c Indian Academy of Sciences
REVIEW ARTICLE
The potential significance of binovular follicles and binucleate
giant oocytes for the development of genetic abnormalities
BERND ROSENBUSCH∗
Department of Gynaecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany
Abstract
Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid
after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first
meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the
occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined
as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined
oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both
female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes
retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase
II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or
triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy
whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism,
chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles
is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the
formation of multiovular follicles.
[Rosenbusch B. 2012 The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic
abnormalities. J. Genet. 91, 397–404]
Introduction
A wealth of information on oocyte fertilization and photographic evidence of early developmental stages has been
accumulated since the advent of human-assisted reproduction (Veeck 1999). Undoubtedly, the techniques of in vitro
fertilization have also helped to shed light on irregularities
during gamete development. Normally, a follicle should contain only one diploid female gamete, resulting in a haploid secondary oocyte after first meiotic division. Conjoined
oocytes arising from binovular follicles and binucleate giant
oocytes are notable exceptions from this rule. Both phenomena are sporadically observed in programmes of in vitro
fertilization but giant oocytes have received more atten-
∗ E-mail: [email protected].
tion concerning their relevance for assisted reproductive outcome. They are now recognized as a potential source of
chromosomal disorders in the embryo (Balakier et al. 2002;
Rosenbusch et al. 2002) and it is recommended to exclude
them from in vitro fertilization attempts. At least, the study
by Machtinger et al. (2011) has suggested that the random
formation of giant oocytes does not affect the genetic constitution of other gametes. In contrast, our knowledge on
conjoined oocytes is restricted to a few case reports, indicating that some of the fertilized gametes have been transferred without or after mechanical separation (Rosenbusch
and Hancke 2012). Pregnancies have not been reported from
these transfers.
The present review outlines the origin, occurrence and
biological significance of binovular follicles and binucleate oocytes with particular emphasis on possible genetic
abnormalities arising from fertilization of the corresponding
gametes.
Keywords. binovular follicles; binucleate giant oocytes; chimaeras; mosaicism; tetraploidy; triploidy.
Journal of Genetics, Vol. 91, No. 3, December 2012
397
Bernd Rosenbusch
Table 1. Selected examples for describing the occurrence of more than one oocyte per follicle in mammals.
Term or description
Investigated species
References
Bi(n)ovular follicle(s)
Human
Biovularity
Binovular zona pellucida
Human
Human
Papadaki (1978), Zeilmaker et al. (1983),
Ben-Rafael et al. (1987), Manivel et al. (1988),
Ron-El et al. (1990)
Muretto et al. (2001)
Safran et al. (1998), Vicdan et al. (1999),
Kousehlar et al. (2009)
Payan-Carreira and Pires (2008)
Gougeon (1981)
Kent (1960, 1962a,b), Collins and Kent (1964),
Thompson and Zamboni (1975),
Telfer and Gosden (1987), Al-Mufti et al. (1988),
Dandekar et al. (1988), Stankiewicz et al. (2009)
Sherrer et al. (1977)
Hartshorne et al. (1990)
Ben-Rafael et al. (1987), Fishel et al. (1989)
Multioocyte or multioocytic follicle(s)
Multiovular follicle(s)
Polyovular follicle(s)
Dog
Human
Mouse, rat, hamster, guinea,
pig, rabbit, cat, dog, pig,
rhesus monkey, human
Polynuclear follicle(s)
Two oocytes with fused zonae pellucidae
Two oocytes surrounded by a single zona pellucida
Human
Human
Human
Follicular development and fertilization
of the oocyte
The cortical region of the ovary harbours follicles in different developmental stages. Primordial follicles start to grow
under the influence of sex hormones during the follicular
phase of the menstrual cycle, and this involves a restructuring
of the follicular epithelium, formation of the zona pellucida,
and completion of the first meiotic division in the oocyte. The
growing follicle normally contains only one oocyte. During
release from the tertiary (or Graafian) follicle, this oocyte is
arrested at metaphase II (MII) and surrounded by the cumulus oophorus. The MII stage is characterized by the presence
of the first polar body (PB), resulting from an unequal division of the ooplasm. As a consequence of meiosis, oocyte
and first PB carry a haploid chromosome set (23,X). Only
after sperm penetration will second meiotic division be continued, leading to a separation of oocyte chromosomes into
single chromatids, extrusion of 23 chromatids into the second PB and inclusion of the remaining chromatids within the
membrane of the female pronucleus. Simultaneously, a paternal pronucleus is formed containing the decondensed sperm
chromatin. DNA replication inside of the two pronuclei and
disintegration of the pronuclear membranes leads to a diploid
one-cell zygote that cleaves mitotically into an embryo.
