Clonal analysis of radiation-induced translocations in stem-cell spermatogonia of normal and T70H translocation heterozygous mice

7 T(1;13)70H/+ and 13 +/+ male mice were given 2 doses of 250 rad acute X-rays separated by 24 h. The +/+ mice were analysed in 2 groups during the first meiotic division for induced translocations, on average 177 and 233 days after irradiation, and the T70H/+ mice were analysed in parallel with the second group of +/+ males. One testis was treated with normal air-drying procedures yielding a random sample of cells. The other testis was processed according to a new technique, which enabled separate analysis of the various locations along the seminiferous epithelium where groups of cells are synchronously in the diakinesis-metaphase I stage of meiosis. The number of cells in such groups was estimated. Both capita epididymes were used for a sperm count. In agreement with an earlier finding, fewer induced translocations were recovered from the T70H/+ mice than from +/+ mice (10.6 versus 19.2%, air-drying technique).Estimates of the group sizes in combination with the occurrence of induced translocations yielded the following information. A synchronously moving group of diakinesis-metaphase I cells originates from, on average, 1.25 stem cells (Appendix). We found an indication for a reduction in group size by 33% when a clone originated from a stem cell carrying an induced translocation compared with a wild-type clone (see Appendix). Both, the data on group size and the sperm counts indicate that, 7 months after the irradiation, the seminiferous epithelium has not totally recovered. Final recovery seems to be slower or absent in the T70H/+ males. The data obtained from the T70H/+ heterozygotes indicate the stem-cell spermatogonia to be responsible for the reduction of the rate of translocation induction with this karyotype, either due to a reduced formation rate or due to a diminished capacity of some of the induced translocation-carrying stem cells to proliferate into a clone reaching the meiotic divisions.     

Mutagenic effect of different types of irradiation on the sex cells of male mice. X. Frequency of recessive lethal mutations and reciprocal translocations in the spermatogonia of mice subjected to fractional gamma-irradiation]

The frequency of recessive lethal mutations and reciprocal translocations was investigated in spermatogonia of CBA male mice which were thrice gamma-irradiated at doses of 300 r with 28 days intervals. The rate of induced recessive lethals was estimated 1) by comparison of embryos survival between the irradiated and control groups in mating of the F1 males with their daughters, and 2) by estimation the frequency of males heterozygotes for recessive lethals in the first generation. In the first case the frequency of recessive lethals was 2,8 +/- 0,8-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete in the second case. The frequency of heterozygotes for reciprocal translocations in the first generations of males was 3,1 +/- 0,9-10(-5) per r per gamete.     

Duration of the cycle of the seminiferous epithelium and its stages in the rhesus monkey (Macaca mulatta)

Doses of 1 Gy or more of X-irradiation killed all B spermatogonia present in the testis, and during the first 3 weeks after irradiation, virtually no new B spermatogonia were formed. The number of Apale spermatogonia decreased during the first cycle of the seminiferous epithelium while the number of Adark spermatogonia only began to decrease during the second cycle after irradiation. In this study, the duration of the cycle of the seminiferous epithelium in the rhesus monkey was estimated to be 10.5 days (SE = 0.2 days). This was determined following the depletion of germinal cells in the seminiferous epithelium during the first 3 weeks after irradiation. The duration of each of the 12 stages of the cycle was also determined. Our observations of the progress of germinal cell depletion revealed that after a dose of X-irradiation sufficient to kill all B spermatogonia, all spermatocytes disappeared from the testis within about 17 days, and all spermatids within about 31 days.     

Effect of follicle-stimulating hormone (FSH) on radiation-induced chromosomal aberrations in mouse germ cells and Chinese hamster cells in vitro

Earlier observations on the induction by X-rays of reciprocal translocations in stem-cell spermatogonia of the rhesus monkey have established a correlation between the level of follicle-stimulating hormone (FSH) in blood at the moment of irradiation and the final recovery of translocations (van Buul, 1980). In the present study, FSH treatment of mice did not induce chromosomal aberrations in bone-marrow cells or stem-cell spermatogonia, nor did it change the radiosensitivity of stem-cell spermatogonia for the induction of chromosomal translocations. Experiments in vitro with Chinese hamster ovary cells (CHO), however, showed a clear radiosensitizing effect of FSH on the induction of structural chromosomal aberrations.     

