Meiotic behaviour and spermatogenesis in male mice heterozygous for translocation types also occurring in man

In this thesis a start was made with meiotic observations of mouse translocation types - a Robertsonian translocation and a translocation between a metacentric and an acrocentric chromosome - which also occur in man. It is generally accepted that, when no chromosomal rearrangements are involved, man shows a higher level of non-disjunction than the mouse. When the meiotic behaviour of translocations in mouse and man would be more similar than that found for the autosomal bivalents (and sex bivalent) of these species Chapter 1 reviews the methodology to establish this difference mouse systems characterized by chromosome aberrations would become more attractive as models for the study of endogeneous and exogeneous factors on the meiotic process and extrapolation from mouse to man would be facilitated. As it appeared in these studies that not only the translocation complex but also the normal bivalents contribute to a significant extent to the non-disjunction rates in the heterozygote for a translocation of the meta/acro type, this material appeared even more favourable as an experimental substitute for man than was initially expected.Meiosis in male mice, heterozygous for the Robertsonian translocation Rb(11.13)4Bnr, has been studied in an attempt to elucidate the mechanism(s) of non-disjunction for the trivalent formed at meiosis I by the Rb chromosome and the two acrocentric homologues 11 and 13 (Chapters 2 and 3). As an exogeneous factor of possible influence, the meiotic effects of two types of radiation, administered at relatively low doses 2 and 3 hours before prometaphase-metaphase II (probably during metaphase-anaphase I), were determined in Rb4Bnr/+-males (Chapter 3).The segregational behaviour of the multivalent at anaphase I has been compared in T(1;11.13S),+/+,RBnr, structurally homozygous for the metacentric Rb(11.13)4Bnr chromosome whereby arm 13 of one of the two metacentrics is involved in a reciprocal translocation with the acrocentric chromosome 1, and T(I113)70H,+/+,Rb4Bnr (Chapter 4). The multivalents formed by the latter karyotype at meiosis I are highly comparable to those of the former one; for details see section III below.The results obtained, and presented below, warrant a further investigation of these mouse meiotic systems as models for the situation in man as is for example indicated by the significant increase of normal bivalent non-disjunction found in the T(1;11.3S),+/+,Rb4Bnr and the T70H,+/+,Rb4Bnr karyotypes.In the next sections the chapters on which the conclusions are based, have been indicated between parentheses.I  Indications for the relation between delay before and around anaphase I and non-disjunction in male Rb4Bnr Robertsonian translocation heterozygotes- For Rb4Bnr/+ males, inter mouse variation was noted with respect to the duration of the meiotic prophase, i.e. the period "end of the premeiotic S phase-metaphase V'. This was concluded on the basis of the frequency of late diplotene/metaphase I cells with labelled chromosomes recorded autoradiographically in mice killed various time intervals after the i.p. administered injection with 3 H-thymidine (3).- For Rb4Bnr/+ males, compared to their +/+ controls, a greater mean number of late diplotene/metaphase 1 cells was counted in the separate groups along the seminiferous epithelium, whereas the mean distance between the subsequent groups was smaller. As these data are in contradiction with the reduction by 58% of the epidydimal sperm count for Rb4Bnr/+ compared to +/+, the greater number was thought to be an accumulation effect due to delay during diplotene/metaphase I for at least a fraction of the cells. Implicitly a causal relation between delay and cell death before the spermatozoal stage is also suggested (2).- The duration of the period "end meiosis I (metaphase I) - end meiosis II'' was approached autoradiographically for Rb4Bnr/+-males with the aid of cells showing a labelled Y chromosome only. The best assessment of the duration of this period was 3 hours or less. Inter mouse variation with respect to the duration of the period "end of the premeiotic S phase-metaphase ill (see above) prevented a more accurate estimate (3).- Irradiation of Rb4Bnr/+ males for 14.5 minutes with a dose of 15.2 rad fast neutrons (mean energy 1.7 MeV) significantlydecreased the incidence of ancuploid secondary spermatocytes, scored 2 and 3 hours after application. This was explained by selective cell killing of those cells giving rise to aneuploid daughter cells after anaphase I, rather than by protection against non- disjunction by the irradiation dose, as follows: For Rb4Bnr/+ during the final period of the primary spermatocyte stage, cells show a continuous distribution with respect to the extent of delay. Cells with a strong delay will die off before the secondary spermatocyte stage, while the fraction showing only minor delay will survive (in the unirradiated situation) but with an increased risk of producing aneuploid daughter cells. The results suggest that, in terms of cell death, the victims of the 15.2 rad neutron irradiation predominantly have to be searched in the last category. A causal relation between delay and ensuing non-disjunction at anaphase I, and between delay and the neutron irradiation effect respectively, is thus suggested but the basis