fusion of their zona pellucida in a defined region. For reasons
explained below, only conjoined oocytes have been included
as examples for true binovularity in the present review.
Binucleate (or binuclear) giant oocytes are sometimes
simply referred to as giant oocytes (Balakier et al. 2002;
Rosenbusch et al. 2002). The term denotes the presence of
two nuclei that are visible in the immature germinal vesicle (GV) stage. In addition, these gametes are larger than
normal ones. The mean diameter of a preovulatory oocyte
including the zona pellucida is ∼150 μm whereas it reaches
∼200 μm in giant oocytes. Therefore, the volume of giant
oocytes was estimated to be ∼1.6-fold larger than normal
(Balakier et al. 2002). Multinuclear oocytes with more than
two nuclei have been found in human ovaries (see below) but
were not observed during assisted reproduction. Normally,
the appearance of binucleate oocytes is associated with the
detection of polyovular follicles in a given species (Hartman
1926).
The terms ‘binucleate/binuclear giant oocytes’ and those
listed in table 1 for multiovular follicles were used as keywords for a computer-based literature search using PubMed
and Scopus to identify relevant literature.
Polyovular follicles and multinucleate oocytes
in the ovary
Nomenclature and definitions
In the following, ‘uniovular follicle’ indicates a follicle that
contains a single oocyte. Follicles with more than one oocyte
have been described by a variety of terms. Without claiming completeness, table 1 gives a survey on corresponding
expressions and also some examples for species in which
multi-ovular or poly-ovular follicles occur. In human, the
majority of these unusual follicles are binovular, i.e. they harbour two oocytes. The most striking examples are conjoined
oocytes that may share a common zona pellucida or show a
398
In a survey on the occurrence of polynuclear ova and polyovular follicles in mammals, Hartman (1926) described the
existing literature on this topic as ‘somewhat extensive’.
Nowadays, it might be astonishing to read this comment in a
review published in 1926 but in fact, histological studies of
the ovary and its inherent abnormalities date back to the 19th
century. These early investigations have already shown that
polynuclear ova and polyovular follicles may appear in very
great numbers in certain individuals of any species and that
the ‘doubling of nuclei and of ova is the most common form
Journal of Genetics, Vol. 91, No. 3, December 2012
Binovular follicles and binucleate oocytes
of these atypical follicles’ (Hartman 1926). Later, the systematic analysis of 117 ovaries by Gougeon (1981) revealed that
multinuclear oocytes and multiovular follicles are present in
98% of 18 to 52-year-old women. The incidence of these
structures varied between 0.06% and 2.44% of the total follicular population of the ovary. In detail, 29% of the patients
(34/117) had only multiovular follicles, 13% (15/117) only
multinuclear oocytes, and 56% (65/117) a combination of
multiovular follicles and multinuclear oocytes. The relative
frequency of both features was not affected by the use of oral
contraceptives, ovulation inducers, the day of the menstrual
cycle and pregnancy. In contrast to Sherrer et al. (1977), who
did not observe ‘polynuclear’ follicles in women older than
40 years, Gougeon (1981) also failed to detect an influence
of age.
