A Pachytene map of the mouse oocyte

A map of the mouse oocyte has been constructed utilizing early and mid/late pachytene stages. Each bivalent was clearly identified. At the mid/late stage 195 chromomeres corresponded directly to precursor chromomeres in the early stage, whilst 84 chromomeres were derived from two chromomeres from the early stage. The X and 13 bivalents were found to have a single chromomere in the early stage which later yielded two sites. There were approximately twice as many total chromomeres as mitotic bands, with good correspondence between pachytene chromomeres and major mitotic bands. Application of the use of mapping to a previously reported bivalent bearing a pleiomorphic region is noted.     

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.     

Pachytene pairing and sperm counts in mice with single Robertsonian translocations and monobrachial compounds

Data on testis weights, sperm counts, and synaptonemal complexes are presented for mice carrying the following Robertsonian translocations: Rb(6.15)lAld; Rb(4.6)2Bnr; Rb(4.15)4Rma; Rb(6.15)lAld/Rb(4.6))2Bnr, which is male-sterile; and Rb(6.15)lAld/Rb(4.15)4Rma, which is male-fertile. In RblAld/Rb2Bnr sperm were absent or sparse, whereas the sperm count in RblAld/Rb4Rma was just over 50% of the parental value. The translocated chromosomes appeared as fully paired bivalents in homozygotes, as trivalents in single heterozygotes, and as quadrivalents in compounds. About 20-40% of trivalents had unsynapsed ends. The proportion of quadrivalents with unsynapsed ends was about 85% in the male-sterile compound, compared with 75% in the male-fertile compound. The proportion of quadrivalents associated with XY was about 70% in both. Testis weights, but not sperm counts, were found to differ in two of three reciprocal crosses. It is concluded that, in addition to pairing failure and autosome/XY association, the effect of translocations on spermatogenesis is affected by other factors, including genetic background, inbreeding, and perhaps environmental factors. It remains to be elucidated whether pairing failure and XY association are primary or secondary effects.     

Chromosome pairing and germ cell loss in male and female mice carrying a reciprocal translocation

Female carriers of the T(5;12)31H reciprocal translocation had an average reduction of 73% in oocyte numbers compared with normal litter mates, which was of a magnitude similar to the reduction in sperm counts of male carriers. Analysis of synaptonemal complexes showed that the translocated chromosomes appeared as quadrivalents, or trivalents and univalents, or bivalents in both sexes. Quadrivalents were of three types: fully synapsed, with asynapsis confined to breakpoints, and with unsynapsed ends. There was more pairing in spermatocytes than in oocytes: 37% of spermatocytes, but only 14% of oocytes, contained a fully synapsed quadrivalent, and trivalents were also more frequently fully synapsed in spermatocytes. When these results are compared with those previously obtained for other chromosome anomalies, it becomes evident that there are considerable differences in chromosome pairing between males and females, and that different chromosome rearrangements differ in the relative amount of pairing failure occurring in male and female carriers.     

Pachytene analysis in a 17;21 reciprocal translocation carrier: role of the acrocentric chromosomes in male sterility

Summary Pachytene analysis was undertaken in an infertile male, heterozygous for a 17;21 reciprocal translocation. The quadrivalent was identified by its configuration and chromomere pattern. A non-random association was found between the quadrivalent and the sex vesicle in 77% of the pachytene nuclei analysed. In 13.1% of the cells the contact with the sex vesicle was established by the terminal chromomere of the two chromosomes 21; in 63.9% of the cells, the entire region of the breakpoints was completely hidden by the sex vesicle. In some nuclei asynapsis was found in the region of the breakpoints. The nature of the contact between the quadrivalent and the sex vesicle is discussed in this paper. It is proposed that the acrocentric chromosome favours the contact between the quadrivalent and the sex vesicle, and increases the risk of sterility in male carriers of Robertsonian translocations and of reciprocal translocations involving one acrocentric chromosome.     

Segregation of chromosomes in sperm of reciprocal translocation carriers: a review

