Patterns of activity of nucleolar organizer regions during spermatogenesis and oogenesis in Kalotermes flavicollis Fabr. (Insecta: Isoptera) analyzed by silver staining

The distribution and the behaviour of the nucleolus organizer regions (NORs) were analysed during the spermatogenesis and oogenesis of K. flavicollis with the silver staining method. The Ag-stainability of the NORs increases in growing spermatocytes up to pachytene and is absent during the remainder of the meiotic prophase. During female meiosis the nucleolar material undergoes a more complex transformation. It is active until pachytene; in early diplotene the mass of silver stainable material progressively increases as an effect of rDNA amplification. By the end of meiotic prophase the nucleolar strands disappear and a large nucleolus is rebuilt in the mature oocyte.     

Multiplication of nucleolar fibrillar centres and absence of rDNA amplification in mouse ooctye during meiotic prophase I

During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of ‘nucleolonema’ type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97–113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitatative analysis of autoradiographs after hybridization in situ with -³H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).     

Cellular organization in the synganglion of the mite Macrocheles muscaedomesticae (Acarina: Macrochelidae)

Summary A large DNA containing body is found in oocytes of the house cricket, Acheta domesticus. Little or no RNA synthesis is associated with the DNA body during the leptotene, zygotene, and pachytene stages of meiotic prophase I. During the early diplotene stage of development, large masses of nucleolar material begin to accumulate at the periphery of the DNA body. The onset of RNA synthesis correlates with a change in the histochemically detectable histone proteins associated with the DNA body. In ovaries of animals injected with uridine-H³, most of the label accumulates in ribosomal RNA. Autoradiographic studies show that the cytoplasm of late diplotene stage cells accumulates uridine label to a greater extent than does the cytoplasm of early diplotene stage cells. Increased transport of nucleolar material through the nuclear envelope of late diplotene stage cells accounts for the increased cytoplasmic labeling.This investigation was supported by PHS Research Grant No. GM 16440 from the Institute of General Medical Sciences, and by Grants No. L-16 and J-1 from the Health Research and Services Foundation.The authors gratefully acknowledge the technical assistance of Mrs. Marcia Andrews and Miss Celeste Malinoski.     

LOCALIZATION OF RIBOSOMAL DNA WITHIN OOCYTES OF THE HOUSE CRICKET, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.     

Ovarian nests in cultured females of the Siberian sturgeon Acipenser baerii (Chondrostei,Acipenseriformes)

Ovaries of Acipenser baerii are of an alimentary type and probably are meroistic. They contain ovarian nests, individual follicles, inner germinal ovarian epithelium, and fat tissue. Nests comprise cystoblasts, germline cysts, numerous early previtellogenic oocytes, and somatic cells. Cysts are composed of cystocytes, which are connected by intercellular bridges and are in the pachytene stage of the first meiotic prophase. They contain bivalents, finely granular, medium electron dense material, and nucleoli in the nucleoplasm. Many cystocytes degenerate. Oocytes differ in size and structure. Most oocytes are in the pachytene and early diplotene stages and are referred to as the PACH oocytes. Oocytes in more advanced diplotene stage are referred to as the DIP oocytes. Nuclei in the PACH oocytes contain bivalents and irregularly shaped accumulation of DNA (DNA‐body), most probably corresponding to the rDNA‐body. The DNA‐body is composed of loose, fine granular material, and comprises multiple nucleoli. At peripheries, it is fragmented into blocks that remain in contact with the inner nuclear membrane. In the ooplasm, there is the rough endoplasmic reticulum, Golgi complexes, free ribosomes, complexes of mitochondria with cement, fine fibrillar material containing granules, and lipid droplets. The organelles and material of nuclear origin form a distinct accumulation (a granular ooplasm) in the vicinity of the nucleus. Some of the PACH oocytes are surrounded by flat somatic cells. There are lampbrush chromosomes and multiple nucleoli present (early diplotene stage) in the nucleoplasm. These PACH oocytes and neighboring somatic cells have initiated the formation of ovarian follicles. The remaining PACH oocytes transform to the DIP oocytes. The DIP oocytes contain lampbrush chromosomes and a DNA‐body is absent in nuclei. Multiple nucleoli are numerous in the nucleoplasm and granular ooplasm is present at the vegetal region of the oocyte.     

