The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities

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1 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, 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 The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities. J. Genet. 91, ] 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- bernd.rosenbusch@uniklinik-ulm.de. Keywords. binovular follicles; binucleate giant oocytes; chimaeras; mosaicism; tetraploidy; triploidy. 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. Journal of Genetics, Vol. 91, No. 3, December

2 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 Papadaki (1978), Zeilmaker et al. (1983), Ben-Rafael et al. (1987), Manivel et al. (1988), Ron-El et al. (1990) Biovularity Human Muretto et al. (2001) Binovular zona pellucida Human Safran et al. (1998), Vicdan et al. (1999), Kousehlar et al. (2009) Multioocyte or multioocytic follicle(s) Dog Payan-Carreira and Pires (2008) Multiovular follicle(s) Human Gougeon (1981) Polyovular follicle(s) Mouse, rat, hamster, guinea, Kent (1960, 1962a,b), Collins and Kent (1964), pig, rabbit, cat, dog, pig, Thompson and Zamboni (1975), rhesus monkey, human Telfer and Gosden (1987), Al-Mufti et al. (1988), Dandekar et al. (1988), Stankiewicz et al. (2009) Polynuclear follicle(s) Human Sherrer et al. (1977) Two oocytes with fused zonae pellucidae Human Hartshorne et al. (1990) Two oocytes surrounded by a single zona pellucida Human Ben-Rafael et al. (1987), Fishel et al. (1989) 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. 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 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 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 398 Journal of Genetics, Vol. 91, No. 3, December 2012

3 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

4 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 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 400 Journal of Genetics, Vol. 91, No. 3, December 2012

5 Binovular follicles and binucleate oocytes Table 2. Conjoined oocytes detected during assisted reproduction and their developmental fate. a Total number of reported cases 18 No. of cases in which both oocytes were mature 1 No. of cases in which both oocytes were 1 normally fertilized No. of cases in which at least one oocyte 16 b was immature No. of cases in which one oocyte was 7 normally fertilized No. of pregnancies resulting after transfer of an 0 embryo from a binovular follicle 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 observations, 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/ Immature or mature Zygotes or Cytogenetic analysis total no. of gametes unfertilized oocytes embryos of zygotes and embryos Reference 4/2010 (0.2%) 0 4 Four triploid or triploid Munné et al. (1994) mosaic embryos after FISH analysis of chromosomes X, Y and 18 or only X and Y. 18/7065 (0.26%) 13 5 Triploidy in all five zygotes Rosenbusch et al. (2002) detected by Giemsa staining of the whole chromosome complement. 44/14272 (0.3%) FISH for chromosomes 9, 22, X and Y Balakier et al. (2002) 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

6 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 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;Riveraet 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 402 Journal of Genetics, Vol. 91, No. 3, December 2012

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