A,B/B,C/C,D/D. B6D2F1 mice are a/
a,B/b,C/C,D/d. If enucleation of B6D2F1 oocytes had failed
and they were fertilized by B6D2F1 spermatozoa, the coats
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WAKAYAMA AND YANAGIMACHI
FIG. 2. Percentages of live polar bodies at various times after ovulation
in a hybrid strain (B6D2F1), an outbred strain (CD-1) and an inbred strain
(C3H) of the mouse.
of all offspring would have been black (a/a,B/1,C/C,D/1),
brown (a/a,b/b,C/C,D/1), and gray (a/a,1/1,C/C, d/d), but
not agouti or white in color. If only C3H polar body chromosomes and B6D2F1 sperm chromosomes had participated in the development of enucleated oocytes, all offspring
would be expected to have agouti coats (A/a,B/1,C/C,D/
1).
On the 19th day postcoitum, recipient females without
apparent signs of pregnancy were killed and their uteri were
examined for the presence of fetuses. Cesarean section was
necessary because a recipient female carrying # 2 fetuses
was unable to deliver by herself. Live fetuses, if any, were
raised by lactating CD1 (albino) foster females. Other females were allowed to deliver and raise their offspring.
RESULTS
Figure 2 shows the proportion of viable first polar bodies
at various times after hCG injection. As ovulation in the
mouse occurs between 10 and 14 h after hCG injection
[11], most first polar bodies seem to have degenerated before or soon after ovulation. Nevertheless, 5–15% of polar
bodies were viable for many hours after ovulation.
When a live first polar body was injected into an enucleated oocyte, the polar body chromosomes aggregated
gradually (Fig. 3, A and B). By 2 h after injection, the
chromosomes were arranged on the metaphase plate of the
second meiotic division (Fig. 3C). Chromosomes in dead
polar bodies never transformed into typical metaphase chromosomes (Fig. 3D).
When a total of 171 enucleated oocytes were injected
with live first polar bodies, about half survived (Table 1).
After single sperm injection (Fig. 4A), the majority of the
oocytes were fertilized normally (Fig. 4B) regardless of the
postovulatory age of the polar bodies (Table 1). Transfer of
74 two-cell embryos to 11 foster mothers resulted in the
FIG. 3. Transformation of the first polar body chromosomes into metaphase II chromosomes within enucleated oocytes. A) Shortly after injection into the oocyte; chromosomes are scattered. B, C) Chromosomes
gradually aggregate and are arranged on the metaphase plate by 2 h after
injection. D) Chromosomes in the dead polar body, injected into the oocyte, cannot be organized to form chromosome-spindle complex.
birth of 27 normal offspring (Table 1). Of these, 3 were
born by cesarean section of two females. Others were delivered naturally. All 27 offspring were raised, and mating
among them was carried out. All 15 females became pregnant and had litters of normal size (8–12).
DISCUSSION
The present results show that chromosomes within the
first polar body have the same genetic and reproductive
potentials as those left in the oocyte after the first meiotic
division. Chromosomes within dead polar bodies did not
become organized after injection into oocytes. Chromosomes in live polar bodies were scattered, stretched, or aggregated. We do not know whether there is a causal relationship between the state of chromosomes within the polar
body and the success/failure of embryonic development after injection.
Under normal conditions, the first polar body of the
mouse degenerates rather quickly. According to Evsikov
and Evsikov [1], more than half degenerate within a few
hours after ovulation, and the vast majority disintegrate during the next 12 h. In humans, by contrast, many first polar
bodies persist for more than 20 h after ovulation [12]. In
TABLE 1. Development of mouse enucleated oocytes injected with first polar body chromosomes (Pb1c) and spermatozoa.
