FOETAL ERYTHROPOIESIS IN GENETICALLY ANAEMIC, FLEXED-TAILED (f/f) MICE

This paper presents measurements of foetal weights, haemoglobin concentrations, red cell counts, blood volumes, erythroblast thymidine labelling indices and cell cycle parameters in genetically anaemic, flexed-tailed ( f/f ) and haematologically normal ( f/+ ) foetuses.
The production of red blood cells from the livers of ( f/f ) foetal mice lags behind that of heterozygote ( f/+ ) foetuses, and the cells that are produced are small and poorly haemoglobinized. The anaemia is not due to an abnormal cell cycle in f/f erythroblasts, which appear to be capable of responding to the anaemia by a small increase in their rates of proliferation. The number of red cells in the foetal circulation can be calculated from the cell cycle time and number of hepatic erythroblasts; the calculated number agrees quite well with the measured number in both f/f and f/+ foetuses. Many characteristics of the anaemia of the f/f foetus can be accounted for if it is assumed that there is a deficiency in the production or activity of an enzyme involved early in the haem synthetic pathway.     

CELL PROLIFERATION IN HAEMATOPOIETIC SPLEEN COLONIES OF MICE: DIFFERENCE BETWEEN COLONIES DERIVED FROM INJECTED ADULT BONE MARROW AND FOETAL LIVER CELLS

Cell proliferation in mouse spleen colonies, derived from injected foetal liver and young adult bone marrow, was studied by measuring incorporation of radio-iodine-labelled 5-iodo-2'-deoxyuridine (IUdR). Foetal liver-derived colonies incorporated significantly more IUdR than marrow-derived colonies on the 8th and 12th days after cell injection. The data are consistent with the view that foetal haematopoietic stem cells are capable, on average, of producing larger descendant populations than are stem cells from young adults.     

Haemopoietic stem cells in the foetal mouse thymus.

The presence of haemopoietic stem cells (HSC) in the foetal mouse thymus was assessed to determine whether all cells which enter the developing organ are precommitted to thymocyte differentiation, or if stem cell multipotentiality still exists. The Till and McCulloch spleen colony assay was used to delineate foetal-thymus derived HSC in lethally irradiated recipients. Of the range examined, between 13 days of gestation to birth, a peak of stem cell activity occurs in 15-day foetal thymus. The surface colonies produced by the thymus-derived HSC are small compared to colonies produced by the liver derived HSC, although well within the range of the latter. Histologically, five types of colonies were identifiable which were produced by the thymus-derived HSC, indicating that these cells retain the potential to form a wide range of differentiated colonies.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver

In the developing mouse embryo the first definitive (transplantable-into-the-adult) haematopoietic stem cells/long-term repopulating units (HSC/RUs) emerge in the AGM region and umbilical vessels on 10-11 days post coitum (d.p.c.). Here, by limiting dilution analysis, we anatomically map the development of definitive HSC/RUs in different embryonic tissues during early colonisation of the liver. We show that by day 12 p.c. the mouse embryo contains about 66 definitive HSC/RUs (53 in the liver, 13 in other tissues), whereas on the previous day the total number of definitive HSC/RUs in the entire conceptus is only about 3. Owing to the length of the cell cycle this dramatic increase in the number of definitive HSC/RUs in only 24 hours is unlikely to be explained purely by cell division. Therefore, extensive maturation of pre-definitive HSCs to a state when they become definitive must take place in the day 11-12 embryo. Here we firstly identify the numbers of HSCs in various organs at 11-13 d.p.c. and secondly, using an organ culture approach, we quantitatively assess the potential of the aorta-gonadmesonephros (AGM) region and the yolk sac to produce/expand definitive HSC/RUs during days 11-12 of embryogenesis. We show that the capacity of the AGM region to generate definitive HSC/RUs is high on 11 d.p.c. but significantly reduced by 12 d.p.c. Conversely, at 12 d.p.c. the YS acquires the capacity to expand and/or generate definitive HSCs/RUs, whereas it is unable to do so on 11 d.p.c. Thus, the final steps in development of definitive HSC/RUs may occur not only within the AGM region, as was previously thought, but also in the yolk sac microenvironment. Our estimates indicate that the cumulative activity of the AGM region and the yolk sac is sufficient to provide the day 12 liver with a large number of definitive HSC/RUs, suggesting that the large pool of definitive HSC/RUs in day 12 foetal liver is formed predominantly by recruiting 'ready-to-use' definitive HSC/RUs from extra-hepatic sources. In accordance with this we observe growing numbers of definitive HSC/RUs in the circulation during days 11-13 of gestation, suggesting a route via which these HSCs migrate.     

Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice

The relative proliferative capacity of haematopoietic cell populations derived from 22-week-old adult bone marrow and 14–18 day foetal liver has been studied in lethally irradiated syngeneic recipients by means of chromosome markers. Although starting at a disadvantage in terms of the number of colony-forming units (stem cells) injected, the foetal liver-derived populations steadily increased their relative numbers in the myeloid and lymphoid tissues over a period of several weeks until a plateau was reached. It is suggested that stem cells in foetal liver have, on average, a higher intrinsic capacity for self-renewal than do those in bone marrow, and that this capacity falls to the adult level within about ten weeks of transfer.     

Experimental histoplasmosis capsulati in athymic nude mice

Granuloma formation in nude (nu/nu) mice and their heterozygous littermates (nu/+ mice) against Histoplasma capsulatum var. capsulatum infection was studied.A culture of H. capsulatum var. capsulatum, isolated from a granuloma in the nasal cavity of a Japanese patient, was used in this experiment. Sixteen specific-pathogen-free male nu/nu and 32 nu/+ mice were used in this study.The nu/+ mice were divided into two groups. Sixteen nu/+ mice in one group and 16 nu/nu mice were inoculated intraperitoneally with 10⁶ yeast cells of the fungus, those in the other group of nu/+ mice were inoculated intravenously with the same number of the yeast cells. Two mice out of each group were sacrificed 2, 3, 7, 11, 14, 18, 25 and 30 days after inoculation, and each of their organs was examined histopathologically. In addition, pieces of these tissues were cultured on Sabouraud's dextrose agar slants.In the nu/+ mice inoculated intraperitoneally, although the fungus was recovered from the spleen, kidney and lymph nodes during the initial course of the infection, lesions were not detected in their histopathological sections. In the nu/+ mice inoculated intravenously, colonies were recovered from all of the organs examined, other than the brain and thymus, 7 days after inoculation.Histopathologically, a few microfoci consisting chiefly of mononuclear cells with or without yeast cells were found in the liver sections 4 days after inoculation. Seven and 11 days after inoculation the number of lesions had increased. They had large accumulations of mononuclear cells. From day 14 on, almost all of the yeast cells had lost most of their staining affinity or were destroyed in the granuloma. From day 25 on, the granulomatous lesions changed gradually to fibrous tissue.In the nu/nu mice the fungus was readily recovered from the spleen, liver, kidney and lymph nodes. Histopathologically, a few microfoci consisting of mononuclear cells were present in the liver sections 4 days after inoculation. That is to say, during the initial course of infection granulomas were formed. In the liver, from day 7 on, the lesions were large and their number increased. However, there was a definite difference between the nu/nu and nu/+ mice. In the former, the yeast cells were not killed, and they continued to multiply within the granulomas. These granulomas were never transformed into fibrous tissue.     

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.     

Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.     

Hepatic stem cells: from inside and outside the liver?

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.     

Haemopoiesis in the beagle foetus after in utero irradiation

On day 33 of gestation, foetal beagles were irradiated in utero (0.9 Gy of 60Co gamma-irradiation, 0.4 Gy/min). Foetal haematocytopoiesis was studied during the third trimester of gestation (days 42-55). Peripheral blood nucleated cell counts were 33 per cent lower than normal on day 44 and continued to be lower until day 49, when values became higher than normal. Splenic cellularities of irradiated pups on day 44 were more than 3 times those of the nonirradiated, but thereafter they were similar to normal. Differences in haemopoietic progenitor cell activity between irradiated and normal foetuses were observed. In comparison with the other foetal tissues, the foetal liver appeared to experience greater radiation injury. For example, on day 44, the irradiated liver BFU-E, CFU-E, and GM-CFC per 10(5) cells were almost fivefold lower than normal values. Spleens of irradiated foetal beagles contained a marked increase in all haemopoietic progenitor cells (BFU-E, CFU-E, and GM-CFC) and recognizable proliferative granulocytic cells and nucleated erythroid cells. The haemopoietic activity of the irradiated bone marrow during days 42-44 was similar to that of the irradiated spleen, and compensated for the damaged liver. However, unlike the irradiated spleen, the irradiated bone marrow had decreased BFU-E activity compared with the values for the nonirradiated bone marrow during days 48-55. Until day 50, the irradiated marrow contained fewer recognizable proliferative granulocytic cells but more nucleated erythroid cells.     

