Pachytene Oocytes Research Articles

Analysis of meiotic chromosome pairing in the female mouse using a novel minichromosome.

Analysis of a radiation-induced minichromosome using classical cytogenetic techniques and fluorescence in situ hybridization (FISH) has shown this element to be composed primarily, if not entirely, of repeat sequence DNAs that are common to the ends of all chromosomes in the mouse genome. This novel chromosome has been used to examine further the role of centromeric and telomeric DNA sequences in the initiation of homologous chromosome pairing and synapsis in female germ cells. In pachytene oocytes of a fetus carrying two copies of the minichromosome, it was found that these elements were collocalized, but had not undergone synapsis, in half of the cells analysed. The minichromosomes formed a synaptonemal complex in approximately 14% of oocytes. Overall, therefore, the minichromosomes showed a surprising ability to achieve the first phase of homologous chromosome pairing, i.e. collocalization, despite their dearth of chromosome-specific DNA sequences. It is suggested that this collocalization largely results from the tendency of mouse chromosome ends to form centromeric and telomeric clusters at zygotene. The observations here provide support for the proposition that clustering of chromosome ends in early meiosis fosters pairing interactions and synapsis.

Germ cell restriction and regular transmission of an accessory chromosome that mimics a sex body in the zebra finch, Taeniopygia guttata.

Mitotic and meiotic analysis with light and electron microscopy was performed in male and female zebra finches (Taeniopygia guttata). Somatic cells from bone marrow have a 2n = 80 and show the usual sex chromosome mechanism of birds ZZ (male)/ZW (female). In the germ lines of both sexes, a single accessory chromosome was regularly present in all the cells examined from all the individual birds. In synaptonemal complex (SC) spreads of pachytene oocytes and spermatocytes, this accessory chromosome forms a single axis, but it behaves differentially in male and female meiosis. While this accessory chromosome is euchromatic in oocytes, it is strongly heterochromatic in spermatocytes. In pachytene spermatocytes, the accessory chromosome adopts a morphology strikingly similar to that of the XY body ('sex vesicle') of mammalian spermatocytes. This accessory chromosome is eliminated during male meiosis and forms a cytoplasmic dense body in young spermatids that shows strong fluorescence with DAPI. The presence of this germ line-restricted chromosome does not affect the behaviour of the ZW pair in oocytes, as the sex chromosomes pair regularly and show a localized recombination nodule. It is suggested that this accessory chromosome has transcriptional activity during oogenesis, and thus it is regularly transmitted through preferential segregation during female meiosis.

Cyclophosphamide-induced aberrations of chromosome pairing in pachytene oocytes

The aim of this study was to characterise damage to synaptonemal complexes in oocytes following cyclophosphamide exposure. Pregnant mice were treated with three different doses of cyclophosphamide (10, 30 and 50 mg/kg body weight) at day 13 of gestation, when oogonia and very early meiotic cells in the female fetuses are found. Primary oocytes were analysed by light- and electron microscopy at gestational day 17 to reveal effects of the alkylating agent on the chromosomal pairing behaviour. Our pachytene analysis demonstrated that the fraction of cells with lesions of synaptonemal complexes, partial asynapsis and desynaptic bivalents were significantly increased over the levels in the control group. The frequency of alterations was similar at doses of 30 and 50 mg/kg. In addition, a significant increase in frequency of univalents over the base level became evident, showing the highest incidence on a dose of 50 mg/kg.

Spatial arrangement of intra-nucleolar rDNA chromatin in amplified Xenopus oocyte nucleoli: structural changes precede the onset of rDNA transcription.