for these interactions remains unknown (3).II  Irradiation induced damage during meiosis in male Rb4Bnr Robertsonian translocation heterozygotes- After irradiation of Rb4Bnr/+ male mice for 14.5 minutes with either a dose of 15.2 rad neutrons or 60 rad X rays, only neutrons affected, viz. significantly decreased (see above), the incidence of aneuploid secondary spermatocytes estimated 2 and 3 hours after irradiation. The reason for this difference remained unknown and could not be explained on the basis of a difference in the levels of radiation induced damage. The two radiation types induced comparable levels of chromosome damage, i.e. numbers of breaks, fragments and deletions per cell, when compared in either late diplotene/metaphase I or metaphase II cells. For these parameters the neutrons: X ray RBE ratios estimated were 5.4 for meiosis 1 and 3.3 for meiosis II cells (3).- After an irradiation-fixation interval of 2 to 3 hours, the chromosome damage (i.e. numbers of breaks, fragments and deletions) induced by either 15.2 rad neutrons or 60 rad X rays was5-10 times higher when scored in metaphase II than in diakinesis/ metaphase I cells. At the moment of irradiation the two categories of cells were, most probably, at prometaphase/metaphase 1 and late diplotene/early diakinesis, respectively. The difference in chromosome damage was argued to be merely the consequence of differences in chromosomal processes taking place during the irradiation-fixation interval and not a reflection of a difference in radiation sensitivity between the two meiosis I stages (3).III  The meiotic behaviour in T(1;11.13S),+/+,Rb4Bnr and T70H,+/+,Rb4Bnr male mice, also with reference to the behaviour of the normal bivalentsThe T(1;11.13S),+/+,Rb4Bnr karyotype includes the metacentric Rb(11.13)4Bnr chromosome in homozygous condition whereby the 13 arm of one of the metacentrics is involved in a reciprocal translocation with the acrocentric chromosome 1. The T70H,+/+,Rb4Bnr karyotype is double heterozygous for the T(1;13)7OH reciprocal translocation and the metacentric Rb(11.13)4Bnr chromosome. Its multivalent at meiosis I differs from the one in the former karyotype in one respect: The (acrocentric) chromosome 11 is here involved once separately and once as one of the arms of the metacentric, i.e. attached to the 13 arm, whereas in the former karyotype the two chromosomes 11 are involved each in a metacentric.- For the short translocated segment 13 t , in both T(1;11.13S),+/+, Rb4Bnr and T70H,+/+,Rb4Bnr males, chiasma terminalization with proceeding meiosis I was observed less frequently compared with T70W+. It was argumented that this is probably due to spermatocyte I degeneration taking place to a higher extent in the former two karyotypes as in both, in comparison to T7OH/+, a greater frequency of meiosis 1 cells with short contracted bivalents was observed. The latter phenomenon was in an earlier study shown to correlate highly with a decreasing sperm count (4).- In T(1;11.13S),+/+, Rb4Bnr a chiasmate association of the long interstitial segment 13 i was absent in 12.5% (N=1000) of the meiosis I cells, but only in 2.7% (N=1000) of these cells in T70H,+/+,Rb4Bnr males. A cause for this discrepancy could not be given the phenomenon could not be contributed to a precocious "disappearance" of the 13 i chiasma - and seems to be an effect of the metacentric chromosome present in homozygous condition in the former karyotype (4).A significant increase of the 3:1 segregation of the multivalent chromosomes after anaphase 1 in comparison to T7OH/+ (3.5%) was found for T(1;11.13S),+/+,Rb4Bnr (10.9%) but most significantly for T70H,+/+,Rb4Bnr (22.6%). In the latter karyotype this refers only to the four chromosomes involved in that part of the multivalent caused by the T70H translocation. In T70H,+/+,Rb4Bnr, a preference of the small marker chromosome 1 13 to segregate ; with the metacentric Rb4Bnr chromosome was indicated and this could be sustained by the high frequency of Ts(1 13 )70H tertiary trisomics among the progeny of males of this karyotype. The results suggest that factors predisposing for 3:1 segregation and life expectancy of the tertiary trisomic product produced among the progeny may coincide (4).In T70H,+/+,Rb4Bnr males the alternate and adjacent 1 segregation products could be distinguished at meiosis II and almost equal frequencies were found for both. In the offspring however, karyotypes resulting from adjacent 1 were found more frequently but this disagreement is probably due to the limited sample size of the offspring karyotyped (4).In both T(1;11.13S),+/+,Rb4Bnr and T70H,+/+,Rb4Bnr male mice, the normal bivalent non-disjunction after anaphase I appeared to be highly elevated in comparison to +/+ and T70H/+ For the former karyotype a correlation was observed, moreover, between nondisjunction for the normal bivalents and adjacent 2 segregation of the multivalent chromosomes. This suggests either an interaction between the two phenomena or, alternatively, their coincidence may be an effect of the general (poor) physiological status of the cells. A sharp distinction between these alternatives cannot be made. The indication of spermatocyte I degeneration (see first point of this section) in both karyotypes puts weight on the latter hypothesis, assuming an interaction between the poorer physiological status of the cell and adjacent 2/normal bivalent non-disjunction.