Spermiogenic Cells Research Articles

Ultrastructural study on cytotoxic effects of cyclosporine A in spermiogenesis in rats.

Cyclosporine A (CsA) is known to have testicular toxicity, leading to male infertility. The occurrence of numerous anomalous spermatids and residual bodies in the epididymal ducts of rats treated with CsA was observed in our previous studies. To examine the fine structural changes of impaired spermiogenesis induced by CsA, rats were treated s.c. with 40 mg/kg/day CsA for 2 weeks. Desquamation of round spermatids still surrounded by a slender Sertoli cell cytoplasm was occasionally observed. A prominent change in the seminiferous tubules was an occurrence of numerous residual bodies containing one or more flagella. They were not phagocytosed by the Sertoli cell, and accumulated in the epididymal ducts. Cellular components in the epididymal lumen included degenerating round spermatids and abnormal spermatozoa and residual bodies. Although separation of the head from the tail of the spermatozoa was rarely seen, various kinds of abnormalities of flagella were frequently encountered; compact aggregation of numerous flagella in a single spermatozoon or residual body, disarrangement of mitochondrial or fibrous sheath with outer dense fibers, and fusion of flagella with a single intervening mitochondrial layer. These findings indicate that CsA gives rise to toxic effects on the spermiogenesis by impairing directly the spermiogenic cell development and by impeding Sertoli cell function including a reduction of its phagocytic activity.

Ultrastructural variations in the spermiogenesis of Triatoma infestans induced by temperature changes.

The reproductive process in insects may be affected by several factors, among which environmental temperature is one of the most important because of the poikilothermic condition of insects. To determine the effect of suboptimal temperature on the spermiogenic process of Triatoma infestans, males were exposed to 12°C for 10 days. In order to determine the reversibility of such alterations, males were returned to 28°C for 10 days after exposure to 12°C. Low temperature caused abnormal changes in the spermiogenic cells such as lack of spermatid orientation, general cyst disorganization, and asynchrony in the development of contiguous cysts. Highly vacuolized areas and pseudomyelinic bodies enclosing isolated or clustered vesicles of different electrodensity were also observed. At the end of the recovery time, cysts with spermatids developed synchronously. However, some presented isolated and clustered vesicles of different size and electrodensity. Some supernumerary organelles were also observed. These abnormal structures were related to processes of autophagy and phagocytosis of degenerating sexual cells. © 1993 Wiley-Liss, Inc.

Paired arrangement of nonhomologous centromeres during vertebrate spermiogenesis

Indirect immunofluorescence staining with human anti-kinetochore antibodies was used to study the position of centromeres during vertebrate spermiogenesis. Many species of Amphibia have a low chromosome number and very large spermatids and spermatozoa. The number of kinetochore dots correlates exactly with the haploid chromosome number. This implies that kinetochore duplication occurs in the interval between meiosis I and meiosis II. The nonhomologous centromeres are arranged in tandem during the entire course of spermiogenesis and in mature spermatozoa. A higher order centromere arrangement was found in spermiogenic cells of Anura and Urodela. In mammals, immunofluorescence analysis is complicated by the extreme condensation of chromatin during spermiogenesis and the high chromosome numbers. Nevertheless, centromere-centromere associations were observed in mammalian round spermatids and sporadically in testicular spermatozoa. This indicates that pair-wise association of centromeres is a universal principle of centromere arrangement at the postmeiotic stage.

Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro.

Dispersed testicular cells prepared from 14-d-old rats were cultured on type 1 collagen gels using a medium composed of a 1:1 mixture of Ham's F12 medium and Leibovitz's L15 medium (F12-L15 medium) containing 10% (vol/vol) fetal bovine serum. The viability of the spermatogenic cells was facilitated by supplementing a rat adrenal extract into the medium. The effective substance(s) (the survival factor) was purified from acid extracts of adrenals by molecular sieve high performance liquid chromatography and identified as epinephrine and norepinephrine. Both epinephrine and norepinephrine promoted the survival of the spermatogenic cells with a half saturating dose of 10 ng/ml. The spermatogenic cells, which could be cultured for 2 wk on a collagen gel by supplementing with the survival factor (epinephrine or norepinephrine), were subjected to Giemsa staining and to DNA flow cytometry. The following results were obtained: a) The spermatogenic cells from 14-d-old rats did not contain spermiogenic cells (1c-cells). b) During a culture period of 2 to 7 d the ratio of meiotic prophase spermatocytes (4c-cells) to premeiotic cells (2c-cells) increased. On Day 7, more than 90% of the surviving cells were meiotic prophase spermatocytes. c) On Day 10, spermatids (1c-cells) appeared for the first time. The time of the first appearance of spermatids in the culture was consistent with that in vivo. These results suggest that both epinephrine and norepinephrine facilitated the viability of meiotic prophase spermatocytes and that a part of the meiotic prophase spermatocytes completed the meiotic divisions in the testicular cell culture.

Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its relationship to genetic damage

Mice received an intraperitoneal injection of 14C-labeled acrylamide (AA) at an exposure of 125 mg/kg to equal that used in genetic studies carried out by Shelby et al. (1986). Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a 3-week period and assayed for the amount of bound AA. A strong increase in the level of binding occurred in late-spermatid to early-spermatozoa stages; these same stages are also genetically most sensitive to the action of AA. At all time points, alkylation of DNA within the sperm accounted for a very small fraction (generally <0.5%) of the total sperm-head alkylation. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm-head alkylation and accounted for essentially all of the AA binding. Two radioactive adducts were found in hydrolysed protamine samples, one of which co-eluted with a standard of S-carboxyethylcysteine. Protamine alkylation appears to be a significant cause of acrylamide-induced genetic damage in spermiogenic cells of the mouse.

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16–20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0–8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed limitedly in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.

The effects of acrylamide on mouse germ-line and somatic cell chromosomes.

The industrial chemical acrylamide is suspected to induce potentially heritable genetic damage. While several studies in rodents have indicated that this substance can damage spermiogenic cells, resulting in dominant lethals and heritable translocations, cytogenetic assessments of premeiotic and meiotic cells after exposure have produced equivocal results. In the present study, various cytogenetic endpoints in both somatic and germ-line cells from acrylamide-treated mice were evaluated. Sister chromatid exchanges and micronuclei, but not chromosome aberrations, were induced in spleen cells; synaptonemal complex irregularities (asynapsis), but not chromosome aberrations, were induced in germ cells.

The effects of gossypol on nuclear protein in rat testes. II. The synthesis of histones and testis-specific proteins after gossypol treatment

This communication is to report the effects of gossypol on the synthesis of chromosomal basic proteins in the spermiogenic cells. A double-labelling experiment has been performed. The control rats received 14C-arginine, whereas the gossypol-treated rats received a 3H-arginine injection. An equal volume of the tissues (from control and gossypol-treated) were combined, and the total nuclear basic proteins (TNBP) from round spermatids (RS) and elongating spermatids (ES) were extracted for electrophoretic separation. By studying the ratio of 3H 14C , it was indicated that the synthesis of H 1, H 3, H 2B plus H 2A and TP 1 in the RS was reduced by gossypol. The amount of inhibition of these proteins were 48%, 19%, 27%, and 11%, respectively. In the ES, the synthesis of H 1, H 3, H 2B plus H 2A, H 4 plus TP 2, TP 3, and TP 1 was reduced by gossypol, the reduction was 33%, 22%, 26%, 14%, 33%, and 8%, respectively. The synthesis of S 1 protein was not inhibited by gossypol in both RS and ES.

The effects of gossypol on nuclear protein in rat testes. I. Reduction in the content of basic proteins in the spermiogenic cells

In rat testes, after 45 days of gossypol treatment, the number of round spermatids (RS) reduced from 9.8 × 10 6 to 6.2 × 10 6 per testis. The number of elongating spermatids (ES) reduced from 6.4 × 10 6 to 3.1 × 10 6 per testis. Total nuclear basic proteins (TNBP, i.e., somatic type of histones H 1, H 2A, B, H 3, and H 4, plus testis-specific proteins TP 1,2,3 and sperm-specific S 1 proteins) were extracted from RS and ES. Gossypol has no detectable effect on the concentration of TNBP in RS. However, significant effect was found in ES, where the TNBP was reduced from 8.7 μg to 6.8 μg/10 6 cells. A two-stage polyacrylamide gel has been used to separate the TNBP and the individual proteins were quantified by a Beckman Model DU-7 computer-monitored spectrophotometer. In the RS cells, the content of the TNBP was not sensitive to gossypol. However, in the ES cells, the content of histones H 1, H 2A, and H 2B were reduced by gossypol treatment (with an inhibition of 50%, 58%, and 31%, respectively). The inhibition on H 3, TP 1, T 3 and S 1was insignificant by gossypol.