Amphibian oocyte nucleoli are a particular suited object for research on nucleolar chromatin organization. By selective rDNA amplification each pachytene oocyte nucleus accumulates 30 pg of extrachromosomal rDNA, this amount corresponds to 2 million rDNA copies. Following the selective amplification stage, the amplified gene copies are finally distributed within more than thousand extrachromosomal nucleoli per individual oocyte nucleus. The aim of the present study was first to obtain a precise documentation of the fate of amplified rDNA during early Xenopus oogenesis until the final functional integration of these copies into individual oocyte nucleoli, and, second, a close correlation of the structural data with determination of rDNA transcription rates by S1 transcript analysis for the subsequent stages of oocyte differentiation. In order to investigate the structural complexity of the intranuclear rDNA translocation process in detail, a confocal laser scan microscope (CLSM) was used, equipped with an external UV-laser. This instrumentation unambiguously allowed (i) the detection of small clusters of rDNA copies and (ii) the precise spatial documentation of the intranuclear position of rDNA clusters in relation to the protein-free pre-nucleolar protein bodies, a specific characteristic of late pachytene/early diplotene amphibian oocyte nuclei. Our results indicate that the major rDNA translocation processes, e.g. the association of rDNA clusters with pre-nucleolar protein bodies, the formation of ribbon-like pre-nucleolar units sensu Van Gansen and Schramm (J. Cell Sci. 10: 339-367, 1972), and, finally, the translocation of fused rDNA units into the interior of pre-nucleolar protein bodies, occur--for the most part--in absence of massive rDNA transcription. As shown by the S1 transcript analysis, the onset of massive rDNA transcription starts concomitantly with an unraveling of the densely packed rDNA clusters into finely dispersed rDNA units, which were shown by CLSM analysis to be distributed throughout the entire nucleolar volume.

Histoenzymological studies on embryonic and posthatching development of the ovary in the tropical oviparous lizard, Calotes versicolor.

The development of the ovary in the lizard Calotes versicolor has been studied histologically and histochemically from the day of oviposition (stage 27) to 2 months after hatching. The gonadal component appears as a genital ridge at stage 27. Sex differentiation of gonads occurs at stage 34, which indicates that the period of sexual bipotentiality is very brief. Oogenesis starts at stage 35, where preleptotene and leptotene oocytes appear for the first time in the ovary. The zygotene and pachytene oocytes appear during stage 36 and 38. Diplotene oocytes occur at stage 40. Folliculogenesis occurs 10 days after hatching. Follicles having a polymorphic membrana granulosa consisting of small, intermediate, and large round cells are found in 1-month-old hatchlings, and pyriform cells are observed in the granulosa of the ovary of 2-month-old hatchlings. The number of germ cells increases as development of the ovary proceeds. At oviposition, ∼236 and 244 germ cells were counted in right and left ovaries, respectively, and there was a manyfold increase in their number (to 4,234 and 4,294) at hatching. Mitotic germ cells are more abundant at stage 34, and no mitotic germ cells are seen after hatching. Histochemical localization of of Δ5-3β-HSDH and G-6-PDH activity is seen in the medulla of the ovary after sexual differentiation (stage 36), which indicates that the medulla of the ovary is the site of steroidogenesis. intensity of the enzyme activity remains more or less the same in the ovary up to 1 month after hatching and begins to increase thereafter. © 1994 Wiley-Liss, Inc.

Sex chromosome pairing and sex chromatin bodies in W–Z translocation strains of Ephestia kuehniella (Lepidoptera)

Structure and pairing behavior of sex chromosomes in females of four T(W;Z) lines of the Mediterranean flour moth, Ephestia kuehniella, were investigated using light and electron microscopic techniques and compared with the wild type. In light microscopic preparations of pachytene oocytes of wild-type females, the WZ bivalent stands out by its heterochromatic W chromosome strand. In T(W;Z) females, the part of the Z chromosome that was translated onto the W chromosome was demonstrated as a distal segment of the neo-W chromosome, displaying a characteristic non-W chromosomal chromomere-interchromomere pattern. This segment is homologously paired with the corresponding part of a complete Z chromosome. In contrast with the single ball of heterochromatic W chromatin in highly polyploid somatic nuclei of wild-type females, the translocation causes the formation of deformed or fragmented W chromatin bodies, probably owing to opposing tendencies of the Z and W chromosomal parts of the neo-W. In electron microscopic preparations of microspread nuclei, sex chromosome bivalents were identified by the remnants of electron-dense heterochromatin tangles decorating the W chromosome axis, by the different lengths of the Z and W chromosome axes, and by incomplete pairing. No heterochromatin tangles were attached to the translocated segment of the Z chromosome at one end of the neo-W chromosome. Because of the homologous pairing between the translocation and the structurally normal Z chromosome, pairing affinity of sex chromosomes in T(W;Z) females is significantly improved. Specific differences observed among T(W;Z)1-4 translocations are probably due to the different lengths of the translocated segments.

Tdy-negative XY, XXY and XYY female mice: breeding data and synaptonemal complex analysis.

