BIOLOGY OF REPRODUCTION 52, 653-657 (1995)
Male and Female Genomes Associated in a Single Pronucleus in Human Zygotes
JACOB LEVRON,' SANTIAGO MUNNE, STEEN WILLADSEN, ZEV ROSENWAKS
and JACQUES COHEN
The Gamete and Embryo Research Laboratory, Centerfor Reproductive Medicine and Infertility
Department of Obstetrics and Gynecology, New York Hospital, Cornell University Medical Center
New York, New York 10021
ABSTRACT
The ploidy of single-pronucleated human zygotes obtained after conventional in vitro fertilization was determined by fluorescent in situ hybridization (FISH) using multiple simultaneous probes for gonosomes and autosomes. After zona removal the
single-pronucleated zygotes were exposed to cytochalasin B, and the pronucleus, surrounded by scant cytoplasm and the plasma
membrane (karyoplast), was divided from the rest of the egg (cytoplast). The karyoplasts and the corresponding cytoplasts were
analyzed separately by FISH. Of the 16 zygotes analyzed, 10 had haploid pronuclei and 6 were diploid. Four diploid pronuclei
contained XY chromosomes, and 2 contained XX chromosomes. These results suggest that during the course of their interaction,
human gamete nuclei can associate together and form diploid, single-pronucleated zygotes. These findings confirm a newly recognized variation of human pronuclear interaction during syngamy.
INTRODUCTION
therefore usually discarded [15, 16]. Although it has been
suggested recently that pronuclei may develop asynchronously [11, 12], the nature of syngamy in unipronucleate zygotes remains obscure. An appreciation of the cellular
mechanism that underlies this phenomenon is important
for better understanding of the human fertilization process,
as well as for improving the clinical outcome of IVF. This
question has been analyzed here by determining the sex
and ploidy of single-pronucleated zygotes following IVF by
means of pronuclear removal and fluorescent in situ hybridization (FISH) with multiple probes.
The concluding event of the fertilization process in animals is the association of male and female pronuclei at
syngamy. For each species studied so far, syngamy between
the male and female pronucleus follows one of two general
patterns [1-6]. In the first, the maternal and paternal pronuclei become closely associated, sometimes with interdigitations of opposing envelopes, but without actual fusion
before these membranes break down. This prototype of
pronuclear interaction was first described by Wilson in 1925
[1] in the round worm Ascaris, and is characteristic of eggs
that are fertilized prior to completion of meiosis. In the
second pattern, fusion occurs after association of the pronuclei with formation of single-nucleated zygotes. Fertilization in the sea urchin is representative of this pattern of
pronuclear interaction [1, 2], which occurs in eggs that have
completed meiosis at the time of fertilization.
In the human, the late interaction of maternal and paternal pronuclei at syngamy is characterized by tight association of both pronuclear envelopes with close alignment
of the nucleoli [7]. Nonetheless, pronuclear membrane
breakdown and commencement of the first mitotic division
ensues without actual fusion of the pronuclear envelopes
[8, 9]. However, it has been recently demonstrated that normal human embryos can develop from zygotes that manifest a single nucleus after in vitro fertilization (IVF) [1013]. In fact, uninucleate human zygotes are relatively common following that procedure (2-5%) [10-15]; until recently, they were considered to result from either parthenogenetic activation or abnormal fertilization and were
MATERIALS AND METHODS
A total of 16 zygotes presenting clear single pronuclei
and two polar bodies were obtained 14-16 h after insemination from consenting patients undergoing IVF treatment.
These zygotes were investigated under protocols (#0689583 and #0692-654) reviewed by the Human Investigation
Committee of the New York Hospital-Cornell University
Medical College.
All patients received luteal phase GnRH agonist (Lupron;
TAP Pharmaceuticals, Deerfield, IL) followed by ovarian
stimulation with intra-muscular gonadotropins (Metrodine
and Pergonal; Serono, Randolph, MA). Human CG (Serono)
was administered for final maturation, and the patients
underwent the standard IVF procedure [12, 13]. The mean
maternal age was 28.6 ± 7.2 yr (range of 27-37 yr). Before
final selection, the zygotes were assessed at least twice over
a 4-7-h period to ensure the presence of only one pronucleus and extrusion of the second polar body by rolling
them with a glass probe under an inverted microscope at
400x (Hoffman interference optics). The zonae were removed by exposure to 0.25% pronase E (Type XIV, Sigma
Chemical Co., St. Louis, MO) in Dulbecco's PBS (D-PBS) for
5-10 min at room temperature. The zona-free zygotes were
Accepted November 8, 1994.
Received July 27, 1994.
'Correspondence: Gamete and Embryo Research Laboratory, Cornell University
Medical College, P.O. Box 30, 1300 York Avenue, New York, NY 10021. FAX: (212)
746-8589.
