Differences in bull spermiograms using eosin-nigrosin stain, feulgen stain, and phase contrast microscopy methods

The Society for Theriogenology recently adopted a minimum standard of 70% normal spermatozoa for the bull breeding soundness examination (BSE). We conducted this study to determine if spermiograms derived by brightfield microscopy of eosin-nigrosin stained semen smears (Society method) overestimated the proportion of normal spermatozoa. Comparison of the above method was made with that of phase contrast microscopy (Phase method). We then evaluated our ability to discern head abnormalities by comparing brightfield microscopy of Feulgen-stained sperm DNA (Feulgen method) with those of the Society and Phase methods. Spermiograms were determined for each of the 181 beef bulls using all 3 methods. Only bulls that were being routinely tested prior to the 1993 breeding season were included. The mean percentage of normal spermatozoa surpassed the minimum standard with the Society (72.8%) but not the Phase (52.5%) method, which identified more distal cytoplasmic droplets that adhered to cells without distal midpiece reflexes. The Phase method also identified more total primary and fewer primary head abnormalities. We conclude that the Phase method is not a suitable substitute for the Society method when applying the minimum standard during a routine BSE. The Feulgen method identified more head abnormalities, especially in the pattern of DNA, than the other methods, however, when compared to the minimum standard that improvement was not clinically important. Both the Phase and Feulgen methods are better than the Society method for monitoring changes in abnormal spermiograms over time.     

DECREASE IN NUCLEAR FEULGEN-POSITIVE MATERIAL (DNA) UPON AGING IN IN VITRO STORAGE OF BOVINE SPERMATOZOA

The Feulgen-DNA content of sperm cells from 5 bulls was studied by means of microspectrophotometry after storage at 5°C for 2, 3, 5, and 10 days in a yolk-citrate diluent permitting slow aerobic metabolism. A subsample of sperm cells from each bull was subjected to the Feulgen technique on each of the storage days selected. The cells sampled on each of these days received a standard 12 minute, 60°C hydrolysis. Absorption measurements at 546 mµof the individual cells indicated a marked progressive decrease in the Feulgen-DNA content of the stored spermatozoa. The loss of 30 per cent of the initial DNA at the end of 5 days' storage was highly significant statistically. This decrease approximately parallels the known decrease in fertility of stored sperm cells, as well as the increase in apparent embryonic mortality resulting from the use of similarly aged spermatozoa for artificial insemination.     

Ultrastructure of nuclear condensation and localization of DNA and proteins in spermatozoa of a dasyurid marsupial,Sminthopsis crassicaudata

In the dasyurid marsupial, Sminthopsis crassicaudata, the mature spermatozoon has an inner homogeneous (C1) and a peripheral indented (C2) region. Using DNase-gold conjugates, and biotinylated genomic DNA probes, DNA was found to occur in both C1 and C2 regions. The morphogenesis of the spermatozoon nucleus was investigated using ultrastructural and cytochemical studies. Spermiogenesis was divided into 15 steps. By step 10, condensation of the C1 region was complete, and at the caudal extremity of the spermatid nucleus, the nuclear envelope enclosed an electron-lucent space. This space and the surrounding nuclear envelope became very enlarged at step 11. At this stage, a plate of approximately 70 nm in thickness was present along the caudal segment of the C1 region; this “nuclear mantle” did not bind DNase-gold conjugates but stained for lysine-rich proteins using alcoholic phosphotungstic acid. Chromatin condensation resumed at step 12 with the appearance of spherical chromatin structures peripheral to the C1 chromatin. These structures then partially coalesced and the indentations of the C2 region were observed. The expanded nuclear envelope at the caudal extremity persisted in caput epididymal spermatozoa. Spherical inclusions within it did not bind to DNase-gold conjugates but stained for lysine-rich proteins. As the sperm traveled down the epididymis, these inclusions amassed near the nuclear pores and were then removed from the nucleus. In addition, the nuclear mantle was found to have disappeared by the time the spermatozoa reached the corpus epididymidis. © 1996 Wiley-Liss, Inc.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

ANTIGEN-INDUCED CHANGES IN LYMPHOID CELL HISTONES : I. Thymus

An acute effect of antigens on the nuclear histones of mouse thymocytes was investigated by means of cytophotometric measurements of thymocytes stained with ammoniacal-silver (A-S) and with fast green (FG). In addition, the DNA content was measured in terms of Feulgen staining. In terms of such staining it appeared that nuclei of control thymocytes contain a greater amount of nuclear histones and a higher histone/DNA ratio than do renal cell nuclei from the same animal. Within 1 hour after the injection of antigen the thymocyte nuclei appear to lose approximately 32 per cent and 20 per cent, respectively, of A-S and FG stainable nuclear proteins, while the Feulgen staining remains unchanged. Since the renal cell nuclei show no antigen-induced change in histone staining, the histone staining and histone/DNA ratios were found to be similar in the thymocytes and renal cells of the antigen-injected mice. The antigen-induced loss of thymocyte histones was also found to be associated with a change in the color of the A-S staining, from yellowish brown to black. This and other findings suggest that thymocyte nuclei contain an antigen-labile, lysine-rich histone. The implication of these observations in regard to the phenomenon of immunological competence is discussed and the need for continued investigation indicated.     

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:

1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.

2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).

3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.

4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.

From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.     

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010)     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

Naegleria fowleri: Light and electron microscopy study of mitosis

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.     

