A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

Identification of Early Oogenetic Cells in the Solitary Ascidians, Ciona savignyi and Ciona intestinalis: An Immunoelectron Microscopic Study

We raised monoclonal antibodies against homogenates of ovaries of Ciona intestinalis . We obtained an antibody named GC-1 which specifically recognized early germ cells in C. intestinalis and C. savignyi . Using GC-1 as a marker in immunoelectron microscopy, we determined the morphological sequence of early oogenesis in the ovaries of Ciona . In the stratified epithelium composing the wall of the ovarian tubes, the oocytes were identifiable at the early stages of meiotic prophase according to nuclear features such as condensed chromatin with synaptonemal complexes. GC-1 recognized these early oocytes. We found round cells with large and homogeneous nuclei clustered at the marginal end of the stratified epithelium. We identified these cells as oogonia on the basis of: (1) features of the nucleus, (2) reactivity to GC-1, and (3) early emergence in the developing ovaries. The oogonia were classified into three types: type A was large (7–9 μm in diameter) and clear, type B was intermediate in size (5–6 μm) and electron-density, and type C was small (4–5 μm) and dark. In the developing ovaries of juvenile C. intestinalis, type A oogonia appeared first (before 11 days after settlement) and types B and C followed (15 days after settlement). Thus we see that the type A is the oogenetic stem cell, type B is the proliferating oogonium, and type C is the final oogonium just before meiosis. The oocytes appeared 18 days after metamorphosis.     

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.     

Premiers stades de l'oogenèse de Rhabdophaga saliciperda (Cecidomyiidae,Diptera)

The females of Rhabdophaga saliciperda have in their somatic cells 8 chromosomes and the males 6. The type of sex determination is therefore: X₁X₁X₂X₂—♀; X₁X₂—♂. The cells of the germinal line have 46 chromosomes, but a variation of their number was observed. In the oogonia and spermatogonia the number of heterochromatic chromosomes may exceed the number of E chromosomes, i.e. 8. In the beginning of the growth stage of the oocytes an incorporation of somatic cells was observed. The nuclei of these somatic cells persist in the cytoplasm of the oocytes until the maturation divisions. The possibility of their participation in the reconstruction of the nucleus of the mature egg is envisaged. The metaphase of the I segmentation division has a complex character. During prophase of the first meiotic division the E chromosomes form 4 bunches of 6–8 chromosomes each. Some univalents may also be present. The 8 S chromosomes form 4 regular bivalents. The 4 groups of E chromosomes persist until metaphase I. During metaphase I a phenomenon of expulsion of the majority of E chromosomes from the metaphase spindle was observed. The 4 bivalents remain in the equatorial plain of the spindle with some E Chromosomes. After this expulsion 2 groups of chromosomes are formed. In connection with them 2 spindles develop. An irregular distribution of E chromosomes follows without their division. The bivalents are probably separated in regular manner. These 2 spindles correspond to the I maturation division. The II maturation division was not observed because of lack of respective stages.     

Histological studies on early oogenesis in barfin flounder (Verasper moseri)

There is much information on oogenesis from the resumption of the first meiotic division to oocyte maturation in many vertebrates; however, there have been very few studies on early oogenesis from oogonial proliferation to the initiation of meiosis. In the present study, we investigated the histological changes during early oogenesis in barfin flounder (Verasper moseri). In fish with a total length (TL) of 50mm (TL 50mm fish), active oogonial proliferation was observed. In TL 60mm fish, oocytes with synaptonemal complexes were observed. Before the initiation of active oogonial proliferation, somatic cells which surrounded a few oogonial germ cells, started to proliferate to form the oogonial cysts that accompanied oogonial proliferation. In TL 70mm fish, however, the cyst structure of the oocyte was gradually broken by the invagination of somatic cells, and finally the oocyte became a single cell surrounded by follicle cells. Upon comparison of nuclear size, DNA-synthesizing germ cells could be divided into two types: small nuclear cells and large nuclear cells. Based on histological observation, we propose that the small nuclear cells were in the mitotic prophase of oogonia and the large nuclear cells were in the meiotic prophase of oocytes, and that the nuclear size increases upon the initiation of meiosis.     