Polyovular follicles
As reported by Gougeon (1981), most of the polyovular follicles (97.1%) contained two oocytes whereas triovular follicles or follicles with more than three oocytes were rare
(2.5% and 0.4%, respectively). Three different assumptions
have been discussed to explain the formation of follicles
containing more than one gamete: i) division of a polynuclear oocyte, ii) fusion of several individual follicles, and
iii) nonseparation of several oocytes at the time of nidation
of oogonia in the sexual cords (Reynaud et al. 2010). The
latter alternative is nowadays considered the most probable
(Vicdan et al. 1999; Reynaud et al. 2010), giving reason to
the concept that polyovular follicles do not represent a pathological phenomenon but a natural polymorphism (Telfer and
Gosden 1987; Reynaud et al. 2010). According to Reynaud
et al. (2010), this polymorphism could favourably be
described as an imbalance between the number of germ and
somatic cells.
It had already been assumed several decades ago that
exogenous gonadotropin stimulation may promote the inclusion of two or more oocytes in a single follicle (Jones 1968)
but clarifying this question obviously requires comparative
histological studies of stimulated and unstimulated ovaries.
At least, the suggested possibility for occasional ovulation of polyovular follicles (Gougeon 1981) could be confirmed by single observations during assisted reproduction
(Rosenbusch and Hancke 2012). Of note, further points at
issue are the association of binovular follicles with polycystic
ovaries (Uebele-Kallhardt and Knoerr 1975; Shettles 1981)
and their role in neoplastic transformation or development of
ovarian teratomas (Manivel et al. 1988; Muretto et al. 2001).
The different patterns of zonal fusion in conjoined oocytes
may be explained by slightly varying distances between the
two precursor cells that subsequently grow and undergo zona
formation (Rosenbusch and Hancke 2012). However, this
would imply that some binovular follicles can contain two
oocytes connected by more or less distinct layers of cumulus cells without zonal fusion. Observations supporting this
view have obviously been made by Ron-El et al. (1990).
Their report includes nine cases of two adjacent oocyte–
cumulus complexes that could easily be separated mechanically and one example for inclusion of two oocytes with
individual corona radiata within a single cumulus complex.
Together with five cases of conjoined oocytes, the overall
frequency of binovular follicles in the study of Ron-El et al.
(1990) amounts to 0.3% (15/4695) what deviates conspicuously from the rate of 8% (76/898) indicated by Dandekar
et al. (1988) for polyovular follicles. According to Dandekar
et al. (1988), there even were five cases with three or four
oocytes but photographic evidence for these findings has
unfortunately not been provided. The divergent data of the
two studies might be due to different methods of oocyte
retrieval: laparoscopy (Dandekar et al. 1988) versus vaginal
ultrasound in 89% of the cases (Ron-El et al. 1990). Ultrasound has been considered more accurate for identifying an
individual follicle (Ron-El et al. 1990), but of course, aspiration of oocytes from two different uniovular follicles can
never definitively be excluded.
Not only because of this uncertainty has the present compilation focussed on conjoined oocytes as the most reliable
indication for binovular follicles. Moreover, the data given
in two previous studies (Dandekar et al. 1988; Ron-El et al.
1990) for attached oocyte–cumulus complexes could not be
used for verifying their further development because information on oocyte maturity and fertilizability was unclear or
incomplete. Finally, in Hartman’s review (1926) there is an
interesting distinction between different types of polyovular
follicles based on histological studies. Type I refers to follicles containing more than two oocytes that are separated by
granulosa cells. In contrast, binovular follicles would usually
correspond to type II characterized by mutual contact of the
gametes over large surfaces (Hartman 1926). This supports
the opinion that in the vast majority of cases, oocytes with a
common or conjoined zona pellucida represent the gametes
grown in binovular follicles.