Cells with natural killer activity in the cerebrospinal fluid of normal mice and athymic nude mice with acute Sindbis virus encephalitis

Sindbis virus causes an acute, nonfatal inflammatory encephalitis in weanling BALB/c mice. Mononuclear inflammatory cells are present in the cerebrospinal fluid (CSF) as well as in the parenchyma of the brain. Both aspects of this inflammatory response were eliminated by treatment with cyclophosphamide. Athymic nude (nu/nu) mice developed no inflammation in the brain, but did develop a CSF pleocytosis that peaked on day 2 after infection. The time course of the appearance of cells in the CSF was earlier in nu/nu mice than their heterozygote (nu/+) littermates. The pleocytosis in nu/nu mice reached a peak on day 2, whereas in nu/+ mice the peak was on day 4, as it is in normal BALB/c mice. To determine whether some of the CSF cells in nu/nu mice may be natural killer (NK) cells, NK activity was measured in a 4-hr assay by using a YAC-1 target cell. NK cell activity in the spleen and peripheral blood was induced by infection with Sindbis virus in nu/nu mice with a similar time course to that of nu/+ mice (peak 1 day after infection). CSF from nu/nu mice had NK activity present 2 days after infection that was greater than that present in either the peripheral blood or spleen. BALB/c and nu/+ mice had insufficient cells present for assay at day 2, but BALB/c mice had NK activity present in the CSF 3 and 5 days after infection that exceeded that in the peripheral blood or spleen. Brain interferon was detectable on day 1 in nu/nu mice, but not until day 2 in nu/+ mice even though the amounts of brain virus were the same in the two groups at all time points. It is concluded that cells with NK activity contribute to the CSF pleocytosis induced by acute Sindbis virus encephalitis.     

Early ontogeny of monocytes and macrophages in the pig

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

Abnormal development of genetically normal fetal hematopoietic stem cells in steel mutant mouse fetuses

The role and possible transplantability of the early hematopoietic microenvironment was investigated by transplacental inoculation of fetal liver cells from normal donors into steel mutant early fetuses. Donor hematopoietic stem cells were able to lodge in the livers of recipients and to progress to the bone marrow postnatally. However, self-renewal of stem cells and production of differentiated blood cells was very limited in extent and duration after transplantation into mildly anemic steel as compared with Wv/+ heterozygotes. The microenvironmental defect known to exist (albeit undefined) in steel and not in W mutants thus adversely affects proliferation and differentiation of stem cells from the very inception of hepatic hematopoiesis and is not correctable by introducing normal stromal cells under the conditions of the experiment.     

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.     

Starvation survival of Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT7137, exopolysaccharide-producing bacterium, was isolated from groundwater contaminated with leachate in a former municipal landfill site (São Paulo, Brazil). The strain was submitted to starvation in phosphate-buffered saline solution for 56 days so as to evaluate its behavior regarding cuturability and cell morphology. As a response to starvation, G. polyisoprenivorans CCT7137 presented reduction in viable cell count, cell size and cell shape alteration. The initial number of viable cells was 1.51 × 10⁷ c.f.u. ml. After 7 days of starvation culturability dropped to 13.70% (2.07 × 10⁶ c.f.u/ml) and, after 56 days, to 3.25% (4.93 × 10⁵ c.f.u/ml). It was also observed that after 7 days of starvation the cell size presented an average reduction in the values of length, length/width ratio, volume and area of 50, 58, 40 and 42%, respectively. The length/width ratio showed a change of shape from rod to coccobacillus. Changes in cell dimensions and distribution of cell into classes were not significant after day 7 of starvation. The results obtained show that it is not necessary for the strain to starve for more than a week to obtain G. polyisoprenivorans CCT7137 size- reduced cells. The results also indicate the potential for its starved forms to be used in future tests in porous medium to study the production of exopolysaccharide in situ.     

Intracerebral transplantation of foetal neural stem cells improves brain dysfunction induced by intracerebral haemorrhage stroke in mice

Intracerebral haemorrhage (ICH) can lead to secondary insults and severe neurological deficits. Transplantation of neural stem cells (NSCs) was suggested as an alternative to improve ICH-induced neurological dysfunction. The present study aimed at investigating the therapeutic role and long-term survival of foetal NSCs and potential role of foetal NSCs-produced factors in ICH. Our results demonstrated that foetal NSCs could differentiate into neural axons and dendrites and astrocytes in both in vitro and in vivo conditions, demonstrated by positive double or triple staining with Hoechst, neuronal specific nuclear protein, neurofilaments and glial fibrillary acidic protein. Intracerebral transplantation of foetal NSCs 3 days after ICH induction by intrastriatal administration of bacterial collagenase could improve the functional performance in the limb-placing test and shorten the duration of the recovery from ICH-induced neural disorders. The foetal NSCs may also produce neurotrophic and/or neuroprotective factors during culture, because the culture medium alone could partially improve functional performance. Thus, our data suggest that the foetal NSCs may be one of the therapeutic candidates for ICH.     

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.