Meiotic prophase I in oocytes without chromosome duplication.
Most of the pachytene and early diplotene oocytes I we previously studied (Spangenberg et al., 2020; 2021) had surprising features for hybrid karyotypes; successful synapsis of all autosomal bivalents composed of pairs of homeologs but not homologous chromosomes. The fact that we detected a homeologous but not homologous synapsis in oocytes without chromosome duplication was previously proven by us (Spangenberg et al., 2021). Visualization of somatic SC-trivalent in the unisexual form with an odd number of chromosomes (D. unisexualis 2n=37; Keti population) confirmed the synapsis of thehomeologs (Spangenberg et al., 2021). Additional evidence for this was the significant divergence of centromeric DNA of the chromosomes involved in meiotic trivalent revealed by comparative genomic hybridization (CGH) (Spangenberg et al., 2021).
Interestingly, in studies of diploid parthenogenetic species of the genus Aspidoscelis (Lutes et al., 2010; Newton et al., 2016), Lepidodactylus, Hemiphyllodactylus and Heteronotia (Dedukh et al., 2022), the authors noted that oocytes without genome duplication necessarily contain unpaired chromosomes — univalents. This pattern is typical for all studied diploid parthenogenetic species, except for unisexual species of the genus Darevskia. The authors justifiably assumed that such oocytes with long regions of asynapsis are doomed to undergo apoptosis due to the zygotene or pachytene checkpoints (Newton et al., 2016; Dedukh et al., 2022). Thus, cells with genome duplication are the only cells that are able to reach the diplotene stage (Newton et al., 2016).
In contrast, in two Darevskia parthenogenetic species we have studied to date (D. unisexualis and D. armeniaca), in addition to nuclei at the pachytene stage without visible synaptic disturbances, we also found a large number of normal diplotene nuclei (Spangenberg et al., 2020, 2021; this study Figure 1A-B). This is an important feature of the unisexual Darevskia species. Theoretically, such cells with only homeologous synapsis could overcome the synaptic checkpoints of meiosis I, similar to what has been observed in the triploid hybrid D. unisexualis х D.valentini we studied before (Spangenberg et al., 2017).
It remains unclear whether such primary oocytes without endoduplication can undergo postmeiotic fusion, as suggested by our previous study (Spangenberg et al., 2020, 21). If so, what contributions do they make to the offspring of unisexual species? Perhaps such oocytes are eliminated from the germ cell line and undergo apoptosis. It is important to recall the findings of previous studies and our own observations of the incubation of egg clutches in parthenogenetic species. It was shown that some parthenogenetic Darevskia lizards do not hatch from eggs. Studies of such embryos have shown multiple developmental anomalies (Darevsky 1960; Darevsky and Kulikova 1961, Danielyan 1970). It cannot be ruled out that some such abnormal embryos are the result of the development of oocytes along the path of postmeiotic fusion of nuclei or a result of unequal segregation of chromosomes during the meiotic I anaphase stage leading to aneuploidy (see below section “Developmental deviation in parthenogenetic species of the genus Darevskia”).
Z and W sex chromosomes in oocytes without genome duplication.
Analysis of the behavior of the sex Z and W chromosomes in the hybrid karyotype of D. armeniaca revealed two variants of their localization during the pachytene and diplotene stages — joint and separate localization on the spread preparation of synaptonemal complexes (Figure 1A, B). Analysis of the nuclei with ZW joint localization revealed that the centromeric regions of the sex chromosomes were the most contiguous regions (Figure SM1). Thus, a pseudoautosomal region (PAR) is likely located in a pericentromeric region (Figure SM1). Nevertheless, the question of whether the true synapsis or Z-W association occurs in the PAR remains open for unisexual forms of Darevskia (Spangenberg et al., 2020a).
Regardless of whether the Z and W sex chromosomes are located separately or together, Z and W univalents often form curved or even circular axial structures (Figure 1A, B). In general, the curved structure of sex univalents is characteristic of elongated asynaptic regions of sex chromosomes in different animals, especially in late prophase I, in diplotene
(Bogdanov et al., 2011; Spangenberg et al., 2022a; Surov and Feoktistova 2023). Thus, the lengths of sex chromosomes in oocyte nuclei without endoduplication do not reflect the real lengths of univalents and do not allow us to accurately determine the position (according to length) of sex chromosomes in the karyotype. On the other hand, we were able to measure the length of the WW sex pseudobivalent (see the section below “WW bivalent formed de novo in the nuclei of oocytes after genomic endoduplication”).