Dose—response relationship for X-ray-induced reciprocal translocations in stem-cell spermatogonia of the rhesus monkey (Macaca mulatta)

The induction of reciprocal translocation in stem-cell spermatogonia of the rhesus monkey (Macaca mulatta) was studied after testicular X-irradiation of mature males (50, 100 and 200 rad) or whole-body irradiation of young males (200 and 300 rad). After the recovery of the germinal epithelium, cytogenetic analysis was carried out on spermatocytes descended from irradiation spermatogonia. Preparations of C-banded diakinesis-metaphase I were screened for translocation configurations. The frequencies of abberations obtained were 0% at 0 rad, 0.36% at 50% rad, 0.86% at 100 rad, 0.99% at 200 rad and 0.68% at 300 rad, suggesting a humped dose—response relationship. There was no evidence for the contribution of a quadratic component to the yield in the lower dose range. A comparison of these results with those obtained for other mammals by a number on investigators shows that the frequencies of translocations in the rhesus monkey are much lower than those published for most other mammalian species.     

No induction of interchromosomal effect in male meiosis of Chinese hamsters with reciprocal translocations

Chinese hamsters from five strains with reciprocal translocations, T(1;3)7Idr, T(1;3)8Idr, T(1;2)9Idr, T(7;9)16Idr, and T(1;5)17Idr, and a karyotypically normal strain, CHS/Idr, were used to look for an interchromosomal effect by chromosomal analysis of meiotic cells and one-cell embryos. The frequencies of nondisjunction at first meiosis in five normal (+/+) males, calculated by doubling the number of hyperhaploid cells, ranged from 0.43% to 1.33%, and there was no significant difference in frequency among individuals. On the other hand, the frequency of hyperhaploid cells in males heterozygous for each translocation ranged from 3.0% to 11.8%, and the frequency of hyperhaploid cells with an extra translocation-unrelated chromosome ranged from 0.2% to 0.4%, which is no different from that estimated from scoring of +/+ males at the second meiotic metaphase. In one-cell embryos from crosses between karyotypically normal females and male heterozygotes for T(1;2)9Idr and T(7;9)16Idr, 1.1% and 0.5% of embryos had an extra translocation-unrelated chromosome. Compared with the control, the frequency of meiotic nondisjunction showed no increase in male heterozygotes for the reciprocal translocations. Therefore, the results suggest that multivalents and rearranged chromosomes existing at first and second meiosis in male Chinese hamsters exert no influence on segregation of normal bivalents and chromosomes unrelated to the rearrangements.     

Meiotic drive of t-haplotypes: segregation of chromosomes in mice with partial trisomy

The properties of the t haplotypes, specific mutant states of the proximal region of chromosome 17 in the house mouse keep renewing interest. One such property is increased transmission of the t haplotype from heterozygous t/+ males to their offspring. By means of reciprocal translocation T (16; 17)43H, we have constructed males with tertiary trisomy 17 (+T43/++/RB7+) carrying Robertsonian translocation Rb(16.17)7Bnr. The offspring of these males was viable when sperm of +T43/++ and Rb7+ was used. The segregation patterns in the offspring of t-bearing trisomics were analysed on days 16-18 of embryonic development. It was found that in the case when the t haplotype is on the normal acrocentric (male male ++T43/+t12+/Rb7++), its presence in the gamete +t12+/++T43 does not produce meiotic drive. However, when t6 is on Rb7, meiotic drive was equal to 80%. It is concluded that the presence of a normal homolog and a t-bearing chromosome in sperm does not result in meiotic drive. Possible mechanisms of meiotic drive of the t haplotypes are discussed.     

Kit ligand mediates survival of type a spermatogonia and dividing spermatocytes in postnatal mouse testes

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c-kit is expressed in type A, intermediate, and B spermatogonia, and kit-ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c-kit function. The present work was undertaken to determine whether blocking c-kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8-day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c-kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SI^d + males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15-fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output. © 1995 wiley-Liss, Inc.     