In this paper we have compared the breeding performance of Tdy-negative XY, XXY and XYY females to assess the relative importance of the lack of a second X chromosome compared with the presence of a Y chromosome, in reducing fertility. The XY females were of poor fertility, although five of twelve produced at least one offspring. The XXY females had larger, more frequent litters, and a longer reproductive lifespan, implicating the lack of a second X as the major cause of the poor fertility of XY females. Nevertheless, XYY females appeared to be more seriously affected than the XY females, suggesting that the presence of the Y may be a contributory factor. Pachytene analysis demonstrated that the Y is a very inefficient pairing partner for the X during female meiosis. In XY females only 11% of pachytene cells had the X and Y paired; in XXY females the two X chromosomes paired and the Y was almost always a univalent, while in XYY females the X paired with a Y in only 15% of pachytene cells. The presence of unpaired sex chromosomes has previously been implicated as a cause of oocyte loss during pachytene, and the proportion of cells with unsynapsed sex chromosomes decreased as pachytene proceeded, suggesting that they were progressively eliminated. Significantly, protection against elimination was afforded not only by synapsis between sex chromosomes, but also by self-synapsis if a sex chromosome remained as a univalent. It is concluded that sex chromosome univalence leading to pachytene oocyte failure is responsible for the postnatal oocyte deficiency seen in XY females. A separate study has shown that XXY females have a similar level of oocyte deficiency. It is suggested that the presence of a second X chromosome improves the fertility of XXY females, compared with XY females, by improving oocyte quality and by eliminating the production of lethal XY and OY zygotes. The genotype frequencies for the offspring of XY and XYY females differed from those predicted from the pachytene data. The XY females showed a marked deficiency of XO offspring compared with XXY and XYY aneuploid offspring, whereas the XXY females had fewer than expected XXY and XYY aneuploid offspring.

Synaptonemal complex karyotype of the pachytene spermatocytes and oocytes of the Turkish hamster (Mesocricetus brandti)

SUMMARYThe complete synaptonemal complex karyotype of the pachytene spermatocytes and oocytes of the Turkish hamster (Mesocricetus brandti) are described. The species has unique polymorphisms of the X and Y. Multiple configurations of the XY bivalent were seen possibly resulting from an autosomal translocation to the sex chromosomes.

The pachytene chromomere map of the oocyte of the Turkish hamster (Mesocricetus brandti).

A study of the chromomere maps of the sex and twenty autosomal bivalents of Turkish hamster pachytene oocytes was carried out. The average total number of chromomeres in early/mid pachytene autosomes was 280 with 91 on the p (short arm) and 189 on the q (long arm). The submetacentric X1 chromosome had 20 chromomeres and the metacentric X2 had 27. Comparisons of the number and location of oocyte chromomeres are made with the pachytene spermatocyte chromomere maps of this species.

A Study of Pairing Behavior of Three Paracentric Inversions in Mouse Pachytene Oocytes.

A light and electron microscopy examination of the pairing behavior of the mouse oocytes carrying paracentric inversions In (X) 1H, In (1) Rk, and In (5) 2Rk revealed loop formation, synaptic adjustment and significant asynapsis of the inversion segments.

The fate of intramitochondrial paracrystalline inclusion bodies in germ line cells of water frogs (Amphibia, Anura)

Numerous intramitochondrial paracrystalline inclusion bodies (ICIB) were observed in the germinal plasm of a mid-blastula, and in primordial germ cells (PGCs) after their migration to the germinal ridges, in Rana ridibunda, R. lessonae and R. esculenta. In oogonia the number of ICIB decreases rapidly. Single ICIB are observed in the germ cells up to the leptotene stage; they have never been observed in pachytene oocytes. In diplotene oocytes that have reached a diameter of about 100 microns ICIB are visible again, and their number increases concomitantly with oocyte growth.

Pachytene pairing and oocyte numbers in mice with two single Robertsonian translocations and the male-sterile compound with monobrachial homology

Oocyte numbers and synaptonemal complexes were studied in two Robertsonian translocations, Rb(6.15)1Ald and Rb(4.6)2Bnr, and their male-sterile compound. Oocyte numbers in the compound were lower than those of either parent, and there was a marked difference between reciprocal crosses. Synaptonemal complexes of homozygous females appeared as 19 bivalents, those of single heterozygotes as 18 bivalents and a trivalent, and those of compound heterozygotes as 17 bivalents and a quadrivalent. Most trivalents were fully paired, whereas the majority of quadrivalents exhibited terminal asynapsis. About one-half of all oocytes had other pairing abnormalities, probably reflecting reduced survivability. Whereas all fully paired quadrivalents were present in cells not showing any pairing anomalies, one-half of the quadrivalents with terminal asynapsis were seen in oocytes with other anomalies. It is suggested that in oocytes destined for atresia, there is a predisposition to synaptic failure of translocation configurations. Additional oocytes are likely to break down because of the deleterious effect of the compound translocation on gametogenesis. This effect seems to be more pronounced in Rb1Ald/Rb2Bnr spermatocytes than in oocytes.