Meiotic prophase I in oocytes with genome endoduplication.
The nuclei after genomic duplication found in the present study contained 38 bivalents (twice more than usual) at the pachytene and early diplotene stages. There are 36 autosomal pseudobivalents and two sex chromosome bivalents: ZZ and WW. All pseudobivalents are composed of identical copies of chromosomes due to premeiotic endoduplication.
The mechanism of premeiotic endoreplication, described for some unisexual animals of hybrid origin, is considered a way to avoid hybrid sterility (Dedukh et al., 2020). Thus, unisexual reptile species can restore and maintain their 2n or 3n ploidy in many generations. Importantly, due to the identity of the chromosomes that are synapsing in the endoduplicated nuclei, crossing over does not lead to the emergence of new genetic combinations. We can talk about the stop of recombination (Grebelnij 2008; Grebelnij 2009), a kind of "freezing” of the initial heterozygous state of the interspecific F1 hybrid.
An important marker in Darevskia karyotypes is a clearly visible heterochromatin block on the W chromosome (Figure 1 C-D, D`), which was previously described in mitotic metaphase plates (Odierna et al., 1993; Kupriyanova, 2010). Since such a heterochromatin block is present only on one of the 38 bivalents, this finding is another one confirmation of the assembly of pseudobivalents but not homeologous bivalents.
Interestingly, the m1 and m2 microchromosomes in the D. armeniaca hybrid karyotype inherited from the two parental species had different centromere positions clearly visible on the immunostained SC preparation (Figure 1D) and on the idiogram (Figure 2B). In oocytes after endoduplication, the corresponding SCs (m1m1 and m2m2) are formed without any shifting of the centromeres in bivalents, in addition confirming the assembly of the pseudobivalents.
On the other hand, in the nonduplicated oocyte nuclei, we detected different variants: the m1m2 homeologous SC (Figure SM1A) possibly formed after synaptic adjustment (Maguire et al., 1984; Lisachov et al., 2014; Stundlova et al., 2022) as well as located nearby or completely asynaptiс m1 and m2 univalents (Figure SM1B).
WW bivalents formed de novo in the nuclei of oocytes after genomic endoduplication.
The temporary appearance of a second copy of the W chromosome in a germ cell line is, of course, an extraordinary fact. However, the ultrastructure of the synaptonemal complex allows normal assembly and synapsis of the newly emerged WW bivalent (Figure 1D`).
Notably, during the evolutionary period of existence of the bisexual maternal species (D. mixta), the W chromosome obviously did not have the possibility to synapse with its homolog to form a WW bivalent. This is a normal situation for the W and Y chromosomes in bisexual animals. Rare cases of polysomy on the X and Y chromosomes are known in humans: 47, XXY; 47, XYY; 48, XXXY; 48, XYYY; 48, XXYY; 49, XXXXY; and 49, XXXYY (Visootsak and Tartaglia 2013). On the other hand, rare cases of Y polyisomy lead to severe developmental abnormalities and do not represent reproductive strategies.
We should separately note an interesting feature of the morphology of sex chromosomes (especially the W chromosome) in the nuclei of oocytes after premeiotic genomic endoduplication. Usually, in animals with heteromorphic pairs of sex chromosomes, in meiotic prophase I, the sex chromosomes Z and W (or X and Y) have lengths that do not correspond to their chromosome numbers in the somatic karyotype. That is, the Z and W axial elements in meiotic prophase I are usually atypically elongated, curved, deformed, or coiled (Figure 1 A, B). For this reason, a heteromorphic pair of sex chromosomes is often removed separately from autosomal pairs on karyotype diagrams (Gil-Fernandes et al., 2021; Surov and Feoktistova 2023). In contrast, in homogametic sex, such as in male reptiles, the sex Z chromosomes form a ZZ bivalent that is difficult to distinguish from autosomes (Spangenberg et al., 2022a).
In the case of premeiotic duplication of chromosomes, the sex W chromosome is copied and can form a normal bivalent. We did not observe any deformations of axial elements in the structure of the WW bivalent in D. armeniaca (Fig. 1 D-D`). That is, in this case, the WW bivalent has a relevant length since a normal synaptonemal complex is formed, similar to the usual ZZ bivalent in male reptiles. According to the constructed idiogram of the SC karyotype of endoduplicated nuclei, the WW bivalent had number 36 in the karyotype of D. armeniaca (Figure 2B).