Meiotic drive of t haplotypes: chromosome segregation in mice with tertiary trisomy

The properties of the t haplotypes, specific mutant states of the proximal region of chromosomes 17 in the house mouse, are of continuing interest. One such property is increased transmission of the t haplotype by heterozygous t/+ males to offspring. Using the reciprocal translocation T(16;17)43H we have constructed males with tertiary trisomy of chromosome 17 (+T43/+ +/Rb7+) carrying the Robertsonian translocation Rb(16.17)7Bnr. Only the progeny of these males which had inherited either T43/+ or Rb7 from their male parent were viable. The segregation patterns in the offspring of t-bearing trisomics were analysed on days 16-18 of embryonic development. It was found that, when the t12 haplotype is in the normal acrocentric (males+ +T43/+ t12 + /Rb7+ +), its presence in the gamete +t12+/+ + T43 does not produce meiotic drive. However, when t6 is in Rb7, meiotic drive was observed: 80% of offspring carried the t haplotype. It is concluded that the meiotic drive is probably inhibited by the presence of a normal homologue of chromosome 17 in the same sperm. Possible mechanisms for the t haplotype effect are discussed.     

Effect of gonadotrophins and testosterone on the seminiferous tubules of the immature rat.

The actions of HCG and PMSG for different periods and of testosterone of the immature rat testis were studied. Short-term administration of HCG (1-3 days) induced an early meiotic and postmeiotic stimulatory effect but a decrease in spermatogonial numbers. Administration of HCG for longer periods (10 days) caused a reduction in numbers of all cell types. Treatment with HCG + PMSG reduced the amount of inhibition, while PMSG alone resulted in histological and humoral signs of stimulation of the interstitial tissue and the meiotic and postmeiotic stages; the numbers of spermatogonia were not affected. Testosterone caused stimulation of the meiotic and postmeiotic stages and a reduced number of spermatogonia. It is concluded that while PMSG directly stimulates spermatogonia, HCG acts through testosterone secretion at the meiotic and postmeiotic stages. The early inhibitory effects of HCG and testosterone on spermatogonial numbers could be ascribed to the inhibition of endogenous FSH by androgens.     

Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia

Unbalanced (duplication/deficiency) sperm from balanced reciprocal translocations induced in spermatogonial stem cells of mice generally lead to embryonic lethality around the time of implantation. In a recent study (Generoso et al., 1985), it was found that the incidence of X-ray-induced embryonic lethality differed markedly between two hybrid stocks of irradiated male mice. A parallel difference in the frequencies of reciprocal translocations was observed cytologically in the meiocytes of irradiated males. In the present report, which is an adjunct to the study by Generoso et al. (1985), it was determined whether or not similar differences between the two stocks exist for congenital defects resulting from genetic damage to stem-cell spermatogonia. The results indicate not only an association between the frequencies of induced reciprocal translocations and congenital abnormalities, but also a parallel greater frequency of induced malformations in the (C3H × 101)F1 stock versus the (SEC × C57BL)F1 stock of males.     

X-ray-induced specific-locus mutation rate in newborn male mice

The specific-locus mutation frequency resulting from 300 R of acute X-irradiation has been determined for the germ cells present in newborn male mice. The frequency is 13.7·10^?8 mutations/locus/R, which is statistically significantly lower than that of 29.1·10^?8 mutations/locus/R found earlier for the same loci in spermatogonia of the adult male by W. L. Russell. The mutation rate for newborn males does not differ significantly from the induced specific-locus frequency reported for fetal males by T. C. Carteret al.The incidence of clusters of specific-locus mutations found following the irradiation of the newborn males was statistically significantly higher than the cluster incidence reported by W. L. Russell for similar irradiation of adult males. This presumably indicates the survival of relatively fewer reproductive cells following irradiation of the day-o testis.Although there are suggestions that the distribution of mutations among the loci following irradiation of the newborn males may be different from that of the irradiated adults, no statistically significant differences are demonstrated.It is quite possible that the testis of the newborn mouse may be comparable to the relatively undifferentiated human testis which persists for approx. 10 years. Until the present research was undertaken, no attempt had been made to determine the specific-locus mutation frequency resulting from X-irradiation of newborn male mice. Although some important questions still remain concerning the explanation for the lower mutational response of the newborn mouse testis, from the hazard standpoint it is reassuring that the mutation frequency of the newborn male is statistically significantly lower than that of the adult.     

