Analysis of the progression of meiosis in dispersed rat testicular cells by flow cytofluorometry.

Initiation and progression of meiosis was followed in dispersed rat testicular cells by flow cytofluorometry and cytology. The DNA content of dissociated testicular cells of rats 6--30 days old, killed at daily intervals, was analysed by flow cytofluorometry using propidium iodide as a DNA-specific and quantitative fluorochrome. Testicular cells of a 6-day-old rat showed one peak of fluorescence. A second peak, at twice the modal channel number, appeared in testicular cells of 9-day-old animals. The number of cells under this peak increased progressively with age. A third peak, at half the channel number of the original one, appeared at 20 days and accounted for an increasing proportion of cells in testes taken from older rats. Cytological examination of the testicular tissue used for flow cytofluorometric analysis showed that preleptotene spermatocytes first appeared at 8 days after birth. Spermatids were first observed cytologically at 20 days after birth. The close temporal appearance of the fluorescence peaks with that of spermatocytes and spermatids, and the close association of the frequency of diploid and tetraploid cells as derived by flow cytofluorometry and cytology, indicated that the fluorescence peaks correspond--in order of increasing fluorescence--to spermatids, spermatogonia and somatic cells, and to spermatocytes. This conclusion was re-examined by analysing the ploidy levels of testicular cells of hypophysectomized or estradiol-treated by flow cytofluorodmetry. There was a loss of the haploid and tetraploid peaks subsequent to hypophysectomy. Estradiol dipropionate-treated rats, given weekly injections starting at 7 days of age, showed no appearance of the haploid peak and the regression of the tetraploid peak after an initial and transitory appearance. These results indicate that changes in ploidy levels that accompany the progression of meiosis in the testis were reflected in the sequential appearance of three fluorescence peaks as detected by flow cytofluorometry. The close correlation between the frequency of cell types as obtained by cytology and flow cytofluorometry indicates that the latter is a sensitive method for studying selected aspects of spermatogenesis in dissociated testicular cells.     

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.     

Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis

Spermatogenesis is a highly ordered process which requires mitotic and meiotic divisions. In this work, we studied the relative changes in the levels of the two components of the M-phase promoting factor (MPF): the regulatory subunit cyclin B1 (CycB1) and its catalytic subunit cdk1, in spermatogenic cells of rats between 16 and 90 days of life. A multivariate flow cytometry analysis of forward scatter (FSC), side scatter (SSC) and DNA content was used to identify six populations of rat germ cells: spermatogonia with preleptotene spermatocytes, young pachytene spermatocytes, middle to late pachytene spermatocytes, secondary spermatocytes with doublets of round spermatids, round spermatids, and elongated spermatids. For any population studied no significant difference in the relative cellular content of CycB1 or cdk1 proteins between animals of different ages was observed. By contrast, CycB1 and cdk1 levels were different between the different populations of germ cells. CycB1 and cdk1 were rather high in young pachytene spermatocytes and culminated in late spermatocytes, i.e. just before the first meiotic division. The relative levels of the two proteins remained high in secondary spermatocytes then decreased in round spermatids at the exit of meiosis. Similar results were obtained by Western-blot analysis of total proteins obtained from lysates of elutriated fractions of spermatocytes and spermatids. MPF activity was assessed in lysates of germ cells from 32-day-old rats or adult animals using p13suc1 agarose and histone H1 as an exogenous substrate. H1 kinase activity was higher in pachytene spermatocytes than in round spermatid fractions from both adult and young rats. These results indicate that the meiotic G2/M transition is associated to high levels of CycB1 and cdk1 leading to high MPF activity irrespective of the age of the animals.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Effects of danazol on spermatogenesis in adult rats

Adult male Wistar rats were treated with Danazol (4 mg/day s.c.) for 52 days. The drug produced a marked, rapid drop in serum testosterone concentrations to very low levels and caused a slower decrease in serum FSH, LH and testis weight. Flow cytometric analysis of testicular cell suspensions showed a decline in the absolute numbers of haploid cells (spermatids), tetraploid cells (mainly pachytene spermatocytes) and of cells in the S-phase of the division cycle, suggesting that Danazol inhibited proliferation of spermatogonia and/or primary spermatocytes. Histological counting of the different types of spermatogonia, however, revealed no significant change in their numbers during Danazol treatment. It is concluded that Danazol inhibited spermatogenesis primarily after the preleptotene stage of primary spermatocytes.     

The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells

The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20- to 22- or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.     

Male meiosis in polyploid silkworms,Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.     

Flow cytometry of DNA in mouse sperm and testis nuclei.

Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.     

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

Summary 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 (lc-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 (lc-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.     

Increased recombination frequency showing evidence of loss of interference is associated with abnormal testicular histopathology

Nondisjunction leading to aneuploid gametes has been linked genetically to both increases and decreases in recombination frequency on the aneuploid chromosome. In the present study, we present physical evidence of increased frequency of recombination nodules as measured by Mut-S-like homologue-1 (MLH1) foci on pachytene chromosomes from sterile male mice homozygous for a mutation in the protein phosphatase 1cgamma (PP1cgamma) gene. The pattern of elevated recombination frequency in PP1cgamma mutant spermatocytes is consistent with a loss of interference. Previous studies demonstrated: (1) spermiogenesis is impaired starting at step 8 with a severe reduction in elongating and condensed spermatids; (2) spermatids and sperm exhibit elevated rates of DNA fragmentation; and (3) haploid gametes exhibit elevated levels of aneuploidy. Morphometric analysis of developing testes revealed that the first wave of meiosis proceeds at a normal rate in mutant testes, a surprising result given that the PP1 inhibitor okadaic acid has been shown to accelerate progression of spermatocytes from pachytene to the first meiotic division (MI). Evidence of abnormal testicular histopathology is apparent at 3 weeks, before the appearance of haploid gametes, eliminating the possibility that the mutant phenotype is caused by the presence of abnormal spermatids, but coincident with the appearance of the first set of mid to late pachytene spermatocytes. These observations lead us to conclude that the PP1cgamma mutation causes a complex phenotype, including subtle adverse effects on meiosis, possibly mediated by defective signaling between germ cells and Sertoli cells.     