653
654
LEVRON ET AL.
FIG. 1. Single-pronucleated human zygote divided into a karyoplast (left) and cytoplast (right). Cytoplasm granularity and irregularity is related to the effect of cytochalasin B.
pipetted through a fine-bore pipette to remove the polar
bodies and were transferred to D-PBS solution containing
10 pLg/ml cytochalasin B for 20 min at room temperature
in order to relax the plasma membrane. The single pronucleus surrounded by the oolemma was drawn into a polished pipette with an opening diameter of 60-70 Vtm by
applying gentle aspiration. The pipette was then rubbed
against a zona-intact egg that was fixed in place by a holding pipette, serving as a cushion for the enucleation procedure. The pronucleus, surrounded by the zygote plasma
membrane, was then divided to form a karyoplast, leaving
the rest of the egg as cytoplast (Fig. 1). The karyoplast and
cytoplast were fixed separately on a slide by use of 1:3 v/v
acetic acid and methanol solution prior to FISH analysis.
Karyoplasts and cytoplasts were analyzed by simultaneous multiple-probe FISH. Probes for X and Y chromosomes in combination with one or more for autosomes (18
and/or 13/21) were used for simultaneous determination
of sex and ploidy [17, 18]. DAPI was used as a counterstain
to detect unlabeled DNA as well as DNA not associated with
the pronucleus.
RESULTS
Sixteen zygotes with one nucleus were successfully analyzed (Table 1). Of the karyoplasts derived from these, 10
were haploid and 6 were diploid. In all zygotes analyzed
all of the nuclear material was confined within a single
structure of DNA matrix. Eight zygotes (numbers 1 to 8)
showed a haploid karyoplast as evidenced by one X, one
18, and/or two 13/21 signals (13/21 is a common probe
for chromosomes 13 and 21), and no DNA signal in the
cytoplasts. Two other zygotes that had haploid karyoplasts
contained DNA in the cytoplasts as well. Zygote 9 had a
haploid karyoplast with one missing signal for 13/21 chromosomes (probably due to FISH failure) and a nucleus in
the cytoplasm displaying a haploid set of X, 18, 13, and 21
chromosome-specific signals, each with a double-dotted
signal indicative of two chromatids per chromosome. Zygote 10 showed a haploid karyoplast, which was paternal
in origin since a Y signal was detected in it, and an additional small haploid nucleus in the cytoplast.
In the other 5 zygotes (numbers 11 to 15), the derived
karyoplasts were diploid and the corresponding cytoplasts
were DNA-free, showing them to be diploid. Two of these
zygotes were XX, and 3 were XY. Finally, zygote 16 also
had diploid karyoplast, but the cytoplast showed an additional nucleus with an XO,18/18 constitution.
DISCUSSION
In the present study, 16 zygotes with only one pronucleus were examined by isolating that pronucleus from the
655
DETERMINATION OF PLOIDY IN SINGLE-PRONUCLEATE HUMAN ZYGOTES
TABLE 1. Ploidy of single nucleated human zygotes after IVF.
FISH signal*
Zygote
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Karyoplast
Cytoplast
Zygote ploidy
XO,180
X0,180,2[13/211
XO,180,2[13/211
XO,180,2[13/211
XO,180,2[13/211
XO,180,2113/211
XO,180,2[13/21
X0,180,2113/211
X0,180,1 [13/211
YO,180,2[13/211
XX,1818,4[13/21]
XY,1818,4113/211
XY,1818,4[13/211
XY,1818
XX,1818
XY,1818
0
0
0
0
0
0
0
0
X0,180,2113/21]'
XO,180,2113/211
0
0
0
0
0
X0,1818
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
7
Diploid
Diploid
Diploid
Diploid
Diploid
Zygote sex
Female
Male
Male
Male
Female
Male
*13/21 is a common probe for chromosomes 13 and 21.
'Each signal had two dots which represent two chromatids of polar body origin.
*Undecondensed nucleus.
FIG. 2. Suggested mechanisms for unipronucleate zygote formation. I. Early fusion: before the formation of pronuclear envelopes. II. Late fusion: after
the formation of pronuclear envelopes.
656
LEVRON ET AL.
rest of the cytoplasm and analyzing it separately for XY, 18,
and 13/21 chromosomes. It was demonstrated that in 10
zygotes the pronucleus was haploid, while in the remaining
6 it was diploid with a male:female sex ratio of 4:2. In 1 of
the 6 diploid zygotes (no. 16 in Table 1), the pronucleus
demonstrated XY, 18,18 signals while the corresponding cytoplast demonstrated additional XO,18,18 signals. Since the
pronucleus was diploid, these cytoplast signals of one gonosome and two autosomes could be related to the polar
bodies or to dispermy where only one spermatozoon participated in syngamy. Among the other zygotes with haploid
karyoplasts, in 2 (nos. 9 and 10 in Table 1), signals were
evident in the corresponding cytoplasts as well. In zygote
9, the latter signals related to the second polar body, since
they were expressed as double-dotted signals that typify its
chromatids (unpublished observations, S. Munne). In zygote 10, the karyoplast was of paternal origin only (YO),
whereas the corresponding cytoplast expressed signals
originating from a compact nucleus that may have represented persistently condensed maternal chromosomes.