Multinucleate oocytes
In Gougeon’s (1981) examination of human ovarian biopsies, the observed multinucleate oocytes were mostly binuclear (96.1%) and the corresponding primordial follicles
had twice the volume than normal ones. Gametes showing
three or more than three nuclei were detected at considerably
lower frequencies (3.4% and 0.5%, respectively). Concerning the origin of binucleate female gametes, it is assumed that
they either arise from fusion of two previously independent
oogonia or from one precursor cell that undergoes nuclear
division without subsequent cytoplasmic cleavage (Hartman
1926; Austin 1960). These mechanisms also explain the
above-mentioned increased size of binucleate gametes.
Initially, the occurrence of polynuclear oocytes seemed to
be restricted to primary oocytes in immature members of
the investigated species (Austin 1960; Kent 1960, 1962a,b;
Collins and Kent 1964) and atresia was considered the
normal fate of the corresponding follicles (Hartman 1926;
Journal of Genetics, Vol. 91, No. 3, December 2012
399
Bernd Rosenbusch
Gougeon 1981). Detailed studies in the Chinese hamster
(Funaki and Mikamo 1980), however, indicated that giant
binucleate oocytes can be ovulated and then undergo fertilization and embryonic development. Interestingly, Funaki
and Mikamo (1980) noted that follicles containing a giant
gamete might indeed have an increased tendency to nonovulation compared to normal follicles but they also suggested
that the selective mechanism could be disordered by exogenous administration of gonadotropic hormones. This view is
in agreement with Kennedy and Donahue (1969) who, having observed two binucleate oocytes in antral follicles, suspected an increased incidence after gonadotropic stimulation
and a role in abortions due to triploidy arising from their
fertilization.
In fact, giant oocytes occur sporadically in programmes of
human-assisted reproduction that depend on induced superovulations for the desired recovery of larger numbers of
fertilizable oocytes. All of the cases reported up to now
have obviously been binucleate. Whereas Balakier et al.
(2002) found significantly higher oestradiol levels in patients
with giant oocytes compared with a group that did not produce such gametes, this tendency could not be confirmed
by Machtinger et al. (2011). As a reason, Machtinger et al.
(2011) stated that they matched for the number of oocytes
retrieved what was not done in the earlier study (Balakier
et al. 2002).
Binovular follicles and genetic abnormalities
Only one pair of conjoined oocytes has been examined cytogenetically by fluorescence in situ hybridization (FISH),
showing that the immature GV-stage gamete was diploid
whereas the associated embryo that developed after ICSI had
a diploid female chromosome constitution. The latter finding allowed concluding that the second gamete was a normal, haploid MII oocyte before fertilization (Safran et al.
1998). Thus, conjoined oocytes represent two individual
female gametes, each with a chromosomal constitution that
corresponds to its stage of maturity.
The documented existence of conjoined oocytes has stimulated speculations on their further development, particularly from a genetic point of view (Zeilmaker et al. 1983;
Fishel et al. 1989; Hartshorne et al. 1990; Rosenbusch and
Schneider 2004). Interestingly, several alternatives are conceivable if both oocytes are mature and independently
undergo monospermic fertilization. The first and most
uncomplicated scenario is that the resulting two diploid
zygotes develop individually into embryos and finally produce a twin pregnancy. On the other hand, the two zygotes
may fuse and cleave into a tetraploid embryo. In our brief
review of mechanisms leading to tetraploidy (Rosenbusch
and Schneider 2004), we surmized that two oocytes enclosed
within a common zona pellucida would be ideal candidates for such an event. However, in instances with merely
400
attached zonal regions, a preceding local dissolution of the
zona has to be postulated.
The third alternative involves formation of a chimaera, that
is, an individual carrying tissue that originated in two different embryos. In this respect, Edwards and Fowler (1970)
already noted that chimaeras might arise through the attachment of two embryos by their zona pellucida and that their
close implantation could lead to the exchange of cells or to
total fusion. The authors further assumed that this could be
a common mechanism in multi-ovulating species whereas
twin ovulations from the same ovary would be a prerequisite
to the formation of chimaeras in man. This discussion was
resumed by Zeilmaker et al. (1983) who observed two simultaneously fertilized, conjoined oocytes and by Aoki et al.