Reciprocal translocations, the most frequent structural aberration in humans, are mainly transmitted by one of the parents. In order to analyze the chromosomal content of the spermatozoa from carriers of chromosomal reorganizations, two methods have been used, karyotyping of sperm chromosomes by the human-hamster system and fluorescence in situ hybridization (FISH) in decondensed sperm nuclei. In this work, we review 92 sperm chromosome segregation studies from 85 different reciprocal translocation carriers, including a triple translocation carrier. Using the human-hamster method, a total of 5,818 spermatozoa from 44 reciprocal translocation carriers have been analyzed, 43 of them carrying a single reciprocal translocation and one was a carrier of a double reciprocal translocation. A segregation analysis in a carrier of a t(2;22;11) has been also reported. Carrying out FISH in sperm nuclei, a total of 237,042 spermatozoa from 46 reciprocal translocation carriers have been analyzed. Six of these were also analyzed by the human-hamster system. Taking into account both methods, a total of 76 different reciprocal translocations have been studied. In 74 of these 76 translocations, the reorganization occurs between autosomes, and in the other two, the Y chromosome is involved. Although along general lines, there are similarities between the results obtained by the two methods of analysis, variations are observed when the distribution of the different types of segregations that produce imbalances is compared. As a general rule reciprocal translocation carriers produce more unbalanced sperm than normal or balanced sperm. The results reported also corroborate that the proportion of unbalanced forms depends on the characteristics of the reorganization and that it varies widely. Thus the importance of performing a detailed meiotic behavior analysis for each particular translocation in order to obtain enough information to give adequate genetic counseling is stressed. Aspects as to the possible overestimation of 3:1 segregations or the presence of interchromosomal effects still need to be elucidated.     

Human sperm chromosome studies in a reciprocal translocation t(2;5)

Summary Sperm chromosome complements have been studied in a man heterozygous for a reciprocal translocation t(2;5)(p11;q15). Human sperm chromosomes were obtained after fertilization of zona-free hamster eggs. A total of 75 human sperm metaphases were analysed. Of the complements studied, 59 (78.6%) resulted from a 2:2 segregation and 16 (21.3%) from a 3:1 segregation, 4:0 segregation was not observed. Our results indicate that at least 36% of sperm complements were unbalanced with respect to the translocation. The frequency of other chromosome anomalies unrelated to the translocation was 16%.     

Spermatogenic effects of male-fertile translocations in the mouse

Four male-fertile translocations, T(2;4)13H, T(2.8)26H, T(7;18)50H and T(1;13)70H were crossed to the inbred strains CBA/H and C57BL/6J. F1 heterozygotes were compared with wild-type litter-mates for signs of spermatogenic impairment, in view of previous reports that the C57BL strain had this effect in the T(14;15)6Ca translocation. There was a general tendency for body-weights to be slightly reduced in translocation carriers vs. wild-type. Mean testis weights were significantly reduced on the C57BL background with all four translocations as compared to wild-type, but also significantly increased in T26H on CBA. Sperm counts were also reduced on the C57BL background in T13H, T50H and T70H (significantly so in the last two) but were significantly increased in T13H on a CBA background. Only in T50H did the frequency of sperm-head abnormalities show any marked change in the translocation heterozygotes, being approximately doubled with both CBA and C57BL backgrounds although still remaining at a low level. It was concluded that the deleterious effects of C57BL strains on spermatogenesis in translocation heterozygotes were not confined to T6Ca but were probably widespread. Some inconclusive evidence suggested that this might be because some genetic factors associated with C57BL tended to reduce chiasma frequencies in translocation heterozygotes.     

Trisomy 20p due to a paternal reciprocal translocation

A mentally retarded boy with multiple malformations was found to have trisomy for the distal two-thirds of the short arm of chromosome 20 (trisomy 20p), resulting from a paternal translocation (5;20)(p15;p11). The patient had a cleft palate, a feature not present in other trisomy 20p patients. A review of the reported trisomy 20p patients indicates that their varied features do no constitute a readily recognizable clinical syndrome.     

Pachytene mapping of the C 9 and acrocentric bivalents in the human oocyte

Summary Provisional maps are presented for all acrocentric bivalents and bivalent 9, according to their chromomere patterns at pachytene in the human oocyte. Each G band is subdivided into several sub-bands whose number varies according to the degree of chromosomal compacting. Chromomere number and sequence are in basic agreement with those observed in late prophase mitotic chromosomes. Thus, metaphase G bands of mitotic chromosomes result from progressive compressing together of smaller chromomeres whose individuality disappears as chromosomal condensation increases with progression of prophase.     

Transmission of X-ray-induced reciprocal translocations in normal male mice and in male mice with a reduced sperm count due to translocation homozygosity

Normal (+/+) and translocation T(1; 11.13S)70H homozygous (T/T) male mice received 2 X 2.5 Gy X-rays with a 24-h interval. After 120 days, the frequency of late diplotene-metaphase I spermatocytes with translocation multivalents was 14.1% for +/+ and 13.7% for T/T males, respectively, in one group of animals of each type. The difference is not significant. A second group was allowed to sire progeny for 60 days with 2 normal females per week. Reciprocal translocations detectable at diakinesis/metaphase I were observed in 2.5% of the 395 male progeny from the irradiated +/+ fathers, and in 2.9% of the 489 male progeny from the irradiated T/T fathers. This leads to a pooled estimated transmission of 0.81 +/- 0.19. Translocations induced in the long 11.13 metacentric chromosome were not transmitted with a different frequency. The rate of heritable induced translocations in this study was 5.4 X 10(-5)/rad/gamete. On the basis of the data of Generoso et al. (1984) for the frequency of the heritable spontaneous translocations in male mice, it is concluded that, because of their low doubling dose (3.3-4.6 rad), the spontaneous translocations are probably of postmeiotic origin.     