It has been proposed that diploid single-pronucleated
zygotes may contain a cryptic pronucleus. This hypothesis
was based on observations of asynchronous pronuclear development in dispermic zygotes [19, 20] and single-pronucleated zygotes after intra-cytoplasmic sperm injection [9],
and on time-dependent pronuclear inflation in bovine oocytes [21]. However, here it is demonstrated that two distinctly different types of single-pronucleated zygotes can
develop after IVF. The first type is parthenogenetic, since
the isolated pronuclei had always an X signal and a haploid
chromosome content and the remaining cytoplast was devoid of nuclear DNA. The second type is monospermic diploid, since the isolation between the karyoplast and the cytoplasm revealed diploid XY- or XX-containing pronuclei.
These findings suggest that the second type of single-pronucleated zygote is produced by a fusion of the paternal
and maternal genomes during the course of syngamy. This
latter phenomenon represents a cellular mechanism that is
considerably different from the normal one (Fig. 2).
The exact sequence involved in the occasional association of the paternal and maternal chromosomes within unipronucleate human zygotes is not clear. Pronuclear fusion
creating single-nucleated zygotes is normal in species where
fertilization occurs after completion of meiosis (e.g., sea urchins) [1, 2]. In humans and most other eutherian mammals
studied, where sperm penetration is the stimulus for completion of meiosis, the pronuclei do not fuse during syngamy but rather stay as separate juxtaposed entities until
nuclear membrane breakdown during the prophase of the
first mitotic division [2,8,9,22-25]. The linkage between
these two patterns is probably related to the time interval
between sperm penetration and its association with the female pronucleus. If this interval is artificially prolonged in
the sea urchin (Arbaica punctulata),syngamy can take on
characteristics typical of that in the Ascaris, i.e., pronuclear
interaction without fusion [26, 27]. In the human egg, the
interval between sperm penetration and association with
the female pronucleus may be affected by the site of penetration in relation to the location of the metaphase spindle. The plasma membrane of the human oocyte appears
to be homogenous throughout its entire surface [28,29],
and penetration close to the metaphase plate may be responsible for this modified form of syngamy. This suggestion is supported by the observation that when the sperm
is injected away from the meiotic spindle during intra-cytoplasmic sperm injection, diploid single-nucleated zygotes
are relatively rare [13]. It is more likely, therefore, that unipronucleate human zygotes are formed by enclosure of
the juxtaposed male and female nuclei in a common pronuclear envelope (Fig. 2, I) rather than by pronuclear
membrane fusion at a later stage (Fig. 2, II).
This and previous studies [12,13] on single-nucleated
human zygotes and embryos suggest that they can be used
for replacement, particularly when other embryos are not
available. At present, however, it is not possible to distinguish between the parthenogenetically activated pronucleus and the diploid one. In the small number of zygotes
analyzed in the present study, we were not able to identify
morphological differences that predict ploidy; but with larger
numbers biometric measurements may prove useful. However, there appears to be an important difference in the
subsequent cleavage performance of the diploid single-nucleated zygotes and the parthenogenetic haploid eggs, which
appear to be selected against during this period. This was
evidenced in the present study by the common incidence
of haploidy at the zygote stage (10 of 16) and its rarity (3
of 21) after three days of culture [12].
In conclusion, we present here what appears to be a
newly recognized variation of human pronuclear association during syngamy. It is likely that this pronuclear behavior represents a normal variant of fertilization, as indicated by previous studies in our laboratory [12, 13] and
also by the delivery of a healthy baby in our program as a
result of such embryo replacement (unpublished data). In
these studies [12, 13], it was postulated that asynchronous
pronuclear inflation may be the mechanism responsible for
the development of diploid embryos from unipronucleate
zygotes. The present findings may suggest, however, that
association of the maternal and paternal genomes in a common pronuclear envelope during the course of syngamy is
more likely to be the underlying mechanism.
ACKNOWLEDGMENTS
We thank embryologists Mina Alikani, Alexis Adler, Cindy Anderson, Adrienne
Reing, Toni Ferrara, Elena Kissin, and Sasha Sadowy for their assistance; Giles Tomkin
for his assistance in preparing the manuscript; and Dr. J. Michael Bedford for his
critical review.
REFERENCES
1. Wilson EB. The Cell in Development and Heredity. New York: Macmillan; 1925.
2. Longo FJ. Fertilization: a comparative ultrastructural review. Biol Reprod 1973;
9:149-215.