SC-tetravalents consisting of sister and homeologous chromosomes in the SC-karyotypes of D. armeniaca.
An interesting finding we made in the endoduplicated nuclei of D. armeniaca was the SC-tetravalents. We found fairly extensive ectopic synapsis at both the distal (Figure 3A) and centromeric (Figure 3B) ends of chromosomes, leading to the formation of SC-tetravalents in nuclei after genome endoduplication. It is likely that such configurations arise between corresponding homeologs. Apparently, during the assembly of SCs in the endoduplicated nucleus of D. armeniaca oocytes, active processes associated with the correction of ectopic synapsis occur; these processes were previously described for many polyploids (Holm and Rasmussen 1979, Jenkins and Jimenez 1995; Martinez-Perez et al., 2001; Moore 2002; Vasil’ev et al., 2022) and for the competitive synapsis between unpaired regions (univalents) of XY sex bivalents and autosomes in male mammals (Spangenberg et al., 2021a). These studies showed that non-homologous partial synapsis often occurs in zygotene in the pericentromeric/peritelomeric regions of chromosomes and can be corrected in subsequent stages of meiotic prophase I. Indeed, it is known that such synaptic associations can be resolved before metaphase I and do not affect normal chromosome segregation (Holm, Rasmussen 1979, Spangenberg et al., 2021).
Premeiotic genome endoduplication and postmeiotic central fusion pathways.
An important question is, after the discovery of premeiotic genome endoduplication in D. armeniaca, can we reject the possibility of a postmeiotic automictic mechanism through central fusion — the fusion of the oocyte and the first polar body (Spangenberg et al., 2020; Ho et al., 2023)? The mechanism of premeiotic endoduplication ensures long-term preservation of heterozygosity of the unisexual form. On the other hand, in numerous nuclei without chromosome duplication, we detected homoeologous synapsis for both unisexual species examined: D. unisexualis (Spangenberg et al., 2020, 2021) and D. armeniaca (Figure 1A, B; Figure SM1A, B). Moreover, we showed loading of the MLH1 protein associated with crossing over into bivalents of homeologues (Spangenberg et al., 2021). Finally, we previously revealed weak meiotic checkpoints in Darevskia hybrid individuals, allowing even triploid males to produce numerous mature but aneuploid spermatids (Spangenberg et al., 2017).
Thus, there is a fairly high probability that oocytes without endoduplication can overcome the synaptic checkpoints of meiosis in parthenogenetic Darevskia. However, what happens next to these oocytes is unknown. If natural populations of unisexual Darevskia species contain individuals born as a result of a central fusion mechanism, then we should expect deviations from strict clonality and some polymorphism in the populations. According to some studies, all unisexual species of Darevskia, especially D. armeniaca, demonstrate some diversity in their morphology. This issue requires detailed analysis in the future. In other words, could the appearance of several clones within one parthenogenetic species be the result of rare cases of automixis through central fusion (fusion of meiotic products) rather than errors in the nuclei after the genome endoduplication?
Developmental deviation in parthenogenetic species of the genus Darevskia.
Numerous findings of embryonic abnormalities have been documented for parthenogenetic Darevskia rock lizards (Darevsky 1960; Darevsky and Kulikova 1961, Danielyan 1970). Data from previous studies indicate that these developmental deviations (up to 10 different types) occur quite often (3-6.8%) in unisexual species of the genus Darevskia (4.5-4.9% for D. armeniaca) compared to bisexual species (1.1-1.8%) (Danielyan 1970).
Additionally, during the egg incubation in our current work, out of the 14 clutches studied, several of the eggs stopped developing, and two cases were associated with the inability of the embryo to hatch from the egg. This finding is consistent with previous findings of developmental embryonic abnormalities previously described in the classification suggested by I.S. Darevsky: unclosed body cavities, complete absence of the lower jaw, disproportion and curvature of the jaws (Darevsky, 1960; Danielyan 1970). We documented two cases of the same abnormalities in late embryos (Figure SM4А, B). These data may indicate a high risk of errors in oogenesis during the process of endoduplication. Further comparative studies of the karyotypes of such individuals may help in understanding which of the mechanisms of ploidy restoration or incorrect synapsis between duplicated or homeologous chromosomes took place in such cases.
In general, one thing is clear: the mechanisms of ploidy restoration in parthenogenetic Darevskia also carry increased risks of the formation of nonviable embryos (Figure SM4А,B). Whether they are associated with the formation of aneuploid karyotypes or loss of heterozygosity is an important question for further research.