Ontogeny of gonadal sex development relative to growth in fathead minnow

Ovarian differentiation of fathead minnow Pimephales promelas occurred at between 10 and 25 days post‐hatch (dph)(8–11 mm fork length, L _F, and 7–12 mg), and was characterized by the presence of meiotic cells in the centre of the gonad, location of the somatic cells at the periphery of the gonad and the formation of an ovarian cavity. In contrast with the developing ovary, in the presumptive testis somatic cells were scattered throughout the gonads and this was evident from 25 dph (fish >10 mm and >11 mg). In males, at 60 dph (15–26 mm and 39–220 mg) the efferent ducts (sperm ducts) were apparent and the testis lobules started to form, but germ cells (spermatogonia) did not enter meiosis until between 90 and 120 dph. Fish of both sexes reached full sexual maturity at between 120 and 150 dph (males: 33–59 mm and 400–2895 mg; females: 24–48 mm and 160–1464 mg). Differences in body size ( L _F and mass) between males and females were only apparent when the fish were approaching full sexual maturity (120 dph).     

Testicular maturation in the sheep bot fly Oestrus ovis

The process of testicular maturation in relation to intrapuparial development was studied in the sheep nasal bot fly, Oestrus ovis L. (Diptera: Oestridae). After formation of the puparium during larval-pupal apolysis and the cryptocephalic pupal stage (approximately 24-72 h), spermatogonia had undergone mitotic divisions and sperm cysts had been formed. Five days after pupariation, spermatogonia transformed into primary spermatocytes during the phanerocephalic pupal stage, and secondary spermatocytes first appeared during the pupal-adult apolysis. Secondary spermatocytes began undergoing the second meiotic division by day 8 (transparent-eye pharate adult stage). By days 9 and 10, round spermatids were present and began to elongate by day 11. By day 12, the first bundles of tailed spermatozoa had appeared. By day 15 (the yellow-orange eye pharate adult stage), round, elongated, tailed and bundled spermatids were predominant and by day 17 differentiating spermatids occupied nearly 35% of the testicular cavity, and 60% was occupied by free sperm. By day 21 (the red-brown eye pharate adult stage), spermatozoa colonized the seminal vesicle. At emergence (approximately day 22), a complement of free sperm occupied the testis and the seminal vesicle, but groups of developing cells frequently remained in certain zones. Spermatogenesis was carried out after pupariation and spermiogenesis occurred during the pharate adult stage. After emergence, males possessed fully formed spermatozoa ready for ejaculation.     

The induction of specific-locus mutations with N-propyl-N-nitrosourea in stem-cell spermatogonia of mice.

A specific-locus test to determine the effect of N-propyl-N-nitrosourea (PNU) on the stem-cell spermatogonia of mice has been performed. Male wild-type mice (C3H/He) were treated with an intraperitoneal injection of 200 mg/kg [corrected] of PNU. Eight weeks after the injections, the males were mated with tester stock females (PW), homozygous for 6 visible recessive genes. Twelve mutants among 8605 offspring were observed. The mutation frequency with PNU was calculated to be 23.2 x 10(-5)/locus/gamete, showing a significant difference from that of the non-treated control. The mutations were all heritable and half of them were viable in homozygous condition. The mutation frequency with PNU was about one-third of that with N-ethyl-N-nitrosourea, a highly potent mutagen for mouse stem-cell spermatogonia.     

Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome- specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere- specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8- specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.     

Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum (II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatogonial stem cells of male (101/E1 x C3H/E1)F1 mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1-13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63-70 days after treatment, representing treated stem-cell spermatogonia. Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Adler, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

Gonadal morphogenesis and sex differentiation in cultured Ussuri catfish Tachysurus ussuriensis