Source of Pb1c
B6D2F1
C3H
Time after hCG
injection when
Pb1c were
collected
15 h
20 h
15 h
No. of enucleated oocytes
Total
Surviving
after Pb1c
injection (%)
Normally
fertilized after
sperm injection
No. of 2-cell
embryos transferred
(no. of
foster mothers)
No. of live
offspring (%)
61
85
25
35 (57)
41 (48)
16 (64)
30
39
10
28 (6)
36 (3)
10 (2)
16 (57)
18 (50)
3 (30)
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FIRST POLAR BODY
FIG. 4. Fertilization of the oocyte with the first polar body chromosomes
instead of its own chromosomes. A) This oocyte was enucleated, and then
first polar body chromosomes were transferred and then injected with a
sperm head. This photograph was taken about 10 min after sperm injection; an arrow indicates a sperm head within the oocyte; c indicates chromosomes of the first polar body origin. B) An oocyte (egg) at 5 h after
sperm injection, with two pronuclei and one (second) polar body (arrow).
general, however, the first polar body in eutherian mammals
has a shorter life than the second polar body. Although
degeneration of polar bodies (and of unfertilized oocytes as
well) is likely to be an apoptotic process [13, 14], the factors that determine the individual and species differences
in the degeneration rates of polar bodies (and of unfertilized
oocytes) are not understood.
As shown here, live first polar body chromosomes are
capable of undergoing the second meiosis after transfer into
mature oocytes regardless of their postovulatory age. Damaged polar body chromosomes, as shown by Rodman [15],
are apparently unable to do so. Thus, chromosomes within
live polar bodies that are destined to degenerate can be
rescued by being transferred into the ooplasm.
The rate of oocyte survival after polar body injection
was not very high (see Table 1), perhaps because of the
large size of the injection pipette. Oocyte survival rate can
probably be increased through technical improvements.
Moreover, the mouse oocyte (about 75 mm in diameter) has
a relatively large polar body (about 10 mm in diameter).
Microsurgical operation as reported here will probably
103
FIG. 5. This diagram illustrates the production of four offspring using the
chromosomes of a single oocyte. The first polar body of oocyte A, from
animal 1 (colored), is transferred into oocyte C from animal 2 (albino),
which has been enucleated. Here, the number (2n, n) refers to ploidy of
chromosomes rather than centromere number per se. After the first polar
body chromosomes (PB I) transform into metaphase II chromosomes, a
single sperm head is injected. This oocyte is activated, forms two pronuclei and the second polar body (PB II), and eventually develops into
offspring C. Oocyte D is first enucleated, then injected with a single sperm
head. After the sperm head transforms into a pronucleus in the activated
oocyte, a second polar body of oocyte C is injected. This oocyte develops
into offspring D. Offspring A develops from oocyte A of animal 1. Offspring B is produced by transferring the second polar body of oocyte A
into oocyte B with the sperm pronucleus only.
prove easier when polar bodies are smaller relative to the
size of the oocyte, such as in animals (e.g., cattle) and in
humans.
Although technically more difficult than blastomere
chromosome analyses because of the smaller size of polar
bodies [16], polar body chromosome analyses have been
used extensively in preconception genetic diagnosis in humans [17–20]. Since polar bodies are readily available, genetic diagnosis using polar bodies would remain valuable
if we kept possible pitfalls in mind [21]. For experimental
animals, genetic diagnosis using polar bodies would be useful in marker-assisted selection of female gametes and identification of transgenic oocytes, for example [22].
Since it is now possible to use both the first and the
second polar body for production of fertile offspring, the
genetic information in one oocyte can be transmitted to four
offspring (Fig. 5). As donor oocytes are enucleated prior to
transfer of polar body chromosomes, all offspring will have
no genetic influence from oocyte donors other than from
maternal (oocyte) mitochondria. Because each oocyte receives a different spermatozoon, this mode of reproduction
does not represent cloning. Four oocytes receive different
maternal and paternal genes. In an extreme situation in
which only a few females of a species remain, the use of
polar bodies in this way might help to increase the genetic
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WAKAYAMA AND YANAGIMACHI
diversity of offspring, provided that oocytes of closely related species are readily available.
11.
ACKNOWLEDGMENTS
12.
Special appreciation is expressed to Dr. J. Michael Bedford for reviewing an early version of the manuscript. We thank Mrs. Charlotte Oser for
her assistance in the preparation of the manuscript.
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