Binding of ethylene oxide in spermiogenic germ cell stages of the mouse after low-level inhalation exposure.

Mice received inhalation exposures of 3H-labeled ethylene oxide (EtO) gas at levels from 0.65 to 3.2 parts per million-hours (ppm-hr), which are below the exposure limits currently allowed for humans. Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a two-week period and assayed for the amount of bound EtO. A strong increase in the level of EtO binding occurred in late spermatid stages; these stages are also genetically sensitive to the action of EtO. The maximum binding of EtO in late spermatids amounted to 6 X 10(3) alkylations/sperm head/ppm-hr of exposure. Alkylation of the DNA within the sperm accounted for a very small fraction of the total sperm head alkylation, averaging about 20 DNA alkylations per sperm per ppm-hr of exposure over the two-week period. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm head alkylation and accounted for nearly all of the EtO binding. Protamine alkylation appears to be a significant cause of EtO-induced genetic damage in spermiogenic cells of the mammal.

Adaptive Biochemical Repair Response Toward Germ Cell DNA Damage

The Final expression of chemical toxicity is determined by pharmacokinetic, pharmacodynamic, homeostatic, and adaptive favors. In order to understand differential toxicity at the cellular and organ levels, one must outsider Ow main factors which may modulate the toxic effects of chemicals. In the male gonads, such modifying factors are the bloodtestis barrier which restricts the testicular penetration of many foreign chemicals, differentially distributed mixed‐function oxidase(s) between the germ cell and interstitial cell compartments, and the presence of differing DNA repair capacities in the various stages of spermatogenic cells. DNA repair systems are present in spermatogonia and spermatocytes, while more mature spermiogenic cells (spermatids and testicular sperms) appear deficient in DNA repair capabilities. Furthermore, strain and species differences in the germ cell's ability to repair DNA suggest a diverse response of germ cells to DNA‐damaging agents. The capacity of prespermiogenic cells to respond to damage induced by certain chemical mutagens accounts for stage‐specific spermatogenic cell toxicity. Mono‐functional alkylating agents such as methylmethane sulfonate (MMS) cause single strand DNA breaks in prespermiogenic cells which are repaired expeditiously, while the same germ cell types are unable to repair DNA damage induced by bifunctional or polyfunctional alkylating agents. DNA damage induced by polyfunctional alkylating agents involves inactivation or DNA template as a result of inter‐ and/or intrastrand DNA, which is more slowly repaired with greater error frequency in the newly‐synthesized DNA. Thus, both bifunctional and polyfunctional alkylating agents are more toxic to male germ cells than are the monofunctional atkylating agents. DNA damage measured by the alkaline elution analysis appears to be a relatively simple, sensitive indicator of germ cell DNA damage. The DNA repair system appears to be another protective mechanism against monofunctional alkylating agents such as MMS, while the same adaptive system is ineffective in overcoming the effects of bifunctional or polyfunctional alkylating agents. DNA repair rates need to be quantified and factored into the phamacokinetic model being developed for extrapolation from laboratory animals to man.

