The First Polar Body Can Be Used for the Production of Normal Offspring in Mice¹

^a Department of Anatomy and Reproductive Biology, University of Hawaii Medical School, Honolulu, Hawaii 96822 ^b Department of Veterinary Anatomy, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

	ABSTRACT

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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 2-cell embryos were transferred to foster females, 30–57% developed into fertile offspring. This outcome supports a long-standing 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.

	INTRODUCTION

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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.

	MATERIALS AND METHODS

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Animals

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. Animals 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.

Media

Oocytes and fertilized eggs were cultured in a bicarbonate-buffered CZB medium [4] at 37.5°C under 5% CO₂ in air. All oocyte manipulations were carried out in Hepes-buffered CZB (Hepes-CZB) [5] at room temperature (23–25°C) 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 µm for oocyte enucleation and was 7–8 µm 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].

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 37.5°C under 5% CO₂ in air for less than 1 h before further treatments.

Enucleation of Recipient Oocytes

Enucleation of mature oocytes was performed in Hepes-CZB containing 5 µg/ml cytochalasin B [8]. The oocytes were kept in this medium for about 10 min (25°C) 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-µm 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 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).

View larger version (124K): [in this window] [in a new window]   FIG. 1. Live (A) and dead (B) polar bodies (arrows) seen with interference-contrast optics. A', B') 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.

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 pipette. 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 37.5°C 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 (23–25°C) rather than at 17–18°C.

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 of all offspring would have been black (a/a,B/+,C/C,D/+), brown (a/a,b/b,C/C,D/+), and gray (a/a,+/+,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/+,C/C,D/+).

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

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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<<014>>15% of polar bodies were viable for many hours after ovulation.

View larger version (25K): [in this window] [in a new window]   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.

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).

View larger version (189K): [in this window] [in a new window]   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.

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 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).

View larger version (107K): [in this window] [in a new window]   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).

	DISCUSSION

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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 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 µm in diameter) has a relatively large polar body (about 10 µm in diameter). Microsurgical operation as reported here will probably 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 diversity of offspring, provided that oocytes of closely related species are readily available.

View larger version (38K): [in this window] [in a new window]   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.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
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; yana{at}hawaii.edu

Accepted: February 25, 1998.

Received: January 16, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Evsikov AV, Evsikov SV. A study of the first and second polar bodies in mouse oogenesis. Russ J Dev Biol 1995; 26:196–200.
2. Wakayama T, Hayashi Y, Ogura A. Participation of the female pronucleus derived from the second polar body in full embryonic development of mice. J Reprod Fertil 1997; 110:263–266.[Abstract/Free Full Text]
3. Feng JL, Hall JL. Birth of normal mice after electrofusion of the second polar body with the male pronucleus: a possible treatment for oocyte-factor infertility. Am Soc Reprod Med Ann Meet 1997; Abstract P-050.
4. Chatot CL, Torres I, Ziomek CA. Development of 1-cell embryos from different stains of mice in CZB medium. Biol Reprod 1990; 42:432–440.[Abstract]
5. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52:709–720.[Abstract]
6. Hogan B, Costantini F, Lacy E. Manipulating the Mouse Embryo: Laboratory Manual. Section D. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1986: 191.
7. Kimura Y, Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development 1995; 121:2397–2405.[Abstract]
8. Kono T, Ogawa M, Nakahara T. Thymocyte transfer to enucleated oocytes in the mouse. J Reprod Dev 1993; 39:301–307.
9. Yanagida K, Yanagimachi R, Perreault SD, Kleinfeld RG. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes. Biol Reprod 1991; 44:440–447.[Abstract]
10. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod 1996; 55:789–795.[Abstract]
11. Edwards RG, Gates AH. Timing and the stages of maturation divisions, ovulation, fertilization and first cleavage of eggs of adult mice treated with gonadotropins. J Endocrinol 1959; 18:292–304.
12. Ortiz ME, Lucero P, Croxatto HB. Postovulatory aging of human ova: II. Spontaneous division of the first polar body. Gamete Res 1983; 7:269–276.
13. Takase K, Ishikawa M, Hoshiai H. Apoptosis in the degenerated process of unfertilized mouse ova. Tohoku J Exp Med 1995; 175:69–76.[Medline]
14. Choi T, Fukasawa K, Zhou R, Tessarollo L, Borror K, Resau J, Woude GFV. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degeneration of the first polar body in maturing mouse oocytes. Proc Natl Acad Sci USA 1996; 93:7032–7035.[Abstract/Free Full Text]
15. Rodman TC. Chromosomes of the first polar body in mammalian meiosis. Exp Cell Res 1971; 68:205–210.[CrossRef][Medline]
16. Egozcue J. Polar body analysis: possible pitfalls in preconception diagnosis of single gene and chromosome disorder. Hum Reprod 1994; 9:1208.[Free Full Text]
17. Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990; 5:826–829.[Abstract/Free Full Text]
18. Munne S, Dailey T, Sultan KM, Cohen J. The use of the first polar bodies for preimplantation diagnosis of aneuploidy. Mol Hum Reprod 1995; 10:1014–1020.
19. Dyban AP, De Sutter P, Dozortsev D, Verlinsky Y. Visualization of second polar body chromosomes in fertilized and artificially activated mouse oocytes treated with Okadaic acid. J Assist Reprod Genet 1992; 9:572–579.[CrossRef][Medline]
20. Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, White M, Lifchez A, Kaplan B, Moise J, Valle J, Ginsburg N, Storm C, Kuliev A. Polar body diagnosis of common aneuploids by FISH. J Assist Reprod Genet 1996; 13:157–162.[CrossRef][Medline]
21. Angell RR. Polar body analysis: possible pitfalls in preimplantation diagnosis of chromosomal disorders based on polar body analysis. Hum Reprod 1994; 9:181–182.[Free Full Text]
22. Wheeler MB, Noble JA, Jarrell VL. Production of live offspring with predicted genotypes using PCR-RFLP analysis of polar bodies from mouse oocytes. Mol Reprod Dev 1995; 40:267–272.[CrossRef][Medline]