(2006) who reported a case of blood chimerism in monochorionic twins. According to Aoki et al. (2006), binovular follicle fertilization could have caused close apposition
of two embryos and monochorionic placentation with vessel anastomoses. However, if the origin of chimaerism is not
allocated exclusively to blastocyst fusion after hatching, it is
again necessary to postulate dissolution of the zona pellucida
prior to implantation in some instances.
Finally, even the formation of a diploid–triploid mosaic
individual has been suggested (Fishel et al. 1989). In this
model, a diploid embryo arising from monospermic fertilization of an MII oocyte would fuse with a digynic triploid
embryo resulting from monospermic fertilization of a conjoined primary (diploid) oocyte. Fishel et al. (1989) further
proposed that the conjoined oocytes were probably derived
from the division of a single precursor cell. Nevertheless,
the term ‘mosaic’ cannot be applied here because two different spermatozoa are involved and because mosaics, by
definition, develop from only one zygote.
The pivotal question is, of course, whether convincing evidence exists to support the above-mentioned assumptions.
The first problem concerns a spontaneous dissolution of the
zona, which is required for the production of tetraploidy or
chimaeras at the zygote or embryonic stage, respectively.
This event seems to remain purely hypothetical because to
our knowledge, it has not yet been observed. Experimentally, cell fusion has been realized in a variety of species but
needs chemical or electrical stimuli. However, even if one
admits a rare possibility for spontaneous fusion of embryos,
particularly in those few cases in which the zona is thin or
absent at the site of junction (Rosenbusch and Hancke 2012),
fertilizability of both conjoined oocytes is the next obstacle. As shown in table 2, this event has been observed only
once (Zeilmaker et al. 1983) whereas one of the gametes
was definitely immature in 16 out of 18 currently known
cases. Normal fertilization with formation of two pronuclei in
only one of the conjoined oocytes occurred seven times and
pregnancies from transferred embryos could not be achieved.
Taken together, these admittedly limited data do not argue
for an eminent role of binovular follicles in the development of twins or genetic abnormalities. Obviously, the crucial point is a discordant maturity of the oocytes that prevents
Journal of Genetics, Vol. 91, No. 3, December 2012
Binovular follicles and binucleate oocytes
Table 2. Conjoined oocytes detected during assisted reproduction
and their developmental fate.a
Total number of reported cases
No. of cases in which both oocytes were mature
No. of cases in which both oocytes were
normally fertilized
No. of cases in which at least one oocyte
was immature
No. of cases in which one oocyte was
normally fertilized
No. of pregnancies resulting after transfer of an
embryo from a binovular follicle
18
1
1
16b
7
0
a Data compiled from Rosenbusch and Hancke (2012).
b Maturity of the second oocyte not indicated in one case.
or impairs simultaneous fertilization. Asynchronous maturation of oocytes enclosed within the same follicle has been
observed, for instance, in the rabbit (Al-Mufti et al. 1988)
and led to the suggestion that completion of maturation
including resumption of meiosis depends on a certain position of the oocyte inside the follicle. In porcine ovaries, polyovular follicles yielded significantly less mature oocytes than
uniovular follicles and their developmental competence after
fertilization was compromised (Stankiewicz et al. 2009).
According to Dandekar et al. (1988), 46 out of 73 (63%)
human polyovular follicles contained oocytes with discordant maturity but there was no difference in the fertilizability of oocytes from polyovular or uniovular follicles when
the gametes were matched for maturity. Again, however, the
findings of this study (Dandekar et al. 1988) must be interpreted under the restriction made above, i.e. the difficult distinction of true binovularity and artefacts occurring during
follicular aspiration.