The ultrastructure of germinal beds in the ovary of Gerrhonotus coeruleus (Reptilia: Anguidae)

A study of ovarian structure in adult Alligator Lizards (Gerrhonotus coeruleus) was conducted by light microscopy and transmission electron microscopy. Particular attention was directed to characterizing the ultrastructure of germ-line cells, prior to follicle formation. General ovarian structure in this lizard is similar to that of other lizards. The paired organs are hollow, thin-walled sacs containing follicles in roughly 3 to 4 size classes. Ovarian germinal tissue consists of oogonia (diploid cells which divide mitotically) and oocytes (meiotic cells), intermixed with ovarian surface epithelial cells. Germ cells reside in two dorsal patches of epithelium per ovary (germinal beds), as is common in lizards. Oogonia in interphase show a highly dispersed chromatin pattern. Within oogonia cytoplasm, Golgi complexes are scarce, rough endoplasmic reticulum is absent, and lipid droplets are rare. Ribosomes are scattered in small clusters. Small, round vesicles are common in all oogonia; glycogen-like granules are present in some. Mitochondria form a juxtanuclear mass within which groups of several mitochondria surround a dense granule. “Nuage” granules also are found unassociated with mitochondria. Oocytes are present in stages of meiotic prophase up to diplotene. Synaptinemal complexes are seen in several (pachytene) cells. The cytoplasm of oocytes differs from that of oogonia in that mitochondria do not form groups, and nuage and glycogen are absent, whereas small round vesicles and large irregular vesicles are common. The ultrastructural similarities in germ cells of a reptile as compared to those of other vertebrates strengthens the notion that germ-line cells possess (or lack) qualities related to the undifferentiated state of these cells.     

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.     

The role of the rete ovarii in meiosis and follicle formation in the cat, mink and ferret.

Ovarian morphology was studied from the inception of meiosis in the cat, mink and ferret. It was shown that "open connections", allowing cellular contact, existed between the intra-ovarian rete cords and the groups of germ cells as well as between the surface epithelium and the germ cells. The germ cells in the innermost part of the cortex and lying in contact with the rete cells were those which were the first to enter meiotic prophase. Later, the more peripheral oogonia transformed to oocytes. The first follicular formations occurred at the innermost part of the cortex. The granulosa cell layers were in open connection with the intra-ovarian rete cords. In the mink and ferret, a certain part of the rete system at the hilus differentiated into the hilar rete body. In all animals, the extra-ovarian rete cells were actively secreting. It is proposed that the rete system interacts with the cortex, initiating the start of meiosis and that the rete cells as well as cells of the surface epithelium contribute to the granulosa cell layer.     

Ribosomal bodies in early oogenetic stages of the lizard Lacerta sicula Raf.

During hibernation, ribosomal bodies are present in the germ cells of Lacerta sicula, which have undergone meiotic prophase, but neither in oogonia nor in stroma cells. These bodies are similar in structure, although smaller in size, to those which have been described in the cytoplasm of growing oocytes; in the germ cells at zygopachytene, they are frequently associated with the nuclear envelope.     

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.     

Details of the female and male pathway of the Keimbahn determined by enzyme histochemical and autoradiographic studies