A timing study of DNA amplification in Xenopus laevis oocytes

The time course of meiotic amplification of nucleolar DNA in Xenopus laevis oocytes has been studied autoradiographically. We find that the process is first detectable in zygotene nuclei less than 7 days after the end of premeiotic S-phase. It is completed 3 1/2 weeks later, towards the end of pachytene. Premeiotic S-phase lasts for 1–2 weeks. We are not certain whether it is followed by a short G2 or whether leptotene commences immediately. Leptotene lasts for 5±2 days, zygotene for 7±2 days and pachytene for about 20 days before the oocyte gradually enters the extended diplotene stage. Various molecular mechanisms for amplification are discussed in the light of a 24±3 day amplification time. All are found to be potentially capable of amplifying sufficient nucleolar DNA in the time available.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

Nucleolar DNA in oocytes of crickets: representatives of the subfamilies Oecanthinae and Gryllotalpinae (Orthoptera: Gryllidae)

A large extrachromosomal mass of Feulgen positive material, the DNA body, has been visualized in early prophase oocytes of crickets (Orthoptera: Gryllidae) representative of the closely related subfamilies Gryllinae and Nemobiinae. A similar structure is present in oocytes of representatives of two subfamilies of crickets (subfamilies Oecanthinae and Gryllotalpinae) which taxonomically and phylogenetically are quite separate from those mentioned previously. In situ hybridization demonstrates that the body contains amplified copies of genes coding for ribosomal RNA. Unlike the DNA body in early diplotene oocytes of representatives of the subfamily Gryllinae, which is closely associated with the developing nucleolar apparatus, the DNA body in oocytes of the Oecanthinae and Gryllotalpinae cannot be demonstrated during diplotene. In the Oecanthinae, the nucleolar apparatus of early diplotene stage oocytes is composed of four to seven separate structures, the ribonucleoprotein of which has a characteristically lamellated appearance. During late diplotene, these nucleoli give rise to many smaller structures which are distributed throughout the germinal vesicle. In early diplotene stage oocytes of Scapteriscus acletus (Subfamily: Gryllotalpinae), the nucleolar apparatus consists of a single compact mass of ribonucleoprotein. In contrast to the oocytes of all other crickets that have been studied, the nucleolus of S. acletus remains single throughout diplotene. In situ hybridization analysis indicates that the amplified genes coding for rRNA which are localized in the DNA body of early prophase oocytes become incorporated into this compact nucleolar mass. Differences in nucleolar structure appear to reflect differences in the organization of amplified genes coding for rRNA.     

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.     

Nucleolar DNA in oocytes of Salmo irideus (Gibbons)

The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 g. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus ( = 1.731 g cm^–3) is 1.715 g cm^–3 and corresponds to a G + C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA.Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.     

Nucleolar organization in oocytes of gryllid crickets: subfamilies Gryllinae and Nemobiinae

A cytological and cytochemical survey was made of nucleolar changes during oocyte development in several different species of crickets (Gryllidae) representing the subfamilies Gryllinae and Nemobiinae. A large mass of extrachromosomal DNA is characteristic of the pachytene stage nuclei of all species examined. Nucleolar material accumulates at the periphery of the DNA body as the cells proceed into the diplotene stage of development. As the oocytes proceed through diplotene, the nucleoli reorganize into many small masses which eventually disperse in the nucleoplasm. These changes reflect both an increase in number and in size of the nucleolar material during the diplotene stage and the mode by which dispersal of nucleolar material is accomplished. These differences probably reflect differences in the organization of extrachromosomal nucleolar DNA.     

Incorporation of H3-thymidine into the oocytes of a sterlet in the early meiotic prophase]

DNA synthesis in meiotic oocytes of the sterlet (A. ruthenus) has been studied during early prophase stages using H3-thymidine. The pattern of H3-thymidine incorporation is similar to that in oocytes of Amphibia and Osteichthyes. In the oogonia as well as in the leptotene and zygotene oocytes, the label is predominantly localized over chromosomes. An intensive incorporation of H3-thymidine into the material of the heterochromatic "cap" has been observed during pachytene. Thus, the main synthesis of extra DNA in the sterlet oocytes occurs during pachytene. No DNA in synthesized by the diplotene oocytes.     

