Abstract
In egg-laying fish, survival of the embryonic and initial larval stages depends on accumulation of substantial reserves of yolk and lipid within the egg during oocyte growth. In sturgeon, a non-teleost basal ray-finned fish, the relationship between lipid physiology and oogenesis has not been investigated. Gonadal development is arrested in the early stages of maturation. However, androgens have been implicated in regulation of early follicular growth in several vertebrate classes. Considering such gaps in the literature, this thesis, therefore, aimed to elucidate key physiological and molecular changes associated with lipid biology during oocyte development in beluga, or great sturgeon. Moreover, the effects of 11-ketotestosterone treatment on ovarian growth and mRNA levels of putative lipidation-related genes were investigated in pre-vitellogenic (PV) ovaries of sturgeon.
To do so, as a first attempt, the developmental stages of oocytes from captive beluga were classified through an analysis of general appearance of intact oocytes after non-lethal biopsy techniques which was followed by histological observations (Chapter 2). The different stages of oocyte growth were standardized based on morphological criteria. As such, ovaries of captive beluga were classified as being in the perinucleolar (PN), oil droplet (OD), primary yolk (PY), secondary yolk (SY) or tertiary yolk (TY) stage.
To focus on sturgeon lipid physiology, an investigation was conducted to determine changes in lipid transport, concentration of sex steroids, and expression of genes that have been implicated in ovarian lipid uptake during the reproductive cycle (Chapter, 3). The lipoprotein profile changes were determined using fast protein liquid chromatography (FPLC). Also, the triglycerides (TGs), and cholesterol changes in the FPLC extracted fractions of lipoprotein profiles were determined. Plasma levels of sex steroid hormones (17β-estradiol (E2) and 11-ketotestosterone (11-KT)) and lipid (TGs, cholesterol and phospholipid) were elevated during gonadal development. Using gel filtration, low levels of TGs and cholesterol were observed in different lipoprotein classes across different stages of oogenesis, but no differences were found. Albumin, vitellogenin (Vtg) AB2b, immunoglobulin light chain precursor and immunoglobulin heavy chain were detected as components of different lipoprotein fractions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). The expression of housekeeping genes was significantly affected by gonadal development. Also, a high abundance of 5S ribosomal RNA transcripts was detected in early stages of gonadal development. In this study the geometric means of ribosomal RNA (5S, 18S and 28S rRNA) was used to normalize the transcript abundance of putative lipidation-related genes. The transcript abundances of lipoprotein lipase (lpl), and apolipoprotein e (apoe) and lipoprotein receptors, i.e., very-low density lipoprotein receptors (vldlr), and low-density lipoprotein receptor-related protein 8-like (lrp8), increased in the early stages of oogenesis (from PN to OD) and decreased thereafter.
To get insights into the association between Lpl, which is a central enzyme in TG metabolism, and sturgeon lipid physiology (Chapter 4), the full-length beluga lpl cDNA encoding a 501 residue (amino acid) protein was isolated. Phylogenetic analysis indicates a monophyletic ancestry of Lpl from beluga with other sturgeon, tetrapods and Latimeria, rather than with modern teleost fish. Phylogenetic analysis indicated that LPL protein is highly conserved during vertebrate evolution. A specific sturgeon polyclonal antibody against bacterially expressed beluga Lpl was successfully developed. The mRNA abundance of lpl by quantitative real-time polymerase chain reaction, identification of the Lpl protein in different tissues by Western blotting and localisation of Lpl in ovarian tissue (immunohistochemistry) was achieved by employing contemporary molecular and biochemical techniques. The lpl mRNA and protein was present in almost all assayed tissues. A higher expression of lpl was detected in liver and heart which would suggest that these tissues synthesize Lpl. Three different-sized proteins, representing putative mature (58 kDa), glycosylated (70 kDa) and broken down fragments of Lpl (38 kDa), were detected in ovarian tissues. The 70 kDa band was also found in liver, intestine, heart and spleen. The immunohistochemistry results showed positive specific staining in the follicle cells surrounding the oocyte.
A positive effect of 11-KT on oocyte growth, facilitating lipid uptake during the early stages of reproduction, has been reported in some fish species. Therefore, to evaluate the effects of 11-KT on ovarian growth of beluga with pre-vitellogenic oocytes, an in vivo 11-KT implant study was conducted (Chapter 5). The results confirmed that 11-KT increased the levels of serum 11-KT, associated with increased oocyte diameters and increased expression of lipidation-related transcripts three weeks after onset of treatment. However, serum E2, TGs and cholesterol levels, ovarian yolk uptake, and ovarian reproductive stage remained unchanged.
To further explore the role of different hormones on the expression of lipidation-related transcripts, a short-term (24 hour) in vitro explant study was conducted, using PV ovarian tissue (Chapter 6). The results indicate that follicle-stimulating hormone, 11-KT, and to some extent E2 are important factors in the regulation of genes believed to be involved in ovarian lipid uptake and transport.
Together, this thesis provide molecular, hormonal and biochemical information on non-teleost ray-finned fish during gonadal development. The research advance the understanding of ovarian pre-vitellogenic growth, focusing on ovarian lipid physiology in sturgeon. The low levels of TGs and cholesterol within different lipoprotein classes and the presence of albumin and vitellogenin AB2b in lipoproteins suggest that lipid physiology in sturgeon may be different from that in some model teleost fish. The transcript abundances of putative lipidation-related genes indicate that these genes were highly expressed at pre-vitellogenic stages, before lipid accumulation into oocytes. This pattern of expression implies that the oocytes need to be fully prepared during early stages of oogenesis before transition into the vitellogenesis growth stages. Although 11-KT increased the oocyte diameter in pre-vitellogenic beluga, it is still to be elucidated whether this effect is direct or indirect. The increase in lpl mRNA expression following both in vivo and in vitro 11-KT treatment shows that the effect of 11-KT in early ovarian growth is likely mediated to some extent by lpl.