Pachytene spermatocytes can achieve meiotic process in vitro

Highly enriched pachytene spermatocytes prepared from adult rats by centrifugal elutriation were tested for their capacity to enter the meiotic process when cocultured with 20-day old rat Sertoli cells. This was traced by phase-contrast microscopy and by DNA flow cytometry. We also performed a Northern blot analysis using the mouse protamine I cDNA as a probe, the expression of which being restricted to spermatids. Our results demonstrate that pachytene spermatocytes cocultured with Sertoli cells developed into spermatids. The number of pachytene spermatocytes entering meiosis was affected neither by growth factors nor by hormones. However these later were required in long term cocultures for the maintenance of cell integrity and viability.     

Efficiency of the process of meiosis in scrotal testes of healthy boars and unilateral abdominal cryptorchid boars.

Unilateral abdominal cryptorchidism has usually been correlated with abnormalities in the spermatogenic activity of the scrotal testis. The present study describes the effects of unilateral abdominal cryptorchidism on the meiotic process in scrotal testes from postpubertal boars. The percentage of primary spermatocytes, secondary spermatocytes, and round spermatids was evaluated in testicular smears from scrotal testes of healthy boars and of right-sided unilateral abdominal cryptorchid boars. As compared to the scrotal testes of healthy boars, the scrotal testes of unilateral abdominal cryptorchid boars showed low transformation from primary to secondary spermatocytes (meiosis I), but normal transformation from secondary spermatocytes to round spermatids (meiosis II). The data obtained indicate that spontaneous unilateral abdominal cryptorchidism on the right side induced partial arrest of spermatogenesis at the primary spermatocyte stage that was attributed to anomalies in Sertoli-cell activity. Abnormal paracrine signals from altered Sertoli cells could have resulted in either disturbed mitosis, which led to the formation of spermatocytes with an abnormal DNA content, or abnormalities in the metabolic activity and the organization of the cytoskeleton of primary spermatocytes.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.     

Annual reproductive cycle and rate of the spermatogenic process in male mosquitofish <Emphasis Type="Italic">Gambusia affinis</Emphasis>

The present study investigates the relationship between the annual cycle of testicular development and external environment and the rate of spermatogenesis in the mosquitofish Gambusia affinis based on histological observations of testes. The annual reproductive cycle of the mosquitofish was divided into two periods, i.e., the spermatogenic period (May–October) and resting period (October–April). In the spermatogenic period, the transition from spermatogonia to spermatocytes begins and meiosis actively progresses. In the resting period, the transition from spermatogonia to spermatocytes ceases, meiosis of spermatocytes that already shifted by this period gradually progresses, and a considerable number of sperm balls are produced. Onset of spermatogenesis seems to be related to both a rise in water temperature and a prolonged photoperiod. 5-bromo-2-deoxyuridine (BrdU) was a useful in vivo marker of DNA synthesizing spermatogenic cells. The results of immunohistochemical detection of injected BrdU indicated that 5 days are needed for the conversion of spermatocytes to spermatids, 5 days for spermatids to spermatozoa, and 10 days for spermatozoa to sperm balls.     

The differential expression of the actins and tubulins during spermatogenesis in the mouse

Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.     

Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.     

Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry

Dual parameter flow cytometry was used to investigate cellular changes in male germinal tissue during normal postpartum maturation in B6C3F1/J mice. Animals were killed at 2-day intervals from 2 to 42 days postpartum and at 48, 64, 72, 93 and 100 days postpartum. Testicular, cauda epididymis and vas deferens cell suspensions were stained with the metachromatic fluorochrome acridine orange and measured by flow cytometry for red and green fluorescence levels after excitation by blue laser light. Intensities of red and green fluorescence reflect amounts of single- and double-strand nucleic acid sites available for acridine orange staining, respectively, and were used to classify cells on the basis of ploidy level, RNA content, and chromatin structure, as defined by susceptibility to acid denaturation of DNA in situ. Sperm from cauda epididymis and vas deferens were examined by light microscopy to determine frequency of abnormal sperm head morphology. Fluorescence data derived from acridine orange-stained testicular cells quantified the sequential changes in 1) proportions of haploid, diploid and tetraploid cell types during the first round of spermatogenesis, and 2) proportions of round, elongating, and elongated spermatids during the first round of spermiogenesis. Ratios of the three major testicular populations (haploid, diploid, and tetraploid) reached adult levels by 48 days postpartum. Sperm cells were first detected in the cauda epididymis and vas deferens on 30 and 36 days postpartum, respectively. Early sperm populations, compared to adult sperm, exhibited up to 89% abnormalities in sperm head morphology that correlated with significant levels of abnormal chromatin structure. Percentage of sperm head abnormalities and chromatin structure in the cauda epididymis and vas deferens approached normal adult levels by 42 and 48 days postpartum, respectively.     

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.     

Micronuclei and chromosome aberrations in Xenopus laevis spermatocytes and spermatids exposed to adriamycin and colcemid

Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.