DNA synthesis,reduction, and elimination during life cycles of the eimeriine coccidian, Eimeria tenella and the haemogregarine, Hepatozoon domerguei

The occurrence of zygotic nuclear reduction and the haploid state of postzygotic stages were demonstrated in Eimeria tenella (Coccidia, Eimeriina) and Hepatozoon domerguei (Coccidia, Adeleina) by direct measurement of DNA by microdensitometry. Nuclear divisions, which were probably mitotic, were demonstrated in the macrogametocytes of a species of Hepatocystis (Coccidia, Haemosporina). The macrogametocytes of E. tenella were Feulgen negative but gametocytes of both sexes of H. domerguei were Feulgen positive and had DNA values estimated at about twice the haploid amount. This latter finding was interpreted as DNA synthesis preceding a maturation process which involved mitotic divisions and led to micro- and macrogamete formation. In macrogametogenesis this may be an atavistic trait, recapitulating the evolution of sexuality or a method for increased RNA synthesis.     

Histochemical properties of spermatozoa and somatic cells. III. Depolymerization and extraction of DNA during Feulgen acid.

The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

Histochemical properties of spermatozoa and somatic cells

Summary The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B

Summary Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.Dedicated to Prof. Dr. E. Schauenstein on the occasion of his 70th birthday     

Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera,Drosophilidae): Cytochemical,structural and ultrastructural characterization

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1–3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.     

A study of DNA depolymerisation during feulgen acid hydrolysis

Summary The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

A study of DNA depolymerisation during Feulgen acid hydrolysis.

The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair.     

Spermatozoa of chromosomally heterozygous mice and their fate in male and female genital tracts

The fate of morphologically normal but chromosomally abnormal spermatozoa derived from mice with variable degrees and complexity of Robertsonian heterozygosity was studied at different sites along the male and female genital tract by Feulgen-DNA measurements. In addition, the percentage frequencies of morphologically abnormal spermatozoa in transit along the male and female genital tracts were studied. It was found that during transit from the epididymis to the vas deferens the distribution of the Feulgen-DNA contents of morphologically normal spermatozoa changed: Spermatozoa with chromatin with the extremely low or high Feulgen staining intensity disappeared. The percentages of morphologically abnormal sperm cells did not change at these levels. In the female genital tracts, the distribution of Feulgen-DNA content of morphologically normal spermatozoa did not show significant changes. This indicates that spermatozoa are able to reach the fallopian tube in spite of gross genome unbalance. There is evidence that unbalanced spermatozoa take part in the fertilization process, producing abnormal zygotes subject to postzygotic loss. Conversely morphologically abnormal spermatozoa were preferentially lost before they reached the fallopian tube, suggesting they had been eliminated prezygotically.     

Cytophotometric determination of genome size in two species of Cyclops of Lake Baikal (Crustacea: Copepoda, Cyclopoida) in ontogenetic development

The genome size of Cyclops in cells at early stages of cleavage (up to the fifth division) and in somatic cells was estimated by static digital Feulgen cytophotometry in order to study quantitative changes in DNA content during chromatin diminution. Described here cytophotometric method was approbated on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation between the data obtained with known from the literature on genome size and high reproducibility, which will allow these systems to be used in future work. We also optimized the conditions for DNA hydrolysis of both blood smears and for two species of Cyclops from the Moscow population as 30 min in 5 N HCl at 24°C. Here, we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus. We estimated the extent of chromatin diminution in Diacyclops galbinus as 95.5–96.2%. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution.     

Generation of a third-order folded structure for chromatin

A model for the initiation of the diffuse-condensed transition of chromatin induced by a change in the conformation of lysine-rich histones is proposed. Three levels of folded structures are discussed. The first-order folded structure refers to the structure of the repeat unit of chromatin, which is called the nucleosome. The nucleosome contains a nuclease resistant region in which 140 base pairs of DNA are wrapped around the surface of a histone aggregated of two copies each of the histones H2A, H2B, H3 and H4. This DNA-histone aggregate is called a core particle. The nuclease accessible region of the nucleosome is approximately 60 base pairs of DNA which link the core particle, hence the terminology “linker DNA.” The lysine-rich histones, (Hl, H5), which are more loosely bound than the core histones, are associated with the linker DNA. The second-order folded structure refers to the conformation of a polynucleosome. Based on neutron scattering and quasielastic light scattering studies the second-order folded structure is assumed to be an extended helix in solution with 5–7 nucleosome units per turn. The third-order folded structure is defined as that structure resulting from the first stage in the condensation process induced by a conformational change in the lysine-rich histones. Generation of the third-order folded structure in the proposed model is effected by an increased affinity of the lysine-rich histones for super-helical DNA in the core particles in adjacent turns of the second-order folded structure. Since the lysine-rich histones preferentially bind to A-T rich regions in DNA, the distribution of these regions would determine the third-order folded structure. The net effect of a non-random distribution of A-T rich regions as in the proposed model is the generation of a helix for the third-order folded structure. The assumption of a non-random distribution of A-T rich regions is indirectly supported by proflavine binding studies reported herein and by the existence of repetitive and non-repetitive DNA regions inferred from renaturation studies. One consequence of the proposed mechanism is that the majority of the A-T rich regions are in the interior of the third-order folded structure. Promoter sites of high A-T content would then be inaccessible to polymerases. The proposed model also suggests a role for spacer DNA in the genome. Higher order folded structures must also be present in the final state of condensed chromatin since the three orders of folded structures considered in this communication accounts for only 2% of that required in the diffuse-condensed transition.