Cytology of the human seminiferous epithelium

The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.     

Development of male germ cells in the fur seal (Callorhinus ursinus)

Three types of spermatogonia have been distinguished in the fur seal: type A, intermediate, type B. The type A spermatogonia are presented by means of five varieties: stem A, A1, A2, A3, A4. The spermatogonia of the stem A type is characterized with presence of equally distributed chromatin in the nucleus. The spermatogonia of the A1-A4 types are characterized with a gradual condensation of chromatin. The intermediate spermatogonia possesses a light nucleus, chromatin as small and large granules is situated near the nuclear membrane. The spermatogonia of B1-B3 types have a round nucleus, their chromatin is granular. Spermatocytes both at the leptotene stage and at the zygotene stage are presented by means of three cellular forms. At the pachytene stage four cellular forms are distinguished. Basing on formation of the acrosomic apparatus, 18 stages of spermatids development are distinguished.     

Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.     

Kinetics of spermatogenesis in megachiropteran bat, Rousettus leschenaulti (Desmarset): seminiferous epithelial cycle, frequency of stages, spermatogonial renewal and germ cell degeneration.

The paper describes in detail the cytomorphology of different types of germ cells, the 10 typical cellular associations or stages of the cycle of seminiferous epithelium (CSE), frequency of appearance of these stages, pattern of spermatogonial stem cell renewal and per cent degeneration of various germ cells in R. leschenaulti. Of the 14 steps of spermiogenesis (stained with PAS-haematoxylin) the first 10 were associated with the stages I-X, whereas, the remaining were found in association with one of the first six stages. The frequency of appearance of the various stages ranged from 3.84% (stage V) to 19.84% (stage I). These observations indicate that stage V is of shortest duration and stage I is of the longest duration in the bat. Five types of spermatogonia (A1, A2, A3, In and B) were identified based on their shape, size and nuclear morphology. Type A spermatogonia are oval with a large nucleus containing 1 or 2 nucleoli. The chromatin showed progressive condensation from A1 to A3 so that the latter appeared darkest among all the A type spermatogonia. The In type derived from A3 are smaller but appear darker than A3 due to heterochromatin crusts along the inner border of the nucleus. The B type spermatogonia derived from In are round and possess single nucleolus. The B type spermatogonia divided mitotically before entering meiosis or the actual production of the primary spermatocytes. The various spermatogonia divided mitotically at fixed stages of the cycle giving rise to their next generations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology of the reproductive system of the armadillo. The spermatogonia

Spermatogonia of the nine-banded armadillo, Dasypus novemcinctus mexicanus, were studied morphologically using light and electron microscopy and examined histochemically using light microscopy. Immature flat type spermatogonia have ovoid or irregular nuclei with loosely condensed chromatin. Free ribosomes are abundant while profiles of rough endoplasmic reticulum are scarce. Smooth endoplasmic reticulum is a prominent feature occasionally taking an unusual cylindrical form. Mature spermatogonia exhibit rounder nuclei with greater degrees of chromatin clumping. Smooth endoplasmic reticulum is no longer prominent whereas profiles of rough endoplasmic reticulum are quite common. Occasional lysosomal configurations are found in mature spermatogonia. The majority of spermatogonial cells exhibit weak to moderate reactivity when stained with the periodic acid-Schiff (PAS) reaction. Certain cells in each tubular cross section stain vividly with this reaction and the PAS positivity is removable with salivary amylase. Because of nuclear characteristics, position of the cell immediately upon the basal lamina, intensity of the PAS reaction and the relative paucity of the vividly staining cells, it is suggested that they are members of the immature spermatogonial cell line, perhaps acting as stem cells. None of the several other histochemical procedures employed was capable of selectively demonstrating these cells.     

c-fos和c-myc在北方山溪鲵精子发生中的表达

用免疫组织化学方法检测原癌基因cf-os和c-myc蛋白在北方山溪鲵(Batrachuperus tibetanus)精子发生中的表达定位。结果显示,在精原细胞缓慢增殖期,8、9月,FOS阳性反应物出现在精原细胞的胞质及核膜外,10、11月,FOS在少量精原细胞的胞核中表达。在精原细胞快速增殖期,即翌年4月,FOS定位在精原细胞的胞质中;5月,FOS在大量的胞核中强阳性表达;6月,FOS定位于部分精母细胞核质和核膜下;7月,FOS在一些精子细胞的核质和核膜下表达。MYC在8、9月的部分精原细胞胞质中表达较弱,在101、1月阳性反应出现在个别精原细胞的核质中。翌年4月,MYC在精原细胞核周围的胞质中表达;5月在大量的精原细胞核膜下有强表达;6月,MYC在一些精母细胞核膜下表达;7月,MYC在部分精子细胞的核膜下弱表达。结果表明,北方山溪鲵的原癌基因cf-os和c-myc表达大强度在生精细胞发育中呈阶段性,表达的强度和细胞数量与细胞增殖的速度相一致。FOS和MYC在精原细胞内从胞质向胞核的转移与细胞快速增殖的时期相吻合。说明cf-os和c-myc对精原细胞有丝分裂有促进作用,并参与精母细胞成熟分裂的调控。     