The objective of this study was to investigate the optimal developmental time to perform sex reversal in Ussuri catfish Tachysurus ussuriensis, to develop monosex breeding in aquaculture. Systematic observations of gonadal sex differentiation of P. ussiriensis were conducted. The genital ridge formed at 9 days post fertilization (dpf) and germ cells begin to proliferate at 17 dpf. The ovarian cavity began forming on 21 dpf and completed by 25 dpf while presumptive testis remained quiescent. The primary oocytes were at the chromatin nucleolus stage by 30 dpf, the peri‐nucleolus stage by 44 dpf and the cortical alveoli stage by 64 dpf. The germinal vesicle migrated towards the animal pole (polarization) at 120 dpf. In presumptive testis, germ cells entered into mitosis and blood vessels appeared in the proximal gonad on 30 dpf. The efferent duct anlage appeared on 36 dpf and formation of seminal lobules with spermatogonia and lobules interstitium occurred at 120 dpf. Therefore, gonadal sex differentiation occurred earlier in females than in males, with the histological differentiation preceding cytologic differentiation in T. ussuriensis. This indicates that undifferentiated gonads directly differentiate into ovary or testis between 17 and 21 dpf and artificial induction of sexual reversal by oral steroid administration must be conducted before 17 dpf.     

Analysis of the testes of H-Y negative XOSxrb mice suggests that the spermatogenesis gene (Spy) acts during the differentiation of the A spermatogonia

H-Y antigen negative XOSxrb mice, like their H-Y positive XOSxra counterparts, have testes; but, in contrast to XOSxra males, XOSxrb testes almost totally lack meiotic and postmeiotic stages of spermatogenesis. The quantitative analysis of the testes of XOSxrb males and their XY +/- Sxrb sibs, described in the present study, identified two distinct steps in this spermatogenic failure. First, there was a reduction in mitotic activity among T1 prospermatogonia, so that approximately half the normal number of T2 prospermatogonia were produced. Second, there was a dramatic decrease in the number of A3 and A4 spermatogonia and no Intermediate or B spermatogonia. These reductions were also largely due to decreased mitotic activity, there being a shortage of A1 and A2 spermatogonial divisions and no divisions among A3 or A4 spermatogonia. Mitotic activity among the T2 prospermatogonia and the undifferentiated A spermatogonia was normal. This means that the spermatogonial stem cells, which are a subset of the undifferentiated A spermatogonia, are unaffected in XOSxrb mice. Sxrb is now known to have arisen by deletion of DNA from Sxra. It is clear from the present findings that a gene (or genes) present in the deleted DNA plays a major role in the survival and proliferation of the differentiating A spermatogonia.     

Male pachytene pairing in single and double translocation heterozygotes and spermatogenic impairment in the mouse

In order to clarify the relationship between meiotic pairing and progress of spermatogenesis, an analysis of male meiotic pairing was carried out in four reciprocal translocation heterozygotes and two double heterozygotes for two semi-identical reciprocal translocations. The reciprocal translocations were chosen to range from fertility (T70H/+) through almost complete sterility (T31H/+) to complete sterility (T32H/+, T42/H+). If meiotic pairing in the translocation multivalent was incomplete, it concerned terminal or probably more often proximal chromosome segments (Chain IV). If both segments failed to pair the multivalent symbol is Chain III+I. Complete pairing is symbolized by Ring IV. To contrast and complement observations of this type, the double heterozygotes were introduced. Males of this type in theory possess two heteromorphic bivalents with a central area of incomplete meiotic pairing (loop formation). Of the T70H/T1Wa double heterozygotes, 36% of the males are capable of inducing at least one decidual reaction in two females whereas for T26H/T2Wa, 79% of the males can do so. For the reciprocal translocations, it was found that proximity of the multivalent to the sex bivalent during pachytene increased in the order Ring IV, Chain IV, Chain III+I. The degree of spermatogenic impairment as measured from cell counts in histological sections and tubular whole mounts, is positively related to the frequency of proximity between the sex chromosomes and the translocation multivalent and thus to lack of meiotic pairing within the multivalent. The meiotic pairing analysis of the double heterozygotes yielded the following findings. For the long heteromorphic bivalents a true loop was never seen in T70H/T1Wa and only rarely observed in T26H/T2Wa. Small marker bivalents of both types were usually recognizable by the following criteria: (i) pairing confined to distal or proximal segments, (ii) both distal and proximal segments pairing and loop formation and (iii) pairing covering the entire length of both homologues but the longer one often with a thickened lateral element. The same positive correlation between the absence of pairing (proximal, distal or central) and the proximity of the small marker bivalent synaptonemal complex to the sex bivalent has been found as for unpaired segments within reciprocal translocation multivalents. One unexpected finding was the occurrence of diploid spermatids and spermatozoa especially in T32H/+ males (70–91%) but also in T31H/+ (3–39%).