Autoradiographic studies and the use of enzyme histochemistry have revealed that early germ line cells (female and male PGC, oogonia, prediplotene oocytes and prospermatogonia) as well as the more advanced germ cells (diplotene oocytes, spermatogonia, spermatocytes and spermatids) show specific patterns of their DNA and RNA synthesis and their enzymatic equipment. The female and male germ lines show similar kinetics up to the arise of oocytes and T prospermatogonia (T for transitional), the final products of a first limited multiplication process of primitive gonia. In former studies we supposed that oocytes and T prospermatogonia are the first exponents of the female and male pathway of the germ line (Hilscher and Hilscher, 1989a). Recently, it could be shown--using the reverse PLM method in slides of plastic embedded material--that the first differences between female and male GC can already be stated at the end of the first proliferation wave of oogonia and multiplying prospermatogonia; that means even before the existence of oocytes and T prospermatogonia (Hilscher and Hilscher, 1989b). Oogonia and M prospermatogonia (M for multiplying) are equipped both with only one active X chromosome. While oocytes traverse the prediplotene stages of meiotic prophase T prospermatogonia prepare for a second extensive proliferation process: spermatogenesis. Oocytes in meiosis are provided with two active X chromosomes, T prospermatogonia possess only one, and the presence of the Y chromosome is not vital for them. However, the Y chromosome is required for the normal course of spermatogenesis characterized by a stock of stem cells, that are responsible for the continuous production of male gamets. The mammalian oocyte--similar as that of insects and amphibia but to a lower degree--acts as pre-embryo.     

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.     

X-chromosome heteromorphism and appearance of meiosis in the rat fetal ovary

Fetal rat oogenesis was examined attempting to test the hypothesis that two functional X chromosomes are required for the onset of meiosis. The presence of a Barr body in germ cells was considered to be evidence for one inactive X chromosome and the detection of leptotene oocytes as the criterion for the establishment of meiotic prophase. It was found that on Day 16 of gestation, 3.9% of the germ cells were leptotene oocytes, but the incidence of Barr body-positive oogonia persisted at 9.9%. On Day 17, the leptotene oocytes had increased to 26.6% and the Barr body-positive oogonia had decreased to 3.5%. It was concluded that X-chromosome reactivation, though occurring at some time during the onset of meiosis, was not the initiating event.     

Activity of the ovarian germinal epithelium in the freshwater catfish,Pimelodus maculatus (Teleostei: Ostariophysi: Siluriformes): Germline cysts,follicle formation and oocyte development

Distinct types of oogonia are found in the germinal epithelium that borders the ovarian lamellae of Pimelodus maculatus: A‐undifferentiated, A‐differentiated and B‐oogonia. This is similar to the situation observed for spermatogonia in the vertebrate testis. The single A‐undifferentiated oogonia divide by mitosis giving rise to A‐groups of single differentiated oogonia, each enclosed by epithelial cells that are prefollicle cells. Subsequently, the single A‐differentiated oogonia proliferate to generate B‐oogonia that are interconnected by cytoplasmic bridges, hence, forming germline cysts. The prefollicle cells associated with them also divide. Within the germline cysts, B‐oogonia enter meiosis becoming oocytes. Meiotic prophase and early folliculogenesis occur within the germline cysts. During folliculogenesis, prefollicle cells grow between the oocytes, encompassing and individualizing each of them. The intercellular bridges disappear, and the germline cysts are broken down. Next, a basement membrane begins to form around the nascent follicle, separating an oocyte and its associated prefollicle cells from the cell nest. Folliculogenesis is completed when the oocyte and the now follicle cells are totally encompassed by a basement membrane. Cells derived from the ovarian stroma encompass the newly‐formed ovarian follicle, and become the theca, thereby completing the formation of the follicle complex. Follicle complexes remain attached to the germinal epithelium as they share a portion of basement membrane. This attachment site is where the oocyte is released during ovulation. The postovulatory follicle complex is continuous with the germinal epithelium as both are supported by a continuous basement membrane. The findings in P. maculatus reinforce the hypothesis that ovarian follicle formation represents a conserved process throughout vertebrate evolution. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

The cytoskeleton organizes germ nuclei with divergent fates and asynchronous cycles in a common cytoplasm during oogenesis in the chordate Oikopleura