Electron microscopic study of endopolyploid nuclei in rat trophoblast giant cells. IV. Changes in nucleolar ultrastructure during cell differentiation

The ultrastructure of the nucleolus of highly differentiated trophoblast giant cells has been studied on the 17th day of the foetus development. Changes in its morphology have been followed in relation to the degree of nuclear chromatin condensation and to the cell differentiation level. The nucleoli have a reticular structure in the nuclei with dispersed and condensed chromatin. In both the cases the nucleoli involve the four components: fibro-granular, fibrillar (of moderate and normal density) and lacunar regions; fibrillar centres are distinguished within the regions. In the nucleoli with condensed chromatin, unlike those with dispersed chromatin, the perinuclear chromatin is clearly seen, and the penetration of nucleolus-organizer threads along lacunae and deep into the nucleolus can be easily followed. The fibrillar centres are more obvious. With the run of a progressive differentiation of the trophoblast cells, the number of granules is reduced; first, the fibro-granular component covers a significant part of the nucleolus, then granules become visible only in the cortical zone of the nucleolus; in the nuclei with strongly condensed chromatin no granules are seen in the nucleolus.     

High-resolution light microscopic characterization of mouse spermatogonia

Characteristics of spermatogonia were determined in the C57BL/6J strain mouse using high-resolution light microscopy of plastic-embedded tissues and identifying cells during stages of the spermatogenic cycle. The frequency of expecting each spermatogonial cell type was a major factor in identifying and categorizing various cell types. Although numerous characteristics were described, several major differences were noted in spermatogonial cell types. The group comprising A(s), A(pr), and A(al) spermatogonia could be differentiated based primarily on mottling of heterochromatin throughout the nucleus in the absence of heterochromatin lining the nuclear envelope. The A(1) cells displayed finely granular chromatin throughout the nucleus and virtually no flakes of heterochromatin along the nuclear membrane. The A(2) through A(4) spermatogonia contained progressively more heterochromatin rimming the nucleus. Intermediate-type spermatogonia displayed flaky or shallow heterochromatin that completely rimmed the nucleus. Type B spermatogonia showed rounded heterochromatin periodically along the nuclear envelope. Use of gray-scale histograms allowed objective quantification of nuclear characteristics and showed a logical shift in the gray scale to a narrower and darker profile, from four cell types leading to A(1) cells. The ability to differentiate spermatogonial types is a prerequisite to studying the behavior and kinetics of the earliest of the germ cell types in both normal and abnormal spermatogenesis.     

The ultrastructure of the nuclei and the behaviour of the sex chromosomes of human spermatogonia

Summary The nuclear structure of human spermatogonia has been studied with electron microscopical and histochemical methods. Type B spermatogonia have chromatin clumps without any special ultrastructure and several nucleoli. Five different types of nuclear bodies, and besides, a nuclear vacuole, have been observed in type A spermatogonia. Type I bodies are typical nucleoli consisting of three regions: amorphous, fibrillar and granular. Type II, III and V are considered to be atypical nucleoli. Type IV bodies are small chromatin condensations. Type I bodies are the only ones in which RNA was demonstrated by light histochemical techniques and no PAS positive material was found inside the nuclei. The absence of any special ultrastructure in the chromatin from spermatogonia, and the small mass of the chromatin condensations, show that the human X chromosome and perhaps the Y chromosome are not heteropycnotic in the interphasic nuclei of human spermatogonia.Abbreviations Used RNA ribonucleic acid - gonia spermatogonia This work has been supported by a grant (No. 2623) of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, and partially by a grant (C.M. 6522) from the Population Council.We wish to thank Professor R. E. Mancini for his suggestions during this investigation and his support for its achievement, and to Dr. J. C. Lavieri for providing the biopsies.     

Quantitative differences between variants of A spermatogonia in man

The area of cytoplasm, nucleus, nucleolus and mitochondria, as well as the elongation and irregular outline of the nucleus were determined, on electron micrographs by using an image analyser, for Ap (pale), Ad (dark with intranuclear vacuole), Ad-like (dark without intranuclear vacuole), Ac (cloudy) and Al (long) human spermatogonia. Ap and Ac spermatogonia had larger nucleus, larger nucleolus, and more cytoplasm than did Ad, Ad-like, and Al spermatogonia. In addition, the nuclei of Ap and Ac spermatogonia were more spherical and had a more distinct outline.     