Binucleate giant oocytes and genetic abnormalities
As discussed elsewhere (Rosenbusch et al. 2002), giant
oocytes in different stages of maturity have repeatedly been
found during assisted reproduction. Besides such single obser-
vations, only few studies (Munné et al. 1994; Balakier et al.
2002; Rosenbusch et al. 2002) have provided detailed figures
on the occurrence of giant oocytes, suggesting an incidence
of up to 0.3% of all gametes retrieved (table 3). However,
another report that evaluated a possible association between
the presence of a giant oocyte and the in vitro fertilization
outcome for the cohort found a lower prevalence of 0.12%
(117/97,556) (Machtinger et al. 2011). The latter article was
not included in table 3 because it neither contained information on the maturity and fertilization of giant oocytes nor on
the chromosomal constitution of giant zygotes and embryos.
Two investigations (Balakier et al. 2002; Rosenbusch et al.
2002) presented comprehensive cytological and cytogenetic
analyses. Based on these data, the development of binucleate giant oocytes before and after fertilization can be summarized as follows. All immature giant oocytes observed up to
now displayed two germinal vesicles, indicative of the presence of two diploid chromosome complements and an overall
tetraploid constitution. However, maturation to MII can proceed in different ways. i) If the binucleate state is maintained,
two haploid first polar bodies will be extruded and the oocyte
itself will retain two separate haploid chromosome complements. This means that fertilization by a single spermatozoon eventually results in the formation of three pronuclei,
one derived from the male and two derived from the female
gamete. This process is accompanied by extrusion of two
second polar bodies, each containing a haploid set of chromatids. ii) Union of the two diploid nuclei during maturation
and subsequent first meiotic division will produce an MII
oocyte with a diploid chromosome complement and a diploid
first PB. In this case, fertilization by a spermatozoon leads to
the formation of a haploid male and a diploid female pronucleus, involving extrusion of a diploid second PB with two
chromatid complements. Thus, it is evident that the size of
the pronuclear stage must always be assessed because counting the number of pronuclei is no absolutely reliable criterion
to prevent the development of triploid zygotes and embryos
(Rosenbusch 2008). In fact, chromosomal studies of giant
embryos by FISH have revealed the presence of triploidy
and/or mosaicism (table 3).
Table 3. Giant female gametes observed during assisted reproduction and the chromosomal constitution of giant zygotes and embryos.
No. of giant cells/
total no. of gametes
Immature or mature
unfertilized oocytes
Zygotes or
embryos
Cytogenetic analysis
of zygotes and embryos
4/2010 (0.2%)
0
4
18/7065 (0.26%)
13
5
44/14272 (0.3%)
29
15
Four triploid or triploid
mosaic embryos after FISH analysis of
chromosomes X, Y and 18 or only X and Y.
Triploidy in all five zygotes
detected by Giemsa staining of the
whole chromosome complement.
FISH for chromosomes 9, 22, X and Y
applied to four embryos. Triploidy
in the majority of the nuclei accompanied
by mosaicism and complex chromosome
constitutions.
Journal of Genetics, Vol. 91, No. 3, December 2012
Reference
Munné et al. (1994)
Rosenbusch et al. (2002)
Balakier et al. (2002)
401
Bernd Rosenbusch
Triploidy and correction of triploidy
Briefly, the presence of an extra set of chromosomes has
severe consequences for the affected pregnancy because the
majority of triploid feotuses end as miscarriage and the rare
infants that survive to term are either stillborn or die soon
after birth. Intrauterine growth retardation and multiple
congenital abnormalities are characteristic for triploid conceptions. For instance, neural tube defects, syndactyly of fingers and toes, malformations of heart and brain, and defects
concerning the urogenital tract have been observed. Besides
autosomal trisomies and monosomy X, triploidy is one of
the most frequent cytogenetic abnormalities found in spontaneous abortions with an incidence that may amount up to
18% of abnormal cases. Among all pregnancies, triploidy
occurs with an estimated frequency of 1–3%.