A new reciprocal translocation in a subfertile bull

Three bulls of the Montbéliarde breed that exhibited fertility rates lower than 30% following more than 400 artificial inseminations were examined. Semen quality (sperm motility and morphology) from these bulls was normal. Fertilizing ability estimated from in vitro embryo production results was studied for two of them. In vitro production rate was very low for one bull (A) and normal for the other (B). Cytogenetic analyses were carried out on the three bulls using chromosome banding techniques. These analyses revealed a reciprocal translocation (12;17)(q22;q14) in bull B. Based on family analyses, the hypothesis of a de novo origin of this rearrangement is proposed.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

Role of guinea-pig sperm autoantigens in sperm binding to the zona pellucida and oocyte penetration

In vitro, binding of acrosome-reacted spermatozoa to the zona pellucida of mature guinea-pig oocytes was inhibited by guinea-pig sperm anti-T IgG and antibodies. Anti-P IgG antibodies prevented oocyte penetration without interfering with sperm-zona binding. The fusion of acrosome-reacted spermatozoa with zona-free oocytes was prevented by anti-T IgG and it was diminished by anti-P IgG. In the same conditions anti-S antibodies had no effect in these in-vitro fertilization events. Immunization of female guinea-pigs with P antigen resulted in a significant decrease of the number of tubal cleaved eggs. T antigens were less clearly implicated in fertilization in vivo. This study provides evidence that well characterized autoantigenic molecules of guinea-pig spermatozoa are involved in fertilization events.     

Secondary trisomy 1 q due to a reciprocal maternal translocation]

The authors report a case of partial trisomy 1 q due to a maternal balanced translocation : t(1 ; 4) (q 32 : p 16). The evocative malformations of trisomy 1 q and monosomy 4 p are discussed and compared to seven others from the literature. Then the interest of the chromosomical prenatal diagnosis and the significance of familial genetic studies are showed.     

Pachytene pairing and metaphase I configurations in a tetraploid somatic Lycopersicon esculentum x L. peruvianum hybrid.

In the tetraploid somatic hybrid between the diploid Lycopersicon species L. esculentum (tomato) and L. peruvianum, synaptonemal complexes formed quadrivalents in 73 of the 120 sets of four chromosomes (60.8%) in 10 cells studied in detail at pachytene. Of these, 43 had one pairing partner exchange, 22 had two, and 8 had three, very close to a Poisson distribution. The points of pairing partner exchange were concentrated at the middle of the two arms. The frequency per arm corresponded with physical arm length. There was a sharp drop around the centromere, and pericentric heterochromatin had a slightly lower probability of being involved in pairing partner exchange than euchromatin. The chromosomes align before pairing and there are several points of pairing initiation, with concentrations at or near the ends and the centromere. From zygotene to late pachytene the quadrivalent frequency decreased considerably. At late pachytene it was lower than expected with the observed high frequency of pairing partner exchange. Pairing affinity between species was only slightly lower than affinity within species, in spite of considerable genetic differentiation. The frequency of recombination nodules increased from early to late zygotene and then decreased strongly to full pachytene. There is a highly significant negative correlation between percent pairing and SC length. At metaphase I the frequency of quadrivalents was 0.444, and branched quadrivalents were rare, probably caused by interference and restriction of chiasma formation to distal euchromatin. Metaphase I quadrivalent frequency is a relatively good indication of pairing affinity in this material.     

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.     

Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.     

Meiotic and sperm chromosome studies in a reciprocal translocation t(1;2)(q32;q36)

Summary Meiotic and sperm chromosomes were studied in a man heterozygous for a reciprocal translocation t(1;2)(q32; q36). Forty-five meiotic metaphase I cells were obtained from semen samples: 86.6% were 22,XY,IV and 13.3% had synaptic anomalies that affected all or some of the bivalents. The quadrivalents observed had a ring configuration (92.3%) or a chain configuration (7.7%). A total of 105 sperm chromosome complements were analyzed: 41% resulted from an alternate segregation, and the percentage of unbalanced sperm was 59%; most of them (71%) resulted from an adjacent 1 segregation. The frequency of anomalies unrelated to the translocation (5.7% numerical and 14.1% structural anomalies) were within the normal range for control donors. There was a good correspondence between the percentage of cells with a ring IV (92.3%) and the proportion of 2:2 segregations (88.6%) and between the percentage of chain IV (7.7%) and the incidence of 3:1 segregations (11.4%).