The histological localization of DNA in rabbit medullary neurons

The nuclei of unfixed isolated rabbit neurons cleared on incubation with DNAse (10 mg/ml), but not RNAse (10 mg/ml). The nuclei stained for DNA with eight chromosomal or nuclear stains more intensely than the cytoplasm, and less intensely after treatment with DNAse (10 mg/ml). On the other hand, when the whole tissue was embedded and sectioned, DNA did not appear to be stained in the nucleus; the nucleolus and the cytoplasm were more heavily stained than the nucleoplasm. Possible explanations for this apparent anomaly are considered. It was concluded that DNA diffused out of the nucleus during embedding and sectioning, and that the colouration of the nucleolus and cytoplasm with the eight staining systems used was due to other nucleotides present.     

The fine structure of nuclei as revealed by electron microscopy

Summary The process of nucleolus formation has been studied by electron microscopy in spermatogonia of new-born, 15-day-old mice. One of two heteropycnotic sex chromosomes is concerned with nucleolus formation in the type A spermatogonia. The evidence for such formation has been presented with regard to behaviour and fine structure of both sex chromosome and nucleolus, Nucleolar material appears at one of two heteropycnotic sex chromosomes which are closely attached to the nuclear envelope. The two sex chromosomes approach each other, and subsequently one of them migrates into the central part of the nucleoplasm, being related to the nucleolar material which develops to show a nucleolar configuration. The sex chromosomes are homogeneously electron dense during the nucleolus formation, but assume a vesicular form at the middle stage of its development. The nucleolus is mostly of fibrillar and amorphous components at early stages of its development, but the granular components increases in amount as development proceeds. The final, mature nucleolus is composed of irregularly twisted nucleolonemata consisting of granular components, separated from fibrillar and amorphous areas. The compactly dense sex chromosome remains closely connected with the mature nucleolus.     

Cytological changes in the testes of vitamin-A-deficient rats. II. Ultrastructural study of the seminiferous tubules.

Ultrastructural study confirmed that, in rats, vitamin A deficiency initially caused the sloughing of some spermatids and spermatocytes into the lumina of the seminiferous tubules around day 3 following the initial decrease of body weight. From days 5 to 10, a considerable number of spermatocytes and spermatids, which still remained in the epithelium, underwent necrosis. Several stages of dying spermatocytes and abnormal spermatids were observed. The latter were distinguished by the presence of chromatin aggregating along the nuclear envelopes and highly vacuolated mitochondria. These cells range from single to multinucleate forms. They were incapable of differentiating further into spermatozoa and ultimately degenerated. Within the same period, Sertoli cells exhibited numerous darkly stained lysosome-like inclusions, and the upper part of their cytoplasm appeared as irregular processes, some of which were broken off and resulted in the thinning of the epithelium. From days 10 to 20, the remaining germ cells comprised mainly spermatogonia and few abnormal spermatocytes. The latter appeared enlarged and were very lightly stained. Their nuclei exhibited unusual blocks of heavily condensed chromatin amidst very highly dispersed chromatin fibers. Though their number was reduced, most of the spermatogonia appeared unaltered. Processes of Sertoli cells became even more irregular and were interrupted at certain sites by large empty spaces. Darkly stained inclusions in their cytoplasm were fewer than observed earlier.     

Silver stained core-like structures in chinese hamster metaphase Chromosomes

Chinese hamster metaphase chromosomes, subjected to prolonged hypotonic pretreatment and subsequently stained with ammoniacal silver, contained a darkly-stained core-like structure in each chromatid, surrounded by a halo of dispersed chromatin which was pale yellow to brown in color. The core was variable in its appearance, ranging from a continuous linear configuration to a spiral structure or a discontinuous, particulate structure. Within the centromeric regions, the cores frequently appeared more intensely stained than elsewhere in the chromosome. The nucleolus organizers also stained darkly and appeared to be attached to the core-like structures. It remains to be determined whether the cores represent a real component of metaphase chromosome structure, or whether they are artifacts resulting from abnormal chromatin aggregation arising at the time of chromosome preparation.     

Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging

Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin.     

Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Nuclear architecture and DNA location in two VAM fungi

Summary The ultrastructural organization of nuclei in Glomus versiforme and G. mosseae hyphae was examined by sampling at different times during the fungus life cycle. In young and active hyphae, the nucleus is chromocentric with dispersed chromatin and a well-developed nucleolus. On aging, and particularly during arbuscule collapse, the chromatin condenses to give a pyk-notic nucleus with blebs in the envelope and conspicuous spaces between this and the nucleoplasm. DNA location by immunogold or enzyme-gold techniques resulted in uniform labelling over the chromatin. Nuclear degeneration is mirrored by decreased gold labelling and may thus be regarded as an important parameter for evaluation of fungus viability and the ephemeral life of the arbuscule.     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌PhysarumpolycophalumSchw的营养生长阶段为没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质是不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩成染色质,分散的核仁物质逐渐合并形成新的核仁。