A. Stahl

Nucleoli, nucleolar chromosomes and ribosomal genes in the human spermatocyte

The formation and development of nucleoli and their connections with the nucleolar chromosomes were studied in human spermatocytes using electron microscopy, silver staining of nucleolus organizer regions (NORs), high resolution autoradiography and in situ hybridization in order to localize rRNA genes and their transcription in the different stages of meiotic prophase I. At leptotene, new nucleoli were formed, consisting of a fibrillar centre surrounded by a cap of dense fibrillar component. Following [3H]uridine uptake, label was found only over the dense fibrillar component. In situ hybridization revealed rDNA mainly in the dense fibrillar component and in the chromatin. During zygotene, nucleoli increased in size. The fibrillar centre was connected with the secondary constriction region of the nucleolar bivalent and was partially surrounded by dense fibrillar component. This shell of dense fibrillar component merged into a fibrillo-granular mesh that extended away from the fibrillar centre. Autoradiography following [3H]uridine uptake again showed the label overlaying the dense fibrillar component and the proximal part of the fibrillo-granular strands. With in situ hybridization in both the light and electron microscope, signal was mainly found in the dense fibrillar component. A small quantity of label was observed in the peripheral region of the fibrillar centre and in the adjacent chromatin. From early to late pachytene segregation of nucleolar components occurred, with a reduction in the dense fibrillar component that formed a narrow rim around the fibrillar centre with small extensions along the granular component. [3H]uridine incorporation progressively decreased. In situ hybridization showed signal located mainly in the dense fibrillar component and in the chromatin corresponding to the condensed short arm of the nucleolar bivalent. Our results indicate that the majority of rDNA is located and transcribed in the dense fibrillar component; only a small amount is present in the peripheral part of the fibrillar centre and may be transcribed there. Moreover, from leptotene to zygotene, rDNA unravels from the nucleolar chromosome into the nucleolar dense fibrillar component. From zygotene to late pachytene a progressive return to the condensed acrocentric short arm is observed.

The association of the nucleolus and the short arm of acrocentric chromosomes with the XY pair in human spermatocytes: Its possible role in facilitating sex-chromosome acrocentric translocations

SummarySex vesicle-nucleolus association was observed in 12% of zygotene and pachytene human spermatocytes using Giemsa and NOR-silver stained preparations. The silver-positive area of the nucleolus, corresponding to the nucleolus organizer (NOR), was usually close to the XY pair. C-banding frequently showed the terminal chromomere, formed by the condensed short arm of an acrocentric bivalent, attached to the sex vesicle. When a nucleolus produced by transcription of rDNA was connected to the short arm, it seemed to be secondarily associated with the sex vesicle. Non-transcribed ribosomal genes, which did not form a nucleolus, were revealed by in situ hybridization. Autoradiographs showed the rDNA-containing short arm of acrocentric bivalents associated with the sex vesicle in 18% of spermatocytes. The difference with the frequency of nucleolus-XY pair association was partially explained by the presence of inactive ribosomal genes. Moreover, electron microscopy showed that the dimensions of the newly formed nucleoli at early zygotene did not exceed 0.5 μm; they can be missed in light microscope investigations. From early zygotene to late pachytene, close relationships were observed between the sex vesicle chromatin and that of the associated acrocentric bivalent, especially in the short arm region. These relationships might explain the frequent involvement of acrocentrics in Y-autosome and X-autosome translocations occurring during male meiosis.

Sex vesicle-associated nucleolar organizers in mouse spermatocytes: localization, structure, and function

Selective silver staining demonstrated that autosomal bivalents containing transcriptively active nucleolar organizers associated with the sex vesicle during pachytene of mouse spermatocytes. Later in pachytene, the nucleolar organizers covered the portion of the sex vesicle furthest from the attachment to the nuclear envelope. Hybridization in situ revealed the presence of rDNA in the silver-positive material. The nucleolus, formed from an autosomal bivalent, exhibited a large fibrillar center surrounded by an electron-opaque fibrillar zone. The nucleolar association with the sex vesicle was studied at early, middle, and late pachytene by hybridization in situ, NOR silver staining, and electron microscopy. These observations enabled us to further define the relationships of the nucleolar components with the X-Y pair.

Nucleolar organizers in human oocytes at meiotic prophase I, studied by the silver-NOR method and electron microscopy

SummaryUse of the silver-NOR method to study the nucleolar organizers in human oocytes demonstrates that topographic and quantitative variations occur during meiotic prophase. In the oogonia nucleolus the nucleolar organizers are dispersed, whereas beginning at leptotene and throughout the remaining stages of meiotic prophase they occupy a marginal position in the nucleolus. At leptotene, a modal number of seven nucleolar organizers can be observed, whereas this number falls to 2.5 at pachytene and rises to ten at diplotene, thus showing that there is intense rRNA synthesis during the latter stage of meiosis. During pachytene, one end of the bivalents containing the ribosomal cistrons is always associated with the Ag-positive zone of the nucleolus. Observation of pachytene in the electron microscope shows that the secondary constriction region of D and G bivalents is constantly associated with the fibrillar center of the nucleolus. Comparison of these two methods of investigation reveals that the silver-stained regions of the nucleolus correspond to the fibrillar centers. The latter are surrounded by a layer of electron-dense fibrils corresponding to the zone of rDNA transcription. This electron-dense layer is absent during pachytene when the nucleolus displays spontaneous segregation of its components; this absence is related to temporary arrest of rDNA transcription. The affinity of the fibrillar centers for silver-NOR staining confirms that these structures contain ribosomal cistrons. During the diplotene stage, numerous micronucleoli are formed outside the nucleolar organizers of D and G chromosomes. Most of these micronucleoli present an Ag-positive granule on one of their margins, thus indicating that they contain an actively transcribed sequence of rDNA. This observation confirms the existence of amplification of ribosomal genes in the human oocyte.

Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus

The nucleolus of the human Sertoli cell displays a spontaneous segregation of its components and has only one or 2 large fibrillar centers. The 3‐dimensional reconstruction and quantitative analysis of its components was undertaken using a Quantimet 900 image analysis system in order to define the spatial relationships between the dense fibrillar component and the fibrillar center and especially to investigate whether threads of dense fibrillar component exist independently, without being linked to a fibrillar center. Our 3D reconstructions demonstrated that the dense fibrillar threads or sheets were never independent of fibrillar centers. These structures belonged to a continuous network that joined the layer of dense fibrils surrounding the fibrillar center. When the nucleolus contained 2 different‐sized fibrillar centers, quantitative analysis showed that there was a proportional relationship between the volume of the dense fibrillar component and the volume of the fibrillar center. These data, compared with those previously obtained by means of autoradiographic techniques, suggest that the rDNA‐containing chromatin passes through the fibrillar center and unwinds from there into the dense fibrillar component.

Meiosis of trisomy 21 in the human pachytene oocyte

Association modalities of the three 21 chromosomes were studied during pachytene in three trisomy 21 fetuses whose chromosomal constitution was identified following amniocentesis. — Three classes of images were observed: a trivalent, a trivalent presenting an important asynaptic region of the long arm, and a bivalent accompanied by a univalent. Such behaviour is analagous to that observed in all trisomic organisms. — We have been able to establish the sequence of chromomeres, whose number varies from 9 to 14 according to the state of contraction in the 21 chromosome. Each band is thus subdivided into several sub-bands: at maximal elongation 2 sub-bands for band p11, 4 for q21 and 3 for q222. In addition, the interchromomeric clear bands q221 and q223 are also subdivided by the presence of a very small chromomere. In this way, the G-bands visible on mitotic metaphase chromosomes result from the compression together of several chromomeres whose individuality disappears as chromosomal condensation increases with progression of prophase.

Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XY-autosome relationships

SummaryA study of XY-autosome relationships at the pachytene stage in an infertile 17–21 reciprocal translocation carrier was undertaken by means of three dimensional reconstruction. Synaptonemal complexes and the sex vesicle were analysed on electron microscopic serial sections and the reconstruction was performed on transparent sheets and on a Samba 2000 (Alcatel TITN) image analysis system. All asynapsed segments were entirely included in the sex vesicle, the chromatin fibre of the autosomes and sex chromosomes being tightly intermingled. In one nucleus, the four arms of the quadrivalent were paired, except around the breakpoints where an interstitial asynapsis was observed. In the other nuclei, a terminal asynapsis involving one or two arms of the quadrivalent was found. In the sex vesicle, autosomal asynapsed segments showed the same morphological characteristics as those of X and Y chromosomes. This observation agrees with the hypothesis of the extension of gene inactivation from sex chromosomes to autosomes.

Nucleolus, nucleolar chromosomes, and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

SummaryThe shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of3H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of3H-uridine uptake showed that the oocyte was not at a “resting” stage. The possible cytogenetic consequences of these observations are discussed.

Random acrocentric bivalent associations in human pachytene spermatocytes

Acrocentric bivalent associations were studied in 232 human male germ cells at pachytene in order to understand better the preferential involvement of chromosomes 13, 14, and 21 in Robertsonian translocations. The tendency of each acrocentric bivalent to associate with another was not correlated with NOR activity, as measured by silver staining. Good agreement was noticed between their ability to associate and the amount of satellite DNA in human acrocentric chromosomes. The distribution of two-by-two acrocentric bivalent associations was random. In order to reconcile this result with the nonrandom distribution of Robertsonian translocations, a molecular hypothesis is proposed. The model is based on homology of recombinational sites, interspersed at regular interval in satellite DNA, which could increase the probability of accidental unequal crossing-over between two specific acrocentric chromosomes.

Sequential changes in the nucleoli of human spermatogonia with special reference to rDNA location and transcription.

The nucleoli of human spermatogonia were studied using electron microscopy, silver staining, radioautography and in situ hybridization. In all types of A spermatogonia, nucleoli were consistently located at the periphery of the nucleus and contained a single fibrillar center associated with the nuclear envelope. In B spermatogonia, nucleoli were centrally located in the nuclei and showed several fibrillar centers or were found to disintegrate. Nucleolar morphology was found to be a good, though not an unequivocal indicator of spermatogonial type. The observed changes in nucleolar morphology reflect the differentiation of spermatogonia: the nucleolar disintegration seen in B spermatogonia corresponds to a pre-leptotene cessation of rDNA transcription. In radioautographs following 3H-uridine uptake, the label was consistently found over the dense fibrillar component, except in the B spermatogonia with disintegrating nucleoli, where no uptake could be detected. In situ hybridization demonstrated that the distribution of rDNA did not correspond to the site of the fibrillar center but to the dense fibrillar component. Compared with radioautographs, this finding clearly established that transcribed units of rDNA were located in the dense fibrillar component. Silver staining was strongly positive in fibrillar centers and in the dense fibrillar component. In Ap spermatogonia the silver deposit was often localized at the edge of the fibrillar threads. The relationships between silver-stained proteins and transcribed and nontranscribed portions of ribosomal genes are reevaluated.