However, it seems to be possible that some tripronuclear
oocytes can undergo a selfcorrection of their abnormal chromosome constitution and thus restore the normal diploid,
heteroparental condition. This interesting aspect has been
investigated recently by comparing the embryonic development of diandric tripronuclear IVF and digynic tripronuclear ICSI zygotes (Grau et al. 2011). It was found that
about 50% of the digynic ICSI embryos are capable of selfcorrection whereas this mechanism mostly fails in diandric
IVF embryos. Obviously, the presence of two centrosomes
delivered by two penetrating spermatozoa impairs the distribution of chromosomes and prevents a return to heteroparental diploidy. A selfcorrection of the chromosomal
constitution in fertilized giant zygotes is conceivable due
to their digynic state but has not yet been investigated and
will need a differentiation of bipronuclear and tripronuclear oocytes. During assisted reproduction, supernumerary
pronuclei can be removed mechanically (‘epronucleation’)
but this approach requires exact knowledge on the different pronuclear patterns and their chromosomal composition
(Rosenbusch 2009, 2010). An extremely rare mechanism for
spontaneous diploidization might be isolation of a pronucleus by premature cytokinesis (Rosenbusch and Schneider
2009).
Conclusions
In the present review, information obtained from humanassisted reproduction was used to evaluate whether binucleate giant eggs and conjoined oocytes may affect embryonic development. Figure 1 is a brief, schematic depiction
of both phenomena versus the normal, uniovular situation.
The available data suggest an unequivocal role of binucleate
giant oocytes in the formation of digynic triploid zygotes and
embryos in vitro and therefore, the need for excluding these
stages from transfer must be accentuated once again. If possible, giant oocytes should a priori be excluded from attempts
of fertilization. Whether giant oocytes participate in natural
triploid conceptions remains obscure at the moment though
there are cases that are consistent with fusion of two oocytes
and fertilization by a single haploid spermatozoon or, more
402
Figure 1. A brief comparison of the different possibilities of
oocyte development up to the one-cell zygote. Polar bodies are
not shown for reasons of simplification. Whereas the existence of
triploid giant zygotes has clearly been documented in programmes
of assisted reproduction, the supposed regular fertilization of both
oocytes from a binovular follicle still requires definite proof. The
two associated diploid zygotes have therefore been provided with a
question mark. For details see text.
likely, fertilization of a diploid giant oocyte originating from
a tetraploid oogonium (Zaragoza et al. 2000).
Judging the significance of binovular follicles is much more
complicated due to the extremely limited data. We recently
mentioned that most of the reported observations are rather
old and that the phenomenon of binovularity may no longer
have attracted attention in the laboratories (Rosenbusch and
Hancke 2012). However, rediscovering this area of research
appears beneficial in view of many unanswered questions.
For instance, the incidence of oocytes without zonal fusion
but included within a common cumulus, their maturity and
fertilizability are still unclear. Increasing and reevaluating
the existing preliminary data (Dandekar et al. 1988; Ron-El
et al. 1990) might constitute a new facet in the discussion on
the role of double ovulation in dizygotic twinning (Boklage
2009). Moreover, it was shown that experimental postnatal
exposure to diethylstilbestrol or bisphenol A induces the formation of polyovular follicles in different mammals (Iguchi
et al. 1990; Rodríguez et al. 2010; Rivera et al. 2011) whereas
environmental pollutants with oestrogenic action have been
suspected to promote the development of multinuclear
oocytes and multioocytic follicles in alligators (Guillette and
Moore 2006). Thus, it is advisable to gather more information on oestrogens of pharmaceutical and/or environmental
Journal of Genetics, Vol. 91, No. 3, December 2012
Binovular follicles and binucleate oocytes
origin and their impact on the endogenous hormonal system
and folliculogenesis in the human.
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Received 7 May 2012, in revised form 14 August 2012; accepted 5 October 2012
Published on the Web: 6 December 2012
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