Assessment of environmental factors affecting male fertility

Exposure to drinking water containing as much as 500 ppm aluminum chloride for periods of 30, 60, and 90 days had no apparent effect on male reproductive processes. In an attempt to correlate enzyme activity with particular spermatogenic cell types, postnatal development of testicular enzymes was studied. Eight enzymes were selected: hyaluronidase (H), lactate dehydrogenase isoenzyme-X (LDH-X), dehydrogenases of sorbitol (SDH), alpha-glycerophosphate (GPDH), glucose-6-phosphate (G6PDH), malate (MDH), glyceraldehyde-3-phosphate (G3PDH), and isocitrate (ICDH). Enzyme specific activities in testicular homogenates were determined. Two types of enzyme developmental patterns were observed. One was represented by H, LDH-X, SDH, and GPDH; and the other by G6PDH, MDH, G3PDH, and ICDH. The former was characterized by a change in enzyme activities from low in newborn to high in adult while in the latter this pattern was reversed. The two complementary enzyme systems crossed each other at puberty. Prior to puberty, only spermatogonial cells are present; sperm differentiation initiated at puberty adds spermatocytes and spermatids to the testicular cell population. Male rats were exposed to borax in their diet for periods of 30 and 60 days. Concentrations of boron were 0, 500, 1000, and 2000 ppm. At the end of each experimental period, the specific activities of the selected enzymes were determined in the testis and prostate. Correlations of enzyme activity with testicular histology and androgen activities of the male accessory organs were sought. In addition, plasma FSH, LH, and testosterone levels were measured to assess pituitary-testicular interaction. Plasma and testicular boron concentrations were determined and a minimum boron concentration which induced germinal aplasia and male infertility was estimated. In both 30 and 60 day feeding studies, male rats receiving 500 ppm failed to demonstrate any significant adverse effects. In contrast, male rats receiving 100 and 2000 ppm boron displayed a significant loss of germinal elements, although most of the Leydig and Sertoli cells appeared normal. Testicular atrophy was associated with a decrease in seminiferous tubular diameter and a marked reduction of spermatocytes and spermatogenic cells. These morphologic alterations were associated with a concomitant reduction of H, SDH, and LDH-X specific activities. In contrast, the specific activities of G3PDH and MDH were significantly elevated above control. The increase in these enzyme activities can be attributed to the relative enrichment of spermatogonial cells during the loss of spermatocytes and spermiogenic cells. Boron-induced male germinal aplasia was also associated with significantly elevated plasma FSH while plasma LH and testosterone levels were not significantly altered. Plasma testosterone levels were unaltered. Male fertility studies demonstrated that at the 500 ppm boron level, fertility was unaffected. However, at 1000 and 2000 ppm boron, male fertility was significantly reduced. Most effects were reversible within 5 weeks. However, the male group receiving 2000 ppm boron for 60 days remained sterile. There was no dose-related decrease in litter size or fetal death in utero. Therefore, the boron-induced infertility was apparently not due to a dominant lethal effect but rather to germinal aplasia. Boron appears toxic to spermatogenic cells at testicular concentrations of 6-8 ppm.

Ultrastructural aspects of gymnosperms reproductive organs tissues due to their functional condition

The ultrastructure of reproductive organs of pine, spruce, larch and ginkgo was investigated. It was found that the male reproductive organs possess similar organization. The most considerable change in the ultrastructure of the microsporocytes occur in meiosis. Sporoderm is being laid at the late tetrad stage. The cells of the male gameto-phyte are distinguished according to the metabolic activity of the or- ganells. They are most weakly developed in the spermiogenic cell. Ta-petum of the gymnosperms is of the periplasmodic - secretorial type. The Ubisch bodies which possess similar structure in the types investigated but are specific in details in different species are produced in tapetum.Parietal and subepidermal layers are distinguished for their high metabolic activity and are capable of the autonomous photosynthesis. Female reproductive organs differ more greatly in their struture and have the most complicated structure in primitive groups. On the first stages of their formation the inner cells of nucellus are transformed into the nucellar tapetum in which the structures similar to the Ubisch bodies taking part in the formation of the sporoderm of female gametophyte have been found.

Factors influencing reproduction and genetic toxic effects on male gonads

The objective of toxicological study of a target organ, such as the testis, is to elucidate the qualitative and quantitative toxic effects of a chemical on that organ. The ultimate objective is to assess the toxic effects of a chemical in laboratory animals and extrapolate the pertinent experimental data to man. To accomplish these objectives, one must consider the main factors which may influence and modulate the toxic effects of chemicals in the organ. In the male gonads, such modifying factors are the pharmacokinetic parameters governing the absorption, distribution, activation and detoxification of indirect carcinogens, covalent bindings to macromolecules, and DNA damage as well as DNA repair of damaged germ cells. All of these factors have been presently studied in our laboratory and are discussed in this paper with the exception of covalent bindings to macromolecules.The pharmacokinetic studies demonstrated that the functional blood-testis barrier (BTB) closely resembles the blood-brain barrier in transport characteristics: the permeability of nonelectrolytes and the acidic drugs with pK(a) values depend upon their molecular size and their partition coefficients, respectively. Thus, the functional BTB, restricts the permeability of many foreign compounds to male germ cells. Studies of mixed function oxidases and cytochrome P-450 system in male gonads demonstrated that the presence of AHH, EH, and GSH-ST implicate activation and detoxification of polycyclic hydrocarbons. Thus, active electrophiles may exert significant toxic effects locally within both interstitial and germ cell compartments. The presence of an efficient DNA repair system in premeiotic spermatogenic cells (and not in spermiogenic cells) can further modify both toxic and mutagenic events in the subsequent differentiation of germ cells to mature spermatozoa.