Germline cysts are conserved structures in which cells initiating meiosis are interconnected by ring canals. In many species, the cyst phase is of limited duration, but the chordate, Oikopleura, maintains it throughout prophase I as a unique cell, the coenocyst. We show that despite sharing one common cytoplasm with meiotic and nurse nuclei evenly distributed in a 1:1 ratio, both entry into meiosis and subsequent endocycles of nurse nuclei were asynchronous. Coenocyst cytoskeletal elements played central roles as oogenesis progressed from a syncytial state of indistinguishable germ nuclei, to a final arrangement where the common cytoplasm had been equally partitioned into resolved, mature oocytes. During chromosomal bouquet formation in zygotene, nuclear pore complexes clustered and anchored meiotic nuclei to the coenocyst F-actin network opposite ring canals, polarizing oocytes early in prophase I. F-actin synthesis was required for oocyte growth but movement of cytoplasmic organelles into oocytes did not require cargo transport along colchicine-sensitive microtubules. Instead, microtubules maintained nurse nuclei on the F-actin scaffold and prevented their entry into growing oocytes. Finally, it was possible to both decouple meiotic progression from cellular mechanisms governing oocyte growth, and to advance the timing of oocyte growth in response to external cues.     

Ultrastructural localization of acid phosphatase in germ cells of chick embryo left ovary

The ultracytochemical localization of acid phosphatase was studied in oogonia and oocytes of the chick embryo left ovary. The reaction products are evident in lysosomes of various types and, in some cells, in the GERL as well. Furthermore, from the onset of the meiotic prophase, the enzymatic reaction also appears in the rough endoplasmic reticulum. Non-incubated sections of the same stages were observed, with the aim of identifying and describing the structure of the organelles, in particular lysosomes which appeared positive in incubated sections. The significance of the presence of the enzyme is discussed.     

Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish

Recent studies in mammals have revealed the heterogeneity of spermatogonial populations which contain differentiated and undifferentiated cells that further divide into actual stem cells and potential stem cells. In fish however, there are no functional definitions, and very few molecular markers, for germ cells. In our present study, specific antibodies were raised against Sycp3, Plzf and Cyclin B3 in zebrafish and then used to determine the localization of these proteins in the testis. We wished to confirm whether these molecules were potential markers for spermatocytes and spermatogonia. Immunohistochemical observations revealed that Sycp3 is specifically localized in spermatocytes in typical nuclear patterns at each meiotic stage. Plzf was found to be localized in the nucleus of both type A and type B spermatogonia until the 8-cell clone, similar to the pattern in Plzf-positive A(single)-A(aligned) undifferentiated spermatogonia in rodents. In addition to Plzf, the localization of Cyclin B3 was predominantly detected in the nuclei of type A and early type B spermatogonia until the 16-cell clone. Additionally, Cyclin B3 protein signals were detected in germ cells in large cysts, possibly corresponding to spermatocytes at the preleptotene stage. Our present data thus show that these molecules have properties that will enable their use as markers of spermatocytes and early spermatogonia in zebrafish.     

Effects of dimethyl sulphoxide on early gametogenesis in Caenorhabditis elegans: ultrastructural aberrations and loss of synaptonemal complexes from pachytene nuclei

In Caenorhabditis elegans, loss of viability and fertility was observed after treatment with dimethyl sulphoxide (DMSO). The decrease in life span is associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens cannot be differentiated. Aging in oocytes at the pachytene stage of meiotic prophase is characterized by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm and decrease in numbers of mitochondria (Goldstein and Curis, 1987). Increasing concentrations of DMSO result in decrease in fertility and increased production of abnormal gametes. At DMSO concentrations higher than 5.0%, synaptonemal comlexes (SC) are absent from the nuclei, thus, effective pairing and segregation of homologous chromosomes is not possible. The absence of SCs may be the result of: (1) a premeiotic colchicine-like effect which influences pairing of chromosomes; (2) changes in the structure of the DNA due to DMSO binding that results in changes in expression of the DNA; and (3) changes in temporal DNA synthesis in response to DMSO. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DMSO treated specimens.