Colcemid effects on homologue pairing and crossing over during fetal mouse oogenesis.

Colcemid was administered to gestational day 13 female mice to test effects on homologue pairing, synapsis and recombination of fetal oogenesis. Pairing abnormalities were detected in pachytene oocytes by light and electron microscopy examination of bivalents and synaptonemal complexes. Reduction of total chiasmata per treated diplotene oocyte (22.74) compared to controls (31.07) was found.

Further examination of the production-line hypothesis in mouse foetal oocytes. II. T(14; 15)6Ca heterozygotes.

Pachytene oocytes from foetal mice heterozygous for the translocation T(14; 15)6Ca were screened for evidence of a "production-line" effect on chromosome pairing. Metaphase I oocytes from adult heterozygotes were also examined to determine whether any such effect on pahytene chromosome pairing is subsequently repeated during adult reproductive life as anticipated by the production-line hypothesis. It was found that as gestation proceeded the proportion of pachytene oocytes with a translocation quadrivalent declined and that with a trivalent and univalent correspondingly increased. That is, there was evidence of variation in pairing behaviour of the translocation at different times of gestation. In contrast, the proportions of metaphase I cells with either a quadrivalent or a trivalent plus univalent did not vary between adult females of different ages. Thus if the variation observed at pachytene was the result of a production-line effect, clearly this was not reflected in the behaviour of the translocation at metaphase I. Our observations therefore do not support the production-line hypothesis for the maternal age effect on nondisjunction.

Meiotic behavior of gonosomically variant females of Akodon azarae (Rodentia, Cricetidae).

The meiotic behavior of sex chromosomes has been investigated in variant females of Akodon azarae, both in pachytene oocytes and metaphase I. In somatic cells, these females have a heteromorphic sex pair, in which the minor chromosome has been previously interpreted as a major deletion of the long arm of the X chromosome (dX). After microspreading for synaptonemal complex analysis, pachytene oocytes show two axes of very different lengths (100:17.1), which correspond to the sex chromosomes X and dX. True synapsis is abnormally restricted (43.3%) between these sex chromosomes; on the other hand, self-synapsis of both the X and dX chromosomes is frequent (60%). Single, nonsynapsed axes or axial segments are thickened. Strong chromatin condensation occurs around nonsynapsed axes or axial segments, giving many of these sex pairs an appearance similar to an XY body ("sex vesicle"). The minor gonosome axis differs from that of the Y chromosome of male meiosis, as the former is shorter (relative to the X) and has a different synaptic behavior. In 17 metaphases I from XdX variant females, only heteromorphic, end-to-end joined sex pairs were observed. These variant females differ from the variant females of the wood lemming Myopus schisticolor in several respects, but a similar mechanism seems to be prevalent in other species of the genus Akodon. Self-synapsis of unequal gonosomes in oocytes is assumed as an escape from functional deterioration, following the hypothesis put forward by others.

The possible role of meiotic pairing anomalies in the atresia of human fetal oocytes.

Following a previous study of human fetal oocytes analysed by light and electron microscope microspreading (Speed 1985), a further and more extensive analysis has now been carried out at electron microscope (EM) level. Some new anomalies not previously observed are described. More than one-third of all pachytene oocytes show degeneration (Z-cells) or synaptic errors which might lead to germ cell death. Meiotic pairing anomalies appear to be much more common among oocytes than spermatocytes, and could be significant factor in the high rate of atresia found between mid-term and birth in the human ovary.