DNA Synthetic Capabilities of Differentiating Sperm Cells

Spermatogenic cells separated by velocity sedimentation were analysed by a micro-procedure for differentiation-associated changes in DNA synthetic capabilities. DNA-dependent DNA polymerase activity is maximal in premeiotic and meiotic cells, sequentially declines in progressively more differentiated spermiogenic cells to a minimum value in testicular spermatozoa which is 1/14 of the maximum. No further decrease of activity is observed during the subsequent process of sperm cell maturation and, at the end-differentiated state, the potential of sperm cells for DNA synthesis is demonstrated by the presence of substantial activities of thymidine and thymidylate kinases as well as DNA polymerase activity, as determined by in vitro assay, are polymerase. Although levels of DNA polymerase activity, as determined by in vitro assay, are negatively correlated with the state of differentiation, the findings support the hypothesis that, in this cell system, DNA synthetic enzymes may not be limiting factors in the control of DNA synthesis.

Single-strand nuclease action on heat-denatured spermiogenic chromatin.

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.

Androgen binding proteins in different testis compartments

Two different high-affinity androgen binding proteins have been demonstrated in the rat testis. The smaller protein (ABP, MW 90,000) is produced in the testis, and transported with the testicular fluid into the epididymis. ABP is found in tubules but not in interstitial tissue or in testicular lymph. Destruction of the spermiogenic cells by physical or chemical agents does not decrease the concentration of ABP in the testis, indicating Sertoli cells to be the site of production. ABP disappears from the testis following hypophysectomy, but reappears following FSH treatment. Other pituitary hormones have no effect. Another androgen binding component, different from ABP, has also been recovered from rat testis tubules. This protein shows characteristics different from ABP but identical to cytoplasmic receptors of epididymis and prostate, indicating the presence of androgen target cells in the testis. Large doses of testosterone or dihydrotestosterone are known to maintain spermatogenesis in the hypophysectomized rat. We postulate that a major action of FSH on spermatogenesis may be mediated by ABP. ABP may facilitate androgen transport into the seminiferous tubules and provide the germ cells with the milieu of androgenic hormones which is required for normal spermatogenesis.

AUTORADIOGRAPHIC STUDIES OF GONADOTROPHIN ACTION ON GERM CELL MATURATION RATE

The influence of exogenous gonadotrophic hormones (GTH) on the rate of spermatogenesis and germinal maturation in the testes of hypophysectomized male mice was measured. Liquid emulsion autoradiographic techniques were used to measure the incorporation of tritiated thymidine into germinal DNA to see if exogenous hormones could alter the rate of development of late spermiogenic cell stages (Gude, 1968; Clermont & Trott, 1969). Two experiments were carried out using hypophysectomized and intact 70-day-old CD-I Charles River albino male mice (10/group). In experiment 1, intact (1A) and hypophysectomized (1B) males were compared with hypophysectomized males injected s.c. on alternate days either with 25 i.u. pregnant mare serum gonadotrophin (PMSG) (Equinex, Ayerst) (group 1C), or with 25 i.u. PMSG plus 25 i.u. human chorionic gonadotrophin (Ayerst) (group 1D). After 2 weeks of hormonal replacement, spermatogenic cells in the S phase (spermatogonia and primary spermatocytes synthesizing DNA) were labelled in vivo by a single

The Effects of γ-rays on Fecundity and Mutagenesis in Oregon-R Males of Drosophila

SummaryOregon-R males were tested in day-by-day sperm samples for dominant visible and hemizygous sex-linked mutations, fecundity (number of offspring produced), and crossing-over after exposure to cobalt-60 γ in the 100 r to 10 kr range. Tests totalled 42 184 X-chromosomes and 210 111 flies. Mutations were most frequent after irradiation during meiosis and spermiogenesis in low doses. Only sperm and gonial cells subsequently produced adult flies after exposure to 5 kr and 10 kr. After 500 r fecundity was reduced chiefly in cells in late mitosis, and in earlier and later stages with increasing doses. Reduced fecundity may not be due to the kind of mutation detected here, but both effects may combine to influence the frequency of mutations recovered, particularly from irradiated meiotic and spermiogenic cells. Males receiving 500 r might contribute more mutations to a population's gene pool than competing males after a substantially higher dose.