Duration of meiotic prophase stages during oogenesis in the newt Triturus cristatus cristatus]

The duration of the early stages of meiotic prophase was determined in the oogenesis of T. cristatus cristatus by means of autoradiography. The oocytes were being investigated during 39 days from the moment of 3H-thymidine injection. It was shown that preleptotene lasts 1--2, leptotene ca. 4, zygotene 5 and pachytene 26 days. When studying the preparations obtained 1 day after the injection of 3H-thymidine, the silver grains were found to be localized over the nuclei at all stages of meiotic prophase; this suggests the amplification of rDNA which begins in leptotene-zygotene and ends in early diplotene.     

Anti-topoisomerase II recognizes meiotic chromosome cores

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II — a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation — is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.     

DNA accumulation during oogenesis in the free-living nematode Panagrellus silusiae

DNA contents of nuclei at different stages of gonadogenesis, gametogenesis and embryogenesis in the free-living nematode Panagrellus silusiae were measured by Feulgen microspectrophotometry. Somatic nuclei of gonadal tissue in both males and females have DNA values that range from 2C to 4C. Relative DNA values identified meiotic processes during spermatogenesis. In the adult female, developing oocytes amass an amount of nuclear DNA in excess of the expected 4C equivalent. The chromatin of these nuclei is diffuse with varyingsized clumps of Feulgen-positive material. Throughout oogenesis maturing oocytes remain uninucleolate. — The extra oocyte DNA is distributed proportionately at each of the first four cleavage divisions; thereafter, its presence is not resolvable. — A standardized microphotometric comparison indicated that the amount of DNA in the unit genome of P. silusiae is about 0.1 pg. The oviduct oocytes accumulate about 3.6 pg DNA in excess of a 4C equivalent. The kind of DNA that builds up during oogenesis in this organism is not known.     

Multiple synaptinemal complexes at the region of gene amplification in Acheta

Ovaries of Acheta domesticus (house cricket) were fixed for electron microscopy at two stages of development: (1) ovaries containing mainly oocytes at interphase and early prophase of meiosis, and (2) ovaries with oocytes mainly at pachytene and diplotene. The E.M. study was accompanied by three types of light microscopy controls consisting mainly of cytochemical tests. Every oocyte contains a DNA body which at pachytene and diplotene acquires the appearance of a puff. In the light microscope two zones can be distinguished inside the body: (1) an inner core of DNA and (2) and outer shell of RNA. In the E. M. the inner core consists of a fibrillar material and the outer shell is composed of areas of high electron opacity consisting mainly of tightly packed particles and fibrils. At these stages synaptinemal complexes are seldom seen associated with the DNA body but are present throughout the nucleus as part of the paired chromosomes. The complexes are present as single units. — In the oocytes at interphase and early prophase of meiosis, where the DNA body is active in DNA replication, the body appears in the light microscope as a large Feulgen positive sphere containing Feulgen negative areas. In the E. M. at these stages the DNA body consists of: (a) the two components found at pachytene, (b) a third electron dense component which is more homogeneous than the other two, and (c) of large assemblies of synaptinemal complexes originating from several centers. The most significant features of the axial complexes are: (1) the circular packing of the complexes, (2) their occurrence in packages of 300 to 400 units and (3) the fact that not all of the DNA body forms complexes but only a part of it.Biochemical experiments (Lima-de-Faria, Birnstiel and Jaworska, 1969) have demonstrated the amplification of ribosomal cistrons in the DNA body of Acheta. The simplest explanation is that the multiple complexes are formed either between the extra gene copies of the two homologues, or between the extra copies of each chromosome as well. There seems to be a correlation between the presence of multiple axial complex formation and gene amplification in Acheta but the exact relation between the two phenomena demands further study.Dedicated to Dr. Sally Hughes-Schrader on the occasion of the seventyfifth birthday on the twentyfifth of January 1970.     

Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes

Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyl-transferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.