Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.     

RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37 degrees C (in the presence of Colcemid) or at 0-4 degrees C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37 degrees C with Colcemid or at 0-4 degrees C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.     

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.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.     

Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid.

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Acridine orange-induced precipitation of mouse testicular sperm cell DNA reveals new patterns of chromatin structure

Precipitate resulting from interaction between certain intercalators, such as acridine orange (AO), and nucleic acids can be detected by electron microscopy. Formation of precipitate in nuclei of live cells is modulated by chromatin structure. Susceptibility of in situ DNA to precipitation was studied in mouse testicular germ cells during various stages of sperm maturation. DNA in round spermatid chromatin, similar to somatic cell euchromatin, was rather resistant to precipitation; the electron-dense precipitate was granular and randomly distributed. DNA in elongated spermatids was more susceptible to precipitation; the products were in the form of fibers. At early stages of spermatid maturation these fibers were distributed uniformly throughout the entire nucleus. At later stages, the products appeared as approximately 25-nm-thick fibers arranged longitudinally in arrays within the nucleus. With further cell maturation, fibers in the anterior portion of the nucleus appeared to fuse, forming homogeneously dense product. These fibrous products likely represent AO interactions with DNA in chromatin in which transition proteins had replaced histones. Changing patterns of these precipitated fibers likely reflect progressive stages of chromatin condensation, which starts at the center and anterior portion of the nucleus where the fibers coalesce. Mature sperm cell DNA, known to be complexed with protamines, was more resistant to AO-induced precipitation. The data suggest that precipitation induced by AO and monitored by electron microscopy may be a useful probe of nuclear chromatin structure.     

Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids.

DNase I-hypersensitivity of rat spermatogenic cells was analyzed 1) to establish overall patterns of hypersensitivity in individual cell types, 2) to correlate these patterns with known changes in chromatin organization and function, and 3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from "checkerboard" in steps 7-8 round spermatid nuclei to "lamellar" in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids. DHR were localized throughout the nuclei or in a graded manner--increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicators the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

Interphase chromatin of human cells with diploid and haploid genomes

A study was made of the chromatin structure of mature sperm cells from healthy males aged 25 to 40 using fluorescent microscopy and acridine orange staining according to the DNP cell thermal denaturation technique modified by the authors. It was shown that normal human sperm cell chromatin melting profiles represent uniform curves with maxima in the following temperature ranges: 43 (+/- 2) degrees, 55 (+/- 1) degrees, 67 (+/- 2) degrees, 77 (+/- 1) degrees, 82 (+/- 0.5) degrees, 89 (+/- 1) degrees, 92 (+/- 2) degrees (P less than 0.01), that are identical to those obtained with lymphocytes of healthy males with certain deviations from the standard normal variant. No heteromorphism was revealed in the sperm cell chromatin. Marked polymorphism in the chromatin structure occurs but at the diploid cell level. A 10-time decrease in the fluorescence of AO bound with sperm cell chromatin as compared to F530 AO bound with lymphocyte chromatin structure of the same individual supports the data on the over condensation of sperm cell nuclear chromatin as compared to that in lymphocytes.     

Hydrodynamic shearing by VirTis blending conserves nucleosome structure of rat liver chromatin

The effects of VirTis shearing on chromatin subunit structure were investigated by enzymatic digestion, thermal denaturation, and electron microscopy. While initial rates of micrococcal nuclease and DNase I digestion were greater postshearing, limit digest values were similar to those for unsheared chromatin. Fractionated chromatin digestion kinetics varied with sedimentation. Digestion of all chromatins produced monomer and dimer DNA fragment lengths, but only unsheared chromatins exhibited higher order nucleosome oligomer lengths. Mononucleosomes and core particles were resolved in digests of sheared and gradient fractions analyzed by electrophoresis. All chromatins exposed to DNase I showed discrete 10-base pair nicking patterns. The presence of nucleosomes was confirmed by electron microscopy. Electron microscopy and histone content of gradient fractions showed that nucleosome density along the chromatin axis increased in rapidly sedimenting fractions. Thermal denaturation detected no appreciable generation of protein-free DNA fragments as a result of shearing. The results indicate that VirTis blending conserves subunit structure with loss of less than 12–15% of nucleosome structure.     

Interactions of nuclear proteins with DNA,during sperm differentiation in the ram

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nuleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous knobby fibers that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30–200 Å region.     

Microelectrophoretic analysis of basic protein changes during spermiogenesis in the dogfish Scylliorhinus caniculus (L.)

Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form ---S---S--- crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.     

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.