DETERMINATION OF PLOIDY IN SINGLE-PRONUCLEATE HUMAN ZYGOTES
3. Austin CR. The Mammalian Egg. Springfield, IL: Charles C. Thomas; 1961.
4. Austin CR, Walton A. Fertilization. In: Parkes AS (ed.), Marshall's Physiology of
Reproduction, Vol. 1, Part 2. London: Longmans, Green; 1960: 360-416.
5. Longo FJ. Regulation of pronuclear development. In: Jagiello G, Vogel C (eds.),
Regulators of Reproduction. New York: Academic Press; 981: 529-557.
6. Longo FJ. Pronuclear events during fertilization. In: Metz CB, Monroy A (eds.)
Biology of Fertilization, Vol 3. Orlando, FL: Academic Press; 1985: 251-258.
7. Wright G, Wiker S, Eisner C, Kort H, Massy J, Mitchell D, Toledo A, Cohen J.
Observations on the morphology of human zygotes, pronuclei and nucleoli and
implications for cryopreservation. Hum Reprod 1990; 5:109-115.
8. Zamboni L, Michell DR, Bell JH, Baca M. Fine structure of the human ovum in
the pronuclear stage. J Cell Biol 1966; 30:579-600.
9. Sathananthan AH, Trounson AO. The human pronuclear ovum: fine structure of
monospermic and polyspermic fertilization in vitro. Gamete Res 1985; 12:385398.
10. Plachot M. Chromosome analysis of oocytes and embryos. In: Verlinsky Y, Kuliev
A (eds.), Preimplantation Genetics. New York: Plenum Press; 1991: 103-112.
11. Staessen C, Janssenwillen C, Devroey P, Van Steirteghem AC. Cytogenetic and
morphological observations of single pronucleated human oocytes after in vitro
fertilization. Hum Reprod 1993; 8:221-223.
12. Munne S, Tang YX, Grifo J, Cohen J. Origin of single pronucleated human zygotes. J Assist Reprod Genet 1993; 10:276-279.
13. Sultan KM, Palermo GD, Alikani M, Cohen J, Rosenwaks Z, Munne S. Ploidy assessment of embryos derived from single pronucleated human zygotes obtained
by regular IVF and intra-cytoplasmic injection. Hum Reprod (in press).
14. Kaufman M. Early Mammalian Development: Parthenogenetic Studies. Cambridge: Cambridge University Press; 1983.
15. Balakier H, Squire J, Casper RF. Characterization of abnormal one pronuclear
human oocytes by morphology, cytogenetics and in situ hybridization. Hum Reprod 1993; 8:402-408.
16. Winston N, Johnson M, Pickering S, Braude P. Parthenogenetic activation and
development of fresh and aged human oocytes. Fertil Steril 1991; 56:904-912.
657
17. Munn6 S, Grifo J, CohenJ, Weier H. Chromosome abnormalities in arrested human preimplantation embryos: a multiple probe fluorescence in situ hybridization (FISH) study. Am J Hum Genet 1994; 55:150-159.
18. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome
aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8:21852191.
19. Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered
cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod
1987; 37:395-401.
20. Kola I, Trounson A Dispermic human fertilization: violation of expected cell
behaviour. In: Schatten H, Schatten G (eds.), The Molecular Biology of Fertilization. San Diego, CA: Academic Press; 1989: 277-293.
21. Xu KP, Greve T. A detailed analysis of early events during in vitro fertilization
of bovine follicular oocytes. J Reprod Fertil 1988; 82:127-134.
22. Gondos B, Bhiraleus P, Conner LA Pronuclear membrane alterations during approximation of pronuclei and initiation of cleavage in the rabbit. J Cell Sci 1972;
10:61-78.
23. Zamboni L. Fine Morphology of Mammalian Fertilization. New York: Harper and
Row; 1971.
24. Yanagimachi R. Requirement of extracellular calcium ions for various stages of
fertilization and fertilization related phenomena in the hamster. Gamete Res 1982;
5:323-344.
25. Zamboni L,ChakrabortyJ, Smith DM. First cleavage division of the mouse zygote:
an ultrastructural study. Biol Reprod 1972; 7:170-193.
26. Longo FJ, Anderson E. A cytological study of the relation of the cortical reaction
to subsequent events of fertilization in the sea urchin Arbaciapunctulata. J Cell
Biol 1970; 47:646-665.
27. Longo FJ, Anderson E. The effects of nicotine on fertilization in the sea urchin
Arbica punctulata. J Cell Blbl 1970; 46:308-325.
28. Dale B. Mechanisms of fertilization. Nature 1987; 325:762-763.
29. Santella L, Alikani M, Talansky BE, Cohen J, Dale B. Is the human oocyte plasma
membrane polarized?. Hum Reprod 1992; 7:999-1003.