Long-term infertility and dominant lethal mutations in male mice treated with adriamycin

Sperm production and fertility were studied in male mice treated with adriamycin (ADR) at 6 or 8 mg/kg. Testicular sperm production and epididymal sperm counts were markedly reduced after ADR treatment. Gradual recovery of counts occurred, but sperm counts had not reached control levels even more than 1 year after treatment. Epididymal sperm showed treatment-induced morphological abnormalities throughout the experiment; the frequencies of sperm with detached tails and the frequencies of sperm with morphologically abnormal heads remained elevated about 2-3-fold above control. According to the frequency of vaginal plugs, treated male mice mated at control rates with untreated females during the post-treatment sterile period. However, after some fertility was regained the fertilization rate (calculated as the fraction of eggs, flushed from the oviduct 2 days after mating, that had been fertilized and had cleaved) was markedly reduced and remained depressed for the remainder of the experiment. The fertilization rate reached only 0.29 at 23-32 weeks after 8 mg/kg ADR and 0.76 at 16-23 weeks after 6 mg/kg ADR; both values were significantly below the control value of 0.94. Dominant lethal mutations in the zygotes flushed from the oviduct were measured in culture by the loss of the zygote's ability to develop to a stage characterized by trophectoderm outgrowths and formation of an inner cell mass. The frequencies of dominant lethal mutations detected in vitro were 1.7 or 7.4% after 6 mg/kg, and 32 or 40% after 8 mg/kg ADR; each value was calculated in two different ways, with 3 of these 4 values significantly different from zero. We conclude that even after mice regain fertility following ADR exposure, the level of fertility remains permanently subnormal as evidenced by a lack of fertilization of eggs that is probably due to the decreased quantity and quality of spermatozoa produced. Furthermore, ADR can induce genetic damage in stem spermatogonia, which can be transmitted through fertile spermatozoa. Thus, there may be a genetic risk to the offspring of cancer patients treated with ADR chemotherapy, but at present we are unable to quantitate that risk.     

Cell proliferation and abnormal nuclei induced by radiation in renal tubule epithelium as an early manifestation of late damage

The time of appearance and the dose response of radiation effects in the mouse kidney were assessed from the determination of increases in labeling index, the appearance of proximal tubule cells with abnormally large nuclei, and kidney weight loss. Increased labeling indices and abnormally large nuclei were observed in the irradiated proximal tubule cells before any other histological changes were seen. The labeling index increased with dose (from 3 to 15 Gy) but not with time (from 1 to 12 months after irradiation). Increased labeling was evident as soon as 1 month after irradiation. Cell depletion as measured by a decrease in kidney weights compared to those of age-matched controls was not significant until 6 or more months after 11-, 13-, or 15-Gy irradiation. The frequency of cells with large nuclei increased steadily during the first 9 months after 15 Gy and tended to decline between 9 and 12 months, coincident with accelerating renal weight loss. These findings are consistent with the hypothesis that the production of these cells is a result of an abortive mitotic division and their loss is an eventual result of such an aberration. The increased proliferation induced by irradiation increases the chance for an abortive mitosis and death, presumably at a subsequent mitosis, of radiation-damaged proximal tubule cells, which is a major factor in the appearance of late radiation damage in the kidney.     

Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes

The presence and biosynthesis of the testis-specific isozyme of lactate dehydrogenase (LDH-X) in cells at various stages of spermatogenesis have been examined. Enrichment of testicular cells in various stages of spermatogenesis has been achieved by two methods: (1) cell separation by velocity sedimentation in the Elutriator rotor and (2) γ irradiation of testes to eliminate specific classes of testicular cells. Separation of cells from immature mice indicated that cells prior to the midpachytene stage contain no LDH-X. Measurement of LDH-X levels in cells separated from adult mice and in testicular homogenates prepared at various times after irradiation indicated that the highest level of LDH-X per cell (normalized for DNA content) was in spermatids. Synthesis of LDH-X was determined, after in vivo injection of [³H]valine, by measurement of the radioactivity in LDH-X precipitated with specific antiserum. After irradiation, the rate of LDH-X synthesis remained constant, despite the loss of early primary spermatocytes. In separated cells, the rate of LDH-X synthesis was highest in late pachytene spermatocytes, lower in round spermatids, and even lower, but still significant, in elongated spermatids. Therefore, the synthesis of LDH-X begins at a specific point during spermatogenesis, the midpachytene stage of spermatocyte development, and continues throughout spermatid differentiation.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

“Cytogenetic” studies of spermatids of mice carrying Cattanach's translocation by flow cytometry

The DNA content of spermatids of mice carrying Cattanach's translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X-and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2±0.1% of the total haploid genome as compared to 3.4±0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanach's translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.     

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.