Immunocytochemical Localization of Methylated DNA in Human Pachytene Oocytes and Questions of Parental Imprinting

SUMMARYA study of immunoreactive sites utilizing an antibody to 5-methylcytidine on human mid-late pachytene oocyte bivalents was made. All centromeres of bivalent four and 70% of bivalent six were negative, while remaining centromeres were always positive. 360 of the entire complement of 607 chromomeres were found to be positive for reaction product. When compared with previous data from this laboratory for human spermatocyte bivalents obtained with the identical antibody and method, it was concluded that the percentage (59.3%) of methylated chromomeres of oocytes was less than that of spermatocytes (70.7%). 135 sites of methylation were found to be identical on the bivalents of both sexes and an additional 44 found to be potentially identical. Unique sites made up 47% of the oocyte complement and 30% of the spermatocyte complement. These findings of over-all methylation at this stage of human germ cell development are discussed with reference to theories of imprinting of the genome at fragile sites and ...

Further examination of the production-line hypothesis in mouse foetal oocytes. I. Inversion heterozygotes.

Chromosome pairing has been examined in foetal oocytes of mice heterozygous either for an X-linked inversion, In(X)1H, or an autosomal inversion, In(2)2H. The patterns of chromosome pairing have been screened systematically in foetuses of different gestational ages in a search for a "production-line" effect particularly affecting the inversion-bearing bivalents. The proportion of pachytene oocytes with a loop fell with increasing gestational age for both inversions. The decrease was linear for In(X)1H but best described by a quadratic function for In(2)2H. Examination of late zygotene cells and a comparison of loop frequency in early, mid and late pachytene oocytes suggested this age-related decrease to be principally due to synaptic adjustment and not to a production-line effect. However, two particular observations were somewhat at variance with this conclusion. Firstly, in In(X)1H heterozygotes, the presence of an inversion loop and the occurrence of partial pairing of long/long-medium bivalents at pachytene were independent of each other only on day 19. Secondly, although the proportion of oocytes with a loop fell overall, there was a rise at 19 days in In(2)2H heterozygotes. Thus in both inversions there is some evidence of a change in pairing behaviour affecting the inversion-bearing bivalents at the latest gestational age, as would be expected under the production-line hypothesis.

Recombination nodules in the oocytes of the chicken, Gallus domesticus.

Chicken oocytes at pachytene were processed with the microspreading technique (Moses, 1977), and their synaptonemal complex (SC) complements were analyzed by electron microscopy. Ellipsoidal nodules, 140 X 120 nm in diameter, were associated with the central space of synaptonemal complexes. The average number of nodules per pachytene oocyte was 57.5. The number of nodules per bivalent showed a clear linear relationship with SC length, except for the microchromosomes, which showed a single obligatory nodule. The distribution of nodules along the 10 longest SCs was nonrandom, with low frequencies in the vicinity of kinetochores and high frequencies near the telomeres. The microchromosomes showed a single nodule whose average location was 1.21 micron from the kinetochore. In the ZW pair there was a single nodule whose average location was 0.31 micron from the paired telomeres and not more than 0.65 micron from them. The total number of nodules per cell and the number of nodules in each of the five major bivalents showed good agreement with the total number of chiasmata and the number of chiasmata of the major bivalents of roosters. Thus, these nodules share the characteristics of recombination nodules described in other organisms. The single, obligatory, strictly localized recombination nodule found in the pairing end of the ZW pair strongly suggests that recombination between the Z and W chromosomes in the female chicken is a regular process that may be similar to the obligatory recombination between the pairing ends of the human X and Y chromosomes that was recently described in studies using DNA probes.

Absence of late effects on survival and developmental abilities of pachytene oocytes X-irradiated during neonatal stages in the Chinese hamster.

In neonatal Chinese hamsters, all oocytes on day 0 after birth are at pachytene and ovarian X-irradiation with 1 Gy causes no acute oocyte killing. The same treatment on day 4, however, kills all diplotene-dictyate oocytes within 48 h, and consequently only a small number of pachytene oocytes survives. We investigated late effects of X-rays on the pachytene oocytes in relation to the reproductive capacity of irradiated females. There was no difference between the 0-day irradiated females and the controls with respect to the age of sexual maturation, fertility span and the rate of age-dependent reduction in the number of oocytes due to follicular atresia. On the other hand, the 4-day irradiated females showed a marked delay in sexual maturation and a very precocious cessation of oestrous cycles. However, the degeneration of the surviving oocytes was not accelerated. Fertility tests on day 18.5 of gestation revealed that there was no difference between both irradiated groups and the controls with respect to the mean number of corpora lutea and to the occurrence of dead and abnormal conceptuses. These results indicate clearly that pachytene oocytes were not affected at all by either acute or late effects of X-rays although the dose was high enough to kill diplotene-dictyate oocytes completely.