Oogenesis in the development of Zebrafish (Danio rerio)

Abstract

One of the central issues in developmental biology concerns the molecular mechanism whereby a multipotent cell give rise to a distinct progeny. This is evidenced by the specific expression of sub-sets of proteins in some cells and their absence in others which reflects variations in their pattern of gene expression. Recently an increasing number of genes that are expressed exclusively during oogenesis and that support fertilization and early zygotic development have been described. The early stages of germ cell development are marked by distinct developmental transitions which must be transversed in order for the primary germ cells to form mature gametes. How these morphological changes take place within the embryo at each developmental transition remains a poorly understood phenomenon. The purpose of this research was to provide a detailed account of the early events of zebrafish gonadogenesis and where possible to establish molecular markers that may help define the developmental phases of oogenesis, such as the mitotic/meiotic transition. <br><br> The main objectives were: (1) to describe the early stages of gonadal development in zebrafishand compare the juvenile events with the adult's known cycle of oogenesis; (2) to determine the molecular mechanisms underlying sex change in the early ovary; (3) to determine the molecular mechanisms controlling early development focusing primarily on the cytoskeletal component the germ cells have adapted to promote gonadal development and the DNA and RNA component; and (4) to study retrospectively the effect of exogenous L- thyroxine on gonadal development. <br><br> In the first study, early gonadal development was examined in fish selected from hatchery tanks at different stages of development by using histological, semi-thin serial sections and TEM. The histological data presented support the evidence that zebrafish is an asynchronous spawner, reproducing over short intervals. Primordial germ cells (PGCs) were first identified at 8 days post fertilization (dpf) and at 13-dpf gonad strings were completed and formed a typical shape in cross section. Some of the PGCs underwent mitotic divisions at 16 dpf. The ovarian cavity was seen in fish at 22 dpf at a time when germ cells were still considered to be undifferentiated. Mitotic-meiotic transition in juvenile zebrafish is a gradual process and occurs after 22 dpf and gonads of all juveniles differentiated as ovaries irrespective of their genetically determined sex. After 40 dpf, female gonads contained lobes with germ cells, including oogonia, post-pachytene and early pre-vitellogenic oocytes whereas in the other type of gonad, degenerative post pachytene oocytes, spermatogonia and cells undergoing mitosis were observed. The findings suggest that the strongest indicator of sex change in juvenile zebrafish is the occurrence of transitional individuals whose gonads contain degenerative ovarian tissues and developing testicular tissues. After 50 dpf, gonads could be distinguished clearly as immature testes or immature ovaries. <br><br> Observations indicate that in zebrafish gonads sex differentiation took place in seven stages characterized by specific germ cell composition. The PGCs remained in a resting stage for up to 2 weeks post fertilization with gametogenesis occurring earlier in females than in males. The gonads developed from an indifferent stage directly into an ovary first and then later into either the ovary or the testis. As a whole, the juvenile ovary consists of primary oocytes and is characterized by a low reproductive status. The adult ovary on the other hand contains primary oocytes, vitellogenic oocytes and mature oocytes and is characterized by a high reproductive status. However, the cellular events and developmental changes taking place within a germ cell as it progresses from one developmental stage to the other were found to be similar in both phases of ovarian growth. <br><br> The second study examined the issue of sex change in juvenile zebrafish as an apoptotic process, the TUNEL method being used to study apoptosis. Histological sections of Paraplast- embedded sections of juvenile fish prior to and during the sex inversion stage of development were labelled by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end- labelling (TUNEL) method. In work arising from the use of the TUNEL method to study apoptosis, it was found that most germ cells during the sex inversion period demonstrated a significant level of oocyte degeneration histologically but were not TUNEL-positive. The TUNEL analyses, however, revealed that in zebrafish pre-vitellogenic and vitellogenic oocytes, thecal/epithelial cells showed fragmented DNA, suggesting that apoptosis is involved in normal ovarian growth in zebrafish, or that the state of DNA seen in cells during zebrafish oogenesis produces breakpoint that respond to TUNEL labeling. <br><br> In juvenile zebrafish gonads the initiation of oogenesis is marked by the changes in the nuclear structures and begins with the pairing and condensation of homologous chromosomes in the first meiotic division (leptotene-zygotene). At pachytene, the homologous condensing chromosomes form synaptonemal complexes. Sooner or later, oocytes are arrested in the lampbrush (diplotene) stage of growth, which is accompanied by cytoplasmic RNA deposition. <br><br> Based on this hypothetical model an immunohistochemical study was carried out using different markers (a monoclonal anti-spectrin antibody, acridine orange and propidium iodide) to verify the staining pattern and any relationship between their expression and the successive phases of oocyte development. The third thematic line of this thesis was to find out whether spectrin could be used as a marker to identify a specific stage in oogenesis. Immunohistochemistry with anti spectrin antibody revealed that spectrin labelling was widespread in tissue sections but localized in the cortex of oogonia and post-pachytene oocytes. Interestingly, pre-vitellogenic and early vitellogenic oocytes showed a remarkable cytoplasmic immunoreactivity for spectrin. Immunoreactivity for spectrin was also seen on the surface of cortical alveoli as well as on that of the nucleoli and the Balbiani's body. These results suggest that spectrin or spectrin related proteins are involved in the architectural changes that accompany oocyte development during the vitellogenetic growth phase. <br><br> The fourth approach of the study was to performed experiments in order to find substantive cytochemical markers to identify specific stages of early gonadal development, focusing primarily on the DNA and RNA component of the germ cells so as to resolve the steps of oogenesis such as the phases of mitotic-meiotic transition. Staining sections with acridine orange and propidium revealed a widespread pattern of staining in a variety of tissues including germ cells. With respect to the organization and the state of the chromatin, it was evident that the low staining seen with both dyes was associated with the diffuse state of the chromatin that occurs prior to the thickening of chromosomes from the pachytene stages of oogenesis. The accumulation of cytoplasmic RNA in diplotene stages of development is coincident with the formation of multiple nucleoli in the nucleus. The pattern of synthesis and appearance of the nucleoli during zebrafish oogenesis is stage specific and may be used to identify the stage of oogenesis. <br><br> The final study examined retrospectively the influence of exogenous thyroxine on developmental events in gonads of juvenile fish. Immersion offish in solutions of thyroxine resulted in high mortality rates and deformity of fish. Furthermore, immersion of fish in solutions of thyroxine and thyroxine with potassium perchlorate resulted in impaired gonadal development making impossible to tell whether exogenous thyroxine promotes growth and differentiation in zebrafish.